WO2008154603A1 - Particules et films biomimétiques destinés à la capture de pathogènes et d'autres usages - Google Patents

Particules et films biomimétiques destinés à la capture de pathogènes et d'autres usages Download PDF

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WO2008154603A1
WO2008154603A1 PCT/US2008/066605 US2008066605W WO2008154603A1 WO 2008154603 A1 WO2008154603 A1 WO 2008154603A1 US 2008066605 W US2008066605 W US 2008066605W WO 2008154603 A1 WO2008154603 A1 WO 2008154603A1
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molecules
film
biomimetic
gal
glycosylated
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PCT/US2008/066605
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Richard Spedden
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Richard Spedden
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Priority claimed from US11/761,045 external-priority patent/US8101274B2/en
Application filed by Richard Spedden filed Critical Richard Spedden
Priority to US12/663,904 priority Critical patent/US20100190690A1/en
Publication of WO2008154603A1 publication Critical patent/WO2008154603A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • the present invention relates generally to the field of glycomics and the identification and immobilization of glycosylated molecules having biomimetic properties, more particularly naturally-occurring, tissue-derived, non-immuno logical glycan sequences or functional equivalents thereof, on solid state surfaces and films or on membranes arising at the interface between non-polar and polar materials.
  • the present invention also relates to the industrial, environmental, diagnostic and/or therapeutic use of such biomimetic films and particles constructed therefrom in the binding, capture, and/or extraction of pathogens, toxins and/or contaminants, in vivo, in vitro or in situ.
  • the present invention further extends to the use of such biomimetic films and particles for the delivery of other therapeutic molecules as well as in the construction of body contacting devices having enhanced biocompatibility and reduced immunogenicity.
  • pathogens have some degree of host and tissue specificity. For example, many of the most virulent pathogens exhibit a degree of tissue tropism, wherein the pathogen recognizes and responds to tissue present in the most advantageous environment for the pathogens to thrive. Consequently, a pathogen which causes gastro-intestinal distress may fail to bind tissues outside this region, for example, mucosal tissues of the nose and throat of the same host, and thus be relatively benign in these other regions. In a similar fashion, a pathogen that is highly contagious in birds may have a limited virulence in humans. This host and tissue tropism is often mediated through glycan recognition.
  • Pathogen binding to host tissue is, in most cases, a prerequisite for the successful infection of the host. This binding is typically achieved by means of adherence factors or "adhesins" expressed on the pathogen surface which are specific to glycans or glycan sequences presented on the surfaces of the cells of the tropic host tissue. These endogenous glycan signatures are often unique to a specific area of the body and utilized by the host to regulate and induce tissue specific biological processes. Glycan signatures can also serve to designate the cell or tissue as "self, thereby insulating it from the immune system recognition.
  • the surface features of the pathogen are recognized by the host immune system as "non-self, thereby generating an antigen specific immune response, namely the production of pathogen/antigen specific antibodies or immunoglobulins.
  • a similar process known as phage display may be used to produce synthetic adhesins to pathogenic antigens.
  • Commercial quantities of antibodies and synthetic adhesins may be economically produced, thereby facilitating their routine use in laboratory scale capture, concentration and detection techniques.
  • the specificity of an antibody or its synthetic analog derived from phage display techniques can extend to a point where two pathogens of equivalent pathogenicity but with minor molecular differences in surface features will not be both recognized by the same antibody.
  • the present invention provides biomimetic films and particles that utilize non-immunological, glycosylated molecules harvested or derived from a relevant biological tissue of interest, particularly a pathogen specific host tissue, immobilized to a solid state support or substrate.
  • the present invention is able to provide improved biomimetic films and particles uniquely suited to the capture of a wide range of pathogens and toxins based on tissue tropism. As discussed in detail below, these improved biomimetic films and particles find both diagnostic and therapeutic applications.
  • the alimentary tract of biological organisms is a preferred tropism for many microorganisms. While many of these microorganisms are symbiotic in nature, others are pathogenic. Ingested pathogenic organisms are the known cause of major health issues in many parts of the world and some are now being implicated in diseases which had previously been thought not to be microorganism based. For example, Helicobacter pylori, is now thought to be a factor in development of gastritis and peptic ulcers, where previously these conditions were thought to be solely a result of diet, stress or genetic predisposition..
  • the immune system of the human body has evolved to be effective in combating many of these pathogens; thus, whether through design or neglect, the most common treatment of pathogenic infection often involves simply letting the disease run its course. However, this option is often unacceptable when dealing with infection in the very young, the very old, in individuals whose immune system is impaired or compromised or when dealing with particularly virulent strains against which the body may not be able to mount an adequate defense.
  • antibiotics may be recommended.
  • antibiotic therapy has its limitations. For example, antibiotics are ineffective against viral pathogens as well as the growing number of antibiotic resistant strains, the onset of which many scientists link to the overuse of antibiotics.
  • antibiotics are not always readily available, particularly in economically disadvantaged locations in the world.
  • antibiotic therapy is not always advisable, even in the treatment of bacterial infections.
  • certain types of bacterial pathogens such as Shiga toxin-producing Escherichia CoIi (STEC), particularly 0157:H7 and its variations, exert their deleterious effects through the production and release of toxic chemicals.
  • HUS hemolytic-uremic syndrome
  • the present invention addresses this need by providing therapeutically administrable, more preferably ingestible, biocompatible pathogen-binding films and particles having a size or geometric configuration so as not to be absorbed by the relevant tissues, said particles capable of
  • molecules such as antibodies may be immobilized to suitable solid substrates that serve as platforms to either allow recovery of the platform, including any attached pathogens, such as in the context of antibodies conjugated to magnetic beads, or to act as stationary capture surfaces, such as with microarrays or capture filters having antibodies attached thereto. Accordingly, by affixing or immobilizing an antibody to a support structure or substrate, one can provide an extremely specific means for capturing anticipated pathogens and facilitating detection.
  • the immobilization of molecules on the surface of a solid substrate is well-studied science and techniques of value can be utilized from many fields.
  • Block copolymers are often used to facilitate such conjugations.
  • Molecules of interest can also be incorporated directly into a material matrix which constitutes the body of the substrate or which is applied as a coating on a substrate. This later method, also known to those skilled in the art, is less efficient in the expression of active moieties at the surface since the molecules of interest may be embedded in a manner where the active moieties are not exposed at the surface.
  • Glycan signatures and other membrane bound molecules tend to be amphiphilic in nature. This feature can be utilized effectively in the context of the present invention for the creation of a biomimetic pathogen-binding films and particles. Accordingly, another aspect of the present invention is to provide novel techniques for immobilizing glycosylated molecules, particularly amphiphilic glycosylated molecules, to solid state supports.
  • Amphiphilic molecules with both hydrophobic and hydrophilic ends tend to self align to form a membrane at the interface of a polar solvent and a non-polar liquid or a solid.
  • the amphiphilic molecules When the amphiphilic molecules are in the presence of a polar solvent and a non-polar liquid, they form micelles or micelle-like structures in which the hydrophobic end of the molecule is embedded in the non-polar liquid.
  • the amphiphilic molecules are in the presence of a polar solvent and a solid, the hydrophobic ends of the amphiphilic compound tend to align against the solid with the hydrophilic end presented to the solvent. In both these situations, when the polar solvent is removed, the electrical forces which maintain the alignment of the amphiphilic compounds are also removed and the membrane structure fails.
  • Biological cell membranes consist of lipid bilayers in which amphiphilic phospholipids and related compounds align with their hydrophobic ends against a lipid layer and their hydrophilic ends facing surrounding or interior sides of the membrane; the resultant construct is a double wall of phospholipids.
  • Amphiphilic compounds have been used in the emulsion polymerization process to produce synthetic rubber and some grades of plastics (PVC, polystyrene, PMMA, polyvinylidene fluoride and PTFE).
  • the emulsion polymerization process is designed to produce small polymer particles that can remain in suspension in products such as paint or other emulsions.
  • the construct of the amphiphilic biomimetic molecules hydrophobically bound to the surface of said latex film or particle may be heated to the glass transition temperature of the latex and then cooled in a manner which results in the hydrophobic tails of the amphiphilic compound becoming embedded in the latex material.
  • the present invention constitutes a marked improvement not only in the area of micelle technology by providing for the secure immobilization of an amphiphilic moiety of interest, thereby enabling long term storage and facilitated transport, but also in the area of pathogen capture, by utilizing amphiphilic biomimetic structures, particularly unique glycosylated amphiphiles which mimic the pathogen activity of native host cells and tissues.
  • a primary objective of the present invention is to provide novel biomimetic films and particles for pathogen capture.
  • the present invention provides a solid state film with biomimetic properties comprised of a solid, preferably non-polar, substrate having a plurality of biomimetic glycosylated molecules immobilized thereon.
  • biomimetic properties comprised of a solid, preferably non-polar, substrate having a plurality of biomimetic glycosylated molecules immobilized thereon.
  • endogenous glycan sequences expressed at the surface of cells, particularly epithelial cells, that represent the principal binding sites for pathogens at the initiation of a host-tropism type infection.
