WO2007063320A1 - Gelificateur polymere - Google Patents

Gelificateur polymere Download PDF

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Publication number
WO2007063320A1
WO2007063320A1 PCT/GB2006/004487 GB2006004487W WO2007063320A1 WO 2007063320 A1 WO2007063320 A1 WO 2007063320A1 GB 2006004487 W GB2006004487 W GB 2006004487W WO 2007063320 A1 WO2007063320 A1 WO 2007063320A1
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Prior art keywords
polymer
temperature
aqueous medium
gelator
monomer
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PCT/GB2006/004487
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English (en)
Inventor
Steven Peter Armes
Peter Jeppe Madsen
Cong-Duan Vo
Chengming Li
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The University Of Sheffield
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Publication of WO2007063320A1 publication Critical patent/WO2007063320A1/fr

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    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C08L101/12—Compositions of unspecified macromolecular compounds characterised by physical features, e.g. anisotropy, viscosity or electrical conductivity
    • C08L101/14—Compositions of unspecified macromolecular compounds characterised by physical features, e.g. anisotropy, viscosity or electrical conductivity the macromolecular compounds being water soluble or water swellable, e.g. aqueous gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L53/00—Compositions of block copolymers containing at least one sequence of a polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/01—Atom Transfer Radical Polymerization [ATRP] or reverse ATRP

Definitions

  • the present invention relates to a polymer gelator which exhibits controllable liquid to gel phase transition behaviour.
  • polymer gelators which undergo a phase transition from a liquid to a gel in response to changes in the local environment, such as temperature, are known.
  • the classic example is poly(N-isopropylacrylamide), but the N- isopropylacrylamide monomer is both toxic and expensive.
  • Another example is poly(propylene oxide), but its thermal transition is relatively weak and ill-defmed.
  • a third example is poly(methyl vinyl ether) but the methyl vinyl ether monomer is both gaseous (i.e. difficult to handle) and relatively expensive.
  • a fourth example is poly(2-(dimethylamino)ethyl methacrylate), but the behaviour of this polymer is highly pH-dependent, as well as thermo-responsive.
  • Stimulus- responsive hydrogels are of particular interest, since the in situ formation of spacefilling gels within complex cavities is important for certain applications, e.g. wound bed dressings.
  • 2 Increasing attention is also being paid to (bio)chemically-responsive hydrogels. For example, Miyata's group reported a new class of hydrogels cross- linked by a specific antigen-antibody interaction that swell in response to excess antigen in solution. 3
  • An object of the present invention is to provide a stimulus and/or chemically responsive polymer gelator which exhibits improved properties over those currently known in the art. According to a first aspect of the present invention there is provided a polymer of formula I
  • A represents a polymer unit which, when polymer I is provided in an aqueous medium, exhibits an increase in hydrophobicity upon changing the temperature of said aqueous medium from a first temperature to a second temperature or upon changing the pH of , said aqueous medium from a first pH value to a second pH value
  • B represents a polymer unit which is more hydrophilic than polymer unit A when polymer I is provided in said aqueous medium and said aqueous medium is at said second temperature or the pH of said aqueous medium is at said second pH value
  • L represents a linking group which is cleavable by hydrolysis, oxidation or reduction, wherein polymer I exhibits a first phase transition from a liquid to a gel upon said change in temperature or pH of said aqueous medium.
  • the polymer of the present invention therefore combines a selectively cleavable linker, which ⁇ s preferably non-polymeric and/or of low molecular weight, and polymer units which bestow on the polymer either temperature or pH dependent gelation behaviour under aqueous conditions.
  • the polymers of the present invention constitute a new class of polymer gelators that are both (biochemically- responsive and stimulus (i.e. thermo or pH)-responsive.
  • Example 1 describes the preparation and characterisation of a specific embodiment of polymer I - PNIPAM 8O -PMPC I25 -S-S-PMPC I25 -PNIPAM 8O .
  • variable temperature DLS and 1 H NMR studies suggests that upon heating to around 37 °C the polymer PNIP AMg 0 - PMPCi 25 -S-S-PMPCj 25 -PNiPAM 80 undergoes a liquid to gel phase transition in which the gel comprises inter-connected PNIPAM-core 'flower' micelles (see Figure 1).
  • the gel is purely physical in nature, since it reverts to a free-flowing liquid below 37 °C.
  • Rheological studies of the gelation behavior of this triblock copolymer in aqueous solution are Summarized below (see Figures 7 to 9).
  • the central disulfide bond is cleaved as shown in Figure 1, the triblock chains are converted into PNIPAMso-PMPC 125 -SH diblocks of half the original copolymer molecular weight and the inter-micelle bridges in the 3D gel network are destroyed, which leads to rapid gel dissolution.
  • the disulfide bond may be considered to act as a 'keystone' for the gel. Since disulfide bonds can be selectively cleaved under mild conditions using various naturally-occurring reagents such as oligopeptides, this specific embodiment exemplifies a new class of gels which is both biochemically responsive and thermo-responsive.
  • a second aspect of the present invention provides a polymer mixture comprising polymer I in accordance with the first aspect of the present invention and a polymer of formula III
  • A' represents a polymer unit which, when the polymer mixture is provided in said aqueous medium, exhibits an increase in hydrophobicity upon said change in temperature or pH of the aqueous medium
  • B 1 represents a polymer unit which is more hydrophilic than polymer unit A' when the polymer mixture is provided in said aqueous medium and the aqueous medium is at said second temperature or the pH of said aqueous medium is at said second pH value.
  • A represents a polymer unit which, when polymer I is provided in an aqueous medium, exhibits an increase in hydrophobicity upon changing the temperature of said aqueous medium from a first temperature to a second temperature or upon changing the pH of said aqueous medium from a first pH value to a second pH value
  • B represents a polymer unit which is more hydrophilic than polymer unit A when polymer I is provided in said aqueous medium and said aqueous medium is at said second temperature or the pH of said aqueous medium is at said second pH value
  • L represents a linking group which is cleavable by hydrolysis, oxidation or reduction, wherein polymer I exhibits a first phase transition from a liquid to a gel upon said change in temperature or pH of said aqueous medium, and further wherein said process comprises the steps of
  • a composition can be prepared which comprises an agent to treat a wound bed together with an embodiment of polymer I which has been selected to undergo a liquid to gel phase transition at close to the temperature of the wound bed environment such that the composition can be conveniently administered to the wound as a relatively cool liquid but then converts to a gel as it warms up so that it remains in place within the wound bed for the desired period of time to deliver the therapeutic agent.
  • the therapeutic agent may then be released in a controlled fashion by conversion of the polymer from the gel back to a liquid simply by lowering the temperature of the polymer composition back down below the cloud point of the polymer I, or by selection of the linker so as to be cleavable under the conditions found within the wound bed so that the polymer degrades in a controlled fashion and returns to a liquid state.
  • a further aspect of the present invention provides a wound bed dressing incorporating a composition comprising polymer I according to the first aspect of the present invention (or the above defined polymer mixture) and a therapeutic amount of a wound bed therapeutic agent.
  • a related aspect of the present invention provides a method for treating a wound bed comprising administering to said wound bed a composition comprising polymer I according to the first aspect of the present invention (or the above defined polymer mixture) and a therapeutic amount of a wound bed therapeutic agent.
  • polymer I is in the targeted release of therapeutic substances. If it- is desired to target delivery of a therapeutic agent to tissue in a specific state, such as hypoxic tissue as is often found in cancerous rum, a composition may be prepared comprising a therapeutic agent together with the polymer according to the first aspect of the present invention in which the linker is chosen to be cleavable under reducing conditions, for example by employing a linker incorporating one or more disulfide groups.
  • Another aspect of the present invention provides a pharmaceutical composition for the treatment of cancer comprising polymer I according to the first aspect of the present invention (or the above defined polymer mixture) and a therapeutic amount of a chemotherapeutic agent.
