Patent Grant
9913926
Proteins described herein are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NO corresponds numerically to the sequence identifiers <400>1, <400>2, etc. The Sequence Listing, in written computer readable format (CFR), is incorporated by reference in its entirety.
Bone fractures are a serious health concern in society today. In addition to the fracture itself, a number of additional health risks are associated with the fracture. For example, intra-articular fractures are bony injuries that extend into a joint surface and fragment the cartilage surface. Fractures of the cartilage surface often lead to debilitating posttraumatic arthritis. The main determining factors in the development of posttraumatic arthritis are thought to be the amount of energy imparted at the time of injury, the patient's genetic predisposition (or lack thereof) to posttraumatic arthritis, and the accuracy and maintenance of reduction. Of the three prognostic factors, the only factor controllable by orthopedic caregivers is achievement and maintenance of reduction. Comminuted injuries of the articular surface (the cartilage) and the metaphysis (the portion of the bone immediately below the cartilage) are particularly challenging to maintain in reduced (aligned) position. This relates to the quality and type of bone in this area. It also relates to the limitations of fixation with titanium or stainless steel implants.
Currently, stainless steel and titanium implants are the primary methods of fixation, but their size and the drilling necessary to place them frequently interfere with the exact manipulation and reduction of smaller pieces of bone and cartilage. A variety of bone adhesives have been tested as alternatives to mechanical fixation. These fall into four categories: polymethylmethacrylates (PMMA), fibrin-based glues, calcium phosphate (CP) cements, and CP resin composites. PMMA cements, which are used in the fixation of protheses, have well-known drawbacks, one of the most serious being that the heat generated from the exothermic setting reaction can kill adjacent bone tissue. Also, the poor bonding to bone leads to aseptic loosening, the major cause of PMMA cemented prothesis failure.
Fibrin glues, based on the blood clotting protein fibrinogen, have been tested for fixing bone grafts and repairing cartilage since the 1970s and yet have not been widely deployed. One of the drawbacks of fibrin glues is that they are manufactured from pooled human donor blood. As such, they carry risk of transmitting infections and could potentially be of limited supply.
CP cements are powders of one or more forms of CP, e.g., tetracalcium phosphate, dicalcium phosphate anhydride, and P-tricalcium phosphate. When the powder is mixed with water it forms a paste that sets up and hardens through the entanglement of one or more forms of CP crystals, including hydroxyapatite. Advantages of CP cements include isothermal set, proven biocompatibility, osteoconductivity, and they serve as a reservoir for Ca and PO4 for hydroxyapatite formation during healing. The primary disadvantages are that CP cements are brittle, have low mechanical strength and are therefore not ideal for stable reduction of small articular segments. CP cements are used mostly as bone void fillers. The poor mechanical properties of CP cements have led to composite cements of CP particles and polymers. By varying the volume fractions of the particulate phase and the polymer phase, the modulus and strength of the glue can be adjusted toward those of natural bone, an avenue that is also open to us.
Given the overall health impact associated with bone fractures and the imperfect state of current fixation methods, new fixation methods are needed.
Described herein is the synthesis of adhesive complex coacervates and their use thereof. The adhesive complex coacervates are composed of a mixture of one or more polycations and one or more polyanions. The polycations and polyanions are crosslinked with one another by covalent bonds upon curing. The adhesive complex coacervates have several desirable features when compared to conventional adhesives, which are effective in water-based applications. The adhesive complex coacervates described herein exhibit low interfacial tension in water when applied to a substrate (i.e., they spread over the interface rather than being beaded up). Additionally, the ability of the complex coacervate to crosslink intermolecularly increases the cohesive strength of the adhesive complex coacervate. The adhesive complex coacervates have numerous biological applications as bioadhesives and drug delivery devices. In particular, the adhesive complex coacervates described herein are particularly useful in underwater applications and situations where water is present such as, for example, physiological conditions.
The advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.
Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase “optionally substituted lower alkyl” means that the lower alkyl group can or cannot be substituted and that the description includes both unsubstituted lower alkyl and lower alkyl where there is substitution.
Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.
Variables such as R1, R2, R3, R4, R5, R13-R22, A, X, d, m, n, s, t, u, v, w, and x used throughout the application are the same variables as previously defined unless stated to the contrary.
The term “alkyl group” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 25 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Examples of longer chain alkyl groups include, but are not limited to, a palmitate group. A “lower alkyl” group is an alkyl group containing from one to six carbon atoms.
Any of the compounds described herein can be the pharmaceutically-acceptable salt. In one aspect, pharmaceutically-acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically-acceptable base. Representative pharmaceutically-acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. In one aspect, the reaction is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0°C. to about 100° C. such as at room temperature. In certain aspects where applicable, the molar ratio of the compounds described herein to base used are chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material, the starting material can be treated with approximately one equivalent of pharmaceutically-acceptable base to yield a neutral salt.
In another aspect, if the compound possesses a basic group, it can be protonated with an acid such as, for example, HCI, HBr, or H2SO4, to produce the cationic salt. In one aspect, the reaction of the compound with the acid or base is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0° C. to about 100° C. such as at room temperature. In certain aspects where applicable, the molar ratio of the compounds described herein to base used are chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material, the starting material can be treated with approximately one equivalent of pharmaceutically-acceptable base to yield a neutral salt.
Described herein are adhesive complex coacervates and their applications thereof. In general, the complexes are a mixture of cations and anions in balanced proportions to produce stable aqueous complexes at a desired pH. The adhesive complex coacervate comprises at least one polycation, at least one polyanion, and at least one multivalent cation, wherein at least one polycation or polyanion is a synthetic compound, and the polycation and/or polyanion are crosslinked with one another upon curing the complex coacervate. Each component of the coacervate and methods for making the same are described below.