  • These same glycan signatures may also constitute "self antigens" that can be used to mask the foreign nature of the film, thereby providing it with reduced immunogenicity.
  • the unique glycan signatures can be either harvested directly from host tissues of interest or derived therefrom, using phage display and recombinant techniques. Functional mimics of these glycan signatures are also useful in the context of the present invention.
  • biomimetic films, surfaces, and particles suitable for pathogen capture for diagnostic, therapeutic or other uses such surfaces composed of a solid substrate having glycosylated molecules expressed on its surface, with the glycans or glycan sequences representative of non-immunological, glycosylated molecules.
  • Molecules of particular interest are those which might be expressed by cells of the potential target host, or mimics of the glycans structures of said molecules or functional analogues.
  • Sialated molecules are of also of particular value in the context of the present invention.
  • biomimetic films and particles for pathogen capture, for example by exposing said surface to a fluid media which may or may not contain pathogens which exhibit the appropriate tissue tropism. It is also an object of the present invention that the construct can subsequently be subjected to analysis techniques to determine the presence and/or nature of any organisms binding to the surface.
  • the magnetic beads can be subsequently subjected to analysis procedures familiar to those skilled in the art to determine the presence and/or nature of material which bound to the beads in solution.
  • biomimetic films and particles of the present invention with one or more type of non-immuno logical, glycosylated molecules, examples of which include, but not limited to, glycoproteins (eukaryotic glycoproteins, proteoglycans, glycomucins), and glycolipids, including natural, synthetic, and recombinant versions thereof as well as homologues, analogues, and functional equivalents thereto.
  • glycoproteins eukaryotic glycoproteins, proteoglycans, glycomucins
  • glycolipids including natural, synthetic, and recombinant versions thereof as well as homologues, analogues, and functional equivalents thereto.
  • non-immuno logical, glycosylated molecules include synthetic molecules with part or all of the glycans structure of naturally occurring glycoproteins (eukaryotic glycoproteins, proteoglycans, glycomucins), and glycolipids, including molecules which are conjugates of different materials.
  • biomimetic films and particles of the present invention with a substrate of a polymer, wax and other hydrocarbon- based substrate, ceramic, glass, metal or other solid material or combinations thereof.
  • a second objective of the present invention is to provide novel biocompatible particles formed from a biomimetic film analogous to that disclosed above, comprised of a solid state film having a plurality of glycosylated molecules immobilized thereon, wherein the particle is of a size or geometric configuration that prevents it from being absorbed by the biological tissue or organs of a living system.
  • the biocompatible particles find utility as ingestible pharmaceutical compositions for the treatment of pathogenic infection, particularly infection with a toxin-producing pathogen.
  • Such particles can also be introduced to other areas of the body as well, according to other conventional administration protocols, such that they can later be removed or expelled.
  • the fibers or worm micelles can be part of a construct of entangled fiber-like particles which in concert provide an effective diameter or configuration to impede absorption into surrounding tissue.
  • the particles can be in the form of a free-standing film as long as at least one dimension is sufficient to prevent absorption of a majority of the particles by the surrounding tissue.
  • the pathogen binding moieties include glycans or glycan sequences, natural or synthetic, which mimic pathogen binding sites in the host body, for example, glycan sequences expressed in glycoproteins (eukaryotic glycoproteins, proteoglycans, glycomucins) or glycolipids (glycosphingolipids), including natural, synthetic, and recombinant versions thereof as well as homologues, analogues, and functional equivalents thereto.
  • glycoproteins eukaryotic glycoproteins, proteoglycans, glycomucins
  • glycolipids glycosphingolipids
  • the pathogen binding glycosylated molecules may be isolated, harvested, or derived from endogenous pathogen host tissues or surrogates thereof (such as tissue cultures). Alternatively, they may be recombinantly or synthetically produced. The population of glycosylated molecules may be homogeneous or heterogeneous.
  • the pathogen binding moieties can comprise transmembrane molecules isolated or derived from host tissues or bacterial intimin binding molecules or mimics as expressed by attaching and effacing lesions. They may also include molecules or mimics of molecules harvested, or otherwise derived, from translocated intimin receptor (TIR) or its interaction with epithelial cells. It is further an object of the present invention to provide a the substrate of natural or synthetic polymers, natural or synthetic waxes, ceramics, metals, materials of biological origin or combinations thereof.
  • compositions suitable for ingestion or administration by other means.
  • Such pharmaceutical compositions may optionally include additional
  • a final objective of the present invention is to provide functionally active solid state films of glycosylated amphiphilic molecules.
  • These properties may be viable either in air or when a polar solvent, such as water, is present.
  • the functionally active solid state films of glycosylated amphiphilic molecules of the present invention are useful in imbuing various surface properties of organic cells onto the surface of solid materials.
  • the present invention represents an improvement in the art that utilized similar membranes formed between a polar solvent and a non-polar liquid. Structures of such membranes traditionally include micelles and liposomes wherein the hydrophobic groups align at the interface between a polar solvent and a non-polar liquid.
  • the solid state films of the present invention differ from conventional micelles and liposomes in that the hydrophobic groups are affixed to or anchored in a solid in such a way that the properties of the hydrophilic groups are preserved on the surface for advantageous use.
  • the polar solvent can be removed and then later reintroduced or then another polar solvent introduced with maintenance of the properties of the hydrophilic groups on the surface.
  • a solid state film having at least one functionally active surface the film composed of a non-polar substrate having a homogenous or heterogeneous population of glycosylated amphiphilic molecules affixed thereto, each glycosylated amphiphilic molecule composed of a hydrophobic tail end and a hydrophilic head end provided with one or more functional groups such that the hydrophobic tail ends are embedded in or chemically or mechanically linked to the non-polar substrate and the functional groups of the hydrophilic tail ends project from the substrate so as to provide at least one surface of said film with functional activity.
  • Such surfaces are important in the fields of glycomics and proteomics for the study of how sugars and proteins react with various biological compounds.
  • the surfaces are also useful in the field of pathogen capture, concentration and detection. Such surfaces capture the pathogen by presenting the same or similar sugars that the specific pathogen, etc. binds to in the body.
  • a surface with this characteristic can concentrate the pathogen to a point where it can be detected through conventional detection means.
  • a material that mimics the surface properties of a biological cell or tissue is less likely to generate an immune response.
  • an appropriate biocompatible glycosylated amphiphilic compound one can provide a biomimetic membrane which would be useful in the formation of or as a film coating for various body contacting, penetrating or implanted devices.
  • Such glycosylated amphiphilic membranes of the present invention, embedded with molecules that mimic biological molecules or express a variety of biological properties can also prove useful in wound dressings and drug delivery.
  • the non-polar substrate may comprise a natural wax or fat isolated from an antigen-matched human donor.
  • Figure 1 depicts a classic micelle structure, in which the hydrophilic "head” regions present outward to contact the surrounding solvent while the hydrophobic "tail” regions are sequestered in the micelle centre.
  • Figure 2 is a schematic of a glycoprotein micelle of the present invention, particularly depicting the spontaneous aggregation and specific alignment of glycoproteins to form a membrane at the interface between polar and non-polar solutions.
  • Figure 3 depicts the successful binding of fluorescent lectins to the sugars of the glycoproteins presented on the surface of the paraffin beads (10-100 microns in diameter). The lectins are glowing under the light of an epi-fluorescent microscope. This type of binding is analogous to the binding of toxins from a fluid sample..
  • Figure 4 depicts the successful binding of Salmonella to the sugars of the glycoproteins presented on the surface of the wax beads (5-50 microns in diameter) with the surface moieties of a biomimetic film derived from glycosylated molecules harvested from porcine small intestine..
  • biomimetic refers to a material which exhibits surface properties, including but not limited to molecular structures such as amino acid and carbohydrate sequences, which provide the surface with characteristics, and in particular molecular binding or biological recognition features, which are in common with or provide functional analogues with biological features of biological materials such as tissue, and in particular cells, which the surface is intended to represent.
  • biomimetic in the context of the present invention does not require that the surface duplicate all functions or binding modalities of the biological material being mimicked. Examples of preferred structures to be mimicked include pathogen binding proteins and immune recognition sequences (e.g., glycan signatures).
  • Whether a particle moiety possesses the requisite biomimetic activity may be routinely assayed using conventional techniques known to those skilled in the art. For example, one may utilize well known immunoassay techniques, such as ELISA, to assay the binding activity of a proposed pathogen-binding biomimic as compared to endogenous host tissue. Likewise, one may utilize conventional immune response assays, such the multiplexed chemokine and cytokine assays available through Meso Scale Discovery (MSD) (Gaithersburg, MD), to assess the risk and assay immunogenic potential of a proposed biomimic as compared to native tissue.
  • immunoassay techniques such as ELISA
  • ELISA immunoassay technique
  • immune response assays such assay the multiplexed chemokine and cytokine assays available through Meso Scale Discovery (MSD) (Gaithersburg, MD), to assess the risk and assay immunogenic potential of a proposed biomimic as compared to native tissue.
