Patent Application
20070190084
The present disclosure generally relates to polymer compositions, methods of forming such polymer compositions, and methods of using such compositions. These compositions have improved properties that make them useful for a variety of applications; in particular, the loading, transport, and delivery of therapeutic agents.
Recognition in nature is a complex orchestration of numerous interactions between individual atoms and cumulative interactions between secondary structures. For example, the active sites of enzymes are composed of several amino acid residues, which noncovalently bind ligand molecules in a very specific manner. However, the activity of these sites is dependent on the stabilization of the three-dimensional structure by the interactions of hundreds of other residues within the structure of secondary and tertiary domains.
The term configurational biomimesis refers to the three-dimensional arrangement of chemical groups that can specifically bind a biomolecule via noncovalent forces. This designed recognition involves analyzing the molecular basis of recognition in biological systems and attempts to mimic similar interactions on a molecular level. For example, analysis of biological systems such as enzyme-substrate, receptor-ligand, antibody-antigen, complementary DNA or RNA strands and protein-protein complexes, nucleic acid-protein recognition, aptamers, and RNA aptamers, can yield much information on the type, number, and arrangement of noncovalent chemical forces needed for aqueous recognition.
Configurational biomimesis is, therefore, a subset of molecular imprinting, which produces precise polymer architectures that can selectively recognize molecules and at times, depending on the matrix structure, differentiate with isomeric specificity.
The concept of molecular imprinting manifests itself from two major synergistic effects, (i) shape-specific cavities or microvacuoles or mesovacuoles or nanovacuoles that match the template molecule and (ii) chemical groups orientated to form multiple complexation points with the template molecule. In terms of selectivity, the resulting polymer networks are selective due to the particular chemistry of the binding site, the orientation of the chemistry, as well as by the size and shape of the site for the template molecule.
The quality of the receptor mechanism of imprinted polymers can be assessed via a number of parameters. The significant parameters in determining how well a polymeric network can recognize a given molecule are binding affinity (i.e., the equilibrium association or dissociation constant between the ligand molecule and the network), selectivity (i.e., the ability to differentiate between the ligand and other molecules), and the binding capacity (i.e., the maximum ligand bound per mass or volume of polymer). To a lesser extent, binding or imprinting ratios (i.e., the ratio of recognitive network template bound compared to control network) highlights the recognition properties at a specific concentration.
Binding affinity is a measure of how well the template molecule is attracted to the binding site or how well a ligand binds or is held to the receptor macromolecule. Considering equilibrium theory of receptor-ligand interactions, the dissociation constant, Kd, provides a quantitative measure of this level of attraction.
According to one embodiment, the present disclosure provides biomimetic polymer networks comprising a heteropolymer network having a cavity, the cavity having a selective affinity for a moiety.
According to another embodiment, the present disclosure provides biomimetic polymer networks formed by a process comprising polymerizing a mixture comprising monomers and crosslinkers in the presence of a moiety or a target molecule or both for which a molecular imprint is to be produced, thereby forming a matrix comprising an imprint of the moiety or target molecule or both, and separating the moiety from the matrix.
According to another embodiment, the present disclosure provides methods for forming biomimetic polymer networks comprising a polymerizing mixture comprising monomers and crosslinkers in the presence of a moiety for which a molecular imprint is to be produced, thereby forming a matrix comprising an imprint of the molecule; and separating the moiety from the matrix.
According to another embodiment, the present disclosure provides biomimetic polymer networks formed by a process of reaction injection molding or casting or extruding a polymerizing mixture comprising monomers, crosslinkers, and one or more moieties, the crosslinkers added at times such that the solidification and crosslinking will occur during the molding or casting or extruding; allowing a matrix to form which has a plurality of molecular imprints; and separating the moiety from the matrix.
According to another embodiment, the present disclosure provides methods for forming biomimetic polymer networks comprising a heteropolymer network having a cavity, the cavity having a selective affinity for a moiety; loading the biomimetic polymer network with a molecule by allowing a moiety present on the molecule to interact with the cavity; delivering the biomimetic polymer network to a desired location; and providing conditions that reduce the affinity of the cavity for the moiety a sufficient amount to release the molecule.
The features and advantages of the present disclosure will be readily apparent to those skilled in the art upon a reading of the description of the embodiments that follows.
Some specific example embodiments of this disclosure may be understood by referring, in part, to the following description and the accompanying drawings.
While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.
The present disclosure generally relates to biomimetic polymer network compositions, methods of forming such polymer compositions, and methods of using such compositions. These compositions and have improved properties that make them useful for a variety of applications; in particular, the loading and delivery of therapeutic agents.
The biomimetic polymer networks of the present disclosure generally comprise a polymer network having architectures that have selective affinity for a moiety. Such biomimetic polymer networks may have shape specific cavities that match the moiety, as well as chemical groups oriented to form multiple complexation points with the moiety. In terms of selectivity, the resulting polymer networks are selective due to the particular chemistry of the binding site, the orientation, and stabilization of the chemistry in a crosslinked matrix, as well as by the size and shape of the site for the template biomolecule.
In some embodiments, the biomimetic polymer networks may further comprise a moiety. This moiety acts as a molecular decoy that can be recognized by the polymer network. Such compositions may be capable of releasing the moiety in a relatively controlled fashion. The moiety may be present on a target compound, for example, a therapeutic agent. Accordingly, the compositions and methods of the present disclosure may be used in the treatment of a disease. For example, the compositions of the present disclosure may be used as a vehicle to deliver a therapeutic agent to a subject (e.g., a human) in need thereof. The compositions of the present disclosure also may be used to form a medical device or an article. The present disclosure also provides methods of forming a biomimetic polymer network of the present disclosure.