The adhesive complex coacervate is an associative liquid with a dynamic structure in which the individual polymer components diffuse throughout the entire phase. Complex coacervates behave theologically like viscous particle dispersions rather than a viscoelastic polymer solution. As described above, the adhesive complex coacervates exhibit low interfacial tension in water when applied to substrates either under water or that are wet. In other words, the complex coacervate spreads evenly over the interface rather than beading up. Additionally, upon intermolecular crosslinking, the adhesive, complex coacervate forms a strong, insoluble, cohesive material.
Conversely, polyelectrolyte complexes (PECs), which can be a precursor to the adhesive complex coacervates described herein, are small colloidal particles. For example, referring to
An exemplary model of the differences in phase behavior between the polyelectrolyte complex and the adhesive complex coacervate is presented in
The polycations and polyanions contain groups that permit crosslinking between the two polymers upon curing to produce new covalent bonds and the adhesive complex coacervate described herein. The mechanism of crosslinking can vary depending upon the selection of the crosslinking groups. In one aspect, the crosslinking groups can be electrophiles and nucleophiles. For example, the polyanion can have one or more electrophilic groups, and the polycations can have one or more nucleophilic groups capable of reacting with the electrophilic groups to produce new covalent bonds. Examples of electrophilic groups include, but are not limited to, anhydride groups, esters, ketones, lactams (e.g., maleimides and succinimides), lactones, epoxide groups, isocyanate groups, and aldehydes. Examples of nucleophilic groups are presented below.
In another aspect, the polycation and polyanion each have an actinically crosslinkable group. As used herein, “actinically crosslinkable group” in reference to curing or polymerizing means that the crosslinking between the polycation and polyanion is performed by actinic irradiation, such as, for example, UV irradiation, visible light irradiation, ionized radiation (e.g. gamma ray or X-ray irradiation), microwave irradiation, and the like. Actinic curing methods are well-known to a person skilled in the art. The actinically crosslinkable group can be an unsaturated organic group such as, for example, an olefinic group. Examples of olefinic groups useful herein include, but are not limited to, an acrylate group, a methacrylate group, an acrylamide group, a methacrylamide group, an allyl group, a vinyl group, a vinylester group, or a styrenyl group.
In another aspect, the crosslinkable group includes a dihydroxyl-substituted aromatic group capable of undergoing oxidation in the presence of an oxidant. In one aspect, the dihydroxyl-substituted aromatic group is a dihydroxyphenol or halogenated dihydroxyphenol group such as, for example, DOPA and catechol (3,4 dihydroxyphenol). For example, in the case of DOPA, it can be oxidized to dopaquinone. Dopaquinone is an electrophilic group that is capable of either reacting with a neighboring DOPA group or another nucleophilic group. In the presence of an oxidant such as oxygen or other additives including, but not limited to, peroxides, periodates (e.g., NaIO4), persulfates, permanganates, dichromates, transition metal oxidants (e.g., a Fe+3 compound, osmium tetroxide), or enzymes (e.g., catechol oxidase), the dihydroxyl-substituted aromatic group can be oxidized. In another aspect, crosslinking can occur between the polycation and polyanion via light activated crosslinking through azido groups. Once again, new covalent bonds are formed during this type of crosslinking.
In certain aspects, the oxidant can be stabilized. For example, a compound that forms a complex with periodate that is not redox active can result in a stabilized oxidant. In other words, the periodate is stabilized in a non-oxidative form and cannot oxidize the dihydroxyl-substituted aromatic group while in the complex. The complex is reversible and even if it has a very high stability constant there is a small amount of uncomplexed periodate formed. The dihydroxyl-substituted aromatic group competes with the compound for the small amount of free periodate. As the free periodate is oxidized more is released from the complex because it is in equilibrium. In one aspect, sugars possessing a cis,cis-1,2,3-triol grouping on a six-membered ring can form competitive periodate complexes. An example of a specific compound that forms stable periodate complex is 1,2-O-isopropylidene-alpha-D-glucofuranose. The stabilized oxidant can control the rate of crosslinking. Not wishing to be bound by theory, the stabilized oxidant slows it down the rate of oxidation so that there is time to add the oxidant and position the substrate before the adhesive hardens irreversibly.
The stability of the oxidized crosslinker can vary. For example, the phosphono containing polyanions described herein that contain oxidizable crosslinkers are stable in solution and do not crosslink with themselves. This permits nucleophilic groups present on the polycation to react with the oxidized crosslinker. This is a desirable feature of the invention, which permits the formation of intermolecular bonds and, ultimately, the formation of a strong adhesive. Examples of nucleophilic groups that are useful include, but are not limited to, hydroxyl, thiol, and nitrogen containing groups such as substituted or unsubstituted amino groups and imidazole groups. For example, residues of lysine, histidine, and/or cysteine can be incorporated into the polycation and introduce nucleophilic groups. An example of this is shown in
In other aspects, the crosslinkers present on the polycation and/or polyanion can form coordination complexes with transition metal ions. For example, a transition metal ion can be added to a mixture of polycation and polyanion, where both polymers contain crosslinkers capable of coordinating with the transition metal ion. The rate of coordination and dissociation can be controlled by the selection of the crosslinker, the transition metal ion, and the pH. Thus, in addition to covalent crosslinking as described above, crosslinking can occur through electrostatic, ionic, or other non-covalent bonding. Transition metal ions such as, for example, iron, copper, vanadium, zinc, and nickel can be used herein.