  • a still further related aspect of the present invention provides a method for treating cancer comprising administering to a patient in need of such treatment a composition comprising polymer I according to the first aspect of the present invention (or the above defined polymer mixture) and a therapeutic amount of a chemotherapeutic agent.
  • the pharmaceutical composition incorporating the chemotherapeutic agent may be administered to a patient as an adjuvant to surgery or radiotherapy. Additionally or alternatively, the agent may be administered to the patient in combination with at least one further chemotherapeutic agent.
  • said first phase transition which is controlled by changes in temperature or pH is reversible.
  • Polymer I may undergo a second phase transition from said gel to a liquid upon cleavage of linking .group L, in which case it is preferred that said second phase transition is non-reversible.
  • The. linking group L may contain a cleavable moiety selected from the group consisting of an acetal group, an ester group, an alkene group, an imine group, a peroxide group, an azo group and a disulfide group.
  • Said ester group is preferably an activated ester group such as a /3-aminoester group.
  • said linking group L contains a cleavable moiety which is cleavable under physiological conditions, which may be taken to be a temperature of around 37 0 C and a pH of about 7.4. It is particularly preferred that the linking group L is cleavable by reductive cleavage using a naturally occurring oligopeptide or polypeptide reducing agent, such as glutathione. Glutathione is an important tripeptide anti-oxidant that is commonly found within cells at millimolar concentrations.
  • a further suitable linking group L is one which is cleavable by reductive cleavage using dithiothreitol (DTT) which contains a pair of terminal thiol groups that can exchange with a disulfide bond.
  • DTT dithiothreitol
  • said linking group L is derived from a radically reactive bifunctional polymerization initiator.
  • the initiator may contain an ester moiety and may further contain at least one alkyl group at the alpha position relative to the carbonyl carbon atom of said ester moiety.
  • said initiator can incorporate a halogen atom at the alpha position relative to the carbonyl carbon atom of said ester moiety.
  • the initiator is bis[2-(2- bromoisobutyryloxy)ethyl] disulfide ((BiBOE) 2 S 2 ).
  • polymer I has a formula II
  • m, n, o and p are integers and at least one of m and p is in the range 20 to 150 and/or at least one of n and o is in the range 100 to 200.
  • m, n, o and p may be chosen within the above defined ranges it is preferred that at least one of m and p is in the range 30 to 120, more preferably at least one of m and p is in the range 40 to 90, and most preferably at least one of rn and p is approximately 80. It is particularly preferred that approximately equal amounts of polymer unit A are present in each half of polymer I located either side of the linking group L and so it is preferred that in formula II m is approximately equal to p.
  • n and o is in the range 110 to 180, more preferably at least one of n and o is in the range 120 to 160, and most preferably at least one of n and o is approximately 125.
  • linking group L is symmetrically substituted with polymer unit B, thus, preferably in formula II n is approximately equal to o.
  • polymer I undergoes said first phase transition upon said change in temperature of said aqueous medium.
  • polymer I exhibits a critical gelation temperature between 0 and 100 0 C, more preferably between 0 and 75 0 C, yet more preferably between 0 and 50 0 C, and still more preferably between 0 and 38 0 C.
  • polymer I exhibits said first phase transition upon a change in temperature that polymer I exhibits a critical gelation temperature in the range 5 to 40 °C, more preferably 5 to 25 °C, and most preferably 5 to 20 0 C.
  • the critical gelation temperature of polymer I is in the range 10 to 38 0 C, more preferably 10 to 25 0 C, and most preferably in the range 12 to 20 °C.
  • hydrophilic character of polymer I decreases upon raising the temperature of the aqueous medium in which polymer I is dispersed. Provided the linker L in polymer I remains intact, i.e. L has not been cleaved such that polymer I retains the formula A-B-L-B-A, it is preferred that the temperature induced change in hydrophilic character of polymer I is reversible.
  • polymer I When polymer I exhibits the desired gelation behaviour and undergoes the first phase transition from a free flowing liquid to a free standing hydrogel this is accompanied with an increase in the viscosity of the medium containing polymer I.
  • said medium Preferably said medium exhibits an increase in viscosity of at least one or two orders of magnitude upon said change in temperature of the aqueous medium.
  • a 9.0 % aqueous solution of said gel containing PBS buffer exhibits a viscosity of greater than 10 Pa.s at a temperature greater than or equal to said second temperature when measured at an applied stress of 3.0 Pa and a frequency of 1.0 rad sec "1 .
  • the first and second temperatures may take any appropriate value and one temperature may be higher than the other or vice versa. It is preferred that the second temperature is higher than the first temperature, such that polymer I exhibits said liquid/gel phase transition upon increasing the temperature of the aqueous medium.
  • At least one of said first and second temperatures is between 0 and 100 0 C, more preferably between 0 and 75 °C, yet more preferably between 0 and 50 0 C and most preferably between 0 and 38 °C.
  • at least one of said first and second temperatures is in the range 5 to 40 °C, more preferably 5 to 38 0 C, still more preferably 5 to 25 °C, and most preferably 5 to 20 °C.
  • at least one of the first and second temperatures is in the range 10 to 38 °C, more preferably in the range 10 to 25 °C, and most preferably in the range 12 to 20 0 C.
  • the difference between the first and second temperatures may be less than 20 0 C, more preferably approximately 1 to 10 °C, and most preferably around 5 °C.
  • the gelation temperature for a particular polymer may be related to the concentration of that polymer in the aqueous medium. It is envisaged that the second temperature for a particular polymer of formula I would increase as the concentration of that polymer in the aqueous medium decreased.
  • a homopolymer of polymer unit A and/or A' exhibits inverse temperature dependent aqueous solubility. It is parti cularly preferred that the lower critical solution temperature of a homopolymer of polymer unit A is lower, preferably significantly lower, than the lower critical solution temperature of a homopolymer of polymer unit B. With regard to polymers III and IV it is preferred that the lower critical solution temperature of a homopolymer of polymer unit A' is lower, preferably significantly lower, than the lower critical solution temperature of a homopolymer of polymer unit B'.
  • a homopolymer of polymer unit A or A' may exhibit a lower critical solution temperature in the range 5 to 50 °C, 8 to 40 °C, or 10 to 37 0 C.
  • Polymer units A and A' should each be chosen not only to provide the desired temperature dependent hydrophobicity behaviour specified above but also to be suitable for incorporation in to the structure of polymer I, III or IV.
  • polymer unit A is derived from a monomer A, which may be a radically reactive vinyl monomer.
  • Polymer unit A' may be derived from a monomer A', which may be a radically reactive vinyl monomer.
  • the relatively hydrophilic polymer unit B is derived from a monomer B, which may be a radically reactive vinyl monomer.
  • Polymer unit B' is preferably derived from a monomer B', which may be a radically reactive vinyl monomer.
  • Monomer A or A' may contain an acrylate or methacrylate moiety. Monomer A or A'may additionally or alternatively contain either a primary or a secondary hydroxyl group. It is particularly preferred that monomer A or A' contains at least one secondary hydroxyl group. Where monomer A or A' constitutes an isomeric mixture of monomers it is preferred that at least one of the isomers contains a secondary hydroxyl group. Particularly preferred compounds for monomer A or A' are selected from the group consisting of 2-hydroxypropyl acrylate (HPA), 2-hydroxypropyl methacrylate (HPMA), t-butylaminoethyl methacrylate, hydroxybutyl acrylate (HBA) and hydroxybutyl methacrylate (HBMA).
  • HPA 2-hydroxypropyl acrylate
  • HPMA 2-hydroxypropyl methacrylate
  • HBA hydroxybutyl acrylate
  • HBMA hydroxybutyl methacrylate
  • a monomer A or A' exists in its monomelic form as a mixture of isomers, e.g. HPMA which is actually a 3:1 mixture of HPMA and 2-hydroxyisopropyl methacrylate (HIPMA) 5 the mixture will be referred to generically by using the name of the predominant component. .