The moiety may be any portion of a molecule recognized by a biomimetic polymer network of the present disclosure, as well as a molecule acting as a molecular decoy. The moiety may be covalently bound to a target compound, for example, a therapeutic agent. In this way, the moiety may be used to associate a target compound with a biomimetic polymer network of the present disclosure. The moiety should either already be present on the target compound or capable of being conjugated to a target compound. Conjugation of moieties to target compounds (e.g., therapeutic agents) is known in the art, for example, as disclosed in A. Wong and I. Toth, Curr. Med. Chem. 8:1123-36 (2001), the relevant disclosure of which is incorporated by reference. The conjugation of moieties to target compounds will depend upon the particular moiety and target, and will be apparent to those of skill in the art. For example, a hydrophilic peptide moiety may be conjugated to a hydrophobic peptide target compound by their respective N- and C-termini to form an amide linkage. Alternatively, a linker may be used. Linkers can be any molecule used by those of skill in the art to link two other molecules. Other suitable linkers include bifunctional reagents, which are known to those of skill in the art and are commercially available. The linker molecule can form a covalent linkage, or the linkage can be non-covalent. The actual choice of linker molecule will depend upon, among other things, the nature of the moiety and/or the target compound, and will be apparent to those of skill in the art. The linkage between the moiety and the target compound may be optionally cleavable. Cleavable linkages are known to those of skill in the art and include linkages that can be cleaved with chemicals, enzymes, electromagnetic radiation, and the like.
Examples of suitable moieties include, but are not limited, to sugars (e.g., glucose), lipids, carbohydrates, peptides, proteins, glycoproteins, proteoglycans, lipoproteins, a portion thereof, organic molecules, small molecules, and functional groups. A specific example of a therapeutic agent that comprises a moiety is streptozotocin (R. R. Herr, et al., J. Am. Chem. Soc. 89:4808-09 (1967)), which has a glucose moiety. Another example is thiazepine (diltiazem) containing a glucose moiety in the 4 position.
In certain embodiments, the moiety is a sugar. For example, the sugar may be a monosaccharide. Monosaccharides have the chemical formula (CH2O)n and the chemical structure H(CHOH)nC=O(CHOH)mH. If n or m is zero, it is an aldose, otherwise it is a ketose. Monosaccharides may include aldoses, trioses (e.g., glyceraldehyde), tetroses (e.g., threose), pentoses (e.g., ribose, xylose), hexoses (e.g. glucose, fructose, mannose, galactose), ketoses, trioses, tetroses, pentoses (e.g., ribulose), hexoses (e.g., fructose). Any of the L and D isomers of a sugar also may be used, although the D isomer may be more preferred for biological applications. Other examples of suitable sugars include polysaccharides. Polysaccharides have a general formula of Cn(H2O)n−1 where n is usually a large number up to 500. Disaccharides, such as, for example, sucrose, lactose, maltose, and the like may be used. Yet another example of suitable sugars includes oligosaccharides and low molecular weight carbohydrates (e.g., having a molecular weight no greater than about 2,000 Da). Further, each carbon atom that supports a —OH group (except for the first and last) is chiral, giving rise to a number of isomeric forms all with the same chemical formula.
Specific embodiments may use the following monosaccharides as moieties: monoses; dioses; trioses; tetroses; pentoses; aldo-pentoses, including arabinose, lyxose, ribose, deoxyribose and xylose; keto-pentoses, including ribulose and xylulose; hexoses, including aldo-hexoses, such as allose, altrose, galactose, glucose, gulose, idose, mannose, and talose, and keto-hexoses, such as fructose, psicose, sorbose, and tagatose; heptoses, including keto-heptoses, such as mannoheptulose and sedoheptulose; octoses, such as octolose, 2-keto-3-deoxy-manno-octonate; and nonoses, such as sialose.
Specific embodiments may use mucopolysaccharides. Mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. This unit consists of an N-acetyl-hexosamine and a hexose or hexuronic acid, either or both of which may be sulfated. Members of this family vary in the type of hexosamine, hexose, or hexuronic acid unit they contain, e.g., glucuronic acid, iduronic acid, galactose, galactosamine, and glucosamine. They also vary in the geometry of the glycosidic linkage. Specific example polysaccharides that may be used as moieties include: chondroitin sulphate; dermatan sulphate; keratan sulphate; heparan sulphate; heparin; sodium heparin; hyaluronic acid; and hyaluronan.
In other embodiments, the moiety may be a short amino acid sequence (e.g., a sequence of about twenty amino acids in length), including non-natural amino acids (e.g., amino acid mimetics). One example of a short amino acid moiety is a 6-His tag, which could be used to both purify a protein expressing the 6-His tag and also as a moiety for imprinting. Another example of a moiety are lectins. Lectins are carbohydrate-binding proteins involved in a variety of recognition processes and exhibit considerable structural diversity. A large variability in quaternary association resulting from small alterations in essentially the same tertiary structure is a property exhibited specially by legume lectins. The strategies used by lectins to generate carbohydrate specificity include the extensive use of water bridges, post-translational modification, and oligomerization. Other carbohydrate-based structures may be used as moieties may be located at http://www.chem.qmul.ac.uk/iupac/2carb/ (accessed Apr. 27, 2006), incorporated by reference herein.
