WO2008074154A1 - Polymers comprising zwitterionic endgroups - Google Patents

Polymers comprising zwitterionic endgroups Download PDF

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Publication number
WO2008074154A1
WO2008074154A1 PCT/CA2007/002337 CA2007002337W WO2008074154A1 WO 2008074154 A1 WO2008074154 A1 WO 2008074154A1 CA 2007002337 W CA2007002337 W CA 2007002337W WO 2008074154 A1 WO2008074154 A1 WO 2008074154A1
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polymer
polymers
formula
cell
group
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PCT/CA2007/002337
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French (fr)
Inventor
Donald E. Brooks
Rajesh K. Kainthan
Jayachandran N. Kizhakkedathu
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The University Of British Columbia
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Publication of WO2008074154A1 publication Critical patent/WO2008074154A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/605—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the macromolecule containing phosphorus in the main chain, e.g. poly-phosphazene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/33306—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group acyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/334—Polymers modified by chemical after-treatment with organic compounds containing sulfur
    • C08G65/3344—Polymers modified by chemical after-treatment with organic compounds containing sulfur containing oxygen in addition to sulfur
    • C08G65/3346—Polymers modified by chemical after-treatment with organic compounds containing sulfur containing oxygen in addition to sulfur having sulfur bound to carbon and oxygen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/335—Polymers modified by chemical after-treatment with organic compounds containing phosphorus
    • C08G65/3353—Polymers modified by chemical after-treatment with organic compounds containing phosphorus containing oxygen in addition to phosphorus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/005—Hyperbranched macromolecules

Definitions

  • the present invention relates to novel zwitterionic polymers, and methods of making such polymers.
  • Biomedical uses of polymers may include coatings on implants or pills, encapsulation and delivery of drugs over time or under particular conditions such as pH, manufacturing of implants and other uses known in the art.
  • the development of some medical devices and implantable devices places mechanical and engineering qualities foremost, with the biocompatibility of such devices and items of secondary consideration.
  • Some plastics and polymers are relatively inert and are tolerated, but these may only be a partial component of a device. Re-engineering of the entire device to incorporate more preferable materials may not be an option.
  • the surfaces of the existing device may be coated with a suitable polymer. It may be desirable to prevent cell adhesion, or to encourage the engraftment of cells on the implant. A variety of surface- modification techniques, materials and biocompatible polymers have been developed to prevent or encourage attachment of cells.
  • ECM extracellular matrix
  • integrin binding proteins proteins that may affect immune response, inflammatory response and other systemic responses. Removing the cells may be difficult, and may damage the cells in the process.
  • Phospholipid polymers for use as biomaterials is described by Hayward et al 1985. Ann. NY Acad Sci 446:267-81.
  • PCT Patent Application WO 92/06719 discloses processes for coating a blood-contacting surface with derivatives of phosphatidyl choline.
  • PCT Patent application WO 01/04201 discloses high molecular weight zwitterionic polymers, and use in the papermaking processes.
  • Japan Patent application 2000186117 discloses various polymers comprising zwitterionic monomers.
  • US Patent No. 6,924,338 discloses polymer blends comprising zwitterionic monomers.
  • Polymers that enable cells to adhere to surfaces in a reversible manner without stimulating undesired cellular or systemic responses may be useful in medical device applications, tissue or cell culture, cell handling and other cell- involved processes, both medical and industrial.
  • the present invention relates to polymers comprising zwitterionic endgroups.
  • This invention is based, in part on the surprising discovery that polymers comprising zwitterionic endgroups that reversibly, stably and without stimulating an adverse cellular response, interact with proteins, lipids or carbohydrates on the outer surface of cells or liposomes. The interaction may be reversed by subsequent exposure to, for example, a cationic compound, for examplephosphatidyl choline or a polymer comprising phosphatidyl choline groups.
  • a cationic compound for examplephosphatidyl choline or a polymer comprising phosphatidyl choline groups.
  • R is a polymer, for example a linear, branched, dendritic or hyperbranched polymer.
  • the polymer R may be a surface or a coating on a surface. , configured so as to present a zwitterionic group comprising A-X-Y a monovalent, multivalent or polyvalent fashion.
  • A is an atom comprising a net positive charge.
  • A may be a quaternary nitrogen, a trivalent sulphur or tetravalent phosphorous.
  • X is a substituted alkyl group comprising at least one carbon.
  • An alkyl group includes any saturated or unsaturated hydrocarbon chain comprising at least one carbons.
  • the alkyl group may comprise substituent groups, such as H, a 1-15 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms.
  • Y is a group comprising a net negative charge, such as a phosphate, phosphonate, phosphinate, sulphate, sulfonate, carbonate or carboxylate group.
  • Y may further comprise a substituted alkyl group (-C-(R , R ,. R ) group where R ,
  • R 5 and R 6 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide or the like.
  • the polymer may be selected from the group comprising but not limited to poly(oxyalkylene) polymers, polyglycerol polymers, polyglycidol polymers, polyglycidol-block polymers, poly(glutamic acid) polymers, polyamidoamine (PAMAM) polymers, polyethyleneimine (PEI), polypropyleneimine (PPI) polymers, polymelamine polymers, polyester polymers, poly(lactic acid) polymers, epsilon poly(caprolactone), poly(lactone), substituted poly(lactones), poly(lactam), substituted poly(lactam), methoxy polyethylene glycol (MPEG or MePEG), polyethylene glycol (PEG), dextran, starch, cellulose, collagen, gelatine, chitosan and deacetylated chitosan.
  • the zwitterionic endgroups include those according to formula 2, formula 3, formula 4, formula 5, formula 6 or formula 7
  • compositions according to Formula 2 3, 4, 5, 6, or 7, A as defined in Formula 1 is N; where R 1 is a polymer; R 1 is a polymer, for example a linear, branched, dendritic or hyperbranched polymer.
  • the polymer R may be a surface or a coating on a surface.
  • R,. and R may
  • compositions according to Formula 2 or 3 independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide.
  • Y as defined in Formula 1 is PO -W.
  • W may be nothing - leaving a single negative charge on the O, or may be
  • R , R and R may independently be H, alkyl, alkene, alkyne,
  • Ri comprises a hyperbranched polyglycidol and the zwitterionic endgroup (A-X-Y, as defined in Formula 1 ) comprises one or more choline phosphate zwitterions.
  • Ri comprises a hyperbranched polyglycidol and the zwitterionic endgroup (A-X-Y, as defined in Formula 1 ) comprises one or more choline-O-sulphate zwitterions.
  • R 1 comprises a hyperbranched polyglycidol and the zwitterionic endgroup (A-X-Y, as defined in
  • Formula 1) comprises one or more dimethyl ammoniopropane sulphonate units.
  • Ri comprises a methoxypolyethyleneglycol polymer (MPEG), and the zwitterionic endgroup (A-X- Y, as defined in Formula 1) comprises one or more choline phosphate zwitterions, one or more choline-O-sulphate zwitterions or one or more dimethylammoniopropane sulphonate.
  • MPEG methoxypolyethyleneglycol polymer
  • A-X- Y as defined in Formula 1
  • Ri may be selected from the group comprising a poly(oxyalkylene) polymer, polyglycerol polymer, polyglycidol polymer, hyperbranched polyglycidol polymer, polyglycidol-block polymer, poly(glutamic acid) polymer, polyamidoamine (PAMAM) polymer, polyethyleneimine (PEI), polypropyleneimine (PPI) polymer, polymelamine polymer, polyester polymer, poly(lactic acid) polymer, epsilon poly(caprolactone), poly(lactone), substituted poly(lactones), poly(lactam), substituted poly(lactam), methoxy polyethylene glycol (MPEG or MePEG), polyethylene glycol (PEG), dextran, starch, cellulose, collagen, gelatine, chitosan or deacetylated chitosan.
  • PAMAM poly(oxyalkylene) polymer
  • PEI polyethyleneimine
  • PPI polypropyleneimine
  • the hyperbranched polyglycidol may have a MW of at least 100,000 or between about 100,000 and 1 ,000,000.
  • the hyperbranched polyglycidol has a MW of about 2,000 to about 20,000, or about 4,000 to about 8,000.
  • compositions according to some aspects of the invention may comprise a liposome.
  • liposomes may comprise a zwitterionic polymer comprising MPEG.
  • the liposome may comprise DPCP.
  • compositions according to some aspects of the invention may further comprise a chemotherapeutic agent.
  • chemotherapeutic agents include mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil; hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), semustine (methyl- CCNU), lomustine (CCNU), streptozocin (streptozotocin), estramustine phosphate, dacarbazine, temozolomide, methotrexate (amethopterin), fluorouracin (5-fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), gemcitabine, mercaptopurine (6- niercaptopurine, 6-MP), thioguanine (6-thiogu
  • a liposome comprising a zwitterionic polymer, and/or DPCP may further comprise a chemotherapeutic agent.
  • compositions comprising zwitterionic polymers may further comprise a pharmaceutically acceptable excipient, to aid in delivery, improve stability or otherwise facilitate administration to a subject.
  • pharmaceutically acceptable excipients include a suspending agent, an aqueous vehicle such as Water for Injection, Ringer's lactate, isotonic saline, salts, buffers, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antimicrobial agents, preservatives, chelating agents, binding agents, surfactants, wetting agents, non-aqueous vehicles such as fixed oils, waxes, creams or polymers or other agents for sustained or controlled release.
  • a suspending agent such as Water for Injection, Ringer's lactate, isotonic saline, salts, buffers, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antimicrobial agents,
  • compositions according to some aspects of the invention may be used to aggregate at least two cells.
  • the cells may be eukaryotic, or prokaryotic.
  • cells or cell types include a red blood cell, white blood cell, dermal cell, epithelial cell, secretory cell, hormone secreting cell, storage cell, barrier function cell such as a cell of the lung, gut, exocrine glands, and/or urogenital tract, a contractile cell such as muscle or cardiac cell, blood or immune system cell, nerve cell, sensory transducer cell, central nervous system cell, lens cell, pigment cell, germ cell, embryonic stem cell, pluripotent stem cell, fibroblast, interstitial cells, mature or immature gamete cell, blastomere, hepatocyte, myoblast, myotube, oocyte, osteoblast, osteoclast, T-cell, zygote or the like.
  • compositions according to some aspects of the invention may comprise a surface for use in the growth of cells.
  • the cells may be prokaryotic, or may be eukaryotic.
  • a 'surface' refers generally to an exterior boundary or outer face of an object, molecule or shape.
  • a surface may be a point of interaction, or an interface, of a first object or molecule with a second object or molecule juxtaposed to the first object or molecule.
  • a 'surface' includes a liquid/solid interface, or an interface between fluids, for example a liquid/liquid interface, or a liquid/gas interface.
  • a surface may be coated or covered by a polymer according to some embodiments of the invention.
  • a surface may be, for example, a metal, or a substance comprising a metal, or may be a surface for growing cells in culture, for example a plastic or glass dish or flask, as are generally known in the field of microbiology, cell culture or the like.
  • a surface may be a glass slide or a bead that is coated with a composition according to some embodiments of the invention.
  • the surface i.e. the surface of a plastic cell culture dish or flask may itself be a polymer.
  • polymers used in cell culture dishes, flasks or other devices used in handling cells in culture include polystyrene, high density polyethylene (HDPE), silicone, Polyvinylidene fluoride (PVDF), low density polyethylent (LDPE), polymethyl methacrylate (PMMA), polycarbonate, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid) or the like
  • a method for growing a cell or cells on a scaffold or surface comprising a zwitterionic polymer comprising providing a scaffold or surface, coating the scaffold or surface with a zwitterionic polymer according to some embodiments of the invention, and seeding the coated scaffold or surface with a cell or cell type that is to be grown. Additionally, the cell or cells may subsequently be released or removed from the scaffold or surface by washing with a cationic compound.
  • Figure 1 shows an illustration of an exemplary hyperbranched polyglycidol-CP polymer, showing examples of zwitterionic endgroups, according to some embodiments of the invention.
  • Illustrated zwitterionic endgroups include choline phosphate (CP), choline O-sulphate (CS) and dimethylammoniopropane sulphonate (APS).
  • Figure 2 shows a plot of a turbidometric titration of HPG-CP-4K (0.2% in 0.15 M NaCI) against HPG-PC-8K in water at a range of concentrations (solid squares), or HPG-CP-8K (0.2% in 0.15 M NaCI) against HPG-PC-8K in water at a range of concentrations (solid circles).
  • X axis shows the concentration of the HPG-PC-8K polymer (wt % in water);
  • Y axis shows the optical density at 540nm.
  • Figure 3 shows a plot illustrating the effect of salt concentration on disaggregation of 1 :1 aggregates of HPG-CP-4K with HPG-PC-4K (solid squares) and HPG-CP-8K with HPG-PC-8K (solid circles).
  • X axis shows the NaCI concentration
  • Y axis shows the optical density at 540nm.
  • Figure 4 shows a plot of electrophoretic mobility of negatively charged DSPC:DOPS:CHOL liposomes after incubation with MPEG-(CP) , MPEG-
  • FIG. 5 shows quantitative analyses of crystal violet (CV) stained biofilms of Campylobacter jejuni grown in the presence of A) HPG-CP-8K; B) HPG-PC-8K: C) HPG-8K (no zwitterion group - HPG only) at polymer concentrations of 0-5 mg/ml. Experiments were done in triplicate. X axis shows concentration of polymer in mg/ml; Y axis shows optical density at 570 nm. Biofilms of the virulent WT Cj strain 81 -176 were allowed to form at the air-liquid interface of borosilicate tubes, in triplicate, in the presence of five concentrations of soluble polymers of varying chemical composition. After two days, biofilms were stained with CV, and staining intensity quantified by dissolution of the CV in DMSO and spectrophotometric analyses
  • Figure 6 shows cell viability upon exposure to HPG-CP-4K (broad hatched bars) or HPG-PC-4K (narrow hatched bars) at various concentrations of polymer from 0-10 mg/ml.
  • biocompatible refers generally to the ability of a material to perform with an appropriate host response in a specific application.
  • biocompatibility of an implantable material refers to the ability of the material to perform its intended function, with a desired degree of incorporation in the host, without eliciting an undesirable level of systemic or localized effect in the host.
  • a biocompatible material such as a polymer, or a polymer comprising a zwitterionic endgroup, may be capable of being degraded or absorbed by the host, or it may not.
  • the biocompatibility of a polymer, or material made from a polymer may be assessed in a variety of ways.
  • assays or tests employed will vary depending on the polymer and the intended use. Examples of assays include the MTT assay, hemolysis test, intercalation assays, and toxicological studies involving animals (see, for example, Kainthan et al, 2006. Biomacromolecules 7:703-709; Klajnert et al 2006. Cell Biol Int. 30:248-252, herein incorporated by reference).
  • a 'surface' refers generally to an exterior boundary or outer face of an object, molecule or shape.
  • a surface may be a point of interaction, or an interface, of a first object or molecule with a second object or molecule juxtaposed to the first object or molecule.
  • a 'surface' includes a liquid/solid interface, or an interface between fluids, for example a liquid/liquid interface, or a liquid/gas interface.