  • HPMA which is actually a 3:1 mixture of HPMA and 2-hydroxyisopropyl methacrylate (HIPMA) 5
  • HIPMA 2-hydroxyisopropyl methacrylate
  • monomer A or A' may incorporate an acrylamide moiety, in which case monomer A or A' may further contain an isopropyl group, and is more preferably N-isopropylacrylamide (NIPAM).
  • NIPAM N-isopropylacrylamide
  • monomer B or B' may contain an acrylate or methacrylate moiety. Since polymer units B and B' should be more hydrophilic than polymer units A and A' respectively it is preferred that at least one of monomers B and B' is a zwitterion under aqueous conditions and approximately neutral at physiological pH, and may optionally incorporate a phosphate group and/or a quaternary amine group.
  • the quaternary amine group preferably comprises at least one N-methyl group, more preferably two or' three N-methyl groups.
  • monomer B and/or B' is 2-(methacryloyloxy)ethyl phosphorylcholine (MPC).
  • monomer B or B' preferably contains at least one hydroxyl group, which may be a primary or secondary hycjroxyl group. Additionally or alternatively, monomer B or B' may contain at least one secondary hydroxyl group.
  • monomer B or B' constitutes an isomeric mixture of monomers it is preferred that at least one of the isomers contains a secondary hydroxyl group.
  • a further preferred compound for use as monomer B or B' is selected from the group consisting of glycercol monomethacrylate (GMA), acrylic acid, methacrylic acid, dimethyl acrylamide, N- vinylpyrrolidone.
  • GMA glycercol monomethacrylate
  • polymer unit B may comprise vinyl alcohol units derived from the polymerisation of vinyl acetate monomer.
  • a monomer B or B' exists in its monomelic form as a mixture of isomers, e.g.
  • GMA which is actually a 92:8 mixture of GMA and 1,3-dihydroxyisopropyl methacrylate (DHIMA), the mixture will be referred to generically by using the name of the predominant component.
  • polymer I undergoes said first phase transition upon changing the pH of the aqueous medium from the first pH value to the second pH value.
  • the difference between the first and second pH values is relatively large.
  • the difference between the first and second pH values may be up to approximately 8 pH points, more preferably up to approximately 5 pH points or, still more preferably, around 2 pH points.
  • said second pH value is higher than said first pH value, i.e. it is preferred that the polymer is a free flowing liquid in acidic aqueous solution and undergoes gelation as the solution is treated so as to become more basic.
  • At least one, more preferably both, of said first and second pH values is in the range 2 to 10, more preferably in the range 4 to 9, and most preferably in the range 5 to 8. It is particularly preferred that at least one of said first and second pH values is about 7 and may lie in the range 7.0-7.5.
  • At least one of said first and second pH values may be around physiological pH.
  • polymer unit A is derived from a monomer A, which may be a radically reactive vinyl monomer.
  • polymer unit B is derived from a monomer B. Any appropriate monomer A may be chosen but it is preferred that monomer A contains a (meth)acrylate moiety.
  • Monomer A may contain a tertiary amine group and may contain at least one N-alkyl group selected from the group consisting of methyl, ethyl, propyl, isopropyl and butyl.
  • monomer A is 2- (diisopropylamino)ethyl methacrylate (DPA) or 2-(diethylamino)ethyl methacrylate (DEA).
  • DPA diisopropylamino
  • DEA 2-(diethylamino)ethyl methacrylate
  • monomer B from which polymer unit B is derived, contains a (meth)acrylate moiety.
  • polymer unit B is more hydrophilic than polymer unit A when the aqueous medium is at the second pH value
  • monomer B is preferably a zwitterion
  • vinyl monomer B may contain a phosphate group and/or a quaternary amine group.
  • the quaternary amine group may comprise at least one N-methyl group and a particularly preferred option for monomer B is 2-(methacryloyloxy)ethyl phosphorylcholine (MPC).
  • monomer B may contain at least one hydroxyl group, which is preferably a primary hydroxyl group. Additionally or alternatively monomer B may contain a secondary hydroxyl group. Where monomer B constitutes an isomeric mixture of monomers it is preferred that at least one of the isomers contains a secondary hydroxyl group. Most preferably monomer B is selected from the group consisting of glycercol monomethacrylate (GMA), acrylic acid, methacrylic acid, dimethyl acrylamide and N-vinylpyrrolidone. Moreover, polymer unit B may comprise vinyl alcohol units ' derived from the polymerisation of vinyl acetate monomer.
  • GMA glycercol monomethacrylate
  • acrylic acid methacrylic acid
  • dimethyl acrylamide dimethyl acrylamide
  • N-vinylpyrrolidone N-vinylpyrrolidone
  • polymer unit B may comprise vinyl alcohol units ' derived from the polymerisation of vinyl acetate monomer.
  • polymer I When polymer I exhibits pH dependent gelation behaviour and undergoes the first phase transition from a free flowing liquid to a free standing hydrogel upon changing the pH of the aqueous medium this phase transition is accompanied with an increase in the viscosity of the medium containing polymer I.
  • the viscosity of the medium increases by at least one, preferably two, orders of magnitude when the pH is changed from the first value to the second value.
  • polymer I exhibits a reduction in number average molecular weight, M n , in the range 40 to 60 % upon cleavage of said linking group L. More preferably polymer I exhibits a reduction in number average molecular weight, M n , of approximately 50 % upon cleavage of said linking group L. It will be appreciated that in order for a specific embodiment of polymer I to undergo the first phase transition from a liquid to a gel it will be necessary to ensure that that embodiment of polymer I is provided in the aqueous medium at a concentration equal to or greater than the critical gelation concentration for that particular embodiment of polymer I.
  • Polymer I may be provided in said aqueous medium at a concentration in the range 1 to 20 w/v%, more preferably 5 to 15 w/v %, still more preferably 7 to 12 w/v % and yet more preferably 8 to 10 w/v %. Most preferably polymer I is provided in said aqueous medium at a concentration of approximately 9 w/v %.
  • the polymer mixture may comprise predominantly polymer I, predominantly polymer III or IV or approximately equal amounts of polymer I and polymer III or IV.
  • the polymer of formula III and/or IV may self-assemble in the aqueous medium to form micelles and/or a gel.
  • Triblock copolymers of formula III preferably form gels at the second temperature.
  • Diblock copolymers of formula IV may be provided which simply form micelles at the second temperature but do not gel (for example see Examples 5, 6 A and 6B below) or polymers of formula IV may be chosen which form gels at the second temperature.
  • the third aspect of the present invention is directed to a process for the production of a polymer of formula I
  • step (a) comprises living radical homopolymerization of monomer B. Furthermore, step (b) preferably comprises living radical copolymerization of polymer V with monomer A.
  • the polymerization step carried out in at least one, preferably both, of steps (a) and (b) comprises atom transfer radical polymerization (ATRP).
  • ATRP atom transfer radical polymerization
  • At least one, preferably both, of steps (a) and (b) is carried out in the presence of a polymerization catalyst, such as copper (I) bromide or copper (II) chloride.
  • the polymerization step carried out in at least one, preferably both, of steps (a) and (b) comprises radical addition fragmentation transfer (RAFT) polymerization.
  • RAFT radical addition fragmentation transfer
  • At least one, preferably both, of steps (a) and (b) is carried out in the presence of a suitable RAFT chain transfer agent, such as a dithi ⁇ ester.
  • the present invention further relates to a method for treating a wound bed employing a composition comprising polymer I and a wound bed therapeutic agent.
  • This composition may further comprise a therapeutic amount of an agent to reduce scarring.
  • a fourth aspect of the present invention provides a temperature dependent polymer gelator comprising polymer units C, D and E, wherein polymer unit C is poly(2- hydroxypropyl methacrylate), which is referred herein generically as PHPMA, and D is a polymer unit which, when said polymer gelator is provided in an aqueous medium, is more hydrophilic than polymer unit C, said polymer gelator exhibiting a phase transition from a liquid to a gel upon changing the temperature of said aqueous medium.