In other embodiments, the moiety may be a functional group, such as a benzene derivative and imidizoles. Many drug molecules contain moieties that are benzene derivatives and thus, benzene derivatives are also excellent candidates as moieties. For example, benzene derivatives that could be utilized as moieties include phenols (e.g., phenol, p-cresol, m-cresol, o-cresol, hydroquinone, resorcinol, catechol, and the like), chlorophenols (e.g., o-chlorophenol, m-chlorophenol, p-chlorophenol, and the like), nitrophenols (e.g., o-nitrophenol, m-nitrophenol, p-nitrophenol), quinines (e.g., o-quinone, p-quinone, and the like), and fluorobenzenes (e.g., fluorobenzene, o-difluorobenzene, m-difluorobenzene, p-difluorobenzene, and the like. Like benzene derivatives, many drugs contain an imidazole ring, such as antifungal drugs and nitroimidazole. Imidizoles also are present in important biological building blocks such as histidine and histamine.
In general the compositions of the present disclosure have enhanced affinities (e.g., impart greater affinity, bound ratios greater than 1) for a chosen moiety, among other things, allowing for increased loading efficiency. Accordingly, the compositions of the present disclosure also may be used to increase the loading of a target compound or control the release rate of a target compound or both. The compositions of the present disclosure also may be used for delivery of a therapeutic agent. For example, the compositions of the present disclosure may be used as an excipient or as a vehicle for a therapeutic agent. Specifically, higher quantities of a therapeutic agent having a moiety can be loaded within the biomimetic polymer networks of the present disclosure, therefore enabling for higher doses to be loaded. The release of a moiety may be tailored to give a desired release profile, for example, for sustained release of a therapeutic agent. Thus, when the moiety is bound to a therapeutic agent, treatment with the therapeutic agent may be optimized.
Example therapeutic agents include water soluble or poorly soluble drug of molecular weigh from 40 to 1,100 including the following: Hydrocodone, Lexapro, Vicodin, Effexor, Paxil, Wellbutrin, Bextra, Neurontin, Lipitor, Percocet, Oxycodone, Valium, Naproxen, Tramadol, Ambien, Oxycontin, Celebrex, Prednisone, Celexa, Ultracet, Protonix, Soma, Atenolol, Lisinopril, Lortab, Darvocet, Cipro, Levaquin, Ativan, Nexium, Cyclobenzaprine, Ultram, Alprazolam, Trazodone, Norvasc, Biaxin, Codeine, Clonazepam, Toprol, Zithromax, Diovan, Skelaxin, Klonopin, Lorazepam, Depakote, Diazepam, Albuterol, Topamax, Seroquel, Amoxicillin, Ritalin, Methadone, Augmentin, Zetia, Cephalexin, Prevacid, Flexeril, Synthroid, Promethazine, Phentermine, Metformin, Doxycycline, Aspirin, Remeron, Metoprolol, Amitriptyline, Advair, Ibuprofen, Hydrochlorothiazide, Crestor, Acetaminophen, Concerta, Clonidine, Norco, Elavil, Abilify, Risperdal, Mobic, Ranitidine, Lasix, Fluoxetine, Coumadin, Diclofenac, Hydroxyzine, Phenergan, Lamictal, Verapamil, Guaifenesin, Aciphex, Furosemide, Entex, Metronidazole, Carisoprodol, Propoxyphene, Digoxin, Zanaflex, Clindamycin, Trileptal, Buspar, Keflex, Bactrim, Dilantin, Flomax, Benicar, Baclofen, Endocet, Avelox, Lotrel, Inderal, Provigil, Zantac, Fentanyl, Premarin, Penicillin, Claritin, Reglan, Enalapril, Tricor, Methotrexate, Pravachol, Amiodarone, Zelnorm, Erythromycin, Tegretol, Omeprazole, and Meclizine.
The compositions of the present disclosure may be formed using configurational biomimetic imprinting (
The network structure depends upon the type of monomer chemistry (i.e., anionic, cationic, neutral, amphiphilic), the association strength and number of interactions between the monomers and template molecule, the association interactions between monomers and pendent groups, the solvent type and the amount of solvent in the mixture, the reactivity ratios of the monomers, and the relative amounts of reacted monomer species in the structure. Since noncovalent forces are weaker than covalent bonds, an increased number of interactions are needed for stable binding and recognition. On a per-bond basis, noncovalent bonds are 1-3 orders of magnitude weaker. Therefore, a greater number of noncovalent bonding with matching structural orientation is needed for aqueous recognition.
In certain embodiments, the heteropolymer network may be a hydrogel. Hydrogels are mainly hydrophilic polymer networks that swell to a high degree due to a high affinity for water, yet are insoluble because of the incorporation of chemical or physical crosslinks. Of note, hydrogels can absorb up to two thousand times their dry weight in water. Due to this high water content and the corresponding rubbery nature, hydrogels are similar to a variety of natural living tissues. This has led to widespread application of hydrogels as biomaterials, such as in contact lenses, sutures, dental materials, and controlled drug delivery devices. See Peppas, N. A., Hydrogels in Medicine and Pharmacy, CRC Press, Boca Raton, Fla., 1986; Hoffinan, A. S., Hydrogels for biomedical applications. Adv. Drug Deliv. Revs., 54, 3-12 (2002).