  • a surface may be coated or covered by a polymer according to some embodiments of the invention.
  • a surface may be, for example, a metal, or a substance comprising a metal, or may be a surface for growing cells in culture, for example a plastic or glass dish or flask, as are generally known in the field of microbiology, cell culture or the like.
  • a surface may be a glass slide or a bead that is coated with a composition according to some embodiments of the invention.
  • the surface i.e. the surface of a plastic cell culture dish or flask may itself be a polymer.
  • the surface may comprise a scaffold for growing cells or tissue in vitro, for tissue engineering.
  • Polymeric biomaterials for guiding tissue growth to produce artificial organs or other structures may be enhanced by the application of zwitterionic polymers according to various embodiments of the invention.
  • tissue engineering biomaterials uses, structures and the like, see, for example, Kim et al 2000. World J Urology 18:2-9; Rather et al 2004. Ann. Rev Biomed Eng. 6:41-75; Falconnet et al 2006. Biomaterials 27:3044-3063.
  • polymers used in cell culture dishes, flasks or other devices used in handling cells in culture include polystyrene, high density polyethylene (HDPE), silicone, Polyvinylidene fluoride (PVDF), low density polyethylent (LDPE), polymethyl methacrylate (PMMA), polycarbonate, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid) or the like.
  • a 'cell' refers generally to any cell or cells, as well as viruses, subcellular particles or other particles having microscopic size e.g. a size similar to that of a biological cell.
  • a 'cell' refers generally to any cell or cells, as well as viruses, subcellular particles or other particles having microscopic size e.g. a size similar to that of a biological cell.
  • the cell may be any cell, for example, which is not to be considered limiting, a red blood cell, white blood cell, dermal cell, epithelial cell, secretory cell, hormone secreting cell, storage cell, barrier function cell such as a cell of the lung, gut, exocrine glands, and/or urogenital tract, a contractile cell such as muscle or cardiac cell, blood or immune system cell, nerve cell, sensory transducer cell, central nervous system cell, lens cell, pigment cell, germ cell, embryonic stem cell, pluripotent stem cell, fibroblast, interstitial cells, mature or immature gamete cell, blastomere, hepatocyte, myoblast, myotube, oocyte, osteoblast, osteoclast, T-cell, zygote or the like.
  • a contractile cell such as muscle or cardiac cell, blood or immune system cell, nerve cell, sensory transducer cell, central nervous system cell, lens cell, pigment cell, germ cell, embryonic stem cell, pluripotent stem cell, fibroblast, inter
  • Cells may have a nucleus, or may be anucleate, such as a platelet, or mature erythrocyte ('red cell'). Cells may be of a variety of shapes, commonly spherical, but may be rod-shaped, ameboid, elongated, flattened, asymmetrical or otherwise irregular. A cell may be alive or dead, or in a form of stasis or rest. The size of a cell may vary widely, with a diameter from about 0.1 microns to about 200 microns, although more typically in the range from about 1 micron to about 100 microns. The size of a cell may vary depending on the stage of growth cycle. The cell may be a transformed cell, for example a cell isolated from a tumor, or a cell infected by a virus. The cell may be a primary cell, or may be an immortalized cell grown in culture conditions.
  • Biocompatible polymers have a myriad of uses known in the art, and incorporation of various functional groups may tailor a polymer or polymers to a particular application.
  • the number of functional endgroups in a polymer is dependent on the quantity of ends - for example, a linear polymer will have two ends, a dendrimeric polymer will have multiple ends and a hyperbranched polymer will have more still, depending on the degree of branching and size of the molecule.
  • the form of the polymer - linear, dendrimeric or hyperbranched, for example - may be influenced by both the monomer, or combination of monomers used, for example in a block copolymer, and by the conditions of synthesis.
  • a "polymer” as used herein may be a linear polymer, dendrimeric polymer or hyperbranched polymer.
  • the polymer may be a homopolymer, comprising a single species of monomer, or may be a heteropolymer, alternatively referred to as a copolymer, comprising at least two species of monomers.
  • the monomers are linked in a chemical reaction referred to as polymerization.
  • a polymer incorporating more than one species of monomer may be referred to as a copolymer, and the chemical reaction that links them as copolymerization.
  • Copolymers may be described as random, alternating or block copolymers, depending on the monomers and reaction conditions used to generate them. Polymers and copolymers may be generally described as crosslinked, thermoplastic, thermoset or elastomeric, or combinations of these.
  • Various references known in the art discuss methods of syntheses, methods of manipulation of polymerization reactions to modify branching, incorporation ratios in copolymer syntheses, and various physical characteristics of the polymers including solubility, net electronic charge, flexibility, physical strength and the like. Examples of such references may include RJ Young and PA Lovell, "Introduction to Polymers", 2nd edition. Chapman and Hall 1991 ; Odian G. "Principles of
  • Ring-opening polymerization refers to a type of addition polymerization. An end of a growing polymer chain serves as a reactive centre capable of reacting with additional monomers to increase the chain length.
  • the monomer is a cyclic compound. Ring-opening polymerizations are described in the art, for example, US Patent 5,582,327; Amecke et al 1992. Clin Mater. 10:47-50; Grubbs and Tumas, 1989. Science 243:907-915; Liggins and Burt, 2002. Adv. Drug. Deliv Rev. 54:191-202;
  • Polydispersity, or polydispersity index (PDI) describes the distribution of the range of molecular weights in a given polymer sample. A narrow PDI has less variation in the molecular weights, while a broader PDI has a greater range. PDI is a value greater than 1. A polymer sample with uniform chain length, for example, would have a PDI approaching unity (1).
  • the PDI of a polymer is dependent on the mechanism of polymerization and reaction conditions, including ratios of reactants, stage of completion of the polymerization reaction, and the like.
  • composition of the polymer may be modified depending on the physical characteristics and use of the polymer. For example, if bioadsorption of the polymer is desired, then a poly(lactic acid) or epsilon poly(caprolactone) polymer may be used. These polymers have been found to degrade under normal physiological conditions over a 3-6 month period. (Andersen and Shive, 1997. Adv. Drug. Del. Rev. 28:5-24).
  • Such polymers may be linear, branched, hyperbranched or dendrimeric,. Examples of these methods may be found in, for example, Sunder et al. 1999
  • PAMAM polyamidoamine
  • PPI polyethyleneimine polypropyleneimine
  • polyester polymers poly
  • a polymer may comprise a zwitterionic functional endgroup, or a 'zwitterionic group'.
  • a 'zwitterion' generally refers to a compound that has both positive and negative charges.
  • a zwitterion, or zwitterionic composition may comprise both a negatively charged anionic group and a positively charged cationic group, providing a net neutral charge on the molecule.
  • zwitterionic compounds include phosphoryl choline (“PC”), choline phosphate (“CP”), buffer compounds useful in biological buffer systems, including
  • HEPES HEPES, PIPES, CAPS or MOPS, and the like; amino acids, or the like .
  • compositions according to some embodiments of the invention may be referred to generally as “zwitterionic polymers”, “zwitterions-polymers”, “zwitterionic compounds” or other similar terms.
  • polymers according to Formula 1 are provided.
  • R is a polymer, for example a linear, branched, dendritic or hyperbranched polymer.
  • the polymer R may be a surface or a coating on a surface, configured so as to present a zwitterionic group comprising A-X-Y, a monovalent, multivalent or polyvalent fashion.
  • A is an atom comprising a net positive charge.
  • A may be a quaternary nitrogen, a trivalent sulphur or tetravalent phosphorous.
  • X is a substituted alkyl group comprising at least one carbon
  • An alkyl chain includes any saturated or unsaturated hydrocarbon chain comprising at least 2 carbons.
  • the alkyl group may comprise substituent groups, such as H, a 1-15 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms.
  • Y is a group comprising a net negative charge, such as a phosphate, phosphonate, phosphinate, sulphate, sulfonate, carbonate or carboxylate group.
  • Y may further comprise a substituted alkyl group (-C-(R 4 , R 5 ,. R 6 ) group where R 4 ,
  • R and R may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal,
  • a as defined in Formula 1 is NR - R 2 -R 3 , where R 1 is a polymer or a surface, as described.
  • R 2 and R 3 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide or the like.
  • Y as defined in Formula 1 is PO -
  • W where W may be nothing - leaving a single negative charge on the O, or W may be -CR 4 R 5 R 6 , where R 4 , R 5 and R 6 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate or peptide.
  • m is an integer value of 2, 3, 4, 5 or more.
  • a as defined in Formula 1 is NR - R -R , where R is a polymer or a surface as described. R and R may
  • 2 3 1 2 3 independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide or the like.
  • Y as defined in Formula 1 is PO -
  • W where W may be nothing - leaving a single negative charge on the O, or W may be -CR 4 R 5 R 6 , where R 4 , R 5 and R 6 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate or peptide.
  • m is an integer value of 2, 3, 4, 5 or more.
  • a as defined in Formula 1 is NR -
  • R and R may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide or the like.
  • X is a substituted alkyl group comprising at least one carbon
  • An alkyl chain includes any saturated or unsaturated hydrocarbon chain comprising at least 2 carbons.
  • the alkyl group may comprise substituent groups, such as H, a 1-15 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms.
  • X is (-CH 2 -), where m is 2, 3, 4, 5 or more.
  • a as defined in Formula 1 is N.
  • R is a polymer or a surface as described. R and R may independently be H,
  • alkyl alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide or the like.
  • X is (-CH 2 -), where m is 2, 3, 4, 5 or more.
  • R is a polymer as described.
  • R and R may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide, or the like.
  • X is (-CH2-), where m is 2, 3, 4, 5 or more.
  • a as defined in Formula 1 is N. .
  • Ri is a polymer as described.
  • R 2 and R 3 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide or the like.
  • X is (-CH 2 -), where m is 2, 3, 4, 5 or more.
  • Reagent (2) is a polymer comprising an amine group at one end, the amine comprising substitutent groups Ffe, R 3 as described supra.
  • Ri is a polymer as described supra.
  • the polymer may comprise an amine as a result of the polymerization reaction, or be aminated following polymerization according to known methods.
  • a polymer comprising an -OH group at an end may be aminated following polymerization, using methods known in the art; for example, by dissolving in a polar aprotic solvent (e.g pyridine, DMF, DMSO, diglyme or the like) and reacting with tosyl chloride.
  • a polar aprotic solvent e.g pyridine, DMF, DMSO, diglyme or the like
  • the resulting polymer-tosylate may be subsequently refluxed with an alkylamine, such as ethylamine in THF.
  • alkylamine such as ethylamine in THF.
  • Other solvents that may be used in this reflux include dioxane, DMF, DMSO, diglyme or the like.
  • Reagent (3) is a phospholane, where Z is -CR 4 -Rs-Re, comprising R4-R5- R 6 as described above.
  • the dioxa-ring may comprise (m) methylene groups, where m is 2, 3, 4, 5 or more.
  • R 7 and R 8 may independently be H, alkyl, alkene, alkyne, benzyl, or aryl.
  • An alkyl group includes any saturated or unsaturated hydrocarbon chain comprising at least 2 carbons.
  • the alkyl group may comprise substituent groups, such as H, a 1 -5 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms.
  • the phosphorylation of reagent (2) by a ring-opening reaction with the phospholane may be done in a non-polar solvent.
  • non-polar solvents include acetonitrile, alcohols such as methanol, ethanol, propanol or isopropanol, diglyme, DMSO and the like. All reagents would need to be soluble in the chosen solvent.
  • the polymer may be a polyglycidol, such as a branched, or hyperbranched, polyglycidol (HPG) in a wide range of molecular weights.
  • the polymer may. for example, have a molecular weight from about 2000 Da to about 20,000 Da, or any amount therebetween. Larger HPGs are also contemplated, having a molecular weight in excess of 100 kDa, or as great as about 1 x 10 6 Da. Such larger HPGs, methods of their synthesis, biological compatibility and other characteristics are described in Kainthan et al 2007, supra.
  • HPG polymers in a wide range of molecular weights, the particular range of molecular weights being employed advantageously for particular functions, such as coating a surface or a device, encapsulating a drug, or other functions described below.
  • Very high molecular weight hyperbranched polyglycerols with narrow polydispersities may be obtained by ring opening multibranching polymerization of glycidol using, for example, dioxane as the reaction medium. Alternately, molecular weight polymers with a broader polydispersity may be obtained using diglyme as a reaction medium. Other solvents that may be used include THF or DMSO. See, for example, Kainthan RK and Brooks, DE. 2007 Biomaterials 28:4779-4787; Kainthan et al 2006 Macromolecules 39:7708-7717; and WO 2006/130978, herein incorporated by reference. These HPGs having molecular weights from about 2000 to about 1x10 6 Da may be used in the synthesis of HPG-CP zwitterionic polymers as described herein.
  • HPG may comprise -OH endgroups, and as such may require amination following polymerization (shown in Scheme 2 reagent 4).
  • Synthesis scheme 2 is an exemplary scheme, illustrating a tertiary amine endgroup comprising two methyl groups, however other alkyl groups or aromatic groups may be contemplated, as described above.
  • Phosphorylation of Formula 4 by 2-methoxy- 1 ,3-2-dioxaphospholane-2-oxide shown in scheme 2, reagent 5
  • HPG-choline phosphate HPG-CP
  • the quantities of reagents used in the methods and examples described may be expressed in a variety of ways.
  • the amount of a particular reagent may be described by, for example, mass (e.g. grams), or by volume (e.g. millilitres), by ratio with another reagent (e.g. 2:1 ), by percentage volume (e.g. 10% (vol/vol), by percentage weight (e.g. 10 wt%, or 10% by wt), and the like.
  • mass e.g. grams
  • volume e.g. millilitres
  • percentage volume e.g. 10% (vol/vol)
  • percentage weight e.g. 10 wt%, or 10% by wt
  • a 10% NaOH solution may be made by dissolving 10 g of NaOH solid in a final volume of
  • the polymer is methoxypolyethylene glycol (MPEG or MePEG).
  • the polymer may, for example, have an average molecular weight from about 200 to about 20,000, or from about 500 to about 10,000, or any amount therebetween.
  • Other embodiments of the invention may contemplate MPEG polymers in a wide range of molecular weights, the particular range of molecular weights being employed advantageously for particular functions, such as coating a surface or a device, encapsulating a drug, or other functions described below.
  • addition of the zwitterionic groups to the polymer may be done on a surface, such as a plastic or glass slide, etc.
  • the polymer coating the surface may comprise an amine group, such as a tertiary amine group, and the reaction may be performed according to scheme 1 , supra.
  • the particular use of the zwitterion-polymer may influence the choice of molecular weight range.
  • branched polymers including hyperbranched polymers
  • the molecular weight, degree of branching and type and quantity of endgroups available for interaction are interdependent.
  • the optimal molecular weight, degree of branching and choice and quantity of endgroups may be determined empirically, and such determinations are within the ability of one of skill in the art.
  • the interaction of a zwitterion with a cell may be used to 'camouflage' the cell.
  • Long-chain polyethylene glycol covalently attached to a red cell reduces the antigenicity of the red cell.