  • glycosylated molecules include, but are not limited to glycoproteins (eukaryotic glycoproteins, proteoglycans, glycomucins), and glycolipids, including natural, synthetic, and recombinant versions thereof as well as homologues, analogues, and functional equivalents thereto.
  • glycoproteins eukaryotic glycoproteins, proteoglycans, glycomucins
  • glycolipids including natural, synthetic, and recombinant versions thereof as well as homologues, analogues, and functional equivalents thereto.
  • glycosylation refers to the addition of a carbohydrate moiety, typically a sugar, to either a biological molecule or a biocompatible synthetic molecule, such as a block copolymer.
  • a carbohydrate moiety typically a sugar
  • glycosylation frequently arises as a result of co-translational or post-translational modification.
  • the glycosylating moiety is frequently selected for its ability to mimic the surface properties of a biological cell or tissue and, in turn, reduce the immunogenic character of the block copolymer.
  • glycosylated lipid is a biological molecule composed of a lipid and a carbohydrate, typically an oligosaccharide
  • a glycosylated protein is a biological molecule composed of a protein and an oligosaccharide.
  • N-glycosylation an asparagine moiety
  • O-glycosylation a hydroxylysine, hydroxyproline, serine or threonine moiety
  • Monosaccharides commonly found in eukaryotic glycoproteins include glucose, N- acetylglucosamine, galactose, N-acetylgalactosamine, mannose, fucose, xylose and N- acetylneuraminic acid (also known as sialic acid).
  • the sugar group(s) often assist in protein folding or improve proteins' stability.
  • the carbohydrate moieties of glycoproteins and glycolipids are often key components in various intercellular recognition processes, particularly immune responses. Accordingly, presentation of glycoproteins on the exposed surface of a micelle, liposome or membrane can be chosen to target appropriate tissue, inhibit uptake by a particular tissue, and/or induce endocytosis of the micelle.
  • Antibodies, apoproteins, and opsonins are examples of glycoproteins that mediate such responses.
  • Soluble glycoproteins are found in a wide range of biological fluids.
  • the glycoproteins “ovotransferrin”, “ovalbumin” and “ovomucoid” are found in certain egg whites (albumen) and the glycoprotein “Glyocophorin A” is commonly found in blood plasma,
  • kappa-Casein is found in cow's milk
  • the "Tamm-Horsfall” protein is found in the urine of mammals.
  • a wide array of glycoproteins are also found in fruit juice and other plant fluids.
  • glycoproteins suitable for use in the context of the present invention include, but are not limited to, human immunoglobulins, such as IgGl; hormones, such as pituitary hormones lutropin (LH), thyrotropin, and pro-opiomelanocortin; proteoglycans, and derivatives thereof.
  • suitable glycoprotein hormones include, but are not limited to, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), human chorionic gonadotropin (hCG), alpha-fetoprotein, and erythropoietin (EPO).
  • Proteoglycans are a special class of heavily glycosylated glycoproteins comprised of a core protein with one or more glycosaminoglycan (GAG) chains.
  • GAG chains are long, linear carbohydrate polymers that are negatively charged under physiological conditions, due to the occurrence of sulphate or uronic acid groups.
  • Proteoglycans are categorized by the nature of their GAG chains, examples of which include, but are not limited to, chondroitin sulfate and dermatan sulfate, heparin and heparin sulfate, keratin sulfate, etc.
  • large proteoglycans include aggrecan, the major proteoglycan in cartilage, and versican, present in many adult tissues including blood vessels and skin.
  • Small leucine rich repeat proteoglycans include decorin, biglycan, f ⁇ bromodulin and lumican.
  • glycosylated transmembrane molecule refers to any molecule produced by a biological organism (either through natural processes or as a result of recombinant techniques) which is amphiphilic in nature, possessing a glycosylated hydrophilic end and a hydrophobic end; these molecules typically, but not necessarily project into or across cell membranes.
  • the term “harvested”, as in, for example 'harvested from biological tissues or cells', refers to any process which separates molecular structures of interest from the tissue or cell membranes in a manner which preserves at least part of said molecular structure's biomimetic properties relative to the source tissue or cell.
  • glycoproteins suitable for use in the instant invention can also be "derived from" biological sources, for example, synthetically produced or produced by genetically engineered plants and animals, including bacteria and other microbes, in accordance with well-known and conventional techniques.
  • glycocan is synonymous with the term “polysaccharide” as referring to compounds composed of a large number of glycosidically linked monosaccharide units (typically ten or more).
  • glycoside generically refers to a molecule in which a sugar moiety, typically through its anomeric carbon, is attached to another non-sugar moiety. When the anomeric carbon is attached via an oxygen atom, the resulting molecule is referred to as an "O-glycoside”. Similarly, when the anomeric carbon is attached via an sulfur atom, the resulting molecule is referred to as an "S-glycoside” or “thioglycosides”.
  • the resulting molecule is preferably designated as a "glycosylamine” rather than an "N-glycoside".
  • amphiphilic glycosides suitable for use in the context of the present invention include, but are not limited to, chitin, chitosan, cellulose, saponin, and derivatives thereof.
  • the present invention makes reference to the immobilization of non-immuno logical, often sialated, glycosylated molecules on solid substrates to create biomimetic films and particles suitable for pathogen capture.
  • the substrate preferably consists of a relatively non- polar material.
  • non-polar refers to a substance or mixture of substances that is relatively uncharged when compared to a polar solvent being used.
  • the concept is also reflected in the references herein to systems of "differing” or “diverging” polarity.
  • the terms “relatively non-polar” “less-polar” can be interchangeably exchanged herein for the term “non-polar”.
  • the non-polar material is typically water insoluble (hydrophobic).
  • a mixture of non-polar and polar substances can be used to form the non-polar material of this invention as long as the resulting combination supports the formation of an amphiphilic film when in the presence of a selected polar solvent.
  • the term "substrate” refers to the relatively non-polar material in the construct of forming a biomimetic film at the interface between a non-polar substrate and a polar solvent.
  • the substrate may be used in conjunction with other materials to provide shape, structural support or other properties of interest.
  • Biocompatible substrate materials are of particular interest in the context of this invention. Biocompatible materials include natural materials extracted from the intended host or appropriate surrogates. Biocompatible substrate materials may also comprise synthetic and other materials which have been found to be benign when introduced in the body. The distinct advantage to biocompatible materials in the context of this invention is that they minimize the potential for adverse reactions when introduced into the intended host.
  • the present invention makes reference to biomimetic surfaces or films composed of solid substrates that find utility in the field pathogen capture.
  • the biomimetic solid surfaces of the present invention are useful for binding target molecules. More particularly, films bearing glycosylated molecules exhibiting appropriate binding components (typically sugars) can be used to bind, isolate and extract target molecules from aqueous samples or solutions, particularly those containing disease-causing organisms or other harmful materials such as biotoxins and heavy metals.
  • the aqueous sample assayed is not particularly limited and includes both environmental samples and biological samples.
  • biological sample includes body fluids, secretions and exudates, examples of which include, but are not limited to, blood, serum, saliva, sputum, urine, plasma, spinal fluid, amniotic fluid, fluids in the gastrointestinal tract, and fluids in the lungs, sweat, breast milk, tears or other lacrimal secretions, pus, and other bodily discharges associated with either normal or diseased conditions.
  • the biomimetic films and particles of the present invention find utility in the field of diagnostic sensing and pathogen capture.
  • Filters made of cellulose or synthetic fibers coated with or formed from the biomimetic films of the present invention are particularly useful for removing certain organisms or biotoxins from aqueous solution.
  • Enzymes can be incorporated into the films of the present invention to catalyze a variety of chemical reactions in aqueous media.
  • Sensors employing such films are also useful in the context of environmental sensing, finding utility in the inspection of foods and in forensic science, for example.
  • target molecule encompasses both endogenous biological entities, such as peptides, proteins, hormones, oligonucleotides, nucleic acid molecules, (e.g., RNA and/or DNA), cellular components, and particulate analytes, as well as foreign materials, including, but not limited to, pathogens, toxins, drugs, contaminants, pollutants, chemical substances, and analytes.
  • endogenous biological entities such as peptides, proteins, hormones, oligonucleotides, nucleic acid molecules, (e.g., RNA and/or DNA), cellular components, and particulate analytes, as well as foreign materials, including, but not limited to, pathogens, toxins, drugs, contaminants, pollutants, chemical substances, and analytes.
  • pathogens e.g., a bacterial infection, heavy metal poisoning, cancer, etc.
  • pathogen is used to refer to an agent of disease or disease producer and encompasses any natural or bioengineered disease-producing agent, particularly viruses, bacteria, and other microorganisms (e.g., amoeba, protozoans, etc.). Accordingly, the term pathogen includes not only infectious organisms, such as bacteria (such as staph), viruses (such as HIV), and fungi (such as yeast), but also noninfectious agents of disease such as a toxins (including molecules of both biological, non-biological (natural or synthetic) origin).