By tailoring the functional groups along the polymer chains, hydrogels can be made sensitive to the conditions of the surrounding environment, such as temperature, pH, electric field, or ionic strength. See Peppas, N. A., Physiologically responsive gels. J. Bioact. Compat. Polym., 6, 241-246 (1991). Such hydrogels may be referred to as “environmentally sensitive.”
Environmentally sensitive hydrogels may be advantageously used according to the present disclosure. For example, a pH-sensitive hydrogels may be used. These pH-sensitive hydrogels are usually based on ionic networks. Anionic networks contain acidic pendant groups, such as carboxylic acid, with a characteristic pKa, while cationic networks contain basic pendant groups, such as amine groups, with a characteristic pKb. In the case of anionic networks, ionization of these acid groups will occur once the pH of the environment is above the acid group's characteristic pKa. With deprotonation of the acid groups, the network will have fixed charges on its chains resulting in an electrostatic repulsion between the chains and, in addition, an increased hydrophilicity of the network. Because of these alterations in the network, water is absorbed into the polymer to a greater degree causing swelling. pH-sensitive hydrogels are made in part from hydrophilic monomers that incorporate separable proton species into the polymer mass. These protons establish, with an aqueous solution, an equilibrium depending on the acidity of the solution. Suitable hydrophilic monomers include unsaturated organic acid monomers, more particularly carboxylic acid monomers such as methacrylic and acrylic acids. Other suitable monomers include glycerolacrylate and glycerolmethacrylate, and acrylic substituted alcohols such as 2-hydroxyethyl methacrylate and 2-hydroxyethyl acrylate. A related utilizable polymer is formed from 2-(dimethylaminoethyl methacrylate (DMAEMA), which introduces an amine group onto the polymer backbone. Here, a lightly or moderately crosslinked poly(HEMA-co-DMAEMA) could be used since it swells at low pH when the amine group is protonated. Further suitable monomers include N-vinyl pyrrolidone and acrylamides such as methacrylamide and N,N-dimethylacrylamide, among numerous others, which will be apparent to those skill in the art.
Another example of an environmentally sensitive hydrogel is a temperature-sensitive or thermosensitive hydrogel. Such hydrogels may volumetrically expand in response to a temperature change. Thus, the degree of expansion or contraction of the hydrogel is dependent on temperature. Temperature sensitive hydrogels are classified as either positive or negative temperature-sensitive systems, depending on whether they are contracted below or above a critical temperature, respectively. Examples of thermosensitive hydrogels include poly(N-isopropyl acrylamide) (PNIAAm), which is a negative temperature-sensitive hydrogel exhibiting a phase transition around 33° C. More generally, a temperature sensitive water absorbing and discharging polymer composition having a predetermined selected temperature sensing point, which controls water absorbability, is obtained by polymerizing in an aqueous solution, N-alkyl acrylamide derivatives with acrylic acid, alkali metal salts of acrylic acid, or mixtures thereof, and diacetone acrylamide. As disclosed in U.S. Pat. No. 5,672,656, the disclosure of which is incorporated by reference, the temperature sensitive water absorbing and water discharging polymer composition may be modified to control the temperature sensing point and has a high water discharging and absorbing property above and below the temperature sensing point.
Another example of an environmentally sensitive hydrogel is an analyte-sensitive hydrogel. These hydrogels contains a chemical component that is in equilibrium with the same chemical component (analyte) in solution or that react with another chemical species (analyte) in solution. Where a hydrogel layer is in contact with a liquid, the presence of a target analyte in the liquid or, more generally, a change in the concentration of the target analyte in the liquid, induces a conformation change of the hydrogel layer. One method is to immobilize into pH-sensitive hydrogel networks enzymes that act on a specific analyte. In one study, activated glucose oxidase was included in pH-sensitive cationic hydrogels. See Podual, K., F. J. Doyle III, and N. A. Peppas. Glucose-sensitivity of glucose oxidase-containing cationic copolymer hydrogels having poly(ethylene glycol) grafts. J. Control Rel., 67, 9-17 (2000). The glucose oxidase converts glucose into gluconic acid lowering the pH of the local environment, which then causes the hydrogel network to swell in the case of a cationic gel. Another approach to impart analyte specificity, which is based on competitive binding, is to load a hydrogel containing the analyte in pendant groups with a corresponding entity that selectively binds the analyte. The entity will bind the pendent analytes and form cross-links in the network, which will then be broken with the competitive binding that takes place in the presence of the analyte. See Lee A. J. and K. Park. Synthesis and characterization of sol-gel phase-reversible hydrogels sensitive to glucose. J. Mol. Recognit., 9, 549-557 (1996).
A wide variety of polymers may be used to form the heteropolymer network. These include polymers produced by reaction of acrylamides and all their substituted structures including: methacrylamide, ethacrylamide, isopropyl acrylamide, acrylic acid, methacrylic acid, ethacrylic acid, all alkyl acrylic acids, any dicarboxylic acid, such as crotonic acid, phthalic and terephthalic acid any tricarboxylic acid with itself another monomer of the above list (forming a copolymer), two other monomers from the above list (forming terpolymers), or three or more monomers from the above list forming higher order coploymers. The above may be in linear, branched, or grafted form, the grafted chains being exclusively one polymer or copolymers of the above, ionically bound, or complexed by hydrogen bonds.