  • a polyethylene glycol polymer comprising one or more zwitterion endgroups that attach noncovalently to a cell surface may provide the same camouflage effect, with the advantage of being reversible by addition of phosphoryl choline alone or covalently attached to another polymer, or by washing the cells with an albumin solution, saline solution, or other suitable salt or buffer solution.
  • the polymer may comprise one, or a plurality of zwitterion endgroups.
  • the zwitterion-polymers may be used in tissue culture surfaces.
  • Tissue engineering to produce three-dimensional living tissue complexes, is dependent on a synthetic scaffold to give shape and support to the cells. While many immortal cell lines in culture are readily adherent to most surfaces, primary cells vary in their need and ability to attach readily to a surface. Further, removing cells from the surface, according to conventional cell culture techniques and methods, may involve use of enzymes, such as trypsin, to digest and weaken the extracellular matrix that allows the cells to adhere. Trypsinization' of cells may adversely affect the viability of the cells.
  • zwitterion-polymers in the manufacture of or to coat surfaces for growth of cells enables adhesion of the cells, with the advantage of being reversible by subsequent exposure to or addition of, for example, a compound comprising a cationic group or functional moiety (a 'cationic compound), for example phosphatidyl choline.
  • a 'cationic compound for example phosphatidyl choline.
  • the cationic compound may be, for example, free in solution, and/or covalently attached to another polymer.
  • the polymer may comprise one, or a plurality of cationic or zwitterionic endgroups.
  • the cell layer or aggregation may be washed with a solution comprising a cationic compound.
  • the solution may comprise, for example an albumin solution, saline solution or other suitable salt or buffer solution.
  • cells may be grown on a synthetic scaffold by providing a surface, coating the surface with a zwitterionic polymer according to some embodiments of the invention, and seeding the coated surface with a cell or cell type that is to be grown. If splitting or reculturing of the cells is necessary or desirable , the cells may be released from the scaffold by washing with a cationic compound, the cationic compound washed away and the cells returned to the scaffold, or to another scaffold or other suitable culture vessel.
  • the zwitterion-polymers may be used to coat implantable medical devices where cell adhesion by non-covalent means is desirable.
  • implantable medical devices may include stents, urinary catheters, biosensors or implants of various types.
  • the zwitterion-polymers may be used to inhibit biofilm formation on surfaces or devices.
  • a polymer comprising, for example, a PC zwitterion may be used to coat a surface and reduce adherence of cells, such as bacterial cells, to the surface.
  • the zwitterion-polymers may be used to aggregate or bind cells in a suspension, or 'trap' cells in a filter.
  • Cells may be, for example, bacterial cells.
  • a filter may comprise, for example, a polymer comprising a CP zwitterion, for example an HPG-CP zwitterionic polymer, that binds bacterial cells to the filter.
  • a zwitterionic polymer may be added to an aqueous bacterial suspension, causing the bacteria to aggregate and be trapped by the filter, thus removing them from the suspension and preventing their discharge as the aqueous phase leaves the filter.
  • a device, a surface or a medical device may be constructed so as to comprise a surface suitable for derivatization and addition of zwitterionic groups according to the general scheme 1 or scheme 2.
  • a surface, device or medical device would comprise, as indicated above, available tertiary amine, hydroxyl or carboxyl groups for the coupling reaction.
  • zwitterion-polymers may be applied in multiple layers on a surface, device or medical device.
  • the layers may comprise different polymers, or comprise the same polymer with same or different zwitterionic ends.
  • the layers may encapsulate or otherwise deliver a drug, and the layers may be produced so as to provide desired hydrophobicity, hydrophilicity or aggregation characteristics.
  • zwitterion-polymers may be used to adhere specific tissue surfaces to each other, but not to other tissue surfaces.
  • a first tissue may be treated or have applied to it a zwitterionic polymer comprising a CP group
  • a second tissue may be treated or have applied to it a zwitterionic polymer comprising a PC group.
  • the zwitterions of these polymers may preferentially interact with each other, and thus prevent undesired tissue adhesions.
  • Such an embodiment may be useful, for example, during or following surgical procedures, or to facilitate implantation of a medical device. See for example Benjamin et al 2003. JACS 125:7494-7495.
  • zwitterionic polymers may be used as coagulating agents.
  • a spray, aerosol, solution or other suitable formulation of zwitterionic polymers may be applied to a wound to aid in coagulating blood.
  • zwitterionic polymers may be provided as a coating or substance associated with a bandage to be placed on a wound.
  • the zwitterionic polymer used to adhere specific tissue surfaces may further comprise a drug or a biologically active molecule.
  • zwitterionic polymers may be used to coat a bead or other surface.
  • the bead may further comprise a fluorescent tag or label, or chromatographic dye, or other tracking or visualization features.
  • the coated, tagged bead may be used to tag or label cells in a suspension,
  • zwitterionic polymers may be used as a transfection reagent to enhance the uptake of another compound or composition by a cell.
  • a zwitterionic polymer according to some embodiments of the invention may be combined with the compoundthat would otherwise have poor or no solubility in a transfection buffer.
  • the association of the zwitterionic polymer with the cell or cell aggregate may act to increase the effective local concentration of the compound and thus increase the efficiency with which the compound is taken up by the cells.
  • the zwitterion-polymers may be used in the production of liposomes, micelles, polymeric aggregates, conjugates, drug conjugates or the like, which may be used to encapsulate or deliver drugs.
  • the adhesive nature of the zwitterionic polymers with respect to organs, cells and tissues may enable liposomes comprising such zwitterionic polymers to adhere to cells or tissues to which they are applied, for example topical application on a surface, delivery by a 'patch', or in the case of treatments for hollow organs (i.e.
  • a solution or suspension of the liposomes may be used to fill the organ and deliver the liposomes comprising a drug to the specific tissue, and also avoid systemic effects.
  • Liposomes according to some embodiments of the invention may provide the further advantage of remaining associated with the tissue to which they are applied and may not be flushed out as readily ie. in the example where the liposomes are used on the bladder, the interaction with the cell surface may prevent their removal when the subject urinates.
  • Localized delivery using zwitterionic polymers according to various embodiments of the invention are not ligand-specific, and thus may be more adaptable to various organ, tissue or cell types.
  • Drugs that may be particularly suitable for encapsulation and/or delivery using the zwitterionic polymers according to some embodiments of the invention include those with significant toxic effects, or poor solubility, or both, such as chemotherapeutic agents.
  • agents may include include mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil; hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), semustine (methyl- CCNU), lomustine (CCNU), streptozocin (streptozotocin), estramustine phosphate, dacarbazine, temozolomide, methotrexate (amethopterin), fluorouracin (5-fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), gemcitabine, mercap
  • Branched polymers, dendrimers or hyperbranched HPGs incorporating zwitterionic groups may be derivatized to enable them to bind and release a drug or drugs in a localized manner, effectively increasing local concentration. Increased localized concentration juxtaposed to a cell membrane, tissue or organ may enhance uptake by the cell, tissue or organ.
  • drugs, peptides, proteins or the like encapsulated or conjugated with zwitterionic polymers may be as administered orally.
  • a zwitterionic polymer designed for such a used would be non- or poorly adhesive in the low pH environment of the stomach, and when transitioned to the alkaline environment of the duodenum and small intestine, gain adhesive properties and 'stick' to the gut lining. This may increase the retention time of the drug, etc in the intestine, and may increase absorption, leading to improved bioavailability.
  • liposomes comprising zwitterionic groups are provided.
  • the liposomes may comprise dipalmitoyl choline phosphate (DPCP).
  • the liposomes may further comprise cholesterol, dipalmitoyl phosphatidyl choline and/or a polyalkylene glycol, such as methoxypolyethyleneglycol. Liposomes generally may be used to improve the efficiency of delivery of poorly water-soluble drugs.
  • a poorly water-soluble drug may be encapsulated in liposomes comprising DPCP, and the ability of the zwitterionic group (CP) on the liposome may delay the dispersion of the liposome encapsulated drug from the site of injection, thus increasing the local concentration of the drug, and providing for a controlled release in situ.
  • CP zwitterionic group
  • the zwitterion-polymers may be used as a topical anti-infective.
  • the zwitterion-polymers may be incorporated into a suitable excipient and when applied to the skin, or a break in the skin, adhere to and aggregate foreign cells at the site of application, preventing spread of the foreign cells to distal locations in or on the body. Foreign cells aggregated in this manner may subsequently be more easily removed from the site of application when the zwitterion-polymer composition is removed.
  • zwitterionic polymers may be used as a physical barrier to an infectious organism.
  • MPEG- (CP)n zwitterionic polymers such as MPEG-(CP)20 may be useful for this application.
  • Such a polymer may be applied to a tissue surface, where the zwitterion group associates with the tissue surface, and the MPEG polymer is oriented away from the tissue surface.
  • a zwitterionic polymer as part of an anti-infective, or as a delivery vehicle for a drug or similar, may necessitate combining the zwitterionic polymer with a pharmaceutically acceptable excipient.
  • excipient will be dependent on the particular use or requirement to be met, for example, if it is to be injected, sterile Water for Injection may be a suitable excipient, while if it is to be administered orally, the excipient may include a suspending agent.
  • compositions include, for example, an aqueous vehicle such as Water for Injection, Ringer's lactate, isotonic saline, salts, buffers, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antimicrobial agents, preservatives, chelating agents, binding agents, surfactants, wetting agents, non-aqueous vehicles such as fixed oils, waxes, creams or polymers or other agents for sustained or controlled release. See, for example, Berge et al. (1977. J. Pharm Sci. 66:1-19) or Remington- The Science and
  • Routes of administration of a zwitterionic polymer as part of an anti- infective, or as a delivery vehicle for a drug or similar may be selected depending on the nature of the drug to be delivered, the nature of the infection to be treated or prevented or the like.
  • routes of administration include, for example, subcutaneous injection, direct injection into a disease site or tissue type, for example direct injection into a solid tumor, intraperitoneal injection, intramuscular injection, intravenous injection, epidermal or transdermal administration, mucosal membrane administration, ophthalmic, orally, nasally, rectally, topically, or vaginally. See, for example, Remington, The Science and Practice of Pharmacy, 21 st edition. Gennaro et al. Editors. Lippincott Williams & Wilkins, Philadelphia. Carrier formulations may be selected or modified according to the route of administration.
  • compositions comprising a zwitterionic polymer according to various embodiments of the invention may be provided in a unit dosage form, or in a bulk form suitable for formulation or dilution at the point of use. Such compositions may be administered to a subject in a single-dose, or in several doses administered over time. Dosage schedules may be dependent on, for example, the subject's condition, age, gender, weight, route of administration, formulation, or general health. Dosage schedules may be calculated from measurements of adsorption, distribution, metabolism, excretion and toxicity in a subject, or may be extrapolated from measurements on an experimental animal, such as a rat or mouse, for use in a human subject. Optimization of dosage and treatment regimens are discussed in, for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics 11 th edition. 2006. LL Brunton, editor.
  • a "subject” may refer to an animal, such as a mouse, rat, dog, cat, pig, sheep, guinea pig, hamster or a primate, such as a monkey, chimpanzee or human.
  • a detailed polymerization procedure is described by Sunder et al. 1999 Macromolecules 32:4240, and modified as follows. After the reaction, the polymer was dissolved in methanol, neutralized by passing three times through a column containing cation exchange resin (Amberlite IRC-150). The polymer was then precipitated into excess of acetone and stirred for 1 hour. Acetone was decanted out and this procedure repeated once. Dialysis of polymers was done for three days against water using cellulose acetate dialysis tubing MWCO 1000 or 10,000 g/mol) with water being changed three times per day. The dry polymer was obtained by lyophilisation.
  • HPG hyperbranched polyglycidols
  • the purified polymer (80 mg) was dissolved in 5 ml methanol and 0.3 ml trimethylamine was added and heated at 55 0 C for 3 days in a pressure bottle. The volatiles were then removed in vacuum and the polymer was precipitated twice from acetone.
  • N,N-dimethyl-2,3-dihydoxypropane (2g) was dissolved in 100 ml anhydrous THF. 4.8 ml triethylamine was added and the reaction mixture was kept at O 0 C. Palmitoyl chloride (9.68 g) was added drop wise. The reaction mixture was stirred overnight and filtered through a filter paper. The filtrate was evaporated to give a while solid powder. The product was purified by flash chromatography (silica gel, eluted first with hexane and then with 20 % ethyl acetate: hexane mixture).
  • L-929 fibroblast cell lines was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.
  • Adherent cell lines were plated into 96-well plates one day prior to adding polymers to allow the cells to adhere to the plate. The cell density was 5000 per well. The plates were left in an incubator overnight. The following day, the polymer solutions (100 ⁇ l) made in the culture medium were added at increasing concentration (0.0001 , 0.001 , 0.01 , 0.1 , 0.5, 1 , 5 and 10 mg/ml) to the wells containing 100 ⁇ l of medium.
  • Fibroblast culture media consisted of Minimum Essential Medium with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate (90%) and fetal bovine serum (FBS) (10%).
  • the polymer HPG-CP showed very little cytotoxicity toward fibroblasts with more than 100 % cell viability observed even at a high concentration of 1 mg/ml after 48 hr incubation whereas the HPG-CP-4K was non toxic up to 0.01 mg/ml.
  • VAI 0 single, separated cells, no aggregation 1 50% single cells : 50% aggregated cells
  • HPG-tosylate 5 g was dissolved in 10 m THF and refluxed under a nitrogen atmosphere for 24 hrs with 2M ethylamine solution in THF (30 ml). The volatiles were then removed to provide the polymer. The residual secondary amines in the polymer were methylated by refluxing with a formaldehyde/formic acid mixture (1 :1). HPG-amine (2.5 g) was taken in 250 ml round-bottom flask, and 50 ml water added, with stirring. To the cooled solution, 0.5 ml concentrated HCI was added dropwise. Formic acid (80%, 10 ml) was then added drop wise followed by formaldehyde (8 ml).
  • the reaction mixture was refluxed for 24 hours. The volatiles were removed under vacuum. The residue was dissolved in 10% (by wt) NaOH solution so that the solution became alkaline. The HPG-Amine was extracted with dichloromethane after saturating the aqueous layer with NaCI.
  • the polymer was precipitated in ether or acetone.
  • the isolated polymer was dissolved in a small amount of methanol and precipitated in ether. This procedure was repeated three more times, and the polymer was dried in vacuum.
  • MPEG-epoxide 500 mg was heated with excess of hyperbranched poly(ethyleneimine) (molecular weight 700) in methanol for 24 hrs. The reaction product was then purified by dialysis (MW cut off 1000) against water and isolated by freeze drying. The polymer was then subjected to methylation and phosphorylation.
  • FIG. 6 shows cell viability upon exposure to HPG-CP-4K (broad hatched bars) or HPG-PC-4K (narrow hatched bars) at various concentrations of polymer from 0-10 mg/ml.
  • Red blood cells were exposed to polymers of HPG-CP or HPG-PC - 4K and -8K as described, and observed for aggregation. Results are summarized in Table 1.
  • VAI visual aggregation index
  • VAI 0 single, separated cells, no aggregation
  • Example 6 HPG-CP aggregation of cells is reversible.