  • biomimetic films and particles of the present invention also find utility in the context of environmental detection and detoxification, for example in the removal of heavy metals.
  • heavy metal refers to a metal having a relatively high density (i.e., a specific gravity greater than 4.0, more preferably greater than 5) or a relatively high atomic weight (i.e., falling on the periodic table between copper and bismuth). Excessive levels of heavy metals are known to be detrimental to living organisms.
  • heavy metals associated with serious illness include, but are not limited to, lead, mercury, copper, cadmium, manganese, aluminum, beryllium, molybdenum, vanadium, strontium, zinc, and iron.
  • the present invention is directed, at least in part, to a solid state biomimetic film composed of a non-polar substrate having a surface to which a plurality of glycosylated molecules are adhered or affixed.
  • film and “membrane” are used interchangeably to refer to the thin (usually a molecule in depth), wall-like structure formed by the specific alignment of amphiphilic molecules in the presence of polar and non-polar media.
  • the film or membrane of the present invention can consist of a mixture of different amphiphilic molecules.
  • the membrane can assume any form that the interface between polar and non-polar media can assume, including curved of any radius (including constant and irregular radius (rough) spheres), flat or a random combination (rough) surfaces.
  • the important property is that once the non-polar media is transformed to its solid state the hydrophobic ends of the amphiphilic molecules or the matrix that the molecules form become affixed to the solid's surface while the hydrophilic ends remain free.
  • Mullen micelles can be formed in the manner taught in the relevant Mullen patents while the non-polar media is in a liquid state. In the context of the present invention, rapid cooling is one method for preserving the micelle form in the transition of the non-polar media to a solid state.
  • non-polar refers to a substance that is relatively uncharged, typically water insoluble (hydrophobic).
  • the non-polar substrate must be capable of stable expression both as a liquid and a solid.
  • the non-polar substrate exists in solid state at a conventional room temperature (typically between 15 and 25 0 C).
  • non-polar substrates with melting points above the boiling point of the desired polar solvent can be used if the films are formed while the liquids are under pressure.
  • non-polar materials suitable for use in the context of the instant invention include, but are not limited to, resins, synthetic and natural waxes, synthetic plastics, polymers, and copolymers, including elastic, thermoplastic and vinyl polymers, nylons, polyethylene and the like. Any mixture of hydrocarbons that results in a non-polar liquid when heated and become solid or semi-solid when cooled are appropriate.
  • Waxes such paraffin, microcrystalline, carnauba, beeswax, candelilla, ceresine, ozokerite, and various other animal, vegetable, and synthetic waxes and blends thereof are particularly suitable for use in the instant invention.
  • Petroleum jelly and many animal and vegetable fats are other examples of appropriate non-polar substrate materials.
  • amphiphilic describes any of many organic and synthetic molecules that possess both hydrophilic and hydrophobic properties. It is used interchangeably herein with the term “amphipathic” to describe molecules that have a polar end that is attracted to water and a non-polar end that is repelled by it. Amphiphilic compounds suitable for use in the context of the instant invention may express a net charge at physiological pH, either a net positive or negative net charge, or may be zwitterionic.
  • the hydrophobic component of an amphiphilic molecule is typically a large hydrocarbon moiety, such as a long chain of the form CHs(CH 2 )D, with n > 4.
  • the hydrophilic component is either comprised of charged groups (e.g., anionic entities such as carboxylates, sulfates, sulfonates, and phosphates, or cationic entities such as amines, amino acids, or peptides) or polar groups such as alcohols.
  • amphiphilic species have several hydrophobic parts, several hydrophilic parts, or several of both. Proteins and some block copolymers are such examples.
  • the present invention includes both glycosylated biological amphiphiles and glycosylated synthetic molecules, such as block copolymers. Illustrative methods for preparing glycosylated block copolymers are disclosed in U.S.
  • Block copolymers can be used to construct a hydrophobic tail with the same functionality as the hydrophobic amine based tails of glycoproteins.
  • Surfactants such as sodium dodecyl sulphate, benzalkonium chloride, octanol, and cocaminopropyl betaine are examples of amphiphilic compounds with known industrial uses.
  • the present invention is directed to glycosylated amphiphiles, examples of which include, but are not limited to, glycoproteins, glycolipids, glycosylated block copolymers and the like.
  • the solid state biomimetic films of the present invention may be used to create useful micelles of novel construction.
  • the term "micelle” refers to an aggregate of molecules dispersed in a liquid colloid in which hydrophilic polar components of the molecules orient themselves toward and interact with the aqueous component while hydrophobic, lipophillic and/or nonpolar components are sequestered in the micelle structure.
  • the hydrophilic "head” regions contact the surrounding solvent while the hydrophobic "tail” regions are sequestered in the micelle centre.
  • This type of micelle is referred to as a normal phase micelle (oil-in-water micelle).
  • Inverse micelles have the "head” groups at the centre with the “tails” extending out (water-in-oil micelle).
  • Micelles are generally spherical in shape, though other phases, including shapes such as ellipsoids, cylinders, bilayers and objects with irregular or planar surfaces are also possible.
  • the shape and size of a micelle is a function of the molecular geometry of its surfactant molecules and solution conditions such as surfactant concentration, temperature, pH, and ionic strength, as well as the magnitude of shear forces present during formation.
  • films and particles of the present invention are useful for binding target molecules. More particularly, membranes or micelles bearing glycosylated molecules, particularly glycoproteins, exhibiting the appropriate binding components (typically sugars) can be used to bind, isolate and extract target molecules from aqueous samples or solutions, particularly those containing disease-causing organisms or other harmful materials such as biotoxins and heavy metals.
  • the aqueous sample assayed is not particularly limited and includes both environmental samples and biological samples.
  • biological sample includes body fluids, secretions and exudates, examples of which include, but are not limited to, blood, serum, saliva, sputum, urine, plasma, spinal fluid, amniotic fluid, fluids in the gastrointestinal tract, and fluids in the lungs, sweat, breast milk, tears or other lacrimal secretions, pus, and other bodily discharges associated with either normal or diseased conditions.
  • biomimetic films and particles find utility in the fields of diagnostic sensing and pathogen capture. Filters made of cellulose or synthetic fibers coated with the glycosylated amphiphilic membranes of the present invention are particularly useful for removing certain organisms or biotoxins from aqueous solution.
  • Enzymes can be incorporated into the micelles of the present invention to catalyze a variety of chemical reactions in aqueous media. Sensors employing such membranes are also useful in the context of environmental sensing, finding utility in the inspection of foods and in forensic science, for example.
  • target molecule encompasses both endogenous biological entities, such as peptides, proteins, hormones, oligonucleotides, nucleic acid molecules, (e.g., RNA and/or DNA), cellular components, and particulate analytes, as well as foreign materials, including, but not limited to, pathogens, toxins, drugs, contaminants, pollutants, chemical substances, and analytes.
  • endogenous biological entities such as peptides, proteins, hormones, oligonucleotides, nucleic acid molecules, (e.g., RNA and/or DNA), cellular components, and particulate analytes, as well as foreign materials, including, but not limited to, pathogens, toxins, drugs, contaminants, pollutants, chemical substances, and analytes.
  • pathogens e.g., a bacterial infection, heavy metal poisoning, cancer, etc.
  • the biomimetic films and particles of the present invention find particular utility in the context of pathogen capture.
  • the biomimetic films and particles of the present invention also find utility in the context of environmental detection and detoxification, for example in the removal of heavy metals.
  • heavy metal refers to a metal having a relatively high density (i.e., a specific gravity greater than 4.0, more preferably greater than 5) or a relatively high atomic weight (i.e., falling on the periodic table between copper and bismuth). Excessive levels of heavy metals are known to be detrimental to living organisms.
  • heavy metals associated with serious illness include, but are not limited to, lead, mercury, copper, cadmium, manganese, aluminum, beryllium, molybdenum, vanadium, strontium, zinc, and iron.
  • the biomimetic films and particles of the present invention find therapeutic utility, as pharmaceutical formulations suitable for in vivo administration.
  • the biomimetic particle takes the form of an ingestible micelle, suitable for oral delivery.
  • other methods well known to those skilled in the art may be used to administer the pharmaceutical composition of the present invention to patients, examples of which include, but are not limited to, intraarterial, intravenous, intramuscular or percutaneous injection or via intranasal, transbronchial, transurethral, peritoneal or oral administration.
  • the dosage and method of administration vary according to the body-weight and age of a patient and the administration method; however, one skilled in the art can routinely select a suitable mode and method of administration.
  • the biomimetic films and particles of the present invention find utility as coatings for medical devices.
  • the term “medical device” encompasses both devices intended for limited introduction (for example angioplasty catheters) as well as devices intended for long term insertion (for example cardiac pacemakers). More particularly, as used herein and in the appended claims, the term “medical device” refers to any apparatus, appliance, instrument, implement, material, machine, contrivance, implant, in vitro reagent, or other similar or related article including a component party or accessory which is intended for the diagnosis, prevention, monitoring, treatment or alleviation of disease, injury or handicap. It further encompasses any article intended to affect the structure or function of the body of humans or other animals, and which does not achieve its principal intended action in or on the body by pharmacological, immunological or metabolic means, but which may be assisted in its function by such means.