The polycation and polyanion are generally composed of a polymer backbone with a plurality of chargeable groups at a particular pH. The groups can be pendant to the polymer backbone and/or incorporated within the polymer backbone. In certain aspects, (e.g., biomedical applications), the polycation is any biocompatible polymer possessing cationic groups or groups that can be readily converted to cationic groups by adjusting the pH. In one aspect, the polycation is a polyamine compound. The amino groups of the polyamine can be branched or part of the polymer backbone. The amino group can be a primary, secondary, or tertiary amino group that can be protonated to produce a cationic ammonium group at a selected pH. In general, the polyamine is a polymer with a large excess of positive charges relative to negative charges at the relevant pH, as reflected in its isoelectric point (pI), which is the pH at which the polymer has a net neutral charge. The number of amino groups present on the polycation ultimately determines the charge of the polyanion at a particular pH. For example, the polycation can have from 10 to 90 mole %, 10 to 80 mole %, 10 to 70 mole %, 10 to 60 mole %, 10 to 50 mole %, 10 to 40 mole %, 10 to 30 mole %, or 10 to 20 mole % amino groups. In one aspect, the polyamine has an excess positive charge at a pH of about 7, with a pI significantly greater than 7. As will be discussed below, additional amino groups can be incorporated into the polymer in order to increase the pI value.
In one aspect, the amino group can be derived from a residue of lysine, histidine, or imidazole attached to the polycation. Any anionic counterions can be used in association with the cationic polymers. The counterions should be physically and chemically compatible with the essential components of the composition and do not otherwise unduly impair product performance, stability or aesthetics. Non-limiting examples of such counterions include halides (e.g., chloride, fluoride, bromide, iodide), sulfate and methylsulfate.
In one aspect, when the polycation is naturally-occurring, the polycation can be a positively-charged protein produced from a natural organism. For example, proteins produced by P. californica can be used as the polycation.
In another aspect, the polycation can be a biodegradable polyamine. The biodegradable polyamine can be a synthetic polymer or naturally-occurring polymer. The mechanism by which the polyamine can degrade will vary depending upon the polyamine that is used. In the case of natural polymers, they are biodegradable because there are enzymes that can hydrolyze the polymers and break the polymer chain. For example, proteases can hydrolyze natural proteins like gelatin. In the case of synthetic biodegradable polyamines, they also possess chemically labile bonds. For example, β-aminoesters have hydrolyzable ester groups. In addition to the nature of the polyamine, other considerations such as the molecular weight of the polyamine and crosslink density of the adhesive can be varied in order to modify the degree of biodegradability.
In one aspect, the biodegradable polyamine includes a polysaccharide, a protein, or a synthetic polyamine. Polysaccharides bearing one or more amino groups can be used herein. In one aspect, the polysaccharide is a natural polysaccharide such chitosan. Similarly, the protein can be a synthetic or naturally-occurring compound. In another aspect, the biodegradable polyamine is a synthetic polyamine such as poly(β-aminoesters), polyester amines, poly(disulfide amines), mixed poly(ester and amide amines), and peptide crosslinked polyamines.
In the case when the polycation is a synthetic polymer, a variety of different polymers can be used; however, in certain applications such as, for example, biomedical applications, it is desirable that the polymer be biocompatible and non-toxic to cells and tissue. In one aspect, the biodegradable polyamine can be an amine-modified natural polymer. For example, the amine-modified natural polymer can be gelatin modified with one or more alkylamino groups, heteroaryl groups, or an aromatic group substituted with one or more amino groups. Examples of alkylamino groups are depicted in Formulae IV-VI
wherein R13-R22 are, independently, hydrogen, an alkyl group, or a nitrogen containing substituent;
As shown in formula IV-VI, the number of amino groups can vary. In one aspect, the alkylamino group is —NHCH2NH2, —NHCH2CH2NH2, —NHCH2CH2CH2NH2, —NHCH2CH2CH2CH2NH2, —NHCH2CH2CH2CH2CH2NH2, —NHCH2NHCH2CH2CH2NH2, —NHCH2CH2NHCH2CH2CH2NH2, —NHCH2CH2CH2NHCH2CH2CH2CH2NHCH2CH2CH2NH2, —NHCH2CH2NHCH2CH2CH2CH2NH2, —NHCH2CH2NHCH2CH2CH2NHCH2CH2CH2NH2, or —NHCH2CH2NH(CH2CH2NH)dCH2CH2NH2, where d is from 0 to 50.
In one aspect, the amine-modified natural polymer can include an aryl group having one or more amino groups directly or indirectly attached to the aromatic group. Alternatively, the amino group can be incorporated in the aromatic ring. For example, the aromatic amino group is a pyrrole, an isopyrrole, a pyrazole, imidazole, a triazole, or an indole. In another aspect, the aromatic amino group includes the isoimidazole group present in histidine. In another aspect, the biodegradable polyamine can be gelatin modified with ethylenediamine.
In one aspect, the polycation includes a polyacrylate having one or more pendant amino groups. For example, the backbone can be a homopolymer or copolymer derived from the polymerization of acrylate monomers including, but not limited to, acrylates, methacrylates, acrylamides, and the like, in one aspect, the backbone of the polycation is polyacrylamide. In other aspects, the polycation is a block co-polymer, where segments or portions of the co-polymer possess cationic groups depending upon the selection of the monomers used to produce the co-polymer.
In one aspect, the polycation is a polyamino compound. In another aspect, the polyamino compound has 10 to 90 mole % tertiary amino groups. In a further aspect, the polycation polymer has at least one fragment of the formula I
wherein R1, R2, and R3 are, independently, hydrogen or an alkyl group, X is oxygen or NR5, where R5 is hydrogen or an alkyl group, and m is from 1 to 10, or the pharmaceutically-acceptable salt thereof. In another aspect, R1, R2, and R3 are methyl and m is 2. Referring to formula I, the polymer backbone is composed of CH2—CR1 units with pendant —C(O)X(CH2)mNR2R3 units. In this aspect, the fragment having the formula I is a residue of an acrylate, methacrylate, acrylamide, or methacrylamide.
Similar to the polycation, the polyanion can be a synthetic polymer or naturally-occurring. In one aspect, when the polyanion is naturally-occurring, the polyanion is a negatively-charged protein produced from P. californica.