The above may be crosslinked in the presence of crosslinking agents to form insoluble but swellable gels or networks, having the ability to absorb water, physiological fluids, buffers, or salt solutions with final swelling as low as 1 weight % of water and as high as 99.9% water.
The above crosslinking may be achieved with ethylene glycol dimethacrylate, ethylene glycol diacrylate, ethylene glycol trimethacrylate, ethylene glycol diacrylate, ethylene glycol multi methacrylate where “multi” stands for n=4 to 200 units ethylene glycol, multi acrylate where “multi” stands for n=4 to 200 units, same as above but propylene glycol multi methacrylate where “multi” stands for n=1 to 200 units, same as above but alkylene glycol multi methacrylate where “multi” stands for n=1 to 200 units. One may also use higher order acrylates and methacrylates including but not limited to 1,1,1 trimethylolethane trimethacrylate (TrMETrMA, Molecular Weight 324.4); 1,1,1 trimethylolpropane triacrylate (TrMPTrA, Molecular Weight 296.3); 1,1,1 trimethylolpropane trimethacrylate (TrMPTrMA, Molecular Weight 338.4); pentaerythritol triacrylate (PETrA, Molecular Weight 298.3); glycerol propoxy triacrylate (GlyPTrA, Molecular Weight 428.5); pentaerythritol tetraacrylate (PETeA, Molecular Weight 353.2); ethoxylated 1,1,1 trimethylolpropane triacrylate (ETrMPTrA, Molecular Weight 428); glycerol propoxylated triacrylate (GlyPTrA, Molecular Weight 428) and glycerol trimethacrylate (GlyTrMA, Molecular Weight 396.3). One may also use “star polymers” or “dendrimers” with up to 72 independent chains ending in acrylates or methacrylates.
The initiator may be Irgacure products of the Ciba Geigy company including Irgacure 184, IRGACURE® 379, Ciba® IRGACURE® 819, and Ciba® IRGACURE® 250. Any other photoinitiator may also be used. The initiator may also be any peroxide including but not limited to benzoyl peroxide, cumyl peroxide, and the like, or Azobis isobutyronitrile.
In some embodiments, the biomimetic polymer network of the present disclosure may be formed using a template molecule (e.g., D-glucose) and functional monomers selected to match corresponding template molecules (e.g., glucose binding protein residues, such as aspartate, glutamate, and asparagine). Template monomers may also be selected to match corresponding biological mechanisms of action that involve recognition. The template molecule may be added in stoichiometric amounts in regard to the functionality of the template molecule. Since solvent interaction can stabilize or destabilize binding in noncovalent systems, functional monomers may be selected based on optimizing specific noncovalent, self-assembly interactions (hydrogen bonding) with the template molecule within an aprotic solvent (e.g., dimethylsulfoxide). Such techniques are generally applicable to template molecules, in which hydrogen bonding, hydrophobic, or ionic contributions will direct recognition of the moiety.
To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. In no way should the following examples be read to limit or define the entire scope of the disclosure.
A model biomimetic polymer network of the present disclosure capable of recognizing and binding D-glucose was formed and tested. This model biomimetic polymer network illustrates, among other things, the applicability of the biomimetic polymer networks of the present disclosure for the sustained release of a target compound.
The moiety used was a D-glucose fluorescent analogue, 2-NBDG, which also models the moiety attached to a target compound. As 2-NBDG is a small molecule, it also serves as an example of a small molecule moiety.
The model biomimetic polymer network was synthesized via biomimetic molecular imprinting techniques using noncovalent complexation interactions, and fluorescent microscopy was utilized as a novel method to characterize the kinetic and equilibrium binding properties of the polymer systems. Specifically, networks based on acrylamide were prepared and characterized using a novel fluorescent microscopy technique, which allowed for microscale observation of the binding and for the direct observation of the analyte uptake within the polymer film. The equilibrium binding characteristics and the kinetic binding and release characteristics of the fluorescent glucose analogue were analyzed.
The relative binding affinity and amount of fluorescent analogue, 2-NBDG, bound within the network was demonstrated to be controlled by the structure and properties of the polymer network. In general, the more rigid networks (shorter crosslinkers and high crosslinking percentages) exhibited higher affinities for 2-NBDG. As crosslinking percentage of the network decreased, the macromolecular recognition of the network chains decreased. Also, as the length of the crosslinker increased, the bound ratio decreased for a given concentration, indicating less imparted affinity. The relative amount of PEG or Aam within the polymer networks did not have a discernible effect on the binding affinity. In addition, 2-NBDG was a model compound to illustrate the applicability of imprinted polymers in novel drug loading and release applications.
D-Glucose Recognitive Network Synthesis. Acrylamide (Aam), 2,2-dimethoxy-2-phenyl acetophenone (DMPA), dimethylsulfoxide (DMSO), ethylene glycol dimethacrylate (EGDMA), and D-glucose, were purchased from Aldrich (Milwaukee, Wis.). PEGnDMA, with n=200, 400, and 600, was obtained from Polysciences, Inc. (Warrington, Pa.). Irgacure® 184, 1-hydroxycyclohexyl phenyl ketone, was purchased from Ciba Specialty Chemicals (Tarrytown, N.Y.). Fluorescent D-glucose analogue, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-glucose (2-NBDG), was purchased from Molecular Probes, Inc. (Eugene, Oreg.).