  • Preferred embodiments of the fourth aspect of the present invention are exemplified below in Examples 2, 4 and 7.
  • the requirement for the incorporation of the PHPMA unit into the polymer gelator of the fourth aspect of the present invention to bestow temperature dependent gelation behaviour on the polymer is demonstrated in Examples 2, 4 and 7 and can be contrasted with the lack of temperature dependent gelation behaviour exhibited by two closely related triblock copolymers described in Comparative Examples 1 and 2.
  • PHPMA offers significant benefits over conventional temperature dependent polymer gelators since the 2-hydroxypropyl methacrylate (HPMA) monomer is relatively cheap and much lower in toxicity than either N-isopropylacrylamide or methyl vinyl ether. Moreover, the fact that the homopolymer of HPMA possesses a lower critical solution temperature (LCST) of just below ambient temperature means that the HPMA monomer is particularly suitable for use in many different applications, particularly biologically related applications.
  • LCST critical solution temperature
  • PHPMA can be incorporated into polymer gelators exhibiting temperature dependent gelation behaviour is especially surprising given that PHPMA has previously been considered to be simply a hydrophobic polymer, but it has now been recognized that PHPMA can exhibit an increase in hydrophobicity when the temperature of the aqueous medium in which it is provided is changed.
  • the marked concentration dependence observed for the critical gelation temperature suggests that degelation may be achieved by dilution, which may be useful in certain biomedical applications.
  • a related aspect of the present invention provides a polymer mixture comprising the above defined polymer gelator in accordance with the fourth aspect of the present invention and a diblock polymer of formula VI
  • C represents a polymer unit which, when the polymer mixture is provided in said aqueous medium, exhibits an increase in hydrophobicity upon said change in temperature of the aqueous medium
  • D' represents a polymer unit which is more hydrophilic than polymer unit C when the polymer mixture is provided in said aqueous medium. It is particularly preferred that C is 2-hydroxypropyl methacrylate.
  • Another aspect of the present invention provides a process for the production of a temperature dependent polymer gelator comprising polymer units C, D and E, wherein polymer unit C is poly(2-hydroxy ⁇ ropyl methacrylate) and D is a polymer unit which, when said polymer gelator is provided in an aqueous medium, is more hydrophilic than polymer unit C, said polymer gelator exhibiting a phase transition from a liquid to a gel upon changing the temperature of said aqueous medium, and further wherein said process comprises copolymerising a monomer C from which, polymer unit C is derived with a monomer D from which polymer unit D is derived and a monomer E from which polymer unit E is derived to produce said polymer gelator.
  • Said composition may further comprise an agent to reduce scarring.
  • Still further aspects of the present invention provide a pharmaceutical composition for the treatment of cancer comprising a temperature dependent polymer gelator according to the fourth aspect of the present invention (or the above defined polymer mixture with the diblock polymer of formula VI) and a therapeutic amount of a chemotherapeutic agent, and a method for treating cancer comprising administering to a patient in need of such treatment a composition comprising a temperature dependent polymer gelator according to the fourth aspect of the present invention (or the above defined polymer mixture with the diblock polymer of formula VI) and a therapeutic amount of a chemotherapeutic agent.
  • said temperature induced phase transition is reversible.
  • the liquid to gel phase transition is induced by raising the temperature then it is preferred that the gel can be converted back to a liquid state by lowering the temperature, and vice versa.
  • the said second temperature is higher than said first temperature, such that the polymer gelator exhibits the liquid to gel phase transition upon increasing the temperature of the aqueous medium.
  • the polymer gelator undergoes the liquid to gel phase transition at its critical gelation temperature. It is particularly preferred that the polymer gelator exhibits a critical gelation temperature between 0 and 100 °C, more preferably between 0 and 75 °C, yet more preferably between 0 and 50 0 C, and still more preferably between 0 and 38 0 C. It is further preferred that the polymer gelator exhibits a critical gelation temperature in the range 5 to 40 °C, more preferably 5 to 25 °C, and most preferably 5 to 20 0 C. In still further preferred embodiments the critical gelation temperature of polymer I is in the range 10 to 38 °C, more preferably 10 to 25 °C, and most preferably in the range 12 to 20 °C.
  • the liquid to gel phase transition of the .polymer gelator is induced by changing the temperature of the aqueous medium in which it is provided from a first temperature to a second temperature.
  • At least one of the first and second temperatures is preferably being between 0 and 100 °C, more preferably between 0 and 75 0 C, yet more preferably between 0 and 50 0 C and most preferably between 0 and 38 °C.
  • at least one of said first and second temperatures is in the range 5 to 40 0 C, more preferably 5 to 38 °C, still more preferably 5 to 25 0 C, and most preferably 5 to 20 0 C.
  • At least one of the first and second temperatures is in the range 10 to 38 °C, more preferably in the range 10 to 25 °C, and most preferably in the range 12 to 20 0 C.
  • the difference between the first and second temperatures may be less than 20 0 C, more preferably approximately 1 to 10 °C, and most preferably around 5 0 C.
  • the polymer gelator comprises a triblock copolymer of formula VII C-D-E (VII)
  • formula VII encompasses both triblocks C-D-C and C-D-F (where F is a polymer unit having a different structure to polymer unit C).
  • polymer unit E has a different chemical structure to polymer unit C, i.e. formula VIII does not encompass triblock C-C-D since this is simply a C-D diblock copolymer.
  • a further preferred embodiment of the fourth aspect of the present invention provides that the degree of polymerization of polymer units C, D and E is denoted q, r and s respectively, and q is in the range 10 to 100, r is in the range 10 to 500 and s is in the range 10 to 100.
  • q is in the range 30 to 80, more preferably in the range 40 to 60 and most preferably around 50.
  • r is preferably in the range 30 to 400.
  • the degree of polymerization of polymer unit D may be selected so as to be towards the lower end of this range, e.g. if D is highly hydrophilic then it may be desirable for r to be in the range 30 to 60, more preferably around 50, or if D is of lower hydrophilicity (although still exhibiting higher hydrophilicity than C) r may be selected so as to be towards the top end of the quoted ranges, e.g.
  • q is in the range 300 to 400, more preferably around 360.
  • s is in the range 30 to 80, yet more preferably 40 to 70 and most preferably s is around 50 to 60.
  • the ratio of q to r to s (q:r:s) may be chosen so as to ensure that the polymer gelator forming the fourth aspect of the present invention exhibits the desired gelation behaviour. Ln a particularly preferred embodiment, q is about 80, r is about 350-360 and s is around 80. In this embodiment it is preferred that C and E are both PHMPA and D is poly(ethylene) glycol.
  • the polymer unit E is preferably selected from the group consisting of poly(N- isopropylacrylamide), poly(propylene oxide), poly(methyl vinyl ether), poly(2- (dimethylamino)ethyl methacrylate), poly(2-hydroxypropyl methacrylate), poly(2- hydroxypropyl acrylate), ⁇ oly(t-butylaminoethyl methacrylate), poly(hydroxybutyl acrylate) and poly(hydroxybutyl methacrylate).
  • diblock polymer VI may or may not be the predominant component of the mixture, or the polymer gelator forming the fourth aspect of the present invention and polymer VI may be present in the mixture in approximately equal amounts.
  • the temperature change required to induce the polymer gelator to exhibit a liquid to gel phase transition may, when the polymer gelator is provided as a component of the above defined polymer mixture, cause the diblock polymer IV to self-assemble in the aqueous medium to form micelles (for example see Examples 5, 6A and 6B below) and/or a gel. If it is desired to increase the critical gelation temperature of the polymer gelator (and/or diblock polymer VI) then a polymer unit D (or D') may be selected which exhibits a higher lower critical solution temperature (LCST).