  • HPG-CP -4K aggregated cells were washed with ethyl phosphatidyl choline (2 wt %), also reversing aggregation. Washing normally aggregated cells with 1% HSA in 150 mM NaCI reverses aggregation, while washing HPG-CP aggregated cells with 150 mM NaCI (no HSA) reverses aggregation only partially.
  • CP multimers (CP) were prepared from permethylated TREN and PEI by phosphorylation in the absence of HPG Me TREN as well as
  • QELS Quasi elastic light scattering
  • QELS is a static light scattering technique used to measure particle sizes.
  • the average hydrodynamic radius (Rh) of low molecular weight HPG-PC- 4K dissolved in methanol (2% solution) was measured to be 4.8 nm and that of MPEG-(CP) o (1 %) was 13 nm.
  • Hydrodynamic analysis of a mixture of these two solutions in water (2 ⁇ l and 100 ⁇ l respectively) equilibrated overnight was measured to be Rh ⁇ 40 nm.
  • Rh ⁇ 40 nm The size depended on the ratio of polymer concentrations and it did not change with dilution. A size increase to 30 nm was observed in the case of MPEG-CP whose size was only 3 nm.
  • DPCP forms a clear solution in water after 5 fireeze-thaw (60 C) o 31 cycles (by heating to 60 C).
  • the P NMR methanol-d4 in a capillary tube was used for locking
  • Dipalmitoylphosphatidylcholine (DPPC) in water is known to form
  • Liposome preparations were made with and without cholesterol and characterized by dynamic light scattering to provide an average size.
  • the results of the size analyses are given in Table 4.
  • Liposomes were prepared using methods known in the art, see for example Olson et al Biochmica et biophysics acta, 1979. 557 :9-23 ; Hope et al Biochmica et biophysics acta 1985. 812 :55-65
  • DPCP alone did not appear to form uniform liposomes in water as evidenced from the size measurements - the majority of the polymer appeared to form micelles, given the size. Addition of cholesterol enabled formation of liposomes of a reasonable size.
  • MPEG-(CP) n polymers affect the mobility of the liposomes.
  • Example 13 HPG-CP aggregation of bacteria An HPG-CP polymer was able to aggregate Camplyobacter jejuni in liquid growth media.
  • Figure 5 A-C HPG-CP-8K, HPG-PC-8K and HPG-8K (no zwitterion groups) were included in bacterial growth media at 0-5 mg/ml.
  • Biofilms of the C. jejuni strain 81 -176 were allowed to form at the air-liquid interface of borosilicate tubes, in triplicate, in the presence of five concentrations of soluble polymers of varying chemical composition. After two days, biofilms were stained with crystal violet, and staining intensity quantified by dissolution of the crystal

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Abstract

A hyperbranched polyglycidol polymer with zwitteπon endgroups of quaternary nitrogen and phosphate or phosphonate are described, as well as methods to prepare said polymers The zwitteπon endgroups allow the polymer to interact with proteins, lipid and carbohydrates on the outer surface of cells or liposomes reversibly, stabily and without stimulating an adverse cell response The polymers may be useful in medical devices applications, tissue or cell cultures, cell handling and other useful cell involved processes

Description

POLYMERS COMPRISING ZWITTERIONIC ENDGROUPS FIELD OF INVENTION
[0001] The present invention relates to novel zwitterionic polymers, and methods of making such polymers.
BACKGROUND OF THE INVENTION
[0002] A myriad of polymers have been developed for industrial and biomedical uses, the polymers being developed and refined to meet particular niche needs in the relevant industry.
[0003] Biomedical uses of polymers may include coatings on implants or pills, encapsulation and delivery of drugs over time or under particular conditions such as pH, manufacturing of implants and other uses known in the art. The development of some medical devices and implantable devices places mechanical and engineering qualities foremost, with the biocompatibility of such devices and items of secondary consideration. Some plastics and polymers are relatively inert and are tolerated, but these may only be a partial component of a device. Re-engineering of the entire device to incorporate more preferable materials may not be an option.
[0004] To work around this limitation, the surfaces of the existing device may be coated with a suitable polymer. It may be desirable to prevent cell adhesion, or to encourage the engraftment of cells on the implant. A variety of surface- modification techniques, materials and biocompatible polymers have been developed to prevent or encourage attachment of cells.
[0005] When cells attach to a surface, they may do so by deposition of proteins to form an extracellular matrix (ECM), to which cells attach via. integrin binding proteins. Cellular interaction with integrins and the ECM may also stimulate signal transduction events that may affect immune response, inflammatory response and other systemic responses. Removing the cells may be difficult, and may damage the cells in the process. [0006] Phospholipid polymers for use as biomaterials is described by Hayward et al 1985. Ann. NY Acad Sci 446:267-81.
[0007] PCT Patent Application WO 92/06719 discloses processes for coating a blood-contacting surface with derivatives of phosphatidyl choline.
[0008] Some polymers with charged endgroups are known in the art(Lowe and
McCormick Chem. Rev. 2002:102, 4177-4189; Kudaibergenov et al Advances in polymer science 2006, 201 : 157-224)
[0009] PCT Patent application WO 01/04201 discloses high molecular weight zwitterionic polymers, and use in the papermaking processes.
[0010] Japan Patent application 2000186117 discloses various polymers comprising zwitterionic monomers.
[0011] US Patent No. 6,924,338 discloses polymer blends comprising zwitterionic monomers.
[0012] Polymers that enable cells to adhere to surfaces in a reversible manner without stimulating undesired cellular or systemic responses may be useful in medical device applications, tissue or cell culture, cell handling and other cell- involved processes, both medical and industrial.
SUMMARY OF THE INVENTION
[0013] The present invention relates to polymers comprising zwitterionic endgroups.
[0014] This invention is based, in part on the surprising discovery that polymers comprising zwitterionic endgroups that reversibly, stably and without stimulating an adverse cellular response, interact with proteins, lipids or carbohydrates on the outer surface of cells or liposomes. The interaction may be reversed by subsequent exposure to, for example, a cationic compound, for examplephosphatidyl choline or a polymer comprising phosphatidyl choline groups. [0015] In accordance with one aspect of the invention, there are provided polymers, or composition comprising such polymers, the polymers comprising zwitterionic endgroups, according to Formula 1.
Formula 1
Figure imgf000005_0001
[0016] R is a polymer, for example a linear, branched, dendritic or hyperbranched polymer. In some embodiments the polymer R may be a surface or a coating on a surface. , configured so as to present a zwitterionic group comprising A-X-Y a monovalent, multivalent or polyvalent fashion.
[0017] A is an atom comprising a net positive charge. In some embodiments, A may be a quaternary nitrogen, a trivalent sulphur or tetravalent phosphorous.
[0018] X is a substituted alkyl group comprising at least one carbon. An alkyl group includes any saturated or unsaturated hydrocarbon chain comprising at least one carbons. The alkyl group may comprise substituent groups, such as H, a 1-15 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms. At least one C of the 1 -15 carbon saturated, unsaturated or cyclic alkyl group may further be replaced by O, S, SO, SO , NH , or NR', or may be optionally substituted with OH, OR', =O, SH, SR', NH 2 , NHR', NR' 2 , OSO 3 H, OPO 3 H 3 , CO 2 H, CO 2 R' and the like.
[0019] Y is a group comprising a net negative charge, such as a phosphate, phosphonate, phosphinate, sulphate, sulfonate, carbonate or carboxylate group. Y may further comprise a substituted alkyl group (-C-(R , R ,. R ) group where R ,
4 5 6 4 R 5 and R 6 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide or the like.
[0020] The polymer may be selected from the group comprising but not limited to poly(oxyalkylene) polymers, polyglycerol polymers, polyglycidol polymers, polyglycidol-block polymers, poly(glutamic acid) polymers, polyamidoamine (PAMAM) polymers, polyethyleneimine (PEI), polypropyleneimine (PPI) polymers, polymelamine polymers, polyester polymers, poly(lactic acid) polymers, epsilon poly(caprolactone), poly(lactone), substituted poly(lactones), poly(lactam), substituted poly(lactam), methoxy polyethylene glycol (MPEG or MePEG), polyethylene glycol (PEG), dextran, starch, cellulose, collagen, gelatine, chitosan and deacetylated chitosan.
[0021] In accordance with other aspects of the invention, the zwitterionic endgroups include those according to formula 2, formula 3, formula 4, formula 5, formula 6 or formula 7
Formula 2
Figure imgf000006_0001
Formula 3
Figure imgf000006_0002
Formula 4
Figure imgf000007_0001
Formula 5
Figure imgf000007_0002
Formula 6
Figure imgf000007_0003
Formula 7
Figure imgf000007_0004
[0022] For compositions according to Formula 2, 3, 4, 5, 6, or 7, A as defined in Formula 1 is N; where R1 is a polymer; R1 is a polymer, for example a linear, branched, dendritic or hyperbranched polymer. In some embodiments the polymer R may be a surface or a coating on a surface. R,. and R may
1 2 3 independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide. [0023] For compositions according to Formula 2 or 3, Y as defined in Formula 1 is PO -W. W may be nothing - leaving a single negative charge on the O, or may be
4
-CR R R , where R , R and R may independently be H, alkyl, alkene, alkyne,
4 5 6 4 5 6 benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide
[0024] In accordance with another aspect of the invention, Ri comprises a hyperbranched polyglycidol and the zwitterionic endgroup (A-X-Y, as defined in Formula 1 ) comprises one or more choline phosphate zwitterions.
[0025] In accordance with another aspect of the invention, Ri comprises a hyperbranched polyglycidol and the zwitterionic endgroup (A-X-Y, as defined in Formula 1 ) comprises one or more choline-O-sulphate zwitterions.
[0026] In accordance with another aspect of the invention, R1 comprises a hyperbranched polyglycidol and the zwitterionic endgroup (A-X-Y, as defined in
Formula 1) comprises one or more dimethyl ammoniopropane sulphonate units.
[0027] In accordance with another aspect of the invention, Ri comprises a methoxypolyethyleneglycol polymer (MPEG), and the zwitterionic endgroup (A-X- Y, as defined in Formula 1) comprises one or more choline phosphate zwitterions, one or more choline-O-sulphate zwitterions or one or more dimethylammoniopropane sulphonate.
[0028] In accordance with other aspects of the invention, Ri may be selected from the group comprising a poly(oxyalkylene) polymer, polyglycerol polymer, polyglycidol polymer, hyperbranched polyglycidol polymer, polyglycidol-block polymer, poly(glutamic acid) polymer, polyamidoamine (PAMAM) polymer, polyethyleneimine (PEI), polypropyleneimine (PPI) polymer, polymelamine polymer, polyester polymer, poly(lactic acid) polymer, epsilon poly(caprolactone), poly(lactone), substituted poly(lactones), poly(lactam), substituted poly(lactam), methoxy polyethylene glycol (MPEG or MePEG), polyethylene glycol (PEG), dextran, starch, cellulose, collagen, gelatine, chitosan or deacetylated chitosan.
[0029] In accordance with another aspect of the invention, the hyperbranched polyglycidol may have a MW of at least 100,000 or between about 100,000 and 1 ,000,000.
[0030] In accordance with another aspect of the invention, the hyperbranched polyglycidol has a MW of about 2,000 to about 20,000, or about 4,000 to about 8,000.
[0031] In accordance with another aspect of the invention, compositions according to some aspects of the invention may comprise a liposome. For example, liposomes may comprise a zwitterionic polymer comprising MPEG. In other aspects, the liposome may comprise DPCP.
[0032] In accordance with another aspect of the invention, compositions according to some aspects of the invention may further comprise a chemotherapeutic agent. Examples of chemotherapeutic agents include mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil; hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), semustine (methyl- CCNU), lomustine (CCNU), streptozocin (streptozotocin), estramustine phosphate, dacarbazine, temozolomide, methotrexate (amethopterin), fluorouracin (5-fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), gemcitabine, mercaptopurine (6- niercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), pentostatin (21- deoxycoformycin, deoxycoformycin), cladribine, fludarabine, amsacrine, vinblastine (VLB), vincristine, paclitaxel, docetaxel , etoposide, teniposide, topotecan, irinotecan, dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin, L-asparaginase, interferon alpha, buserelin, prednisone, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethinyl estradiol, tamoxifen, anastrozole, testosterone propionate, fluoxymesterone, flutamide, bicalutamide, leuprolide, thalidomide, cisplatin (czs-DDP), oxaliplatin, carboplatin, mitoxantrone, hydroxyurea, procarbazine (N-methylhydrazine, MIH), mitotane, aminoglutethimide, bexarotene, imatinib or the like.
[0033] For example, a liposome comprising a zwitterionic polymer, and/or DPCP may further comprise a chemotherapeutic agent.
[0034] Compositions comprising zwitterionic polymers may further comprise a pharmaceutically acceptable excipient, to aid in delivery, improve stability or otherwise facilitate administration to a subject. Examples of pharmaceutically acceptable excipients include a suspending agent, an aqueous vehicle such as Water for Injection, Ringer's lactate, isotonic saline, salts, buffers, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antimicrobial agents, preservatives, chelating agents, binding agents, surfactants, wetting agents, non-aqueous vehicles such as fixed oils, waxes, creams or polymers or other agents for sustained or controlled release.
[0035] In accordance with another aspect of the invention, compositions according to some aspects of the invention may be used to aggregate at least two cells. The cells may be eukaryotic, or prokaryotic. Examples of cells or cell types include a red blood cell, white blood cell, dermal cell, epithelial cell, secretory cell, hormone secreting cell, storage cell, barrier function cell such as a cell of the lung, gut, exocrine glands, and/or urogenital tract, a contractile cell such as muscle or cardiac cell, blood or immune system cell, nerve cell, sensory transducer cell, central nervous system cell, lens cell, pigment cell, germ cell, embryonic stem cell, pluripotent stem cell, fibroblast, interstitial cells, mature or immature gamete cell, blastomere, hepatocyte, myoblast, myotube, oocyte, osteoblast, osteoclast, T-cell, zygote or the like.
[0036] In accordance with another aspect of the invention, compositions according to some aspects of the invention may comprise a surface for use in the growth of cells. The cells may be prokaryotic, or may be eukaryotic. A 'surface' refers generally to an exterior boundary or outer face of an object, molecule or shape. A surface may be a point of interaction, or an interface, of a first object or molecule with a second object or molecule juxtaposed to the first object or molecule. A 'surface' includes a liquid/solid interface, or an interface between fluids, for example a liquid/liquid interface, or a liquid/gas interface. A surface may be coated or covered by a polymer according to some embodiments of the invention. A surface may be, for example, a metal, or a substance comprising a metal, or may be a surface for growing cells in culture, for example a plastic or glass dish or flask, as are generally known in the field of microbiology, cell culture or the like. For example, a surface may be a glass slide or a bead that is coated with a composition according to some embodiments of the invention. In some embodiments, the surface, i.e. the surface of a plastic cell culture dish or flask may itself be a polymer. Examples of polymers used in cell culture dishes, flasks or other devices used in handling cells in culture include polystyrene, high density polyethylene (HDPE), silicone, Polyvinylidene fluoride (PVDF), low density polyethylent (LDPE), polymethyl methacrylate (PMMA), polycarbonate, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid) or the like
[0037] In accordance with another aspect of the invention, there is provided a method for growing a cell or cells on a scaffold or surface comprising a zwitterionic polymer, the method comprising providing a scaffold or surface, coating the scaffold or surface with a zwitterionic polymer according to some embodiments of the invention, and seeding the coated scaffold or surface with a cell or cell type that is to be grown. Additionally, the cell or cells may subsequently be released or removed from the scaffold or surface by washing with a cationic compound.