Novel heteropolymer films of differing composition and percentage of crosslinking monomer were synthesized in an aprotic, polar solvent via UV free-radical polymerization in a nitrogen atmosphere. In a typical experiment (e.g. PEG200DMA as crosslinking monomer), 15 mmoles of Aam and 60, 30, or 10 mmoles of PEG200DMA (e.g., 80, 67, or 30% mole crosslinking monomer/mole all monomers, respectively) were allowed to complex with 3 mmoles of D-glucose mixed with 6 mL DMSO. After mixing and checking mutual solubilities, Irgacure® 184 initiator was added in the amount of 1-2 wt %. Control polymers were made with exactly the same composition except D-glucose was not added.
After preparation, the solution was placed in a nitrogen atmosphere and nitrogen was bubbled for 30 minutes to remove oxygen, which is a free-radical scavenger and inhibits the free-radical polymerization. The monomer mixtures were pipetted between two clamped 6″ by 6″ glass plates with a Teflon® spacer that was 790 microns thick. Next, the glass plate assembly was placed under a UV source (Dymax Ultraviolet Flood Cure System) and exposed to UV light with an intensity of 10.0-15.0 mW/cm2 for 15 minutes to initiate the free-radical polymerization. Polymers were placed in deionized water immediately after preparation, were carefully separated from the slides, and then were cut into various diameter discs using a cork borer. Discs were then placed in 50 mL centrifuge tubes and placed on a rotating mixer (25 RPM, 70 degree angle, Glas-Col, Terre Haute, Ind.) and resuspended within multiple 24 hour wash steps to remove glucose and excess monomer. The resulting discs were then dried in air at ambient conditions and placed in a vacuum oven (T=26° C., 28 mm Hg vacuum) until a constant weight was obtained (less than 0.1 wt % difference). The discs were then stored in a dessicator until testing. ,
Analysis of Equilibrium Binding via Fluorescent Microscopy. Equilibrium binding experiments were conducted to examine the relative equilibrium binding affinity and bound amount of D-glucose within the networks. These studies were conducted utilizing a fluorescent D-glucose analogue, which allowed for microscale observation of the binding and for the direct observation of the uptake within the film. For the characterization of the binding isotherm, a dry polymer disc of known mass was placed in known concentrations of 2-NBDG (1×10-3, 5×10-4, 1×10-4, and 1×10-5 mg/ml) in deionized water. The amount of bound glucose was determined by fluorescent microscopy of the polymer films, and the equilibrium solution concentration was determined by measurement of the resulting supernatant via fluorescent microplate reader (Bio-Tek Instruments, HT Multi-Detection Microplate Reader, Winooski, Vt.).
Equilibrium binding behavior, qualitative and quantitative, was probed by fluorescent microscopy. By analyzing fluorescent intensity values from polymer discs of equal thickness, quantitative analysis of relative amount bound in network can be made. In using fluorescent methods for quantitative analysis, it is critical that all experimental parameters are matched during analysis (objective and field of view, camera integration time, etc.) including excitation times since fluorophore bleaching could drastically alter intensity profiles.
Binding results in water were visualized using 2-NBDG (maximum absorption 466 nm; maximum emission 542 nm) (
A Nikon Eclipse ME600L fluorescent microscope with a FITC filter set was used and images were acquired with Coolsnap digital camera. Meta-View software from Universal Imaging was utilized to analyze a large amount of pixels within these images for calculation of an average fluorescent intensity and standard deviation across the image.
Results and Discussion of Equilibrium Binding Efficiency. 2-NBDG is suitable for analyzing network binding properties. The biomimetic polymer systems studied examined and their identifiers are listed in Table 1.
The fluorescent analogue molecule, 2-NBDG, was also applied to determine relative binding affinity for polymer networks with varying crosslinking percent (30, 67, and 80%) and crosslinker length (PEGnDMA, with n of 44, 200, and 600). The relative polymer fluorescent intensity due to the binding of 2-NBDG can be used to determine quantitative binding ratios that are a valuable measure to the affinity of a network for an analyte. In these studies, the 2-NBDG concentration was 1×10-4 mg/ml (2.9×10-4 mM).
In
Analysis of Kinetic Binding and Release via Fluorescent Microscopy. Kinetic binding and release experiments were conducted to examine the relative rates of uptake and release from the polymer networks. The effect of the network structure on the diffusion coefficient of the D-glucose fluorescent analogue, 2-NBDG, was examined. By analyzing fluorescent intensity values from polymer discs of equal thickness, quantitative analysis of relative amount bound in network can be made. An aliquot of known concentration of 2-NBDG (1×10-4 mg/ml (2.9×10-4 mM)) was added to washed, solution-swollen polymer discs (preparation described in section 8.2.1) in 50 mL of solution within centrifuge tubes. The tubes were covered with aluminum foil and placed on a rotating mixer (Glas-Col., Terre Haute, Ind.; 70 degree angle, 25 RPM). At various time points, kinetic analysis of the binding was carried out. For the release studies, polymer films that had reached equilibrium binding were placed in 50 ml centrifuge tubes filled with DI water. The water was frequently replaced with fresh DI water, allowing for an infinite sink condition to be assumed. Analysis of fluorescent intensity values from polymer discs of equal thickness provided quantitative analysis of binding and release.
A Nikon Eclipse ME600L fluorescent microscope with a FITC filter set was used and images were acquired with Coolsnap digital camera. Meta-View software from Universal Imaging was utilized to analyze a large amount of pixels within these images for calculation of an average fluorescent intensity and standard deviation across the image.