  • LCST critical solution temperature
  • Polymer unit D (or D') is preferably derived from a monomer D (or D'), which may be radically reactive vinyl monomer.
  • Monomer D (or D') may contain an acrylate or methacrylate moiety.
  • Monomer D (or D') is preferably a zwitterion under aqueous conditions and approximately neutral or physiological pH, and may optionally incorporate a phosphate group and/or a quaternary amine group.
  • the quaternary amine group preferably comprises at least one N-methyl group, more preferably two or three N-methyl groups.
  • monomer D (or D') is 2- (methacryloyloxy)ethyl phosphorylcholine (MPC).
  • monomer D (or D') contains at least one hydroxyl group, which may be a primary or secondary hydroxy! group. Additionally or alternatively, monomer D (or D') may contain at least one secondary hydroxyl group. Where monomer D (or D') constitutes an isomeric mixture of monomers it is preferred that at least one of the isomers contains a secondary hydroxyl group.
  • a further preferred compound for use as monomer D (or D') is selected from the group consisting of glycerol monomethacrylate (GMA), acrylic acid, methacrylic acid, dimethyl acrylamide, N-vinylpyrrolidone.
  • polymer unit D may comprise vinyl alcohol units derived from the polymerisation of vinyl acetate monomer.
  • GMA glycerol monomethacrylate
  • DHIMA 1,3- dihydroxyisopropyl methacrylate
  • polymer unit D of the polymer gelator forming the fourth aspect of the present invention has the same chemical structure as polymer unit D' of the diblock polymer of formula VI present in the above defined polymer mixture. It is still more preferred that C is the same as C, i.e. both the polymer gelator and polymer VI comprise poly(2-hydroxypropyl methacrylate).
  • the polymer gelator forming the fourth aspect of the present invention to undergo the desired liquid to gel phase transition the polymer gelator should be provided in the aqueous medium at a concentration equal to or greater than its critical gelation concentration, which is likely to vary depending upon the nature of polymer units D and E and the absolute and relative degrees of polymerization of each of polymer units C, D and E. Given that this is the case it will be appreciated that dilution may be used as a trigger to gelation, i.e. changes in concentration of polymer I may be used to induce a liquid to gel or gel to liquid phase transition.
  • the polymer gelator may be provided in said aqueous medium at a concentration in the range 1 to 20 w/v%, more preferably 5 to 15 w/v %, still more preferably 7 to 12 w/v % and yet more preferably 8 to 10 w/v %. Most preferably polymer I is provided in said aqueous medium at a concentration of approximately 9 w/v %.
  • said copolymerisation step preferably comprises living radical copolymerization and may comprise atom transfer radical polymerization (ATRP) or radical addition fragmentation transfer polymerization (PvAFT). Certain monomers which may be incorporated into the polymer gelator of the present invention are not suitable for radical polymerization in which case other more appropriate polymerization methods should be used.
  • ATRP atom transfer radical polymerization
  • PvAFT radical addition fragmentation transfer polymerization
  • methyl vinyl ether monomer or the like it is preferred that cationic polymerization is utilized, and where propylene oxide monomer or the like is used an anionic ring opening reaction may be used. It is preferred that the polymerization step is carried out in the presence of a polymerization catalyst, such as copper (I) bromide or copper (II) chloride. Where RAPT polymerization is utilized it is preferred that the polymerization step is carried out in the presence of a suitable RAFT chain transfer agent, such as a dithioester.
  • a polymerization catalyst such as copper (I) bromide or copper (II) chloride.
  • RAFT chain transfer agent such as a dithioester.
  • Figure 1 is a schematic representation of a theoretical model for the biochemical degradation of the free-standing micellar gel formed by the PNIPAM 80 -PMPC 125 -S- S-PMPCi 25 -PNIPAM 8 o triblock copolymer produced in accordance with Example 1 after cleavage of the disulfide bonds using the naturally-occurring tripeptide, glutathione;
  • Figure 2 is a semi-logarithmic plot of conversion vs. time for the homopolymerization of MPC in methanol via ATRP at ambient temperature using (BiBOE) 2 S 2 as polymerisation initiator.
  • MPC 3.72 g, 12.5 mmol
  • target DP n 250
  • the [MPC]: [(BiBOE) 2 S 2 ]: [Cu(I)]: [bpy] relative molar ratios were 250 : 1: 2: 4;
  • Figure 3 is a graphical representation of the evolution of molecular weight and polydispersity with conversion for the Br-MPCi 25 -S-S-MPCi 25 -Br macro-initiator obtained under the conditions stated in respect of Figure 2;
  • Figure 6(a) is the chemical structure of PNIPAM 8O -PMPC 125 -S-S-PMPC I25 - PNIPAM 80 ;
  • Figure 6(b) depicts variable temperature 1 H NMR spectra recorded for an 8.1 % aqueous solution of the PNIPAM 8 O-PMPC 125 -S-S-PMPCI 25 -PNIPAM 8 O triblock copolymer in D 2 O. Note the disappearance of the isopropyl proton signal at 4.2 ppm due to the PNIPAM blocks on heating at 37 0 C;
  • Figure 7 is a viscosity vs. temperature plot obtained for a 9.0 % aqueous solution of the PNIPAM 8O -PMPC 125 -S-S-PMPC I25 -PNIPAM 8O triblock copolymer in PBS solution at an applied stress of 3.0 Pa and a frequency of 1.0 rad sec "1 ;
  • Figure 8 shows the temperature dependence of the storage modulus (G') (•) and loss modulus (G") (o) obtained for the same copolymer solution as that used in Figure 7.
  • the cross-over point for the G' and G" curves was approximately 36 0 C, which corresponds to the critical gelation temperature;
  • Figure 9 shows the frequency dependence of the storage modulus (G') and loss modulus (G") at a constant applied stress of 3.0 Pa for a 9.0 % PNIP AM 80 -PMPC 125 -
  • Figure 10(a) is a series of aqueous GPC traces obtained for the Br-PMPC 125 -S-S- PMPC 125 -Br macro-initiator (solid line) and the glutathione-degraded Br-PMPC 125 - SH homopolymer obtained after 4 h in aqueous solution at 37 0 C;
  • Figure 10(b) is a series of non-aqueous GPC (3:1 chloroform/methanol eluent) traces for the original PNIPAM 8O -PMPC I25 -S-S-PMPCJ 25 -PNIPAM 8O triblock copolymer (solid line) and the glutathione-degraded Br-PNIPAM 8O -PMPC 125 -SH diblock copolymer obtained after 24 h in aqueous solution at 37 0 C;
  • Figure 11 illustrates a series of viscosity vs. time plots for the glutathione-treated triblock copolymer aqueous gels/solutions obtained at a constant applied stress of 5.0 Pa for varying glutathione/disulfide molar ratios at 37 0 C;
  • Figure 12 is a series of GPC traces recorded for the MPCi 2S -S-S-MPCi 2 S macro- initiator and also for the final PHPMA 5O -PMPC I25 -S-S-PMPC I25 -PHPMA 5O triblock copolymer;
  • Figure 13 is a viscosity vs. temperature plot of 1 a 10.0 wt. % aqueous PHPMA 50 - PMPC 125 -S-S-PMPC I2S -PHPMA 5O copolymer solution obtained at an applied shear stress of 3.0 Pa and shear frequency of 1.0 rad sec "1 ;
  • Figure 14 shows the temperature dependence of the storage modulus (G') and loss modulus (G") for a 10.0 wt. % aqueous PHPMA 5O -PMPC I25 -S-S-PMPC 125 - PHPMA 50 copolymer solution;
  • Figure 15 shows the shear rate dependence of G' and G" (G' A, G" ⁇ ) at a constant applied shear stress of 3.0 Pa for a 10.0 wt. % PHPMA 5O -PMPC 125 -S-S- PMPC I25 -PHPMA 5O copolymer solution at various temperatures;
  • Figure 16 shows GPC traces of a PHPMA 5 O-PMPC 125 -S-S-PMPCI 25 -PHPMA 5 O triblock copolymer solution and the same solution 14 h after treatment with 100 equivalents of DTT;
  • Figure 17(a) is a photograph of a free-standing 10.0 % PHPMA 5O -PMPC I25 -S-S- PMPC I25 -PHPMA 5O copolymer gel obtained at 30 0 C;
  • Figure 17(b) is a photograph of a solution of PHPMA 5 O-PMPC I25 -SH formed by cleavage using 100 equivalents of DTT of the disulfide bond present in the PHPMA 50 -PMPC 125 -S-S-PMPC I25 -PHPMA 5O copolymer depicted in Figure 17(a);
  • Figure 18 left hand image shows an inverted PDPA 5O -PMPC 125 -S-S-PMPCI 25 - PDPA 50 copolymer gel and the right hand image shows a free-flowing liquid solution OfPDPA 5O -PMPCi 25 -SH formed by cleavage using 100 equivalents of glutathione of the disulfide bond in the PDPA 5O -PMPC I25 -S-S-PMPC I25 -PDPA 5O copolymer;
  • Figure 19 shows viscosity vs. temperature plots of a 5.0 wt. % and a 10.1 wt. % aqueous PHPMA 5o -PMPC 25 o-PHPMA 5 o copolymer solution obtained at an applied shear stress of 3.0 Pa and shear frequency of 1.0 rad sec " ;
  • Figure 20 A shows the temperature dependence of the storage modulus (G') and loss modulus (G") for a 5.0 wt. % and a 10.1 wt. % aqueous PHPMA 44 -PMPC 25 O- PHPMA 44 copolymer solution;
  • Figure 20 B shows the cross-over temperature of G' and G" as a function of concentration for the same copolymer solution.