  • the pathogens and, to a certain extent, toxins exhibit tissue tropism based on adhesins which bind to specific combinations, sequences and/or orientations of glycans expressed on molecules at the surface of tissues in a prospective host organism.
  • These molecules can be harvested from said tissues, or cultures of said tissues, or homologues, analogues or functional equivalents thereof through any number of techniques for rupturing the cell membrane known to those skilled in the art.
  • These molecules can also be produced by other cells or organisms through recombinant techniques. In certain instances these molecules are also accessible in bodily fluids.
  • the resultant structures are unique in their ability to capture pathogens and toxins on the basis of tissue tropism.
  • One aspect of the present invention relates to the extraction of glycosylated structures from tissue and tissue analogues (e.g., glycan signatures), particularly those suitable for pathogen capture or immune system modulation.
  • tissue and tissue analogues e.g., glycan signatures
  • the present invention is unique in the use of a variety of non-immunological molecules which mimic the glycans signatures of epithelial and other cells of a pathogens target host, such that these molecules are immobilized on solid surfaces such that they can affect pathogen binding to the surfaces.
  • the present invention is also unique in the method of using the resulting constructs.
  • Other unique aspects of the present invention include the use of a variety of molecules from a body tissue to form biomimetic surfaces.
  • the present invention provides a unique method for creating biomimetic films, particles, and constructs thereof utilizing glycosylated molecules harvested, or otherwise derived, from tissues of interest. Consequently, it is further an object of the present invention to teach both the constructs and the method for utilizing molecules obtained from tissues from a biological organism.
  • the molecules can be extracted from tissues obtained from the potential target organism of an infection, the host.
  • the host tissues of particular interest are from the specific body area of potential infection, thus taking advantage of the same properties which permit pathogens to demonstrate host tropism.
  • a gastrointestinal infection may exhibit adhesins to glycoproteins (particularly glycomucins) present in a specific section of the small intestine.
  • glycoproteins extracted from tissue in the affected region of the intestine can be immobilized on a solid substrate which, in turn, maybe be used for pathogen capture.
  • the present invention also provides for molecules of interest to be obtained from cultures of tissues derived or extracted from the host organism, from biological surrogates, or from other organisms genetically engineered to produce tissue mimics or functional analogues.
  • porcine tissue is often used as a biological surrogate for human tissue.
  • Avian eggs have been demonstrated to produce glycosylated molecules of interest.
  • Glycosylated molecules with the desired glycans or glycans sequences can also be constructed through chemical synthesis, and are therefore also part of the present invention. Recombinant techniques and genetic engineering can also be used to induce other organisms, such as yeast and bacteria, to produce the glycosylated molecules (and particularly sialated glycosylated molecules) of interest in this invention in commercial quantities. 4
  • Glycosylated molecules of interest can also be harvested, or otherwise derived, from fluids present in or produced by an organism, including but not limited to body fluids such as mucous, blood, saliva, urine, synovial fluid, breast milk, tears, fluids of the reproductive system (including those related to birth).
  • body fluids such as mucous, blood, saliva, urine, synovial fluid, breast milk, tears, fluids of the reproductive system (including those related to birth).
  • urine contains the glycoprotein, Tamm-Horsfall Protein (THP), also known as Uromodulin (UMOD), and Tamm-Horsfall mucoprotein, THP exhibits a broad range of glycans structures useful in the capture of potential urinary tract infections.
  • Blood contains albumin, which is widely known to bind to a variety of pathogens and is readily available in the form of Bovine Serum Albumin (BSA).
  • BSA Bovine Serum Albumin
  • Glycosylated molecules in bodily fluids may be present as individual molecules and readily available for extraction and immobilization on a surface.
  • the molecules of interest may be membrane bound, as with the glycosylated transmembrane molecules which populate cell walls (this can be true in both tissues and in bodily fluids).
  • the cell must be lysed or ruptured in a manner whereby the molecules of interest can be captured and immobilized.
  • Methods for cell lysis include, but are not limited to, mechanical means (such as sonication, freeze/thaw and high shear techniques), chemical means (for example, using detergents for whole cell lysis and cell fractionation) and biochemical means (for example, by means of enzymes and/or protease inhibitors).
  • Solutions of bodily fluids or solutions containing lysed cell material often include a wide variety of constituents, some of which are extraneous to the purposes of this invention.
  • Many of the glycosylated molecules are amphiphilic in nature, particularly those which are produced as transmembrane molecules. These amphiphilic molecules tend to form micelles or micelle-like structures or to align at the interface between systems of divergent polarity, for example between a polar solvent and a relatively less polar material.
  • this trait can be used to extract molecules of interest from a solution of bodily fluids or solutions containing lysed cell material.
  • the molecules can be immobilized directly on to the less-polar material to form the desired biomimetic constructs through the formation of self-aligning films and then the immobilization of those films through the transition of the less-polar material from a liquid to a solid.
  • the extracted molecules can be introduced into a system where they are conjugated to other molecules with appropriate binding affinities which are already immobilized on the substrates of this invention. In either case, when molecules of interest are extracted from bodily fluids using constructs of the present invention, additional molecules not of interest may be present in the final construct.
  • the presence of these additional molecules can be complimentary or extraneous to the goal of the invention, which is to present a biomimetic selection of glycosylated molecules on a solid substrate.
  • the unique advantage of the present invention in the procedure of extracting materials directly from bodily fluids or tissues is that the identity or nature of the glycosylated molecules of a tissue of interest need not be known to effectively use the molecules in the constructs of the present invention, this represents a significant advantage over techniques which seek to mimic a specific, defined glycans structure, since the broad range of potential pathogen affinities to tissue have not been defined.
  • Techniques for binding biological compounds to other materials are well-known to those skilled in the art. These techniques include means dependent on chemical, mechanical and electrical forces (including weak forces such as van der Waals forces). Techniques utilized for immobilizing antibodies on surfaces are also applicable to immobilization of the biomimetic molecules of the present invention.
  • biomimetic molecules utilized are either themselves amphiphilic in nature or can be conjugated with hydrophobic molecules to create an amphiphilic molecule, they may be immobilized using methods of the present invention for the creation of solid state films from amphiphilic molecules described in further detail below.
  • Substrates of interest suitable for use in the context of the present invention include, but are not limited to, polymers, plastics, resins, natural and synthetic waxes, metals, glasses, natural and synthetic fats, ceramics and combinations thereof. Particularly useful forms or configurations of interest include flat or formed surfaces, filter type membranes, fibers, and beads (including micelles and micelles- like structures). Molecules of interest can readily be adsorbed on to any of these substrates given an appropriate electrical charge difference between the substrate and the desired binding surface of the molecule. Substrate charge can be a result of induced voltage, inherent charge, embedded materials, or chemical or other surface treatment (such as plasma treatment). The relative charge of oil droplets in water can be adjusted through pH changes. The advantage of using adsorptive techniques is the relative simplicity of implementation and the minimization of disruption of the chemical properties of the molecule. The disadvantage is the potential of the bond to be disrupted when changing environments during use.
  • Shastri et.al. teach methods and constructs for solid lipid nanoparticles, with particular focus on delivery of active agents across the blood-brain barrier, across a cellular lipid bilayer and into a cell, and to a subcellular structure.
  • the present invention extends the use of these methods in the creation of biomimetic surfaces through the immobilization of glycosylated molecules and the use thereof for pathogen and toxin capture.
  • Conjugates of heparin with bioactive peptides and a hydrophobic component to create an amphiphilic peptide compound have been described by Zamora and Stupp (both reference above). These same techniques are useful in the conjugation of biomimetic materials harvested from biological tissue to heparin and a hydrophobic component to create amphiphilic molecules of use in the constructs of the present invention. These synthetic compounds are useful in creation of self-assembled structures.
  • Stupp in the reference above has taught the formation of nano fibers, also worm micelles.
  • the nanofiber structures consist solely of amphiphlic compounds aligning to exclude other material from the hydrophobic centerline of the fiber. These nanofibers can entangle to create a hydrogel structure.
  • the hydrogel structure has proven useful for entrapping other materials. It is the object of the present invention to utilize these hydrogel structures to form a construct which does not readily absorb into surrounding biological tissue. Stupp also has taught the creation of amphiphilic peptide compounds through the conjugation of a hydrophobic component and a growth factor recognition product of a phage display process to a peptide in patent application US 2005/0209145, the entire contents of which are included herein by reference. It is an object of the present invention to utilize pathogen and toxin recognition products of a phage display process in a similar technique to form a construct of the present invention.
  • Create of said constructs can either be through conjugation of said product of a phage display process with a hydrophobic molecule to form an amphiphilic molecule of use in the present invention, or it could be through direct conjugation of said product of a phage display process with surface molecules of substrates to form constructs of the present invention.