Results and Discussion of Kinetic Binding and Release. In
For the 67% systems, the power law fit (equation 5.18) and early time fit for Fickian diffusion in a slab geometry (equation 5.24) were applied to determine the power law exponent, n, and the diffusion constant of 2-NBDG in these polymer networks with varying crosslinker length, and these values are included in Table 2. The diffusion analysis for a solute uptake is analogous to that of the solvent uptake. All systems exhibited n values of approximately 0.5, and therefore, can be described by Fickian diffusion. The calculated diffusion constants for the 2-NBDG decreased with decreasing crosslinker length, with the diffusion constant of PEG600DMA network being 5 times larger than for the EGDMA network. For the networks crosslinked with EGDMA and PEG200DMA, the 2-NBDG diffusion constant of the control networks were approximately half of the imprinted networks. This enhancement of the diffusion constants in the imprinted networks is caused by the increased porosity resulting from polymerization in the presence of a template molecule, which acts as a porogen to certain degree. This effect is not observed in the PEG600DMA networks.
In
Biomimetic polymer networks capable of recognizing and binding the moiety D-glucose attached a thiazepine (diltiazem) containing a glucoside moiety in the 4 position was formed and tested.
Thiazepine containing a glucoside moiety in the 4 position was prepared and used for imprinting. The model biomimetic polymer network was synthesized via biomimetic molecular imprinting techniques using noncovalent complexation interactions. Gels based on acrylamide were prepared and characterized. The equilibrium binding characteristics and the kinetic binding and release characteristics of the fluorescent glucose analogue were analyzed.
Acrylamide (Aam), 2,2-dimethoxy-2-phenyl acetophenone (DMPA), dimethylsulfoxide (DMSO), ethylene glycol dimethacrylate (EGDMA), and thiazepine (ditiazem) containing a glucoside moiety in the 4 position, were used with PEGnDMA, with n=600 as a crosslinking agent and IRGACURE® 184, 1-hydroxycyclohexyl phenyl ketone as an initiator. Novel heteropolymer recognitive films were synthesized DMSO via UV free-radical polymerization in a nitrogen atmosphere. In a typical experiment 21 mmoles of Aam and 40 mmoles of PEG200DMA were allowed to complex with 4 mmoles of glucoside containing thiazepine mixed with 8.3 mL DMSO. After mixing, Irgacure® 184 initiator was added in the amount of 2.6 wt %. Two control polymers were made with exactly the same composition except D-glucose was added in the first, and no glucose was present in the second.
The solution was placed in a nitrogen atmosphere and nitrogen was bubbled for 60 minutes to remove oxygen, the monomer mixtures were pipetted between two clamped 6 by 6 inch glass plates with a Teflon® spacer and the glass plate assembly was placed under a UV source and exposed to UV light with an intensity of 12.0 mW/cm2 for 20 minutes to initiate the free-radical polymerization. Polymers were then were cut into various diameter discs which were then placed in 50 mL centrifuge tubes and placed on a rotating mixer and resuspended within multiple 24 hour wash steps to remove he template and excess monomer. The resulting discs were then dried in air at ambient conditions and stored in a dessicator until testing.
Equilibrium recognitive experiments were conducted to examine the relative equilibrium binding affinity and bound amount of thiazepine within the networks. Binding was determined in water. The relative binding affinity and amount of the glucoside containing thiazepine was demonstrated to be controlled by the structure and properties of the polymer network.
Since many classes of drugs have a certain degree of structural homology depending on mechanism of action, the approach described in the present disclosure yields a generalize scheme for glucose moiety containing drugs. For example, the anti-cancer drug streptozotocin has a glucose moiety as a major component of its structure. Streptozotocin or deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranose is approved by the U.S. FDA for treating metastatic cancer of the pancreatic islet cells, as it is particularly toxic to the insulin-producing beta cells of the pancreas in mammals, and was originally used as an antibiotic. Due to its structural similarity to glucose, it is transported into the cell by the glucose transport protein GLUT2.
A composition containing 10 mmoles of Aam and 60 mmoles of PEG200DMA was allowed to complex with 2 mmoles of D-glucose mixed with 6 mL DMSO. After mixing and checking mutual solubilities, Irgacure® 184 initiator was added in the amount of 1-2 wt %. Control polymers were made with exactly the same composition except D-glucose was not added.
In the resulting moiety-imprinted hydrogel, streptozotocin was recognized. Also, this scheme is suitably represented and highly viable for prodrugs, or pharmacologically inactive derivatives of active drugs, that are designed to maximize bioavailability and converted to the active form in vivo. For instance, glycoconjugates or glucose-containing prodrugs have shown improved blood-brain barrier transport by using compounds bearing the sugar moiety. Lastly, this scheme can work for glucose-derivatized drugs that still retain biological activity without enzymatic or non-enzymatic conversion. In most cases, the use of the drug itself to produce and optimize recognition sites within polymer gels has limited applicability due to the high cost of many pharmaceutical compounds.
As mentioned above, many drug molecules contain moieties that are benzene derivatives, and thus, benzene derivatives may be advantageously used as moieties. Using benzene derivatives as moieties yields a generalized scheme for using biomimetic polymer networks with drugs containing benzene derivatives.
A composition containing 10 mmoles of Aam (or other functional monomers) and 60 mmoles of PEG200DMA (or other crosslinkers) can be allowed to complex with 2 mmoles of a target molecule having a benzene derivative or a benzene derivative moiety mixed with 6 mL DMSO (or other appropriate solvent). After mixing and checking mutual solubilities, IRGACURE® 184 initiator can be added in the amount of 1-2 wt %. Control polymers can be made with exactly the same composition except the target molecule having a benzene derivative or a benzene derivative moiety will not be added.