  • Figure 21 shows the shear rate dependence of G' and G" at a constant applied shear stress of 3.0 Pa for a 10.1 wt. % PHPMA 50 -PMPC 250 -PHPMA 50 copolymer solution at various temperatures;
  • Figure 22 shows a series of GPC curves recorded for the PEG 45 -Br macro-initiator (right) and the PEG 45 -PHPMA 49 diblock copolymer (left) using THF eluent and PMMA calibration standards;
  • Figure 23 is a 1 H NMR spectrum of the PEG 45 -PHPMA 49 diblock copolymer recorded in CD 3 OD;
  • Figure 24 shows a series of GPC traces (from right to left) of PHPMA 50 , PHPMA 50 - PGMA 30 (Example 6A) and PHPMA 50 -PGMA 50 (Example 6B) respectively.
  • the GPC chromatograms were recorded in DMF eluent containing 0.01 mmol LiBr at 7O 0 C using PMMA calibration standards;
  • Figure 25 is a 1 H NMR spectrum of the PHPMA 50 -PGMA 30 diblock copolymer (Example 6A) recorded in CD 3 OD;
  • Figure 26 is a 1 H NMR spectrum of a PHPMA 37 -PEG 361 -PHPMA 37 triblock copolymer recorded in CD 3 OD;
  • Figure 27 shows a series of GPC traces (from right to left) of the Br-PEG 36 I-Br macro-initiator and PHPMA 8 o-PEG 36 i -PHPMAs 0 respectively.
  • the GPC curves were recorded using a DMF eluent containing 0.01 mmol LiBr at 7O 0 C with PMMA calibration standards; and
  • Figure 28 is a photograph of a free-standing gel formed by a 20 % aqueous solution of a PHPMA 8O -PEG 36I -PHPMA 8O triblock copolymer at 2O 0 C.
  • Figure 29 shows the temperature-dependent viscosities of 10.0 wt % copolymer solutions of PMMA 55 -PMPC 24 O-PMPC 55 , PHPMA 44 -PMPC 25 O-PHPMA 44 and PHEMA 55 -PMPC 25 O-PHEMA 55 .
  • the triblock copolymer PNIPAM 80 -PMPC 125 -S-S-PMPCI 25 -PNIPAM 8 O was prepared according to the reaction scheme shown above via a two step 'macro-initiator' Atom Transfer Radical Polymerization (ATRP) reaction using a disulfide-based bifunctional ATRP initiator [(BiBOE) 2 S 2 ].
  • ATRP Atom Transfer Radical Polymerization
  • 2-(Methacryoyloxy)ethyl phosphorylcholine monomer (MPC, 99.9 % purity) was kindly donated by Biocompatibles Ltd., UK.
  • iV-Isopropylacrylamide (NIPAM, 97 %) was purchased from Sigma- Aldrich and recrystallized from a mixture of 3:2 benzene/n-hexane prior to use.
  • 2-(Diisopropylamino)ethyl methacrylate was purchased from Scientific Polymer Products, Inc. and passed through a DHR-4 inhibitor removal column provided by the manufacturer prior to use.
  • the silica gel 60 (0.063 - 0.200 ran) used to remove the spent ATRP catalyst was purchased from E. Merck (Darmstadt, Germany) and was also used as received.
  • the disulfide-based bifunctional ATRP initiator (BiBOE) 2 S 2 was synthesized according to a literature protocol. 4 Bis(2-hydroxyethyl) disulfide (15.4 g, 0.1 mol) was dissolved in 200 ml dry THF, excess triethylamine (42.0 ml, 0.30 mol) was added under a nitrogen atmosphere and this solution was cooled in an ice bath. 2- . Bromoisobutyryl bromide (59.8 g, 0.26 mol) was added dropwise from a dropping funnel over a 1 h period so as to minimize the reaction exotherm and the reaction solution slowly turned reddish brown. The solution was allowed to warm up to ambient temperature and stirred for 24 h.
  • the insoluble triethylammonium bromide salt was removed by filtration and the resulting colorless solution was concentrated under vacuum.
  • the concentrated solution was stirred with 0.10 M aqueous Na 2 CO 3 to hydrolyze any residual 2-bromoisobutyryl bromide.
  • the crude product was then extracted three times with dichloromethane using a separating funnel.
  • the MPC conversion was typically more than 98 % after 5 h, as judged by 1 H NMR spectra (disappearance of vinyl group signals at ⁇ 5.5-6.5).
  • the reaction flask was immersed in liquid nitrogen to quench this first-stage polymerization, excess methanol was added to the frozen solution and the resulting solution was then passed though a silica gel column [silica gel 60 (0.063 - 0.200 nm)] to remove the spent ATRP catalyst. After solvent evaporation, the solid polymer was dissolved in de- ionized water and freeze-dried overnight. The resulting Br-PMPCi 2 S-S-S-PMPCi 2 S- Br macro-initiator was obtained as an off-white powder (3.3 g).
  • the second-stage polymerization to prepare the PNIPAM 8O -PMPC 12S -S-S-PMPC I2S - PNIPAMgo triblock copolymer was carried out as follows. iV-Isopropylacrylamide (NIPAM, 1.13 g, 10 mmol) and CuBr/Me 4 cyclam catalyst (7.2 mg, 0.05 mmol CuBr; 12.8 mg, 0.05 mmol Me 4 cyclam) were added to 10 ml degassed methanol in a Schlenk flask and stirred in an ice bath to form a homogeneous solution under nitrogen atmosphere.
  • the Br-PMPC 125 -S-S-PMPCi 25 -Br macro-initiator (1.86 g, 0.05 mmol Br) was added to the methanolic solution of NIPAM monomer and ATRP catalyst under a nitrogen blanket.
  • the NIPAM polymerization was allowed to continue for 3 h until 1 H NMR analysis (CD 3 OD) indicated no further change in conversion (monomer conversions were calculated by comparing the vinyl signals at ⁇ 5.5-6.5 to the single isopropyl proton signal at ⁇ 3.8-4.2). Excess methanol was then added to dilute the reaction solution, which was passed through a silica gel 60 column to remove the spent ATRP catalyst.