  • Antibodies are a specific type of immunological glycoprotein. For commercial purposes, they are typically immobilized on magnetic beads and on surfaces constructed of block copolymers. For analytical purposes, they can also be immobilized on glass slides. In the context of the present invention, it is an objective to immobilize antibodies on solid substrates which are of a size and configuration that is not absorbed by surrounding tissue such that an antibody bearing construct can bind to a targeted pathogen or toxin and then the construct can be removed or expelled from the body.
  • Magnetic beads are readily available with a variety of available binding sites for the conjugation of molecules.
  • Commercially available beads include Amine-terminated Beads, DADPA-terminated Beads, Carboxy-terminated Beads, Carboxy-terminated Beads, Epoxy- activated Beads, Epoxy-activated Beads, Aldehyde-modified Beads, Aldehyde-modified Beads, Hydrazide-modified Beads, IDA-modified Beads and Silica-modified Beads. Kits are readily available to conjugate a variety of molecules to these beads.
  • Block copolymers have also been used to immobilize antibodies in solid surfaces.
  • antibodies were immobilized on polycarbonate surfaces using Poly-L-Lysine and the resultant surfaces were used for pathogen capture to facilitate detection. 7
  • This type of immobilization is also applicable to biomimetic molecules harvested from biological tissue and the use in that context is an object of the present invention.
  • Block copolymers have been used to construct super-amphiphilic molecules and polymersomes. These constructs are described in U.S. Pat. Nos. 6,835,394 and 7,217,427 and U.S. Pat. Appl. No. 2007/0218123, all to Ennis E. Discher, et.
  • Alkan-Onyuksel, et. al. teach the use of lipid-polymer conjugates in the creation of micelle structures followed by an incubation step and the subsequent incorporation of biologically active amphiphilic compounds into the ⁇ 20 nanometer diameter construct.
  • the present invention can utilize techniques presented by Alkan-Onyuksel.
  • the present invention is unique in the creation of constructs which incorporate epithelial cell moieties and other pathogen binding site molecules and their functional analogues in lieu of the various hormonal peptides envisioned by Alkan-Onyuksel. Additionally, in the context of the therapeutic constructs described below, it is an object to provide for constructs which are
  • Neoglycoproteins The Preparation and Application of Synthetic Glycoproteins, ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY (VoI 37), 1980, Academic Press, Inc., pp.225-281
  • the present invention is not limited to a particular construction method. However, the following methods are suitable for constructing biomimetic films and particles of the present invention and are therefore provided for illustration purposes.
  • An illustrative method for making biomimetic films and particles of the present invention comprises the following steps, with resultant films on solid substrates: a. providing a relatively polar solvent having sufficient quantities dissolved therein of one or more amphiphilic molecules which include glycosylated molecules with biomimetic properties, or conjugates of such, or molecules which can be conjugated with glycosylated molecules with biomimetic properties, or molecules with other pathogen binding moieties; b. exposing the polar solvent to a relatively non-polar liquid, the non-polar liquid being immiscible in the polar solvent; the relatively non-polar liquid can optionally comprise a material which can reversibly experience a state change from solid to a relatively non-polar liquid under stimulus; c.
  • amphiphilic molecules allowing the amphiphilic molecules to align so as to form a membrane which separates the polar solvent from the non-polar liquid; and d. inducing or allowing transformation of the non-polar liquid to a corresponding non- polar solid having upper and lower surfaces, wherein the hydrophobic tail ends of the amphiphilic molecule are embedded in or chemically, electrically or mechanically linked to the upper surface of the non-polar substrate and the hydrophilic tail ends project from the upper surface into the polar solvent so as to yield a film having functional activity; and/or e. if additional properties are required: adsorbing or conjugating molecules with biomimetic or other properties to the functional surface of the film if the desired functionality is not present from the preceding steps. f.
  • the construction method may further include a step wherein the non-polar liquid and the liquid containing the amphiphilic molecules are manipulated prior to or during transition of the non-polar liquid to a solid state in order to yield a film having a geometric form.
  • An exemplary manipulation method can comprise the step of agitating the film so as to form micelles, worm micelles or micelle-like constructs with non-polar substrates.
  • the transformation of the non-polar liquid to a corresponding non-polar solid can be performed in a manner so as to maintain the approximate geometric form achieved by the non-polar liquid prior to or during the transition to a solid form, and that this transformation can be achieved through methods which include, but are not limited to, rapid cooling of or removal of pressure force on the non-polar liquid.
  • An alternate method for making biomimetic films and particles of the present invention comprises the following steps, with resultant films on solid substrates: a. providing a liquid having sufficient quantities dissolved therein of one or more molecules with biomimetic properties or pathogen or toxin binding properties; b. exposing said liquid to a solid with surface moieties appropriate for conjugation with said molecules; c. allowing or inducing the conjugation of said molecules to said surface moieties in a manner which results in the biomimetic moieties or pathogen or toxin binding moieties expressed on the surface of the substrate. d. Optionally, removing said film from any surrounding liquid to form a dry construct.
  • Biomimetic films from biological cell- or membrane-bound glycosylated molecules may be constructed according to the following steps: a. Isolating biological tissue extracted from human, animal or plant tissue, a culture of human, animal or plant tissue, a biological surrogate for human, animal or plant tissue, a culture of a biological surrogate for human, animal or plant tissue or recombinant versions of human, animal or plant tissue or human, animal or plant tissue biological surrogates. b.
  • Subjecting the biological cell or membrane to a process which ruptures or lyses the cell or other membrane including but not limited to, mechanical means (for example, but not limited to, sonication, freeze/thaw and high shear techniques), chemical means (for example, but not limited to, using detergents for whole cell lysis and cell fractionation) and biochemical means (for example, but not limited to, by means of enzymes and/or protease inhibitors), to form a liquid volume with biomimetic molecules present.
  • mechanical means for example, but not limited to, sonication, freeze/thaw and high shear techniques
  • chemical means for example, but not limited to, using detergents for whole cell lysis and cell fractionation
  • biochemical means for example, but not limited to, by means of enzymes and/or protease inhibitors
  • the biomimetic films from biological cell- or membrane-bound glycosylated molecules may be constructed as follows: a. Biological tissue is isolated, the biological tissue, including, but not limited to, biological tissue extracted from human, animal or plant tissue, a culture of human, animal or plant tissue, a biological surrogate for human, animal or plant tissue, a culture of a biological surrogate for human, animal or plant tissue or recombinant versions of human, animal or plant tissue or human, animal or plant tissue biological surrogates.
  • suitable biological tissues include, but are not limited to epithelium, connective tissue, muscle tissue, nerve tissue and associated bodily systems and organs and material associated with or contained in amniotic fluid surrounding a fetus, aqueous humour, blood and blood plasma, interstitial fluid, breast milk, mucus, pus, saliva, serum, tears, urine, cerebrospinal fluid, synovial fluid, intracellular fluid, aqueous humour and vitreous humour and other bodily fluids, material from avian species, including material from eggs and tissues from plant origins. b.
  • the biological tissue may then be lysed or otherwise processed using techniques known to those skilled in the art to rupture cell membranes in manner where non-immunological, glycosylated molecules of cell membranes and fluids and surrounding tissue fluids are liberated into a solution.
  • the solution may then subjected to procedures to permit some or all of the non-immunological, glycosylated molecules to bind to a substrate to form the constructs of the present invention; this procedure can include, but does not have to include, intermediate steps of refinement or isolation of the molecules, or enrichment of the solution with other amphiphilic molecules to achieve critical micelle concentration.
  • non-immuno logical, glycosylated molecules of an amphiphilic nature can be extracted from solution prior to or during the binding process by a method that includes the steps of: a. Exposing the solution containing biological tissue, which can comprise, in part, lysed or otherwise ruptured cell material containing molecules of interest in the present invention to a less-polar material; b. If necessary, adding amphiphilic molecules to achieve critical micelle concentration, before or after exposing said solution to said less-polar material; c. Allowing the amphiphilic compounds to form a film at the interface between said solution and said less-polar material; d.
  • biomimetic films resulting of the present invention can be utilized in the construction of a surface having enhanced biocompatibility with particular applicability to a medical device, in a manner analogous to that described in detail below.
  • the biomimetic surface molecules can be obtained from the tissue of the intended medical device host organism (also recipient) or an antigen-matched donor thereof or from a culture of tissue from said recipient or donor.
  • the present invention further provides a method for using the biomimetic films for pathogen capture, the method comprising the steps of a. exposing said film to a liquid sample which may or may not contain pathogens or toxins, b. allowing sufficient time for said film to bind to target pathogens or toxins present in said sample; c. optionally separating said film, to which said target pathogens or toxins may be bound, from said sample. d.
  • analyzing said film to detect the presence or type of pathogen or toxin, or subjecting said surface, to which said target pathogens or toxins may be bound, to an agent which effectively separates the pathogens or toxins from the film and subjecting said pathogen or toxins, or media containing such, to analysis techniques to detect presence or type.
  • the above method of use can further comprise the step of calculating the amount of target molecule (pathogen or toxin) present in the sample based on the amount of target molecule bound to the film.