[0038] Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.
[0039] This summary of the invention does not necessarily describe all features of the invention. Other aspects, features and advantages of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
[0041] Figure 1 shows an illustration of an exemplary hyperbranched polyglycidol-CP polymer, showing examples of zwitterionic endgroups, according to some embodiments of the invention. Illustrated zwitterionic endgroups include choline phosphate (CP), choline O-sulphate (CS) and dimethylammoniopropane sulphonate (APS).
[0042] Figure 2 shows a plot of a turbidometric titration of HPG-CP-4K (0.2% in 0.15 M NaCI) against HPG-PC-8K in water at a range of concentrations (solid squares), or HPG-CP-8K (0.2% in 0.15 M NaCI) against HPG-PC-8K in water at a range of concentrations (solid circles). X axis shows the concentration of the HPG-PC-8K polymer (wt % in water); Y axis shows the optical density at 540nm.
[0043] Figure 3 shows a plot illustrating the effect of salt concentration on disaggregation of 1 :1 aggregates of HPG-CP-4K with HPG-PC-4K (solid squares) and HPG-CP-8K with HPG-PC-8K (solid circles). X axis shows the NaCI concentration, Y axis shows the optical density at 540nm.
[0044] Figure 4 shows a plot of electrophoretic mobility of negatively charged DSPC:DOPS:CHOL liposomes after incubation with MPEG-(CP) , MPEG-
3
(CP) 4 or MPEG-(CP) 20.
[0045] Figure 5 shows quantitative analyses of crystal violet (CV) stained biofilms of Campylobacter jejuni grown in the presence of A) HPG-CP-8K; B) HPG-PC-8K: C) HPG-8K (no zwitterion group - HPG only) at polymer concentrations of 0-5 mg/ml. Experiments were done in triplicate. X axis shows concentration of polymer in mg/ml; Y axis shows optical density at 570 nm. Biofilms of the virulent WT Cj strain 81 -176 were allowed to form at the air-liquid interface of borosilicate tubes, in triplicate, in the presence of five concentrations of soluble polymers of varying chemical composition. After two days, biofilms were stained with CV, and staining intensity quantified by dissolution of the CV in DMSO and spectrophotometric analyses
[0046] Figure 6 shows cell viability upon exposure to HPG-CP-4K (broad hatched bars) or HPG-PC-4K (narrow hatched bars) at various concentrations of polymer from 0-10 mg/ml.
DETAILED DESCRIPTION
[0047] Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the devices, methods and the like of embodiments of the invention, and how to make or use them. It will be appreciated that the same thing may be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Recital of one or a few synonyms or equivalents does not exclude use of other synonyms or equivalents, unless it is explicitly stated. Use of examples in the specification, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments of the invention herein.
[0048] The term "biocompatible", as used herein, refers generally to the ability of a material to perform with an appropriate host response in a specific application. For example, the biocompatibility of an implantable material refers to the ability of the material to perform its intended function, with a desired degree of incorporation in the host, without eliciting an undesirable level of systemic or localized effect in the host. A biocompatible material, such as a polymer, or a polymer comprising a zwitterionic endgroup, may be capable of being degraded or absorbed by the host, or it may not. The biocompatibility of a polymer, or material made from a polymer may be assessed in a variety of ways. The particular assays or tests employed will vary depending on the polymer and the intended use. Examples of assays include the MTT assay, hemolysis test, intercalation assays, and toxicological studies involving animals (see, for example, Kainthan et al, 2006. Biomacromolecules 7:703-709; Klajnert et al 2006. Cell Biol Int. 30:248-252, herein incorporated by reference).
[0049] As used herein, a 'surface' refers generally to an exterior boundary or outer face of an object, molecule or shape. A surface may be a point of interaction, or an interface, of a first object or molecule with a second object or molecule juxtaposed to the first object or molecule. A 'surface' includes a liquid/solid interface, or an interface between fluids, for example a liquid/liquid interface, or a liquid/gas interface. A surface may be coated or covered by a polymer according to some embodiments of the invention. A surface may be, for example, a metal, or a substance comprising a metal, or may be a surface for growing cells in culture, for example a plastic or glass dish or flask, as are generally known in the field of microbiology, cell culture or the like. For example, a surface may be a glass slide or a bead that is coated with a composition according to some embodiments of the invention. In some embodiments, the surface, i.e. the surface of a plastic cell culture dish or flask may itself be a polymer. In some embodiments, the surface may comprise a scaffold for growing cells or tissue in vitro, for tissue engineering. Polymeric biomaterials for guiding tissue growth to produce artificial organs or other structures may be enhanced by the application of zwitterionic polymers according to various embodiments of the invention. For examples of tissue engineering biomaterials, uses, structures and the like, see, for example, Kim et al 2000. World J Urology 18:2-9; Rather et al 2004. Ann. Rev Biomed Eng. 6:41-75; Falconnet et al 2006. Biomaterials 27:3044-3063.
[0050] Examples of polymers used in cell culture dishes, flasks or other devices used in handling cells in culture include polystyrene, high density polyethylene (HDPE), silicone, Polyvinylidene fluoride (PVDF), low density polyethylent (LDPE), polymethyl methacrylate (PMMA), polycarbonate, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid) or the like.
[0051] As used herein, a 'cell' refers generally to any cell or cells, as well as viruses, subcellular particles or other particles having microscopic size e.g. a size similar to that of a biological cell. There are 100's of cell types and subtypes currently known or named. Cells may be named according to the organ in which they are found, the biological role they perform, their embryonic origin, or the like. In some examples, The cell may be any cell, for example, which is not to be considered limiting, a red blood cell, white blood cell, dermal cell, epithelial cell, secretory cell, hormone secreting cell, storage cell, barrier function cell such as a cell of the lung, gut, exocrine glands, and/or urogenital tract, a contractile cell such as muscle or cardiac cell, blood or immune system cell, nerve cell, sensory transducer cell, central nervous system cell, lens cell, pigment cell, germ cell, embryonic stem cell, pluripotent stem cell, fibroblast, interstitial cells, mature or immature gamete cell, blastomere, hepatocyte, myoblast, myotube, oocyte, osteoblast, osteoclast, T-cell, zygote or the like. Cells may have a nucleus, or may be anucleate, such as a platelet, or mature erythrocyte ('red cell'). Cells may be of a variety of shapes, commonly spherical, but may be rod-shaped, ameboid, elongated, flattened, asymmetrical or otherwise irregular. A cell may be alive or dead, or in a form of stasis or rest. The size of a cell may vary widely, with a diameter from about 0.1 microns to about 200 microns, although more typically in the range from about 1 micron to about 100 microns. The size of a cell may vary depending on the stage of growth cycle. The cell may be a transformed cell, for example a cell isolated from a tumor, or a cell infected by a virus. The cell may be a primary cell, or may be an immortalized cell grown in culture conditions.
[0052] Biocompatible polymers have a myriad of uses known in the art, and incorporation of various functional groups may tailor a polymer or polymers to a particular application. The number of functional endgroups in a polymer is dependent on the quantity of ends - for example, a linear polymer will have two ends, a dendrimeric polymer will have multiple ends and a hyperbranched polymer will have more still, depending on the degree of branching and size of the molecule. The form of the polymer - linear, dendrimeric or hyperbranched, for example - may be influenced by both the monomer, or combination of monomers used, for example in a block copolymer, and by the conditions of synthesis.
[0053] A "polymer" as used herein (Ri of Formula 1-7, infra) may be a linear polymer, dendrimeric polymer or hyperbranched polymer. The polymer may be a homopolymer, comprising a single species of monomer, or may be a heteropolymer, alternatively referred to as a copolymer, comprising at least two species of monomers. The monomers are linked in a chemical reaction referred to as polymerization. A polymer incorporating more than one species of monomer may be referred to as a copolymer, and the chemical reaction that links them as copolymerization. Copolymers may be described as random, alternating or block copolymers, depending on the monomers and reaction conditions used to generate them. Polymers and copolymers may be generally described as crosslinked, thermoplastic, thermoset or elastomeric, or combinations of these. Various references known in the art discuss methods of syntheses, methods of manipulation of polymerization reactions to modify branching, incorporation ratios in copolymer syntheses, and various physical characteristics of the polymers including solubility, net electronic charge, flexibility, physical strength and the like. Examples of such references may include RJ Young and PA Lovell, "Introduction to Polymers", 2nd edition. Chapman and Hall 1991 ; Odian G. "Principles of
Polymerization", New York, Wiley Press 1991 ; Carraher CE, "Introduction to Polymer Chemistry" Boca Raton, Taylor & Francis; Lowe and McCormick 2002. Chem. Rev, 102:4177-4189.
[0054] "Ring-opening polymerization" as used herein, refers to a type of addition polymerization. An end of a growing polymer chain serves as a reactive centre capable of reacting with additional monomers to increase the chain length. For a ring-opening polymerization, the monomer is a cyclic compound. Ring-opening polymerizations are described in the art, for example, US Patent 5,582,327; Amecke et al 1992. Clin Mater. 10:47-50; Grubbs and Tumas, 1989. Science 243:907-915; Liggins and Burt, 2002. Adv. Drug. Deliv Rev. 54:191-202;
Albertsson and Varma, 2003. Biomacromolecules 4:1466-86, all of which are incorporated herein by reference. Other variant reactions, necessary reaction conditions, monomers and methodologies will be known to one of skill in the art. Polydispersity, or polydispersity index (PDI) describes the distribution of the range of molecular weights in a given polymer sample. A narrow PDI has less variation in the molecular weights, while a broader PDI has a greater range. PDI is a value greater than 1. A polymer sample with uniform chain length, for example, would have a PDI approaching unity (1). The PDI of a polymer is dependent on the mechanism of polymerization and reaction conditions, including ratios of reactants, stage of completion of the polymerization reaction, and the like.
[0055] The composition of the polymer may be modified depending on the physical characteristics and use of the polymer. For example, if bioadsorption of the polymer is desired, then a poly(lactic acid) or epsilon poly(caprolactone) polymer may be used. These polymers have been found to degrade under normal physiological conditions over a 3-6 month period. (Andersen and Shive, 1997. Adv. Drug. Del. Rev. 28:5-24).
[0056] Various other polymers, monomers and monomer derivatives from which they may be synthesized, and methods of synthesis will be familiar to those of skill in the art. See, for example, Lee et al 2005. Nature Biotechnol. 23:1515- 1526; Klajnert et al 2006. Cell Biol Int. 30:248-252. Methods are described in the art for the preparation of polyglycidol polymers. Polyglycidol may alternatively be referred to as polyglycerol - polymerization of glycidol (CaH6O2) by a ring-opening reaction, or polymerization of glycerol, yields the same polymer, illustrated generally in Figure 1.
[0057] Such polymers may be linear, branched, hyperbranched or dendrimeric,. Examples of these methods may be found in, for example, Sunder et al. 1999
Macromolecules 32:4240; Sunder et al. 2000. Chemistry 6 :2499-2506; Sunder et al. 1999. Angew Chem lnt Ed Engl 3 :3552-3555; Sunder et al. 1999. Angew Chem lnt Ed Engl 38:2928-2930; US Patent No. 6822068; PCT Patent Application WO 03/37532, the entirety of which are incorporated herein by reference. Methods for making high molecular weight polyglycerol polymers, for example 20,000 and above, are described in, for example, Kainthan et al 2006. Macromolecules 39:7708-7717, incorporated herein by refrence..
[0058] Examples of polymers that may comprise embodiments of the invention include but are not limited to poly(oxyalkylene) polymers, polyglycerol polymers, polyglycidol polymers hyperbranched polyglycidol polymers, polyglycidol-block polymers, poly(glutamic acid) polymers, polyamidoamine (PAMAM) polymers, polyethyleneimine polypropyleneimine (PPI) polymers, polymelamine polymers, polyester polymers, poly(lactic acid) polymers, epsilon poly(caprolactone), poly(lactone), substituted poly(lactones), poly(lactam), substituted poly(lactam), methoxy polyethylene glycol (MPEG or MePEG), polyethylene glycol (PEG), dextran, starch, cellulose, collagen, gelatine, chitosan deacetylated chitosan, or the like.
[0059] In one embodiment of the invention, a polymer may comprise a zwitterionic functional endgroup, or a 'zwitterionic group'. A 'zwitterion', as used herein, generally refers to a compound that has both positive and negative charges. At certain pH, a zwitterion, or zwitterionic composition, may comprise both a negatively charged anionic group and a positively charged cationic group, providing a net neutral charge on the molecule. Several examples of zwitterionic compounds are known in the art, including phosphoryl choline ("PC"), choline phosphate ("CP"), buffer compounds useful in biological buffer systems, including
HEPES, PIPES, CAPS or MOPS, and the like; amino acids, or the like .
[0060] Compositions according to some embodiments of the invention may be referred to generally as "zwitterionic polymers", "zwitterions-polymers", "zwitterionic compounds" or other similar terms.
[0061] In one embodiment of the invention, polymers according to Formula 1 are provided.
Formula 1
Figure imgf000019_0001
[0062] R is a polymer, for example a linear, branched, dendritic or hyperbranched polymer. In some embodiments the polymer R may be a surface or a coating on a surface, configured so as to present a zwitterionic group comprising A-X-Y, a monovalent, multivalent or polyvalent fashion.
[0063] A is an atom comprising a net positive charge. In some embodiments, A may be a quaternary nitrogen, a trivalent sulphur or tetravalent phosphorous.
[0064] X is a substituted alkyl group comprising at least one carbon An alkyl chain includes any saturated or unsaturated hydrocarbon chain comprising at least 2 carbons. The alkyl group may comprise substituent groups, such as H, a 1-15 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms. At least one C of the 1 -15 carbon saturated, unsaturated or cyclic alkyl group may further be replaced by O, S, SO, SO2, NH2, or NR', or may be optionally substituted with OH, OR', =O, SH, SR', NH2, NHR', NR'2, OSO3H, OPO3H3, CO2H, CO2R' or the like.
[0065] Y is a group comprising a net negative charge, such as a phosphate, phosphonate, phosphinate, sulphate, sulfonate, carbonate or carboxylate group. Y may further comprise a substituted alkyl group (-C-(R4, R5,. R6) group where R4,
R and R may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal,
5 6 aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide or the like. Formula 2
Figure imgf000020_0001
[0066] For compounds according to Formula 2, A as defined in Formula 1 is NR - R 2 -R 3 , where R 1 is a polymer or a surface, as described. R 2 and R 3 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide or the like.