  • the disulfide-based MPC macro-initiator, Br-PMPC] 25 -S-S- PMPC 125 -Br (0.88 g, 1.5 x 10 "5 mol disulfide)
  • was dissolved in 8 ml deoxygenated de-ionized water and purged for 15 min using nitrogen, followed by addition of dithiothreitol (DTT, 0.24 g, 1.5 x 10 "3 mol, DTT/disulfide molar ratio 100).
  • DTT dithiothreitol
  • M n number-average molecular weights
  • MPD molecular weight distributions
  • the M n and MWD of both the triblock copolymers and the corresponding disulfide- cleaved diblock copolymers were assessed by gel permeation chromatography (GPC) using a Hewlett Packard HP 1090 Liquid Chromatograph as the pumping unit and two Polymer Laboratories PL Gel 5 ⁇ m Mixed-C 7.5 x 300 mm columns in series with a guard column at 4O 0 C connected to a Gilson Model 131 Refractive Index Detector.
  • the eluent was a 3:1 v/v % chloroform/methanol mixture containing 2.0 mM LiBr at a flow rate of 1.0 ml min "1 .
  • a series of near-monodisperse poly(methyl methacrylate) [PMMA] samples were used as calibration standards. Data analysis was carried out using CirrusTM GPC Software supplied by Polymer Laboratories.
  • the MPC conversion was typically more than 98 % prior to isolation of the macro- initiator.
  • the activity of a Br-PMPCi 25 -S-S-PMPC 125 -Br macro-initiator was confirmed by self-blocking experiments using a second charge of MPC monomer in methanol at 20 0 C. This second-stage polymerization proceeded to almost 100 % conversion.
  • the relatively high final polydispersity simply reflects the difficulty in preparing high molecular weight polymers using ATRP.
  • the polymerization of NIPAM using this macro-initiator was not as efficient as for the MPC monomer.
  • a NIPAM conversion of approximately 80 % was achieved in the synthesis of the Br- PNIPAM S o-PMPCj 25 -S-S-PMPC 125 -PNIPAM 8O -Br triblock copolymer gelator.
  • Nonaqueous GPC analysis indicated a unimodal GPC trace, which suggested that a well- defined triblock architecture was obtained (see later, Figure 10).
  • the PNIPAM 8O -PMPC 125 -S-S-PMPC I25 -PNIPAM 8O triblock copolymer was dissolved in D 2 O at 8.1 wt. % and examined both as a free-flowing solution and a free-standing gel using variable temperature 1 H NMR spectroscopy (see Figures 6(a) and 6(b)).
  • the expected PNEPAM signals were observed, indicating a high degree of solvation and mobility for these blocks under these conditions.
  • the apparent block composition at 33°C was essentially the same as that at 20 0 C, which suggests little or no contamination by PNIPAM homopolymer (since this impurity, if present, is expected to.
  • Figure 11 illustrates the results of an assessment of the kinetics of de-gelation of a 9.0 w/v % aqueous solution of the PNIPAM 8 O-PMPC I25 -S-S-PMPC 125 -PNIPAM 8O copolymer using glutathione/disulfide molar ratios of 2:1, 5:1 and 10:1 at 37 0 C in PBS buffer by monitoring the solution viscosity using a rheometer operating at a fixed shear stress of 5.0 Pa and a shear frequency of 1 rad sec "1 .
  • the reaction mixture was diluted with methanol and passed through a silica column to remove the spent catalyst. After evaporation of the solvent, water was added and the polymer was obtained as a white powder by freeze drying. Yield: 7.49 g copolymer, 84%.
  • Chromatograms were assessed using a Hewlett Packard HP 1090 Liquid Chromatograph as the pumping unit and two Polymer Laboratories PL Gel 5 ⁇ m Mixed-C 7.5 x 300 mm columns in series with a guard column at 4O 0 C connected to a Gilson Model 131 Refractive Index Detector.
  • the eluent was a 3:1 v/v % chlorofo ⁇ n/methanol mixture containing 2.0 mM LiBr at a flow rate of 1.0 ml min "1 .
  • a series of near-monodisperse poly(methyl methacrylate) [PMMA] samples were used as calibration standards. Toluene (2 ⁇ L) was added to all samples as a flow rate marker. Data analysis was carried out using CirrusTM GPC Software supplied by Polymer Laboratories.
  • Figure 12 shows the GPC traces recorded for the MPC 125 -S-S-MPCi 25 macro- initiator and also for the final PHPMA 50 -PMPC 125 -S-S-PMPC 125 -PHPMA 5O triblock copolymer.
  • the latter chromatogram elutes earlier than that of the macro-initiator, as expected. This is reflected in the calculated number-average molecular weights.
  • a typical viscosity vs. temperature plot of a 10.0 wt. % aqueous copolymer solution obtained at an applied shear stress of 3.0 Pa and shear frequency of 1.0 rad sec "1 is shown in Figure 13.
  • the copolymer solution behaved as a fluid at low temperature and its viscosity was less than 0:25 Pa.s at 5 0 C. Gelation was indicated by an increase in viscosity starting at around 5 0 C. The gel viscosity exceeded 100 Pa.s at 30 0 C where a stable value was reached.
  • the temperature dependence of the storage modulus (G') and loss modulus (G") for the same aqueous copolymer solution is shown in Figure 14.
  • G' was lower than G" for all temperatures below 21 0 C, which is the typical behavior for a Newtonian fluid.
  • the critical gelation temperature is where G' is equal to G" and is approximately 21 0 C. Above this temperature, G' is greater than G", which is characteristic of an elastic gel. 7
  • Figure 16 shows the GPC traces of the original triblock copolymer and the same solution 14 h after treatment with 100 equivalents of DTT (relative to the molar disulfide content) was added to a 1 % aqueous solution.
  • DTT relative to the molar disulfide content
  • This one-pot synthesis was carried out in two successive steps using sequential monomer addition without purification of the intermediate PMPC-based macroinitiator.
  • MPC (7.3969 g, 25.1 mmol, 250 eq.) was placed under nitrogen.
  • the (BiBOE) 2 S 2 initiator (55.0 mg 0.12 mmol) was dissolved in 12.1 mL methanol. 10 mL of this solution (0.10 mmol, 1 eq.) was added to the solution of MPC and this was degassed for further 30 minutes to remove. oxygen.
  • the same GPC protocol as described above in Example 2 was employed.
  • the PDPA 5O -PMPCI 25 -S-S-PMPC 125 -PDPA 5O copolymer had an M n of 48,000 and an M w /M n of 1.31.
  • the relatively low polydispersity indicated a well-controlled polymerisation.
  • Variable temperature DLS studies (see Table 3) were performed in aqueous solution at pH 8 using the same Brookhaven instrument described above.
  • the diameter of the diblock copolymer micelles (obtained after the in situ cleavage of the disulfide bond in the triblock copolymer micelles using 100 equivalents of glutathione) is approximately half of that of the original triblock copolymer micelles, while the polydispersity remains unchanged.
  • Figure 18 (left hand image) shows the inverted gel. To this gel were added 100 equivalents of glutathione relative to the disulfide bond. After 10-20 min. of hand-shaking at 2O 0 C, the gel became a free- flowing liquid as shown in Figure 18 (right hand image).
  • Example 2 The high resolution NMR method described in Example, 2 for the disulfide-based copolymer was employed. Assuming a DP of 250 for the central PMPC block, the overall copolymer composition was calculated to be PHPMA 44 -PMPC 25O -PHPMA 44 .
  • the same GPC protocol used for the disulfide-based triblock copolymer was employed.
  • the PHPMA 50 -PMPC 25 o-PHPMA 5 o copolymer had an M n of 85,000 and a .
  • MwZM n 1.39.
  • Typical viscosity vs. temperature plots of a 5.0 wt. % and a 10.1 wt. % aqueous PHPMA 5O -PMPC 25 o-PHPMA 5O copolymer solution obtained at an applied shear stress of 3.0 Pa and shear frequency of 1.0 rad sec "1 are shown in Figure 19.