When the polyanion is a synthetic polymer, it is generally any polymer possessing anionic groups or groups that can be readily converted to anionic groups by adjusting the pH. Examples of groups that can be converted to anionic groups include, but are not limited to, carboxylate, sulfonate, phosphonate, boronate, sulfate, borate, or phosphate. Any cationic counterions can be used in association with the anionic polymers if the considerations discussed above are met.
In one aspect, the polyanion is a polyphosphate. In another aspect, the polyanion is a polyphosphate compound having from 10 to 90 mole % phosphate groups. For example, the polyphosphate can be a naturally-occurring compound such as, for example, highly phosphorylated proteins like phosvitin (an egg protein), dentin (a natural tooth phosphoprotein), casein (a phosphorylated milk protein), or bone proteins (e.g. osteopontin).
In other aspects, phosphorous containing polymers can be converted to polyanions. For example, a phospholipid or phosphosugar is not a polyanion but it can be converted into a polyanion by creating a liposome or a micelle with it. Thus, in this aspect, the complex coacervate is a charged colloid. Alternatively, the colloid can be produced by any of the polyanions or polycations described herein.
In another aspect, the polyphosphate can be a synthetic compound. For example, the polyphosphate can be a polymer with pendant phosphate groups attached to the polymer backbone and/or present in the polymer backbone. (e.g., a phosphodiester backbone). In one aspect, the polyphosphate can be produced by chemically or enzymatically phosphorylating a protein (e.g., natural serine-rich proteins).
In one aspect, the polyanion includes a polyacrylate having one or more pendant phosphate groups. For example, the backbone can be a homopolymer or copolymer derived from the polymerization of acrylate monomers including, but not limited to, acrylates, methacrylates, acrylamides, and the like. In one aspect, the backbone of the polyanion is derived from the polymerization of polyacrylamide. In other aspects, the polyanion is a block co-polymer, where segments or portions of the co-polymer possess anionic groups depending upon the selection of the monomers used to produce the co-polymer. In a further aspect, the polyanion can be heparin sulfate, hyaluronic acid, chitosan, and other biocompatible and biodegradable polymers typically used in the art.
In one aspect, the polyanion is a polyphosphate. In another aspect, the polyanion is a polymer having at least one fragment having the formula II
wherein R4 is hydrogen or an alkyl group, and n is from 1 to 10, or the pharmaceutically-acceptable salt thereof. In another aspect, wherein R4 is methyl and n is 2. Similar to formula I, the polymer backbone of formula II is composed of a residue of an acrylate or methacrylate. The remaining portion of formula II is the pendant phosphate group.
As described above, the polycation and polyanion contain crosslinkable groups. In one aspect, the polycation and polyanion includes an actinically crosslinkable group defined herein. Any of the polymers described above (synthetic or naturally-occurring) that can be used as the polycation and polyanion can be modified to include the actinically crosslinkable group. For example, the polycation can be a polyacrylate having one or more pendant amino groups (e.g., imidazole groups). In the case of the polyanion, in one aspect, a polyphosphate can be modified to include the actinically crosslinkable group(s). For example, wherein the polycation and polyanion includes at least one fragment having the formula VII
wherein R1, R2, and R3 are, independently, hydrogen or an alkyl group, X is oxygen or NR5, where R5 is hydrogen or an alkyl group, and m is from 1 to 10, or the pharmaceutically-acceptable salt thereof, wherein at least one of R2 or R3 is an actinically crosslinkable group. In one aspect, referring to formula VII, R1 is methyl, R2 is hydrogen, R3 is an acrylate or methacrylate group, X is NH, and m is 2.
In another aspect, the polyanion can include one or more groups that can undergo oxidation, and the polycation contains on or more nucleophiles that can react with the oxidized crosslinker to produce new covalent bonds. In one aspect, the polyanion includes at least one dihydroxyl aromatic group capable of undergoing oxidation, wherein the dihydroxyl aromatic group is covalently attached to the polyanion. Examples of dihydroxyl aromatic groups include a DOPA residue or a catechol residue. Any of the polyanions described above can be modified to include one or more dihydroxyl aromatic residues. In one aspect, the polyanion is polymerization product between two or more monomers, where one of the monomers has a dihydroxyl aromatic group covalently attached to the monomer. For example, the monomer can have an unsaturated group capable of undergoing free-radical polymerization with the dihydroxyl aromatic group attached to the monomer. For example, the polyanion can be the polymerization product between (1) a phosphate acrylate and/or phosphate methacrylate and (2) a second acrylate and/or second methacrylate having a dihydroxyl aromatic group covalently bonded to the second acrylate or second methacrylate. In another aspect, the polyanion is the polymerization product between monoacryloxyethyl phosphate and dopamine methacrylamide. Polymers 3 and 7 in
Not wishing to be bound by theory, the polyanion with the dihydroxyl aromatic group(s) are stable in that they react slowly with itself in solution. Thus, the polyanion reacts with the polycation primarily via intermolecular cross-linking (e.g., polycation has a nucleophilic group or a dihydroxyl aromatic group) to produce the complex coacervate. This provides numerous advantages with respect to the use and administration of the complex coacervate. For example, the polycation and polyanion can be premixed and administered to a subject instead of the sequential administration of the polymers. This greatly simplifies administration of the complex coacervate that is not an option with currently available bioadhesives.
It is contemplated that the polycation can be a naturally occurring compound (e.g., protein from P. californica) and the polyanion is a synthetic compound. In another aspect, the polycation can be a synthetic compound and the polyanion is a naturally occurring compound (e.g., protein from P. californica). In a further aspect, both the polyanion and polycation are synthetic compounds.