  • the pathogen or toxin can be an agent of disease or disease producer and encompasses any natural or bioengineered disease-producing agent, particularly viruses, bacteria, other microorganisms (for example, but not limited to, amoeba and protozoans), fungi (for example, but not limited to yeast), and toxins (including molecules of both biological, non-biological (natural or synthetic) origin), additionally said toxin can comprise a heavy metal.
  • the analysis step can utilize a detection technology selected from, but not limited to, the group consisting of PCR, immunoassays, DNA microarrays, protein microarrays, spectral analysis, and laser based and other optical techniques.
  • the liquid which may or may not contain pathogens or toxins can be derived from a food product, the processing of a food product or a potable water supply.
  • the liquid which may or may not contain pathogens or toxins can be from an environmental water sample, process water sample, process effluent sample, wastewater sample or is from a water sample from an HVAC or air scrubber system.
  • the liquid sample can be a biological sample selected from the group consisting of blood, serum, saliva, sputum, urine, plasma, cerebrospinal fluid, amniotic fluid, fluids in the gastrointestinal tract, and fluids in the lungs, sweat, breast milk, tears or other lacrimal secretions, pus, and other bodily discharges associated with either normal or diseased conditions. It is also an object of the present invention that said method can be performed in vivo.
  • the liquid which may or may not contain pathogens or toxins can include sample preparation either before and/or after exposure to biomimetic films of the present invention, said sample preparation comprising none or one or more of the following steps: a. Enrichment of the sample with a nutrient compound, b. Incubation of the sample at a temperature between 35 degrees and 40 degrees centigrade for a period of time, c. Incubation in an anaerobic environment.
  • the sample can comprise a continuous fluid stream and the vessel through which said fluid stream sample is passed can have a fluid stream contacting surface which comprises a biomimetic surface of the present invention.
  • the fluid sample which may or may not contain pathogens or toxins, can be subjected to a force which promotes contact between the pathogens or toxins and the biomimetic surface, said force selected from the group consisting of, but not limited to, agitation, mechanical acceleration, centrifugal force, electrical force, magnetic force, and hydraulic force.
  • pathogen and toxin capture constructs described above, as well as similar biocompatible particles constructed with other pathogen and toxin binding molecules, such as antibodies, lectins, and other molecules also have a therapeutic use.
  • molecules when said molecules are immobilized on a substrate which does not degrade in a host organism environment, including for example, wax, and when such constructs are of a size and/or geometric configuration where they are not readily absorbed into the surrounding tissue of the host organism.
  • a minimum nominal diameter greater than something in the range of 50-500 nanometers is considered optimal, though a dosage can comprise a distribution of particle sizes, and it is considered advantageous, but not required, that the average nominal diameter be 500 nanometers or greater.
  • the present invention is directed to a therapeutic biocompatible particle or material (also referred to as a carrier or substrate) in a size and geometric configuration which is not readily absorbed into the biological tissue of the host organism being treated, wherein said biocompatible particle has affixed to its surface molecules which exhibit properties which bind to pathogens.
  • a therapeutic biocompatible particle or material also referred to as a carrier or substrate
  • a carrier or substrate in a size and geometric configuration which is not readily absorbed into the biological tissue of the host organism being treated, wherein said biocompatible particle has affixed to its surface molecules which exhibit properties which bind to pathogens.
  • Illustrative host organism biological tissue include, but are not limited to the walls or membranes (either inside or outside) of the organs of a body, alimentary tract (also gastrointestinal tract), urinary tract, pulmonary tract, blood vessels, amniotic sac, ocular sac, nervous system (for example, but not limited to, the brain or spine), membranes of the musculoskeletal system or cell walls.
  • a plurality of biocompatible particles and associated pharmaceutical formulations thereof can be ingested or introduced into a host for therapeutic benefit resulting from said pathogens binding to the constructs and that the constructs are expelled or removed from the host body with the effect of reducing the concentration of pathogen within the host body.
  • the present invention represents an improvement over anti-adhesion drugs of the prior art, wherein pathogen binding molecules in solution are introduced in an attempt to coat the pathogen and blind all the pathogen's tissue binding sites. In the absence of an immobilizing carrier structure, such molecules are of a size and often a nature where they are readily absorbed through the intestinal (or other system) wall and thus do not achieve the goal of pathogen removal.
  • the binding molecules have to be introduced in high concentrations.
  • the high concentration of said molecules can also have a dehydration effect on the host due to osmotic pressures (i.e., water from surrounding tissues moves through membranes to dilute the concentration of the introduced binding molecules); this dehydration effect can exacerbate stress conditions on the host.
  • the dehydration effect is particularly significant in the case of gastro-intestinal illnesses where dehydration is already an issue.
  • the pathogen binding moiety is immobilized on a substrate which in not absorbed by the tissues of the body and can readily be removed or expelled from the body, only a sufficient number of pathogen adhesin sites need to be engaged to bind the pathogen to the substrate, the pathogen does not need to be completely coated for there to be a therapeutic effect. Additionally, since in the present invention the binding molecules are immobilized on a substrate, they are not lost through absorption into surrounding tissue. Finally, the immobilization of the binding molecules on a substrate also dramatically reduces any osmotic pressure issues and thus treatment with the present invention does not exacerbate dehydration of the host as much as introducing non- immobilized molecules might.
  • the present invention also represents an improvement over the use of antibiotics which can trigger an immediate release of high concentrations of toxin.
  • the use of antibiotic treatment is not recommended in the case of E.coli 0157:H7 infection.
  • toxin-producing bacteria including strains of Vibrio cholerae, Salmonella, Shigella, Clostridium, Helicobacter, cntcropalbo ⁇ cnic I- 1 coh (j PLXX diid eiHefoaggrcgatne Il cob (E ⁇ K 1 )
  • the present invention binds the bacteria in a manner where they can be expelled, rather than stress the bacteria in a manner where toxin production is increased.
  • the present invention also represents an improvement over antibiotic treatment in the ability of the technology to bind and remove both the bacteria producing the toxin, as well as the toxin itself.
  • the molecular moieties represented on the surface of the present invention constitute glycans structures which are natural to the host body (and thus identified by the immune system as "self antigens")
  • the present invention has the potential to avoid adverse immune response effects on or reactions by the host body.
  • the structures of the present invention may have less of a detrimental effect on beneficial bacteria present in the gastro-intestinal or other bodily systems.
  • 5,849,714 Treatment of bacterial dystentery.
  • 5,858,698 Methods for detection of enteropathogenic e. coli.
  • 5,891,860 Treatment of traveller's diarrhea.
  • 5,939,397 Treatment of cholera.
  • 5,955,449 Diagnosis and treatment of bacterial dysentery.
  • 6,224,891 Compounds and methods for the treatment of bacterial dysentery using antibiotics and toxin binding oligosaccharide compositions.
  • 6,262,037 Pharmaceutical compositions for the amelioration of enteropathogenic E. coli infection.
  • 6,291,435 Treatment of diarrhea caused by enteropathogenic Escherichia coli.
  • 6,310,043 Treatment of bacterial infections.
  • 6,358,930 Treatment of C. difficile toxin B associated conditions.
  • 6,465,435 Treatment of C. difficile toxin B associated conditions.
  • Dr. Armstrong, et. al. describe the therapeutic use for gastro-intestinal infections of a monosaccharide or oligosaccharide sequence covalently attached through a non-peptidyl compatible linker arm to an inert substrate, such as diatomaceous earth or silica particles, wherein said monosaccharide or oligosaccharide sequence binds a specific toxin.
  • the techniques and constructs taught by Armstrong, et. al. are applicable to certain aspects of the present invention.
  • the present invention is unique and represents a significant improvement to the above mentioned patents in various areas that will be apparent to those skilled in the art.
  • those unique aspects of the present invention in the field of therapeutic use for treatment of infection in comparison to said patents are: 1.
  • the present invention utilizes glycosylated molecules from potential host organism tissues or their homologues, analogues or functional equivalents. This permits the potential for binding pathogens and toxins by the same mechanisms which permit those entities to demonstrate tissue tropism. This also permits constructs of the present invention to bind pathogens and toxins by multiple mechanisms, even unanticipated or not previously understood ones. Additionally, synthetically produced glycans structures may lack important components or molecular orientations which are inherent in tissue produced glycosylated molecules.
  • the present invention teaches the use of amphiphilic compounds which self-align at the interface between a polar solvent and a non-polar surface to form a film which can incorporate glycosylated moieties at the surface.
  • These self-aligned surfaces can be immobilized on wax or polymer substrates to form economical constructs for therapeutic use from materials which are generally regarded as safe and which can incorporate a broad range of natural and synthetic glycosylated molecules.
  • certain elements of the patents referenced above can be utilized in conjunction with the present invention to provide unique constructs with additional value.
  • the present invention is taught for use in cases beyond the oral treatment of gastro-intestinal infections.
  • the present technology also has utility in areas, such as, but not limited to, the urinary system, ocular fluid, blood and other circulatory fluids, saliva and any other bodily fluid or system where the material of the present invention can be introduced and then extracted.