  • the viscosity increases with temperature from 0 0 C to 54 0 C.
  • the initial viscosity is seen to be high at both concentrations at low temperatures and to be highly dependent on the concentration. Gelation is indicated by an increase in viscosity. The initial increase is seen to be dependent on the concentration.
  • PEG 45 OH (30.0 g, 15.0 mmol) was dissolved in toluene (300 mL) in a 500 mL one- neck flask. After azeotropic distillation of toluene (30 mL) at reduced pressure to remove traces of water, the solution was cooled to room temperature and triethylamine (3.0 mL, 22.5 mmol) was added. The mixture was cooled using an ice bath, then 2-bromoisobutyryl bromide (5.28 g, 22.5 mmol) was added dropwise over 30 min. The reaction mixture was stirred at room temperature (2O 0 C) for 6 days, before treatment with activated decolourising charcoal.
  • the solid was removed by filtration and the solvent was evaporated by rotary evaporation before precipitation into a ten-fold excess of diethyl ether.
  • the esterified polymer was filtered and dried under vacuum to remove solvent. Yield: 28.62 g (88.30 %).
  • the purified PEG 45 -Br macro-initiator was characterized by 1 H NMR and THF GPC (using PMMA calibration standards, see Figure 22). The degree of esterification as judged by NMR was 98 %.
  • PEG 45 -Br macro-initiator (2.00 g, 0.9255 mmol).
  • the macro-initiator was first degassed by three vacuum/nitrogen cycles, followed by the addition of HPMA (6.67 g, 46.27 mmol) and bpy (0.289 g, 1.581 mmol).
  • HPMA 6.67 g, 46.27 mmol
  • bpy 0.289 g, 1.581 mmol
  • the mixture was purged with nitrogen and stirred for 20 min.
  • degassed methanol (8.8 mL) was added and the solution was stirred until homogeneous.
  • Cu(I)Cl was added and the reaction mixture was heated at 5O 0 C using an oil bath for 6 hours under iiitrogen atmosphere with continual stirring.
  • the HPMA monomer conversion reached 98 % as determined by 1 H NMR spectroscopy (see Figure 23).
  • the polymerization was terminated by exposing to the air.
  • the diblock copolymer solution was diluted with methanol (200 mL) and passed through a silica column to remove the spent catalyst. The solvent was then evaporated under vacuum and the solid copolymer was dissolved in THF and precipitated into excess n-hexane so as to remove residual monomer.
  • a PHPMA SO -PEG 36I -PHPMA 8O triblock copolymer was synthesized by ATRP using a bifunctional Br-PEG 36I -Br macro-initiator, Cu(I)Cl and bpy ligand using a relative molar ratio of Br-PEG 36J -Br.
  • CuCl: bpy 1: 2: 4.
  • the same GPC protocol used for the disulf ⁇ de-based triblock copolymer was employed.
  • the PMMA 55 -PMPC 24O -PMMA 55 copolymer had an M n of 89,000 and a
  • the same GPC protocol used for the disulfide-based triblock copolymer was employed.
  • the PHEMA 55 -PMPC 25O -PHEMA 55 copolymer had an M n of 91,900 and a MwZM n of 1.62.
  • thermoresponsive behaviour of the PHEMA chains may be suppressed significantly when they are attached to the much more hydrophilic PMPC block.

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Abstract

L'invention concerne un gélificateur polymère dont le comportement de transition de phase liquide-gel est contrôlable. Le polymère précité peut être de la formule A-B-L-B-A dans laquelle A représente une unité polymère qui, lorsque le polymère (I) est formé dans un milieu aqueux, présente une hydrophobicité augmentée si l'on modifie la température ou la valeur de pH dudit milieu aqueux en la faisant passer d'une première température ou valeur de pH à une seconde température ou valeur de pH. B représente une unité polymère qui est plus hydrophile que l'unité polymère A lorsque ledit milieu aqueux se trouve à la seconde température ou à la seconde valeur de pH. L représente un groupe de liaison qui peut être clivé par hydrolyse, oxydation ou réduction. Le polymère peut être un gélificateur polymère dépendant de la température renfermant des unités polymères C, D et E, où l'unité polymère C est un (2-hydroxypropyl méthacrylate) et D est une unité polymère qui, lorsque ledit gélificateur polymère est formé dans un milieu aqueux, est plus hydrophile que l'unité polymère C. Chaque polymère peut être formé dans un mélange de polymères, combiné à un copolymère biséquencé thermosensible renfermant du (2-hydroxypropyl méthacrylate).
PCT/GB2006/004487 2005-12-03 2006-12-01 Gelificateur polymere WO2007063320A1 (fr)

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GB0524740.8 2005-12-03
GB0524740A GB0524740D0 (en) 2005-12-03 2005-12-03 Polymer gelator

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WO2007063320A1 true WO2007063320A1 (fr) 2007-06-07

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WO2014128500A1 (fr) * 2013-02-21 2014-08-28 The University Of Sheffield Milieu pour cellules souches
CN109568648A (zh) * 2018-11-27 2019-04-05 合肥工业大学 一种具有抗菌和促进伤口愈合功能的热诱导不可逆复合水凝胶的制备方法及其应用
CN113226376A (zh) * 2018-10-26 2021-08-06 内布拉斯加大学董事会 作为新型药物递送平台的基于大分子前药的热敏性可注射凝胶
CN115404062A (zh) * 2022-08-30 2022-11-29 长江大学 一种pH值和温度双响应超分子凝胶暂堵剂及其制备方法和应用、暂堵转向压裂的方法
WO2023210671A1 (fr) * 2022-04-26 2023-11-02 京セラ株式会社 Copolymère, film polymère, dispositif de mesure et support de mesure

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WO2004069888A2 (fr) * 2003-02-05 2004-08-19 Biocompatibles Uk Limited Copolymeres sequences
US20050031575A1 (en) * 2001-12-31 2005-02-10 Stephen Brocchini Block copolymers
US20050220880A1 (en) * 2002-03-07 2005-10-06 Lewis Andrew L Drug carriers comprising amphiphilic block copolymers

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US20050031575A1 (en) * 2001-12-31 2005-02-10 Stephen Brocchini Block copolymers
US20050220880A1 (en) * 2002-03-07 2005-10-06 Lewis Andrew L Drug carriers comprising amphiphilic block copolymers
WO2004069888A2 (fr) * 2003-02-05 2004-08-19 Biocompatibles Uk Limited Copolymeres sequences

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014128500A1 (fr) * 2013-02-21 2014-08-28 The University Of Sheffield Milieu pour cellules souches
US10150946B2 (en) 2013-02-21 2018-12-11 The University Of Sheffield Media for stem cells
CN113226376A (zh) * 2018-10-26 2021-08-06 内布拉斯加大学董事会 作为新型药物递送平台的基于大分子前药的热敏性可注射凝胶
CN109568648A (zh) * 2018-11-27 2019-04-05 合肥工业大学 一种具有抗菌和促进伤口愈合功能的热诱导不可逆复合水凝胶的制备方法及其应用
CN109568648B (zh) * 2018-11-27 2021-01-29 合肥工业大学 一种具有抗菌和促进伤口愈合功能的热诱导不可逆复合水凝胶的制备方法及其应用
WO2023210671A1 (fr) * 2022-04-26 2023-11-02 京セラ株式会社 Copolymère, film polymère, dispositif de mesure et support de mesure
CN115404062A (zh) * 2022-08-30 2022-11-29 长江大学 一种pH值和温度双响应超分子凝胶暂堵剂及其制备方法和应用、暂堵转向压裂的方法
CN115404062B (zh) * 2022-08-30 2023-10-27 长江大学 一种pH值和温度双响应超分子凝胶暂堵剂及其制备方法和应用、暂堵转向压裂的方法

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