The adhesive complex coacervates can optionally contain one or more multivalent cations (i.e., cations having a charge of +2 or greater). In one aspect, the multivalent cation can be a divalent cation composed of one or more alkaline earth metals. For example, the divalent cation can be a mixture of Ca+2 and Mg+2. In other aspects, transition metal ions with a charge of +2 or greater can be used as the multivalent cation. In addition to the pH, the concentration of the multivalent cations can determine the rate and extent of coacervate formation. Not wishing to be bound by theory, weak cohesive forces between particles in the fluid may be mediated by multivalent cations bridging excess negative surface charges. The amount of multivalent cation used herein can vary. In one aspect, the amount is based upon the number of anionic groups and cationic groups present in the polyanion and polycation. For example, when the multivalent cation is a mixture of calcium and magnesium, the polycation is a polyamine, the polyanion is a polyphosphate, and the ratio of calcium to amine/phosphate groups can be from 0.1 to 0.3, and the ratio of magnesium to amine/phosphate groups can be from 0.8 to 1.0. In the Examples, the selection of the amount of multivalent cations with respect to producing adhesive complex coacervates and other physical states is addressed.
The adhesive complex coacervate can be synthesized a number of different ways. In one aspect, the polycation, the polyanion, and at least one multivalent cation, can be mixed with one another to produce the adhesive complex coacervate. By adding the appropriate amount of multivalent cation to the mixture of polyanion and polycation, the adhesive complex coacervate can be produced. In another aspect, the adhesive complex coacervate can be produced by the process comprising:
The adhesive complex coacervates produced herein can undergo subsequent phase changes that ultimately lead to the formation of an adhesive. In one aspect, the adhesive can be produced by the process comprising
In other aspects, the adhesive is produced by the process comprising
In these aspects, once the adhesive complex coacervate is converted to an insoluble solid, the insoluble solid is crosslinked to produce a strong adhesive. As discussed above, the polycation and polyanion possess one or more crosslinkable groups capable of forming covalent bonds. For example, the polycation and/or polyanion can possess at least one dihydroxyl aromatic group capable of undergoing oxidation. In this aspect, the dihydroxyl aromatic group can be oxidized by a variety oxidants such O2, NaIO4, a peroxide, a transition metal oxidant, or stabilized oxidant as described above. In the case when the polycation or polyanion has dihydroxyl aromatic group, the other polymer can possess a nucleophilic group that can react with the oxidized form of the dihydroxyl aromatic group to produce a new covalent bond. In other aspects, when the polycation and polyanion possess an actinically crosslinkable group, the insoluble solid can be irradiated with light to crosslink the polycation and polyanion to produce the adhesive. In this aspect, the insoluble solid (and complex coacervate precursor) can include a photoinitiator to facilitate crosslinking between the actinically crosslinkable groups. Examples of photoinitiators useful herein include, but are not limited to, a phosphine oxide, a peroxide, an azide compound, an α-hydroxyketone, or an α-aminoketone. Upon crosslinking, a strong adhesive is produced having numerous applications.
The adhesive complex coacervates and adhesives produced therefrom described herein have numerous benefits with respect to their use as biological glues and delivery devices. For example, the coacervates have low initial viscosity, specific gravity greater than one, and being mostly water by weight, low interfacial tension in an aqueous environment, all of which contribute to their ability to adhere to a wet surface. An additional advantage with respect to the bonding mechanism (i.e., crosslinking) of the adhesive complex coacervates includes low heat production during setting, which prevents damage to living tissue. The components can be pre-polymerized in order to avoid heat generation by in situ exothermic polymerization. This is due for the most part by the ability of the adhesive complex coacervates to crosslink intermolecularly under very mild conditions as described above.
The adhesive complex coacervates described herein can be applied to a number of different biological substrates. The substrate can be contacted in vitro or in vivo. The rate of crosslinking within the adhesive complex coacervate can be controlled by for example pH and the presence of an oxidant or other agents that facilitate crosslinking. One approach for applying the adhesive complex coacervate to the substrate can be found in
In another aspect, referring to
The properties of the adhesive complex coacervates described herein make them ideal for underwater applications such as the administration to a subject. For example, the adhesive complex coacervates and adhesives produced therefrom can be used to repair a number of different bone fractures and breaks. The coacervates adhere to bone (and other minerals) through several mechanisms (see
Examples of such breaks include a complete fracture, an incomplete fracture, a linear fracture, a transverse fracture, an oblique fracture, a compression fracture, a spiral fracture, a comminuted fracture, a compacted fracture, or an open fracture. In one aspect, the fracture is an intra-articular fracture or a craniofacial bone fracture. Fractures such as intra-articular fractures are bony injuries that extend into and fragment the cartilage surface. The adhesive complex coacervates may aid in the maintenance of the reduction of such fractures, allow less invasive surgery, reduce operating room time, reduce costs, and provide a better outcome by reducing the risk of post-traumatic arthritis.
In other aspects, the adhesive complex coacervates and adhesives produced therefrom can be used to join small fragments of highly comminuted fractures. In this aspect, small pieces of fractured bone can be adhered to an existing bone. It is especially challenging to maintain reduction of the small fragments by drilling them with mechanical fixators. The smaller and greater number of fragments the greater the problem. In one aspect, the adhesive complex coacervate or precursor thereof may be injected in small volumes to create spot welds as described above in order to fix the fracture rather than filling the entire crack. The small biocompatible spot welds would minimize interference with healing of the surrounding tissue and would not necessarily have to be biodegradable. In this respect it would be similar to permanently implanted hardware.
In other aspects, the adhesive complex coacervates and adhesives produced therefrom can be used to secure scaffolds to bone and other tissues such as, for example, cartilage, ligaments, tendons, soft tissues, organs, and synthetic derivatives of these materials. Using the complexes and spot welding techniques described herein, the adhesive complex coacervates and adhesives produced therefrom can be used to position biological scaffolds in a subject. Small adhesive tacks composed of the adhesive complex coacervates described herein would not interfere with migration of cells or transport of small molecules into or out of the scaffold. In certain aspects, the scaffold can contain one or more drugs that facilitate growth or repair of the bone and tissue. In other aspects, the scaffold can include drugs that prevent infection such as, for example, antibiotics. For example, the scaffold can be coated with the drug or, in the alternative, the drug can be incorporated within the scaffold so that the drug elutes from the scaffold over time.