[0067] For compounds according to Formula 2, Y as defined in Formula 1 is PO -
4
W, where W may be nothing - leaving a single negative charge on the O, or W may be -CR 4 R 5 R 6 , where R 4 , R 5 and R 6 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate or peptide.
[0068] m is an integer value of 2, 3, 4, 5 or more.
Formula 3
Figure imgf000020_0002
[0069] For compounds according to Formula 3, A as defined in Formula 1 is NR - R -R , where R is a polymer or a surface as described. R and R may
2 3 1 2 3 independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide or the like.
[0070] For compounds according to Formula 3, Y as defined in Formula 1 is PO -
3
W, where W may be nothing - leaving a single negative charge on the O, or W may be -CR 4 R 5 R 6 , where R 4 , R 5 and R 6 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate or peptide.
[0071] m is an integer value of 2, 3, 4, 5 or more.
Formula 4
Figure imgf000021_0001
[0072] For compounds according to Formula 4, A as defined in Formula 1 is NR -
R -R , where R is a polymer as described. R and R may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide or the like. [0073] X is a substituted alkyl group comprising at least one carbon An alkyl chain includes any saturated or unsaturated hydrocarbon chain comprising at least 2 carbons. The alkyl group may comprise substituent groups, such as H, a 1-15 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms. At least one C of the 1 -15 carbon saturated, unsaturated or cyclic alkyl group may further be replaced by O, S, SO, SO2, NH2, or NR', or may be optionally substituted with OH, OR', =O, SH, SR', NH2, NHR', NR'2l OSO3H, OPO3H3, CO2H, CO2R' and the like.
[0074] According to some embodiments of the invention, X is (-CH2-), where m is 2, 3, 4, 5 or more.
Formula 5
Figure imgf000022_0001
[0075] For compounds according to Formula 5, A as defined in Formula 1 is N. R is a polymer or a surface as described. R and R may independently be H,
1 2 3 alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide or the like.
[0076] According to some aspects of the invention, X is (-CH2-), where m is 2, 3, 4, 5 or more.
Formula 6
Figure imgf000022_0002
[0077] For compounds according to Formula 6, A as defined in Formula 1 is N. R is a polymer as described.. R and R may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide, or the like.
[0078] According to some embodiments of the invention, X is (-CH2-), where m is 2, 3, 4, 5 or more.
Formula 7
Figure imgf000023_0001
[0079] For compounds according to Formula 7, A as defined in Formula 1 is N. . Ri is a polymer as described. R2 and R3 may independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate peptide or the like.
[0080] According to some embodiments of the invention, X is (-CH2-), where m is 2, 3, 4, 5 or more.
Generic synthesis of polymer-zwitterion
Scheme 1
Figure imgf000024_0001
(2) <3) (Formula 8)
[0081] Reagent (2) is a polymer comprising an amine group at one end, the amine comprising substitutent groups Ffe, R3 as described supra. Ri is a polymer as described supra. The polymer may comprise an amine as a result of the polymerization reaction, or be aminated following polymerization according to known methods.
[0082] A polymer comprising an -OH group at an end may be aminated following polymerization, using methods known in the art; for example, by dissolving in a polar aprotic solvent (e.g pyridine, DMF, DMSO, diglyme or the like) and reacting with tosyl chloride. The resulting polymer-tosylate may be subsequently refluxed with an alkylamine, such as ethylamine in THF. Other solvents that may be used in this reflux include dioxane, DMF, DMSO, diglyme or the like.
[0083] Reagent (3) is a phospholane, where Z is -CR4-Rs-Re, comprising R4-R5- R6 as described above. The dioxa-ring may comprise (m) methylene groups, where m is 2, 3, 4, 5 or more. R7 and R8 may independently be H, alkyl, alkene, alkyne, benzyl, or aryl. An alkyl group includes any saturated or unsaturated hydrocarbon chain comprising at least 2 carbons. The alkyl group may comprise substituent groups, such as H, a 1 -5 carbon saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic forms. At least one C of the 1-5 carbon saturated, unsaturated or cyclic alkyl group may further be replaced by O, S, SO, SO2, NH2, or NR', or may be optionally substituted with OH, OR1, =O, SH, SR', NH2, NHR', NFP2, OSO3H, OPO3H3, CO2H, CO2R'.
[0084] The phosphorylation of reagent (2) by a ring-opening reaction with the phospholane may be done in a non-polar solvent. Examples of non-polar solvents include acetonitrile, alcohols such as methanol, ethanol, propanol or isopropanol, diglyme, DMSO and the like. All reagents would need to be soluble in the chosen solvent.
[0085] Other general methods, reagents and the like for isolation and analysis of the products will be known to those of skill in the art.
Synthesis of HPG-CP
[0086] In another embodiment of the invention, the polymer may be a polyglycidol, such as a branched, or hyperbranched, polyglycidol (HPG) in a wide range of molecular weights. The polymer may. for example, have a molecular weight from about 2000 Da to about 20,000 Da, or any amount therebetween. Larger HPGs are also contemplated, having a molecular weight in excess of 100 kDa, or as great as about 1 x 106 Da. Such larger HPGs, methods of their synthesis, biological compatibility and other characteristics are described in Kainthan et al 2007, supra. Other embodiments of the invention may contemplate HPG polymers in a wide range of molecular weights, the particular range of molecular weights being employed advantageously for particular functions, such as coating a surface or a device, encapsulating a drug, or other functions described below.
[0087] Very high molecular weight hyperbranched polyglycerols with narrow polydispersities may be obtained by ring opening multibranching polymerization of glycidol using, for example, dioxane as the reaction medium. Alternately, molecular weight polymers with a broader polydispersity may be obtained using diglyme as a reaction medium. Other solvents that may be used include THF or DMSO. See, for example, Kainthan RK and Brooks, DE. 2007 Biomaterials 28:4779-4787; Kainthan et al 2006 Macromolecules 39:7708-7717; and WO 2006/130978, herein incorporated by reference. These HPGs having molecular weights from about 2000 to about 1x106 Da may be used in the synthesis of HPG-CP zwitterionic polymers as described herein.
[0088] HPG may comprise -OH endgroups, and as such may require amination following polymerization (shown in Scheme 2 reagent 4). Synthesis scheme 2 is an exemplary scheme, illustrating a tertiary amine endgroup comprising two methyl groups, however other alkyl groups or aromatic groups may be contemplated, as described above. Phosphorylation of Formula 4 by 2-methoxy- 1 ,3-2-dioxaphospholane-2-oxide (shown in scheme 2, reagent 5) yields HPG- choline phosphate (HPG-CP) shown as reagent 6 of scheme 2.
Scheme 2
HPOHWf1 *
Figure imgf000027_0001
(4) {5) (6)
[0089] The quantities of reagents used in the methods and examples described may be expressed in a variety of ways. The amount of a particular reagent may be described by, for example, mass (e.g. grams), or by volume (e.g. millilitres), by ratio with another reagent (e.g. 2:1 ), by percentage volume (e.g. 10% (vol/vol), by percentage weight (e.g. 10 wt%, or 10% by wt), and the like. For example a 10% NaOH solution may be made by dissolving 10 g of NaOH solid in a final volume of
100 ml water.
[0090] According to another embodiment of the invention, the polymer is methoxypolyethylene glycol (MPEG or MePEG). The polymer may, for example, have an average molecular weight from about 200 to about 20,000, or from about 500 to about 10,000, or any amount therebetween. Other embodiments of the invention may contemplate MPEG polymers in a wide range of molecular weights, the particular range of molecular weights being employed advantageously for particular functions, such as coating a surface or a device, encapsulating a drug, or other functions described below.
[0091 ] In another embodiment of the invention, addition of the zwitterionic groups to the polymer may be done on a surface, such as a plastic or glass slide, etc. The polymer coating the surface may comprise an amine group, such as a tertiary amine group, and the reaction may be performed according to scheme 1 , supra.
Uses of zwitterion-polymers
[0092] The particular use of the zwitterion-polymer may influence the choice of molecular weight range. With branched polymers, including hyperbranched polymers, the molecular weight, degree of branching and type and quantity of endgroups available for interaction are interdependent. The optimal molecular weight, degree of branching and choice and quantity of endgroups may be determined empirically, and such determinations are within the ability of one of skill in the art.
[0093] In another embodiment of the invention, the interaction of a zwitterion with a cell may be used to 'camouflage' the cell. Long-chain polyethylene glycol covalently attached to a red cell reduces the antigenicity of the red cell. A polyethylene glycol polymer comprising one or more zwitterion endgroups that attach noncovalently to a cell surface may provide the same camouflage effect, with the advantage of being reversible by addition of phosphoryl choline alone or covalently attached to another polymer, or by washing the cells with an albumin solution, saline solution, or other suitable salt or buffer solution. The polymer may comprise one, or a plurality of zwitterion endgroups.
[0094] In another embodiment of the invention, the zwitterion-polymers may be used in tissue culture surfaces. Tissue engineering, to produce three-dimensional living tissue complexes, is dependent on a synthetic scaffold to give shape and support to the cells. While many immortal cell lines in culture are readily adherent to most surfaces, primary cells vary in their need and ability to attach readily to a surface. Further, removing cells from the surface, according to conventional cell culture techniques and methods, may involve use of enzymes, such as trypsin, to digest and weaken the extracellular matrix that allows the cells to adhere. Trypsinization' of cells may adversely affect the viability of the cells.
[0095] Use of the zwitterion-polymers according to various embodiments of the invention in the manufacture of or to coat surfaces for growth of cells enables adhesion of the cells, with the advantage of being reversible by subsequent exposure to or addition of, for example, a compound comprising a cationic group or functional moiety (a 'cationic compound), for example phosphatidyl choline. The cationic compound may be, for example, free in solution, and/or covalently attached to another polymer. The polymer may comprise one, or a plurality of cationic or zwitterionic endgroups. To reverse the interaction of the polymer comprising zwitterionic endgroups according to some embodiments of the invention, the cell layer or aggregation may be washed with a solution comprising a cationic compound. The solution may comprise, for example an albumin solution, saline solution or other suitable salt or buffer solution.
[0096] As an example, cells may be grown on a synthetic scaffold by providing a surface, coating the surface with a zwitterionic polymer according to some embodiments of the invention, and seeding the coated surface with a cell or cell type that is to be grown. If splitting or reculturing of the cells is necessary or desirable , the cells may be released from the scaffold by washing with a cationic compound, the cationic compound washed away and the cells returned to the scaffold, or to another scaffold or other suitable culture vessel.
[0097] In another embodiment of the invention, the zwitterion-polymers may be used to coat implantable medical devices where cell adhesion by non-covalent means is desirable. Examples of such medical devices may include stents, urinary catheters, biosensors or implants of various types.
[0098] In another embodiment of the invention, the zwitterion-polymers may be used to inhibit biofilm formation on surfaces or devices. A polymer comprising, for example, a PC zwitterion may be used to coat a surface and reduce adherence of cells, such as bacterial cells, to the surface.
[0099] In another embodiment of the invention, the zwitterion-polymers may be used to aggregate or bind cells in a suspension, or 'trap' cells in a filter. Cells may be, for example, bacterial cells. A filter may comprise, for example, a polymer comprising a CP zwitterion, for example an HPG-CP zwitterionic polymer, that binds bacterial cells to the filter. Additionally, a zwitterionic polymer may be added to an aqueous bacterial suspension, causing the bacteria to aggregate and be trapped by the filter, thus removing them from the suspension and preventing their discharge as the aqueous phase leaves the filter. In other embodiments of the invention, a device, a surface or a medical device, for example, may be constructed so as to comprise a surface suitable for derivatization and addition of zwitterionic groups according to the general scheme 1 or scheme 2. Such a surface, device or medical device would comprise, as indicated above, available tertiary amine, hydroxyl or carboxyl groups for the coupling reaction.
[00100] In other embodiments of the invention, zwitterion-polymers may be applied in multiple layers on a surface, device or medical device. The layers may comprise different polymers, or comprise the same polymer with same or different zwitterionic ends. The layers may encapsulate or otherwise deliver a drug, and the layers may be produced so as to provide desired hydrophobicity, hydrophilicity or aggregation characteristics.
[00101] In other embodiments of the invention, zwitterion-polymers may be used to adhere specific tissue surfaces to each other, but not to other tissue surfaces. For example, a first tissue may be treated or have applied to it a zwitterionic polymer comprising a CP group, and a second tissue may be treated or have applied to it a zwitterionic polymer comprising a PC group. The zwitterions of these polymers may preferentially interact with each other, and thus prevent undesired tissue adhesions. Such an embodiment may be useful, for example, during or following surgical procedures, or to facilitate implantation of a medical device. See for example Benjamin et al 2003. JACS 125:7494-7495.
[00102] In another embodiment of the invention, zwitterionic polymers according to some embodiments may be used as coagulating agents. For example, a spray, aerosol, solution or other suitable formulation of zwitterionic polymers may be applied to a wound to aid in coagulating blood. In another embodiment, zwitterionic polymers may be provided as a coating or substance associated with a bandage to be placed on a wound.
[00103] In another embodiment, the zwitterionic polymer used to adhere specific tissue surfaces may further comprise a drug or a biologically active molecule.
[00104] In other embodiments of the invention, zwitterionic polymers may be used to coat a bead or other surface. The bead may further comprise a fluorescent tag or label, or chromatographic dye, or other tracking or visualization features. The coated, tagged bead may be used to tag or label cells in a suspension,
[00105] In other embodiments, zwitterionic polymers may be used as a transfection reagent to enhance the uptake of another compound or composition by a cell. As an example, a zwitterionic polymer according to some embodiments of the invention may be combined with the compoundthat would otherwise have poor or no solubility in a transfection buffer. The association of the zwitterionic polymer with the cell or cell aggregate may act to increase the effective local concentration of the compound and thus increase the efficiency with which the compound is taken up by the cells.
Drug delivery
[00106] In another embodiment of the invention, the zwitterion-polymers may be used in the production of liposomes, micelles, polymeric aggregates, conjugates, drug conjugates or the like, which may be used to encapsulate or deliver drugs. The adhesive nature of the zwitterionic polymers with respect to organs, cells and tissues may enable liposomes comprising such zwitterionic polymers to adhere to cells or tissues to which they are applied, for example topical application on a surface, delivery by a 'patch', or in the case of treatments for hollow organs (i.e. a bladder), a solution or suspension of the liposomes may be used to fill the organ and deliver the liposomes comprising a drug to the specific tissue, and also avoid systemic effects. Liposomes according to some embodiments of the invention may provide the further advantage of remaining associated with the tissue to which they are applied and may not be flushed out as readily ie. in the example where the liposomes are used on the bladder, the interaction with the cell surface may prevent their removal when the subject urinates. Localized delivery using zwitterionic polymers according to various embodiments of the invention are not ligand-specific, and thus may be more adaptable to various organ, tissue or cell types. [00107] Drugs that may be particularly suitable for encapsulation and/or delivery using the zwitterionic polymers according to some embodiments of the invention, include those with significant toxic effects, or poor solubility, or both, such as chemotherapeutic agents. Examples of such agents may include include mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil; hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), semustine (methyl- CCNU), lomustine (CCNU), streptozocin (streptozotocin), estramustine phosphate, dacarbazine, temozolomide, methotrexate (amethopterin), fluorouracin (5-fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), gemcitabine, mercaptopurine (6- niercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), pentostatin (21- deoxycoformycin, deoxycoformycin), cladribine, fludarabine, amsacrine, vinblastine (VLB), vincristine, paclitaxel, docetaxel , etoposide, teniposide, topotecan, irinotecan, dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin, L-asparaginase, interferon alpha, buserelin, prednisone, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethinyl estradiol, tamoxifen, anastrozole, testosterone propionate, fluoxymesterone, flutamide, bicalutamide, leuprolide, thalidomide, cisplatin (czs-DDP), oxaliplatin, carboplatin, mitoxantrone, hydroxyurea, procarbazine (N-methylhydrazine, MIH), mitotane, aminoglutethimide, bexarotene, imatinib. Alternate names and trade-names of these and additional examples of chemotherapeutic agents, may be found in, for example Goodman & Gilman's "The Pharmacological basis of therapeutics", 11th edition. LL Brunton, editor. McGraw-Hill, New York.