The target molecule can be a lipid molecule, such as fatty acids, triacylglycerols, glycerophospholipids, sphingolipids, and steroid hormones. The moiety will be chosen with similar rational as described herein, and for example: for fatty acids (e.g., stearic acid, oleic acid, linoleic acid, and the like), the moiety could be carboxylic acids with short chain (e.g., 1 to 10 repeat units) hydrocarbon side group or groups; for triacylglycerols, the moiety could be glycerol, triacylglycerol, and the like; for glycerophospholipids, the moiety could be sn-glycerol-3-phosphate, phosphatidic acids, and the like; for sphingolipids, moiety examples include a phosphocholine or phosphoethanolamine moiety for sphingomyelins, sugar residues for cerebrosides, and the like; for steroid hormones, the moiety could be cholesterol and cholesterol-derivatives. In general, when the moiety is a lipid, the hydrophobic functional monomers will be of particular importance, for including hydrophobic interactions in the recognition site.
For example, a composition containing 10 mmoles of Aam (or other functional monomers mentioned earlier), MMA (or other hydrophobic monomer) and 60 mmoles of PEG200DMA (or other crosslinkers mentioned earlier) is allowed to complex with 2 mmoles of a moiety of a lipid molecule mixed with 6 mL DMSO (or other appropriate solvent). After mixing and checking mutual solubilities, IrgacureO 184 initiator is added in the amount of 1-2 wt %. Control polymers is made with exactly the same composition except the moiety or lipid molecule will not be added.
The target molecule can be a glycoprotein molecule. For proteoglycans, a core protein is combined with at least one glycosaminoglycan chain (e.g, hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, heparin, and the like). The moiety will be chosen with similar rational as described above. For example, proteoglycans, the moiety could be keratin sulfate, chondroitin sulfate, and the like, as well as a glucose-derivative subunit of the glycosaminoglycan chain.
For example, a composition containing 10 mmoles of Aam (or other functional monomers mentioned earlier) and 60 mmoles of PEG200DMA (or other crosslinkers mentioned earlier) is allowed to complex with 2 mmoles of a glycoprotein molecule mixed with 6 mL DMSO (or other appropriate solvent). After mixing and checking mutual solubilities, IRGACURE® 184 initiator is added in the amount of 1-2 wt %. Control polymers are made with exactly the same composition except the glycoprotein molecule will not be added.
In one example, recognitive hydrogels were prepared for use in a biomimetic polymer network. Recognitive hydrogels were prepared by UV-initiated free radical solution polymerization. The monomer mixture contained methacrylic acid (MAA) (Sigma-Aldrich, St. Louis, Mo.), poly(ethylene glycol) monomethylether monomethacrylate (PEGMMA, molecular weight 1000) (Polysciences, Warrington, Pa.), tetraethylene glycol dimethacrylate (TEGDMA) (Sigma-Aldrich), Irgacure 184® (1-hydroxycyclohexyl phenyl ketone) (Sigma-Aldrich), water, and ethanol.
The monomers were mixed in a 1:1 molar feed ratio of MAA:ethylene glycol units, thus in a typical reaction 3.6 g of MAA and 2.0 g of PEGMMA were used. The crosslinker, TEGDMA, was added in the amount of 1.0 mol % of total monomers and Irgacure® 184 was added in the amount of 1.0 wt % of total monomers to initiate the reaction. The solvent was a 50:50 w/w mixture of water and ethanol and was added in a 50:50 w/w ratio of total monomer to solvent.
Monomers, crosslinker, initiator, and solvent were added to an amber bottle and sonicated for 15 minutes prior to polymerization. A lectin WGA was added as a template. The monomer solution was then placed in a sealed glove box environment and purged with nitrogen for 20 minutes to remove oxygen, which is a free radical scavenger. After purging, the glove box remained sealed to ensure a nitrogen environment during polymerization. Two glass plates (15×15 cm×0.3 cm) were separated by a 0.7 mm Teflon spacer and the monomer solution was carefully pipetted between the glass slides. The glass plates were exposed to UV light (Dymax 2000-EC Light Curing System, Torrington, CT) for 30 minutes at an intensity of 17.0 mW/cm2. The resulting polymer was removed from the glass plates and washed in deionized water for 7 days to remove any unreacted monomer. Drying of the polymer was done in a vacuum oven at 35° C. for 2 days. The polymer was then crushed and sieved into microparticles of various sizes. During the course of the experiment, samples were taken of the supernatant and analyzed by HPLC (Waters 2695 Separations Module, Milford, Mass.) to determine avidin and B-WGA concentration.
Therefore, the present disclosure is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this disclosure as defined by the appended claims.
This application is a continuation in part of PCT/US2006/016425 filed Apr. 28, 2006, which claims the benefit of U.S. Provisional Application Ser. No. 60/675,811 filed on Apr. 28, 2005.
This disclosure was developed at least in part using funding from the National Science Foundation, Grant Numbers CTS-03-29317 and DGE-03-33080, and National Institutes of Health, Grant Number EB000246-13A. The U.S. government may have certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60675811 | Apr 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US06/16425 | Apr 2006 | US |
| Child | 11555432 | Nov 2006 | US |