The adhesive complex coacervates and adhesives produced therefrom have numerous dental applications. For example, the adhesive complex coacervates can be used to repair breaks or cracks in teeth, for securing crowns, or seating implants and dentures. Using the spot weld techniques described herein, the adhesive complex coacervate or precursor thereof can be applied to a specific points in the mouth (e.g., jaw, sections of a tooth) followed by attaching the implant to the substrate.
In other aspects, the adhesive complex coacervates and adhesives produced therefrom can adhere a metal substrate to bone. For example, implants made from titanium oxide, stainless steel, or other metals are commonly used to repair fractured bones. The adhesive complex coacervate or a precursor thereof can be applied to the metal substrate, the bone, or both prior to adhering the substrate to the bone. In certain aspects, the crosslinking group present on the polycation or polyanion can form a strong bond with titanium oxide. For example, it has been shown that DOPA can strongly bind to wet titanium oxide surfaces (Lee et al., PNAS 103:12999 (2006)). Thus, in addition to bonding bone fragments, the adhesive complex coacervates described herein can facilitate the bonding of metal substrates to bone, which can facilitate bone repair and recovery.
It is also contemplated that the adhesive complex coacervates and adhesives produced therefrom can encapsulate one or more bioactive agents. The bioactive agents can be any drug that will facilitate bone growth and repair when the complex is applied to the bone. The rate of release can be controlled by the selection of the materials used to prepare the complex as well as the charge of the bioactive agent if the agent is a salt. In certain aspects, when the adhesive complex coacervate is converted to an insoluble solid by a change in temperature and/or pH, the complex coacervate can be administered to a subject and produce an insoluble solid in situ. Thus, in this aspect, the insoluble solid can perform as a localized controlled drug release depot. It may be possible to simultaneously fix tissue and bones as well as deliver bioactive agents to provide greater patient comfort, accelerate bone healing, and/or prevent infections.
The adhesive complex coacervates and adhesives produced therefrom can be used in a variety of other surgical procedures. For example, adhesive complex coacervates and adhesives produced therefrom can be used to repair lacerations caused by trauma or by the surgical procedure itself. In one aspect, the adhesive complex coacervates and adhesives produced therefrom can be used to repair a corneal laceration in a subject. In other aspects, the adhesive complex coacervates and adhesives produced therefrom can be used to inhibit blood flow in a blood vessel of a subject. In general, the adhesive complex coacervate is injected into the vessel followed by converting to an insoluble solid (e.g., heating the complex coacervate) and adhesive to partially or completely block the vessel. This method has numerous applications including hemostasis or the creation of an artificial embolism to inhibit blood flow to a tumor or aneurysm.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
I. Synthesis and Characterization of Adhesive Complex Coacervates
Mimetic Copolymer Synthesis and Characterization.
Pc3 analogs. The dopa analog monomer (dopamine methacrylamide, DMA) was prepared by slight modification of a published procedure. (Lee B P, Huang K, Nunalee F N, Shull K R, Messersmith P B. Synthesis of 3,4-dihydroxyphenylalanine (DOPA) containing monomers and their co-polymerization with PEG-diacrylate to form hydrogels. J Biomater Sci Polym Ed 2004; 15(4):449-464). Briefly, a borate-dopamine complex was reacted at pH>9 with methacryloyl chloride. After disrupting the borate-catechol bond by acidification, the product was washed with ethyl acetate, recrystallized from hexane, and verified by 1H NMR (400 MHz, DMSO-TMS): d8.8-8.58 (2H, (OH)2—Ar—), 7.92 (1H, —C(═O)—NH—), 6.64-6.57 (2H, C6HH2(OH)2—), 6.42 (1H, C6H2H(OH)2—), 5.61 (1H, —C(═O)—C(—CH3)═CHH), 5.30 (1H, —C(═O)—C(—CH3)═CHH), 3.21 (2H, C6H3(OH)2—CH2—CH2(NH)—C(═O)—), 2.55 (2H, C6H3(OH)2—CH2—CH2(NH)—C(═O)—), 1.84 (3H, —C(═O)—C(—CH3)═CH2).
Before polymerization monoacryloxyethyl phosphate (MAEP, Polysciences) was diluted in MeOH and extracted with hexane to remove dienes. Copolymer 1 was prepared by mixing 90 mol % MAEP, 8 mol % DMA, 2 mol % acrylamide (Aam, Polysciences), and 0.1 mol % FITC-methacrylamide in MeOH at a final monomer concentration of 5 wt %. Free radical polymerization was initiated with azobisisobutyronitrile (AIBN) and proceeded at 60° C. for 24 Ins in sealed ampules. A similar procedure was used to make polymers 3-7 as shown in
The MW and polydispersity index (PDI) of 1 were determined by SEC in DMF on a PLgel column (Polymer Labs) connected to a small angle light scattering detector (Brookhaven BI-MWA) and refractive index monitor (Brookhaven BI-DNDC). The column was calibrated with polystyrene standards. The MW of 1 was 245 kda with a PDI of 1.9. The dopamine sidechain concentration and reactivity was verified by UV/VIS spectroscopy (e280=2600 M−1 cm−1). The phosphate sidechain concentration were determined by titration with 0.005 M NaOH using an automated titrator (Brinkmann Titrando 808). The UV/vis spectrum of 1 contained a single absorption peak at 280 nm characteristic of the catechol form of dopamine (
Pc1 analogs. The lysine sidechains of Pc1 were mimicked with N-(3-aminopropyl)methacrylamide hydrochloride (APMA, Polysciences). Copolymer 2 (
Coacervate formation and characterization. A 5 wt % aqueous solution of 2 was added dropwise while stirring to a 5 wt % aqueous solution of 1 until reaching the target amine/phosphate ratio. Total copolymer concentration was 50 mg/ml. After mixing for 30 min the pH was adjusted with NaOH (6M). Compositions at pH (<4) conducive to polyelectrolyte complex (PEC) formation were diluted to 1 mg/ml in DI H2O and the zeta potentials and size distribution of PECs were measured on a Zeta-Sizer 3000HS (Malvern Instruments). At higher pH, coacervated compositions were centrifuged at 2500 rpm in a microfuge (Eppendorf), at 25° C. for 2 min to collect the coacervate phase. The volume of both phases was measured. The coacervate phases were freeze dried and weighed to determine their mass and concentration.