[00108] Branched polymers, dendrimers or hyperbranched HPGs incorporating zwitterionic groups may be derivatized to enable them to bind and release a drug or drugs in a localized manner, effectively increasing local concentration. Increased localized concentration juxtaposed to a cell membrane, tissue or organ may enhance uptake by the cell, tissue or organ. [00109] The pH sensitive cell adhesive property of zwitterionic polymers (non adhesive at < pH = 3 and cell adhesive at > pH = 7) may be used for increasing the bioavailability of hydrophilic drugs in oral delivery or local delivery drugs to the small or large intestine. In some embodiments of the invention, drugs, peptides, proteins or the like encapsulated or conjugated with zwitterionic polymers and may be as administered orally. A zwitterionic polymer designed for such a used would be non- or poorly adhesive in the low pH environment of the stomach, and when transitioned to the alkaline environment of the duodenum and small intestine, gain adhesive properties and 'stick' to the gut lining. This may increase the retention time of the drug, etc in the intestine, and may increase absorption, leading to improved bioavailability.
[00110] In some embodiments of the invention, liposomes comprising zwitterionic groups are provided. The liposomes may comprise dipalmitoyl choline phosphate (DPCP). In some embodiments, the liposomes may further comprise cholesterol, dipalmitoyl phosphatidyl choline and/or a polyalkylene glycol, such as methoxypolyethyleneglycol. Liposomes generally may be used to improve the efficiency of delivery of poorly water-soluble drugs. In some examples, a poorly water-soluble drug may be encapsulated in liposomes comprising DPCP, and the ability of the zwitterionic group (CP) on the liposome may delay the dispersion of the liposome encapsulated drug from the site of injection, thus increasing the local concentration of the drug, and providing for a controlled release in situ.
Anti-lnfectives [00111] In another embodiment of the invention, the zwitterion-polymers may be used as a topical anti-infective. The zwitterion-polymers may be incorporated into a suitable excipient and when applied to the skin, or a break in the skin, adhere to and aggregate foreign cells at the site of application, preventing spread of the foreign cells to distal locations in or on the body. Foreign cells aggregated in this manner may subsequently be more easily removed from the site of application when the zwitterion-polymer composition is removed. [00112] In another embodiment of the invention, zwitterionic polymers may be used as a physical barrier to an infectious organism. For example, MPEG- (CP)n zwitterionic polymers, such as MPEG-(CP)20 may be useful for this application. Such a polymer may be applied to a tissue surface, where the zwitterion group associates with the tissue surface, and the MPEG polymer is oriented away from the tissue surface.
[00113] Application or administration of a zwitterionic polymer as part of an anti-infective, or as a delivery vehicle for a drug or similar, may necessitate combining the zwitterionic polymer with a pharmaceutically acceptable excipient. The choice of excipient will be dependent on the particular use or requirement to be met, for example, if it is to be injected, sterile Water for Injection may be a suitable excipient, while if it is to be administered orally, the excipient may include a suspending agent. Pharmaceutically acceptable excipients include, for example, an aqueous vehicle such as Water for Injection, Ringer's lactate, isotonic saline, salts, buffers, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antimicrobial agents, preservatives, chelating agents, binding agents, surfactants, wetting agents, non-aqueous vehicles such as fixed oils, waxes, creams or polymers or other agents for sustained or controlled release. See, for example, Berge et al. (1977. J. Pharm Sci. 66:1-19) or Remington- The Science and
Practice of Pharmacy, 21st edition. Gennaro et al editors. Lippincott Williams & Wilkins Philadelphia, (both of which are herein incorporated by reference).
[00114] Routes of administration of a zwitterionic polymer as part of an anti- infective, or as a delivery vehicle for a drug or similar may be selected depending on the nature of the drug to be delivered, the nature of the infection to be treated or prevented or the like. Examples of routes of administration include, for example, subcutaneous injection, direct injection into a disease site or tissue type, for example direct injection into a solid tumor, intraperitoneal injection, intramuscular injection, intravenous injection, epidermal or transdermal administration, mucosal membrane administration, ophthalmic, orally, nasally, rectally, topically, or vaginally. See, for example, Remington, The Science and Practice of Pharmacy, 21st edition. Gennaro et al. Editors. Lippincott Williams & Wilkins, Philadelphia. Carrier formulations may be selected or modified according to the route of administration.
[00115] Compositions comprising a zwitterionic polymer according to various embodiments of the invention may be provided in a unit dosage form, or in a bulk form suitable for formulation or dilution at the point of use. Such compositions may be administered to a subject in a single-dose, or in several doses administered over time. Dosage schedules may be dependent on, for example, the subject's condition, age, gender, weight, route of administration, formulation, or general health. Dosage schedules may be calculated from measurements of adsorption, distribution, metabolism, excretion and toxicity in a subject, or may be extrapolated from measurements on an experimental animal, such as a rat or mouse, for use in a human subject. Optimization of dosage and treatment regimens are discussed in, for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics 11th edition. 2006. LL Brunton, editor.
McGraw-Hill, New York, or Remington, The Science and Practice of Pharmacy, 21st edition. Gennaro et al. Editors. Lippincott Williams & Wilkins, Philadelphia.
[00116] A "subject" may refer to an animal, such as a mouse, rat, dog, cat, pig, sheep, guinea pig, hamster or a primate, such as a monkey, chimpanzee or human.
METHODS AND MATERIALS
[00117] All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON) and used without further purification except the following. Glycidol (96 %) was purified by vacuum distillation and stored over molecular sieves in a refrigerator (2 - 4 0C). Anhydrous diglyme and dioxane were obtained from Aldrich and used without further drying. Molecular weights and polydispersities of polyglycerol samples were determined by gel permeation chromatography (GPC) on a Waters 2690 separation module fitted with a DAWN EOS multiangle laser light scattering (MALLS) detector from Wyatt Technology Corp. with 18 detectors placed at different angles (laser wavelength = 690 nm) and a refractive index detector (Optilab DSP from Wyatt Technology Corp.). An Ultrahydrogel linear column with bead size 6-13 μm (elution range 103 - 5 x 106 Da) and an Ultrahydrogel 120 with bead size 6 μm (elution range 150 - 5 x 103 Da) from Waters were used. An aqueous 0.1 N NaNO3 solution was used as the mobile phase at a flow rate of 0.8 mL/min. The dn/dc value for polyglycerol was determined to be 0.12 in aqueous 0.1 N NaNO3 solutions and was used for molecular weight calculations. The data were processed using Astra software provided by Wyatt Technology Corp.
[00118] Intrinsic viscosity, hydrodynamic radii, Mark-Houwink parameters and radius of gyration (Rg) were obtained from a triple detector from Viscotek
Corp. connected to the GPC system, which utilizes refractive index, 90-degree light scattering and intrinsic viscosity determinations. The data were processed using the software provided by Viscotek. Hydrodynamic radii (Rh) were also obtained from a quasi elastic light scattering (QELS) detector (Wyatt Technology Corp.) which was connected to the MALLS detector using the Astra software.
Synthesis of high molecular weight hyperbranched polyglycerols
[00119] A detailed polymerization procedure is described by Sunder et al. 1999 Macromolecules 32:4240, and modified as follows. After the reaction, the polymer was dissolved in methanol, neutralized by passing three times through a column containing cation exchange resin (Amberlite IRC-150). The polymer was then precipitated into excess of acetone and stirred for 1 hour. Acetone was decanted out and this procedure repeated once. Dialysis of polymers was done for three days against water using cellulose acetate dialysis tubing MWCO 1000 or 10,000 g/mol) with water being changed three times per day. The dry polymer was obtained by lyophilisation.
Synthesis of hyperbranched HPG-PC-4K and -8 K
[00120] Two hyperbranched polyglycidols (HPG) of molecular weights 4000 (4K) and 8000 (8K) were used. The polymer (500 mg) was dissolved in 5 ml pyridine and 2-chloro-1 ,3-2-dioxa phospholane-2-oxide (1 ml) was added drop wise while keeping the reaction mixture at O0C. After overnight stirring, the reaction mixture was added to acetone to precipitate the polymer out. The precipitation was repeated twice after dissolving the polymer in a small amount of methanol. Polymer was dried in vacuum and characterized by 1H and 31P NMR. The purified polymer (80 mg) was dissolved in 5 ml methanol and 0.3 ml trimethylamine was added and heated at 550C for 3 days in a pressure bottle. The volatiles were then removed in vacuum and the polymer was precipitated twice from acetone.
Synthesis of Dipalmitoyl CP (DPCP)
[00121] N,N-dimethyl-2,3-dihydoxypropane (2g) was dissolved in 100 ml anhydrous THF. 4.8 ml triethylamine was added and the reaction mixture was kept at O0C. Palmitoyl chloride (9.68 g) was added drop wise. The reaction mixture was stirred overnight and filtered through a filter paper. The filtrate was evaporated to give a while solid powder. The product was purified by flash chromatography (silica gel, eluted first with hexane and then with 20 % ethyl acetate: hexane mixture).
[00122] The dipalmitoyl ester of N,N-dimethyl-2,3-dihydoxypropane (1 g) and
0.22 g of 2-methoxy-1 ,3-2-dioxa phospholane-2-oxide were dissolved in 15 ml THF and stirred at 550C for 3 days. The solvents were then removed and the solid was washed with acetonitrile to remove unreacted methyl phospholane.
[00123] Cytotoxicity testing: The cell lines were obtained from American Type Culture Collection (ATCC), USA. The in vitro cytotoxicity of HPG-A against
L-929 fibroblast cell lines was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Adherent cell lines were plated into 96-well plates one day prior to adding polymers to allow the cells to adhere to the plate. The cell density was 5000 per well. The plates were left in an incubator overnight. The following day, the polymer solutions (100 μl) made in the culture medium were added at increasing concentration (0.0001 , 0.001 , 0.01 , 0.1 , 0.5, 1 , 5 and 10 mg/ml) to the wells containing 100 μl of medium. Following two days of incubation at 370C, 40 μl of MTS solution was added to the wells. The absorbances were measured using a spectrophotometer at 570 nm and compared with those of controls (200 μl of medium). Fibroblast culture media consisted of Minimum Essential Medium with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate (90%) and fetal bovine serum (FBS) (10%). The polymer HPG-CP showed very little cytotoxicity toward fibroblasts with more than 100 % cell viability observed even at a high concentration of 1 mg/ml after 48 hr incubation whereas the HPG-CP-4K was non toxic up to 0.01 mg/ml. Preparation of RBC for aggregation assay
[00124] The response of red blood cells (RBC) cells to HPG-CA polymers added to human blood in vitro was determined by microscopic examination. 10 ul of 10 wt % polymer solutions were added to 40 ul of EDTA -treated blood and incubated for 10 minutes. Aggregation was assessed under microscopic examination, using a qualitative index - a "visual aggregation index" (VAI). A VAI score relates to the following observations:
VAI = 0 single, separated cells, no aggregation 1 50% single cells : 50% aggregated cells
2 rouleaux : >90% cells in mainly linear aggregates
3 rouleaux in side-to-side aggregates
4 small clumps: distorted and crenated cells
5 large clumps: distorted and crenated cells
EXAMPLES
Example 1
Synthesis of HPG-CP-4k Synthesis of HPG-CP-4K and -8 K
Amination of HPG
[00125] To a solution of 5g HPG prepared as described (average polymer mw 4K or 8K) in 25 ml pyridine, tosyl chloride was added in small quantities
O keeping the reaction mixture at 0 C and was stirred for 24 h at room temperature. The pyridinium salt was filtered off and the viscous solution was poured in to large volume of 1 N HCI. The polymer was then collected, dissolved in chloroform and washed with water three times. The solution was dried over anhydrous MgSO and evaporated. The product, HPG-tosylate, was verified and characterized by 'H
NMR. The HPG-tosylate (5 g) was dissolved in 10 m THF and refluxed under a nitrogen atmosphere for 24 hrs with 2M ethylamine solution in THF (30 ml). The volatiles were then removed to provide the polymer. The residual secondary amines in the polymer were methylated by refluxing with a formaldehyde/formic acid mixture (1 :1). HPG-amine (2.5 g) was taken in 250 ml round-bottom flask, and 50 ml water added, with stirring. To the cooled solution, 0.5 ml concentrated HCI was added dropwise. Formic acid (80%, 10 ml) was then added drop wise followed by formaldehyde (8 ml). The reaction mixture was refluxed for 24 hours. The volatiles were removed under vacuum. The residue was dissolved in 10% (by wt) NaOH solution so that the solution became alkaline. The HPG-Amine was extracted with dichloromethane after saturating the aqueous layer with NaCI.
Synthesis of 2-methoxy-1 ,3-2-dioxa phospholane-2-oxide
[00126] A 1.12 g aliquot of methanol and 5.25 ml triethylamine were added
O to 25 ml ether taken in a round-bottom flask and cooled to 0 C. 2-chloro-1 ,3-2- dioxa phospholane-2-oxide (3.22 ml) was added drop wise and stirred for 2 hours. The precipitate was removed by filtration and the solid the washed with more ether. The product was isolated from the combined filtrate by evaporation of the volatiles.
Phosphorylation of HPG-amine (general phosphorylation procedure)
[00127] The HPG amine and 2-methoxy-1 ,3-2-dioxa phospholane-2-oxide (2 eq) were dissolved in acetonitrile in a round bottom flask with a condenser and
O stirred at 55 C for 3 days. After the reaction the polymer was precipitated in ether. The isolated polymer was dissolved in a small amount of methanol and precipitated in ether. This procedure was repeated three more times, and the polymer was dried in vacuum. Tris(2-dimethylaminoethyl)amine (Me 6 TREN) and a permethylated polyethylimine (PEI) were also phosphorylated using the same procedure.
Figure imgf000041_0001
Synthetic Scheme for HPG-CP-4K
Example 2
Sulfonation of HPG-amine
[00128] The HPG amine of Example 1 (50 mg) and 1 ,3-propane sultone (20 mg) or 1 ,3,2-Dioxathiolane 2,2-dioxide (20 mg)) were dissolved in acetonitrile or
0 methanol in a round bottom flask with a condenser and stirred at 55 C for 3 days.