The phase behavior of 1 and 2 mixed at a 1:1 molar ratio of phosphate to amine sidechains (50 mg/ml combined concentration) over the pH range 3-10 is shown in
Extraction of divalent cations with the chelator EDTA resulted in a 50% decrease in compressive strength of P. californica tubes, a ten-fold decrease in adhesiveness, and collapse of the glues porous structure. The effect of divalent cations on the phase behavior of the mimetic polyelectrolytes was investigated by mixing 1 and 2 at amine to phosphate sidechain ratios ranging from 1:1 to 0:1 with divalent cation to phosphate sidechain ratios ranging from 0:1 to 1:1 to create a coacervate phase diagram (
Mechanical bond testing. Bone test specimens, ˜1 cm3, were cut with a band saw from bovine femur cortical bone, obtained from a local grocery store, sanded with 320 grit sandpaper, and stored at −20° C. NaIO4 at a 1:2 molar ratio to dopa sidechains was evenly applied to one face each of two wet bone specimens. Forty ml, a volume sufficient to completely fill the space between 1 cm2 bone interfaces, of the test coacervate solution was applied with a pipette, the bone specimens were pressed together squeezing out a small excess of adhesive, clamped, and immediately wrapped in PBS (20 mM PO4, 150 mM NaCl, pH 7.4) soaked gauze. The applied coacervate contained ascorbate at a 1:5 molar ratio to dopa to prevent premature crosslinking. The bonded specimens were incubated at 37° C. for at least 24 hr in a sealed container containing soaked sponges to maintain 100% humidity. Reference specimens were bonded with 40 ml Loctite 401 superglue in exactly the same manner. A commercial non-medical grade cyanoacrylate was used because there are no hard tissue medical adhesives available for comparison. Mechanical tests were performed on a custom built material testing system using a 1 kg load cell. The instrument was controlled and data acquired using LabView (National Instruments). One bone of a bonded pair was clamped laterally 1 mm from the bond interface. The second bone was pressed with a cross-head speed of 0.02 malts against a dull blade positioned 1 mm lateral to the bond interface. Bond strength tests were performed at room temperature immediately after unwrapping the wet specimens to prevent drying. After testing, the bonds were examined for failure mode. The bonded area was measured by tracing an outline of the bone contact surface on paper, cutting out the trace, and determining its area from the weight of the paper cut-out. At least 6 specimens were tested for each condition.
The shear modulus and strength at failure were measured with bovine cortical bone specimens bonded while wet with the three coacervating compositions marked with an asterisk in
Dopamine-mediated copolymer crosslinking. Addition of NaIO4 to solutions of 3 at a 1:1 molar ratio immediately and quantitatively oxidized DOPA (280 nm) to dopaquinone (392 nm). Within a few minutes the quinine peak decayed into broad general absorption as the reactive quinones formed covalent diDOPA crosslinks (
pH triggered DOPA-mediated crosslinking. To explore, the pH dependence and kinetics of DOPA oxidation, crosslinking of the dopamine copolymers were evaluated by UV-Vis spectroscopy. Results with p(EGMP[92]-DMA[8]) (3) are shown in
The results show that the dopaquinone is stable at low pH and diDOPA crosslinking was inhibited at higher pH in the phosphodopamine copolymers. In the presence of the polyamine, the covalent crosslinking was channeled toward intermolecular amine-DOPA bonds. This is an important observation because it lays out a path to controlled delivery and setting of the synthetic adhesive.
In vitro cytotoxicity. Solutions of 3 and 4, 40 wt % each, were mixed at low pH to form a polyelectrolyte complex. The solution was partially oxidized with NaIO4 and basified with NaOH just before application to sterile glass coverslips. The adhesive-treated coverslips were placed in the bottom of culture plate wells and human foreskin fibroblasts, human tracheal fibroblasts, and rat primary astrocytes in serum containing media were added to separate wells at 30K cells/well (
In the representative images (
Rat calvarial defect model. Production of the fragmented defect and repair with an adhesive complex coacervate is shown in
II. Adhesive Complex Coacervates Produced from an Amine-Modified Polymer
A. Materials and Methods
Materials. Low endotoxin, non-gelling, gelatin (MW 15 kDa) was provided by Gelita Inc. (Souix City, Iowa). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and ethylenediamine dihydrochloride were purchased from Thermo Scientific Inc. Monoacryloxyethyl phosphate (MAEP), 2,2′-azobisisobutyronitrile (AIBN) were purchased from Polysciences, Inc. Sodium periodate (NaIO4), Sephadex LH-20, dopamine hydrochloride was obtained from Sigma-Aldrich.
Polyphosphodopamide synthesis. The polyphosphodopamide copolymer (poly(MAEP85-DMA15)) was synthesized by free radical polymerization of MAEP and dopamine methacrylamide (DMA) using azobisisobutyronitrile (AIBN) as initiator. The copolymer was recovered by size exclusion chromatography (SEC) in MeOH on a Sephadex LH-20 column (Sigma-Aldrich). MeOH was removed, the copolymer resuspended in water, lyophilized, and stored at −80° C. The mol % dopamide side chains in the copolymers were determined by UV/vis spectroscopy: the catechol form of dopamide has an absorption peak at 279 nm (λ279=2600 M−1 cm−1).