After the reaction the polymer was precipitated in ether or acetone. The isolated polymer was dissolved in a small amount of methanol and precipitated in ether. This procedure was repeated three more times, and the polymer was dried in vacuum.
Example 3
Synthesis of methoxypolyethylene glycol-(CP) polymers (MPEG-CP) n n
Synthesis of MPEG-epoxide [00129] MPEG-OH (5 g) was stirred with NaOH (2 g) at 80°C for half an hour. Epichlorohydrin (3 mL) was added drop wise and stirred for 3 days. After completion of reaction the dichloromethane was added and filtered. The polymer was purified by precipitation in ether.
Synthesis of MPEG-CP, MPEG-(CP) , MPEG-(CP)
3 4
[00130] MPEG-mesylate (1 g) dissolved in 50 mL dioxane was refluxed with excess ethylamine (2 M solution in THF) (MPEG-CP) or diethylenetriamine ((MPEG-CP) ) or TREN (MPEG-(CP) ) under nitrogen atmosphere. After completion of the reaction the polymer was isolated and purified by repeated precipitation in ether. The amine groups in the polymers were then methylated by refluxing with a mixture of formaldehyde and formic acid for 24 hours. The volatiles were removed in vacuum and the polymers were isolated and purified by repeated precipitation in ether. The purified polymers were then reacted with methyl phospholane as previously described.
MPEG-(CP)2
[00131 ] MPEG-epoxide (500 mg) was heated with excess of hyperbranched poly(ethyleneimine) (molecular weight 700) in methanol for 24 hrs. The reaction product was then purified by dialysis (MW cut off 1000) against water and isolated by freeze drying. The polymer was then subjected to methylation and phosphorylation.
Figure imgf000043_0001
Figure imgf000043_0002
Figure imgf000044_0001
MPEG- (CP)2O
Figure imgf000044_0002
Example 4 Polymer effect on cell viability [00132] Figure 6 shows cell viability upon exposure to HPG-CP-4K (broad hatched bars) or HPG-PC-4K (narrow hatched bars) at various concentrations of polymer from 0-10 mg/ml.
Example 5
Interaction of HPG-PC and HPG-CP polymers with red cells
[00133] Red blood cells were exposed to polymers of HPG-CP or HPG-PC - 4K and -8K as described, and observed for aggregation. Results are summarized in Table 1.
Table 1. Red cell aggregation response to various polymer treatments
Figure imgf000045_0001
Figure imgf000046_0001
VAI= visual aggregation index.
VAI = 0 single, separated cells, no aggregation
1 50% single cells : 50% aggregated cells
2 rouleaux : >90% cells in mainly linear aggregates
3 rouleaux in side-to-side aggregates
4 small clumps: distorted and crenated cells
5 large clumps: distorted and crenated cells
[00134] Cells treated with HPG-PC appeared normal, with no aggregation. Surprisingly, the cells treated with HPG-CP were aggregated.
[00135] A solution of 2% HPG-PC-4K caused aggregation of red cells at a level similar to that of control (4:1 plasma: 150 mM NaCI). A solution of 10% human serum albumin in isotonic saline did not cause aggregation - separated single cells were observed. A 2% solution of HPG-CP-4K demonstrated a more pronounced aggregation of red cells. [00136] The extent of aggregation is dependent on concentration. Decreasing degrees of cell aggregation is observed from 2%, 0.2% and 0.02% HPG-CP-4K. Washed cells, and cells from alternate donors gave similar aggregation results. An equimolar mixture of HPG-PC and HPG-CP did not cause aggregation.
Example 6 HPG-CP aggregation of cells is reversible.
[00137] Results of these experiments are shown in Table 1. Cells aggregated with 2% HPG-CP-4K were subsequently incubated with 2% HPG-PC-
4K, partially reversing the aggregation. Subsequent washings with additional HPG-PC-4K further reduced aggregation.
[00138] In another experiment, HPG-CP -4K aggregated cells were washed with ethyl phosphatidyl choline (2 wt %), also reversing aggregation. Washing normally aggregated cells with 1% HSA in 150 mM NaCI reverses aggregation, while washing HPG-CP aggregated cells with 150 mM NaCI (no HSA) reverses aggregation only partially.
[00139] Cells aggregated with 2% HPG-CP (high or low MW polymers) were subsequently incubated with 2 wt % (CP) and (CP) . Both visual and microscopic examination showed clear reversal of the aggregates when incubated with (CP)4, while (CP)20 was ineffective in reversing aggregation. Neither of these molecules caused any aggregation of red cells.
[00140] CP multimers (CP) (CP) were prepared from permethylated TREN and PEI by phosphorylation in the absence of HPG Me TREN as well as
6 permethylated PEI (>1000 Da) were also found to be effective to reverse aggregation. This was surprising as they did not comprise a CP zwitterion, instead are amine-based polymers. Additionally, Me 6 TREN prevents aggregation of cells by HPG-CP: a 1 :1 mixture of Me 6 TREN and HPG-CP did not cause aggregation of cells (Table 1 ). [00141 ] The HPG-CP, HPG-PS, HPG-APS were all found to aggregate red cells, platelets, fibroblasts and DSPC liposomes.
Example 7 Interaction of MPEG-(CP) n polymers with red cells.
[00142] Whole blood (pre-treated with anticoagulant), or washed red cells resuspended in 0.5 wt % HSA solution in 150 mM saline were used in aggregation studies with a series of MPEG-(CP) polymers (n = 1 , 3, 4, 20). Controls included PEI, and HPG-amines. None of the smaller MPEG-(CP) n polymers (n=1 ,3,4) demonstrated aggregation of cells greater than controls. MPEG-(CP) exhibited moderate aggregation. Significant aggregation similar to that caused by HPG-CP was observed when washed cells (no plasma) were used. However this aggregation could be reversed by the addition of HSA solution. No difference was observed in the case of washed cells resuspended in
HSA solution.
Example 8 Electrophoretic mobility of red cells incubated with polymers
[00143] Adsorption of the various charged polymers to the cell surface affects the electrophoretic mobility of the cells (Table 2). Cells were incubated with 2% polymer in 150 mM NaCI, diluting the incubated red cells with additional 150 mM NaCI (10 μl to 2 ml_) and electrophoresed. See Bradley 2002. Biochmica et Biophysica Acta, Biomembranes 1561 :147-158 for methods.
Table 2 Electrophoretic mobility of red cells incubated with polymers (2%).
Figure imgf000048_0001
Figure imgf000049_0001
[00144] Electrophoretic mobility of red cells in polymer solution were compared with PEG 5000. The results are shown in Table 3.
Table 3. Electrophoretic mobility of red cells incubated with MPEG-(CP) n solution
(0.5% by wt)
Figure imgf000049_0002
Example 9
Quasi elastic light scattering (QELS) studies of interaction of MPEG-PEI-CP with HPG-PC.
[00145] QELS is a static light scattering technique used to measure particle sizes. The average hydrodynamic radius (Rh) of low molecular weight HPG-PC- 4K dissolved in methanol (2% solution) was measured to be 4.8 nm and that of MPEG-(CP) o (1 %) was 13 nm. Hydrodynamic analysis of a mixture of these two solutions in water (2 μl and 100 μl respectively) equilibrated overnight was measured to be Rh~40 nm. The size depended on the ratio of polymer concentrations and it did not change with dilution. A size increase to 30 nm was observed in the case of MPEG-CP whose size was only 3 nm.
Example 10 Liposomes made from DPPC and DPCP
O
[00146] DPCP forms a clear solution in water after 5 fireeze-thaw (60 C) o 31 cycles (by heating to 60 C). The P NMR (methanol-d4 in a capillary tube was used for locking) showed a narrow peak at 2.27 ppm (peak width at half height was 48 Hz). Dipalmitoylphosphatidylcholine (DPPC) in water is known to form
31 bilayers and demonstrate Chemical shift anisotropy (CSA) in P NMR, as reported in the literature.
[00147] Liposome preparations were made with and without cholesterol and characterized by dynamic light scattering to provide an average size. The results of the size analyses are given in Table 4.
Table 4. Characteristics of particles made from DPPC and DPCP
Figure imgf000050_0001
Notes: DSPC- Disteroyl phosphatidyl choline, DOPS- Dioleyl phosphatidyl serine.
Liposomes were prepared using methods known in the art, see for example Olson et al Biochmica et biophysics acta, 1979. 557 :9-23 ; Hope et al Biochmica et biophysics acta 1985. 812 :55-65
[00148] DPCP alone did not appear to form uniform liposomes in water as evidenced from the size measurements - the majority of the polymer appeared to form micelles, given the size. Addition of cholesterol enabled formation of liposomes of a reasonable size.
[00149] The interaction between DPPC liposomes and DPCP particles was studied by IR spectroscopy. The peak corresponding to the P=O group was at 1230.66 cm-1 for the DPPC and was at 1223.78 for the DPCP liposomes. A mixture of these two showed a peak at 1216.54 which indicates an interaction between the phosphate group and the quaternary ammonium group of the other molecule.
Example 11 Interaction of zwitterionic polymers
[00150] It was found that when HPG-PC and HPG-CP polymers were mixed in equimolar concentration, a precipitate was formed that settled out. A turbidometric titration was conducted and the results are shown in Figure 2. When a 0.2 wt % solution of HPG-CP-4K was titrated with HPG-PC-4K, maximum molecular aggregate formation was observed at ~ 0.05 wt % and it was 0.15% for the high molecular weight analogue. In order to prove that the interaction is electrostatic in nature, we conducted a turbidometric titration of the aggregate formed from an equimolar mixture by adding NaCI (Figure 3). It was found that the NaCI causes disaggregation indicating that the interaction is electrostatic.
[00151] These findings indicate that the two functional groups interact with each other, possibly through electrostatic interactions.
Example 12 Electrophoretic mobility of DSPC liposomes
[00152] The electrophoretic mobility of negatively charged DSPC liposomes after incubation with these various MPEG-(CP) was investigated (Figure 4). n
MPEG-(CP) n polymers affect the mobility of the liposomes.
Example 13 HPG-CP aggregation of bacteria [00153] An HPG-CP polymer was able to aggregate Camplyobacter jejuni in liquid growth media. (Figure 5 A-C) HPG-CP-8K, HPG-PC-8K and HPG-8K (no zwitterion groups) were included in bacterial growth media at 0-5 mg/ml. Biofilms of the C. jejuni strain 81 -176 were allowed to form at the air-liquid interface of borosilicate tubes, in triplicate, in the presence of five concentrations of soluble polymers of varying chemical composition. After two days, biofilms were stained with crystal violet, and staining intensity quantified by dissolution of the crystal
O violet in DMSO and spectrophotometric analyses of growth at 37 C with agitation, the optical density of the suspension culture was measured at 570 nm. The HPG- CP-8K polymer at concentrations at/ above 0.05 mg/ml resulted in a decrease in
OD.
[00154] While specific embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not as limiting the invention.
[00155] All citations are herein incorporated by reference.
[00156] One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

WHAT IS CLAIMED IS:
1. A composition according to Formula 1
Figure imgf000053_0001
where Ri is a polymer or a surface; R2 and R3 are independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate or peptide; A is a quaternary nitrogen, a trivalent sulphur or a tetravalent phosphorous;
X is an alkyl group comprising at least one carbon and at least one substituent group selected from the group comprising: H, a 1 -15 atom saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic form; Y is a group having a net negative charge, selected from the group comprising phosphate, phosphonate, phosphinate, sulphate, sulfonate, carbonate or carboxylate group.
2. The composition of claim 1 , wherein one or more than one carbon of the substituent group is replaced by O, S, SO, SO , NH , or NR', OH, OR', =O, SH, SR', NH , NHR', NR' , OSO H, OPO H , CO H, CO R';
2 2 3 3 3 2 2
3. A composition according to Formula 2 or 3 Formula 2
Figure imgf000054_0001
Formula 3
Figure imgf000054_0002
where Ri is a polymer or a surface;
R2 and R3 are independently be H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide;
W is selected from the group comprising an unpaired electron or - CR 4 R 5 R 6 ; and
R , R and R are independently selected from the group comprising H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid carbohydrate or peptide; and m is 2, 3,
4, 5 or more. A composition according to Formula 4, 5, 6 or 7 Formula 4
Figure imgf000055_0001
Formula 5
Figure imgf000055_0002
Formula 6
Figure imgf000055_0003
Formula 7
Figure imgf000055_0004
where R2 and R3 are independently selected from the group comprising H, alkyl, alkene, alkyne, benzyl, aryl, acetal, aldehyde, ketone, sulfone, primary amine, secondary amine, tertiary amine, thiols, disulfide, carboxylic acid, hydroxyls, alcohol, sulfate, amide, acrylate, methacrylate, methacrylamide, ester, epoxide, halides, amino acid, carbohydrate, peptide; X is an alkyl group comprising at least one carbon and at least one substituent group selected from the group comprising: H, a 1-15 atom saturated, unsaturated or cyclic alkyl group, benzyl or other aryl groups, or an alkylene oxide in linear, branched or cyclic form;
5. The composition of claim 3 according to Formula 2, where W is CH3 ,R2 is CH3; R3 is CH3; and Ri is a hyperbranched polyglycidoL
6. The composition of claim 4 according to Formula 4 or 5 where X is -CH2- CH2-; R2 is CH3; R3 is CH3; and Ri is a hyperbranched polyglycidol.
7. The composition of any of claims 1 to 4, where Ri is selected from the group comprising a poly(oxyalkylene) polymer, polyglycerol polymer, polyglycidol polymer, hyperbranched polyglycidol polymer, polyglycidol- block polymer, poly(glutamic acid) polymer, polyamidoamine (PAMAM) polymer, polyethyleneimine (PEI), polypropyleneimine (PPI) polymer, polymelamine polymer, polyester polymer, poly(lactic acid) polymer, epsilon poly(caprolactone), poly(lactone), substituted poly(lactones), poly(lactam), substituted poly(lactam), methoxy polyethylene glycol (MPEG or MePEG), polyethylene glycol (PEG), dextran, starch, cellulose, collagen, gelatine, chitosan or deacetylated chitosan.
8. The composition of claim 7 where R1 is a hyperbranched polyglycidol, or methoxypolyethylene glycol (MPEG).
9. The composition of claim 8 where the hyperbranched polyglycidol has a MW of at least 100,000.
10. The composition of claim 8 where the hyperbranched polyglycidol has a MW between about 100,000 and about 1 ,000,000.
11. The composition of claim 8 where the hyperbranched polyglycidol has a MW of 2-20,000.
12. The composition of claim 8 where the hyperbranched polyglycidol has a MW of about 4,000 to about 8,000.
13. The composition of claim 8 where Ri is MPEG.
14. A composition according to any one of claims 1 -13, further comprising a chemotherapeutic agent.
15. A liposome comprising the composition of claim 8.
16. The liposome of claim 15, further comprising a chemotherapeutic agent.
PCT/CA2007/002337 2006-12-18 2007-12-18 Polymers comprising zwitterionic endgroups WO2008074154A1 (en)

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