Gelatin modification. The general reaction scheme for producing amine-modified gelatin is provided in
Gelatin coacervate formation. A 50 mg/ml aqueous solution of amine-modified gelatin (pH 5.0) was added dropwise while stirring to a 50 mg/ml aqueous solution (pH 5.0) of poly(MAEP85-DOPA15) containing various ratios of divalent cation (Ca2+ Mg2+) until reaching the target amine/phosphate ratio. The pH of the mixture was raised to 7.4 with NaOH. The coacervate phase was allowed to settle for 24 hrs. The coacervate and equilibrium phases were separated and their volumes measured. The coacervate phases were lyophilized and weighed to determine their mass and concentration.
Dynamic theology. The elastic (G′) and storage (G″) moduli were measured with a cone and plate configuration (20 mm diameter, 4° C. cone) on a stress-controlled rheometer (TA Instruments, AR 500). To compare coacervate compositions the measurements were made with a constant frequency of 1 Hz and dynamic strain of 0.1% as the temperature was ramped from 0° C. to 40° C. at a rate of 0.5° C./min.
Adhesive bond strength. Aluminum test adherends, 0.12×0.6×5 cm, were cut from 5052 aluminum sheet (0.050 in) with a water saw. The adherends were polished with 600 grit super fine sandpaper and then cleaned following the procedure of ASTM D2651. Briefly, the adherends were sonicated twice in MeOH, air-dried, dipped into a solution of sulfuric acid and nochromix for 15 mins, then rinsed thoroughly with DI water and stored in DI water until bonded. The adherends were bonded within 12 hr of cleaning. For each adhesive sample, 9 wet aluminum test specimens were bonded. NaIO4 at 1:2 molar ratio to dopamide sidechains was evenly applied to the bond area of two aluminum adherends. The test coacervate solution (6 μl) was applied to wet adherends with a pipette, which were then pressed together with an overlap of about 25 mm, clamped, and immediately submerged in water adjusted to pH 7.4 with NaOH. The bonded specimens cured fully submerged in water for ˜24 hr at the specified temperature. Shear strengths were measured while the adherends where fully submerged in a temperature-controlled water bath mounted on an Instron 3342 materials testing system with a 100 N load cell. The instrument was controlled and data acquired using Bluehill Lite software (Instron, Inc.).
B. Results
An adhesive complex coacervate was created using a low MW (3-5 kda) non-gelling collagen hydrolysate as the polycation. As received the collagen hydrolysate did not form complex coacervates with the phosphodopa copolymer (poly(MAEP)85-co-dopamide15)) at physiological pH. Amination of carboxylic acid sidechains with ethylenediamine increased the amine concentration to ˜16 mol % and shifted the pI from 5.5 to 10.4. The aminated collagen formed dense coacervates at 25° C. over a broad range of compositions. At pH 5, concentrated coacervates formed at amine to phosphate sidechain ratios from 0.5-1.0 and Ca2+ to phosphate ratios up to 0.8 (
Investigation of the separate effect of Mg2+ on coacervation of the polyelectrolytes revealed significant differences compared with Ca2+. At pH 5 the coacervated region was larger. At ratios up to 1:1 Mg2+ to phosphate none of the compositions precipitated (
The phase diagrams in
At 0° C. the coacervated region in
Bonds formed with Ca2+ ratios ranging from 0 to 0.3 with an amine ratio fixed at 0.6 were tested with polished aluminum adherends fully submerged in a temperature controlled water bath at 37° C., well above the transition temperatures of the compositions. The lap shear strength increased with increasing Ca2+ up to a ratio of 0.3 (
Next, oxidative coupling between the polyphosphate dopamide sidechains and the gelatin amines was initiated by adding 0.5 equivalents NaIO4 relative to the dopamide sidechains during the bonding procedure in order to investigate the contribution of covalent crosslinking to bond strength of the synthetic adhesive. The bonds were cured and tested at 37° C. while fully submerged in water adjusted to pH 7.4. The bonds strengths increased with increasing divalent cation ratio for both Ca2+ and Mg2+ (
In conclusion, the adhesive complex coacervates were dense, partially water-immiscible fluids precariously balanced between soluble polymers and insoluble polymeric salts (see white arrow in
III. Preparation of Photocrosslinkable Polymers
Synthesis of methacrylate-grafted polyphosphate (
Synthesis of methacrylate-grafted polyamine (
The methacrylate terpolymers was dissolved in DMF then, relative to the free amine group, 2.1 equivalents of triethylamine followed by 1 equivalent of methacryloyl chloride was added. The reaction was stirred for 30 min. The t-BOC group was removed by adding 5 equivalents of TFA. The deprotected terpolymer was precipitated with diethyl ether, resuspended in DI water and lyophilized. The degree of methacrylolyl substitution was calculated by 1H NMR using the ratio of the vinyl proton signals to ethyl and propyl proton signals.
Photocrosslinking (
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the compounds, compositions and methods described herein.
Various modifications and variations can be made to the compounds, compositions and methods described herein. Other aspects of the compounds, compositions and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions and methods disclosed herein. It is intended that the specification and examples be considered as exemplary.
This application is a continuation of U.S. patent application Ser. No. 12/508,280, filed Jul. 23, 2009 (now U.S. Pat. No. 8,283,384, issued Oct. 9, 2012), which is a continuation-in-part of International Application No. PCT/US2008/083311, filed Nov. 13, 2008, which claims the benefit of U.S. Provisional Application No. 61/023,173, filed Jan. 24, 2008, all of which are incorporated herein by reference.
This invention was made with government support under EB005288 awarded by the National Institutes of Health. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20130189313 A1 | Jul 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61023173 | Jan 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12508280 | Jul 2009 | US |
| Child | 13617882 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2008/083311 | Nov 2008 | US |
| Child | 12508280 | US |