  • the present invention is also taught for introduction into other areas of infections through other means, such as through catheters or surgically.
  • the present invention is also taught for the removal of pathogens and toxins from bodily fluids which are extracted from and then returned into the source body.
  • the present invention also teaches unique methods for extraction of glycosylated molecules from solutions, and particularly tissues and bodily fluids, for use in pathogen and toxin capture constructs.
  • a solid state biocompatible film or particle comprised of a substrate material having a homogenous or heterogeneous population of pathogen-binding molecules affixed thereto, the substrate material which is of a physical size and shape such that it can be readily introduced into a host through ingestion or the use of a device, such as a catheter but also such that the resulting film or particle is not readily absorbed by surrounding tissue.
  • the pathogen binding moieties are composed of glycans or glycan sequences, natural or synthetic, which mimic pathogen binding sites in the host body. Such pathogen binding moieties may be harvested or derived for a target tissue of interest
  • illustrative glycan sequences of interest include, but are not limited to those expressed in glycoproteins (eukaryotic glycoproteins, proteoglycans, glycomucins) or glycolipids (glycosphingolipids), including natural, synthetic, and recombinant versions thereof as well as homologues, analogues, and functional equivalents thereto.
  • glycoproteins eukaryotic glycoproteins, proteoglycans, glycomucins
  • glycolipids glycosphingolipids
  • the pathogen binding moieties further include, but are not limited to, glycosylated molecules harvested from biological tissue, including, but not limited to, biological tissue extracted from human, animal or plant tissue, a culture of human, animal or plant tissue, a biological surrogate for human, animal or plant tissue, a culture of a biological surrogate for human, animal or plant tissue or recombinant versions of human, animal or plant tissue or human, animal or plant tissue biological surrogates.
  • suitable biological tissues include, but are not
  • glycosylated molecules include but are not limited to those expressing the following moieties:
  • Streptococcus suis This same structure has been found to be important in the binding of bacterial enterotoxins, such as verotoxin and Shiga- like toxin 1 (Stxl), both from E. coli strains, and enterotoxin B from Staphylococcus aureus; 11
  • Ibld Surface molecules of the present invention may also include one or more known pathogen target sequences, examples of which are set forth in Appendix A.
  • the pathogen binding molecules may be moieties derived from or representative of potential binding sites on the tissue of a target host by a pathogen.
  • the pathogen binding glycosylated molecule is not limited to those that mimic endogenous pathogen binding structures.
  • any number of glycosylated molecules may be utilized as the pathogen binding moiety, including antibodies and products of a phage display process.
  • particles of the present invention may comprise more complex mixtures, incorporating more than one type of binding moiety, for example a phage display product for binding bacterial spores, a tissue biomimetic for binding bacteria expressing pili and a toxin binding moiety for binding bacterial toxins.
  • pathogen binding molecules suitable for use in the instant invention include, but are not limited to, sialic acid in any of its various forms; glycosylated molecules present in pigeon eggs; antibodies, particularly those expressing a degree of specificity for spores of bacteria; lectins; lactoferrin (also lactotransferrin) and functional mimics or analogues thereof. These examples are described in further detail below.
  • Anti-adhesion drugs represent a class of therapeutics in which molecules presenting specific glycan structures are introduced into an infected host body to preferentially bind to adhesins on the pathogens and consequently interfere with the pathogen's ability to bind to host tissue. 14
  • the same molecules which are used in the fabrication of anti-adhesion drugs are also of value in the context of the present invention, in the construction of ingestible biomimetic films and particles for pathogen immobilization and removal.
  • glycodendrimers which have been reported as useful as anti-adhesion drugs, are of interest in pathogen capture in the context of the present invention because of reported increased affinity of target pathogens for these constructs.
  • the glycodendrimers in the Krippner constructs can possess biomimetic properties and the constructs can be utilized in novel ways in the formation of the ingestible biomimetic films and particles of the present invention.
  • Glycodendrimer and protein constructs also known as
  • glycodendrimers 16 Yiwen Li, Yiyun Cheng, Tongwen Xu, Design, synthesis and potent pharmaceutical applications of glycodendrimers: a mini review. Curr Drug Discov Technol. Dec. 4, 2007 (4):246-54 glycodendriproteins, developed by Rendle, et. al. at the University of Oxford, have been constructed in a manner which has been shown to have inhibitory effects on pathogens. 17 Accordingly, both glycodendrimers and glycodendriproteins may be used in the construction of the ingestible biomimetic films and particles of the present invention. Antibodies are also of interest in the therapeutic aspects of the present invention due to their pathogen binding properties and the potential to immobilize them using techniques taught herein.
  • synthetically produced molecules, molecules from biological surrogates and molecules produced as a result of genetic (also recombinant) engineering of organisms and molecules may find utility in the context pathogen binding.
  • One example of such technology is in the use of recombinant Factor C peptide as disclosed in United States Patent 6,719,973 by Jeak L Ding, et.al., the entire contents of which are incorporated herein by reference. Ding describe the use of a recombinant peptide to induce bacteriostasis by binding of the lipopolysaccharide (LPS or endotoxin) generated by gram-negative bacteria with the LPS present either in the cell membrane or as a liberated molecule.
  • Pathogen binding can also be achieved through the use of lectins (also known as
  • agglutinins The therapeutic use of lectins in the binding of pathogen receptors to diminish the pathogen's ability to bind to a host is described by Krivan, et. al. in U.S. Patent Application Serial Nos. 11/413826, 10/654,104, 10/097,409, 10/038,645, 08/861,596, 08/640,693, and 08/385,306, all of which are incorporated herein by reference in their entirety.
  • Krivan, et. al. describe the use of a broad range of lectins to blind receptors for a range of pathogens and thereby prevent binding and subsequent infection. Accordingly, the present invention contemplates the use of Krivian's blinding lectins for pathogen binding in the construction of the ingestible biomimetic films and particles of the present invention.
  • Lactoferrin also known as lactotransferrin also has pathogen binding properties of interest. 18192021 Studies have shown that some pathogens exhibit host tropism based on lactoferrin properties specific to a single host species. 22 A method for immobilization of
  • Enteropathogenic Escherichia coli EEC
  • EHEC enterohemorrhagic Escherichia coli
  • Both EPEC and EHEC are thought to initiate attachment to the host cells through carbohydrate recognition/binding of glycolipids expressed by the intestinal mucosal cells.
  • Biomimetics of the mucosal cell carbohydrates and specifically these glycolipids (glycosphingolipids) are useful in the context of the biomimetic films and particles of the present invention.
  • the bacteria follow the initial binding with injection of species specific molecules, translocated intimin receptors (tir), into the cytoplasmic membrane of the epithelial cells.
  • Tir is an enabling protein that initiates a complex change in the epithelium which results in the creation of attaching and effacing (A/E) lesions and ultimately the expression of trans-membrane molecules to which the bacteria initiate a very secure intimin-based binding.
  • A/E lesions are typical of, but not limited to, EPEC and EHEC infections of the intestinal epithelium and the surface moieties include bacterial-initiated binding sites, and are known by those knowledgeable in the science of translocated intimin receptors (tir).
  • the surface moieties of attaching and effacing (A/E) lesions may be harvested, for example by rupturing the cell membranes.
  • synthetic equivalents of such surface expressing moieties may be synthetically derived, for example by means of phage display to develop an intimin recognition product.
  • the resulting transmembrane and other surface expressing molecules or their synthetic equivalents may then be immobilized on a solid state support or substrate for use in the construction of the pathogen binding biomimetic films and particles of the present invention.
  • Trans-membrane molecules tend to be amphiphilic in nature and, as discussed in greater detail below, that feature can be utilized effectively in the context of the present invention for the creation of a pathogen-binding surface.
  • the constructs of the present invention may include a number of divergent pathogen binding moieties as well as therapeutic molecules with other therapeutic purposes, for example, but not intended as limiting, a heterogeneous mixture of amphiphilic molecules or conjugates with pathogen and/or toxin binding properties is present in the film in a manner which provides for multiple pathogen and/or toxin binding mechanisms or moieties.
  • the resulting construct can provide functionality beyond what can be achieved with a homogeneous population.
  • the construct contains a mixture of amphiphilic molecules
  • this mixture can be adjusted in a manner which effectively controls the surface density of a specific biomimetic molecule due to the presence of other displacing material in the surface film.
  • the other amphiphilic molecules can be selected so as the mixture forms a more stable amphiphilic film than one of just biomimetic molecules.
  • therapeutic particle preferably has a minimum nominal diameter sufficient to inhibit absorption of a majority of the particles by the surrounding tissue; in some instances, such particles may have a minimum nominal diameter of greater than 20 nanometers, in other instances a minimum nominal diameter of 50 nanometers or greater may be required, in other instances where inhibiting absorption is of particular concern, a preferred minimum nominal diameter of 500 nanometers or greater may be desired.
  • the maximum particle size is based on the constraint of what can be effectively delivered by ingestion or introduction through a catheter, however efficiency as measured by effective surface area to volume of substrate material decreases with increased particle size for solid particles (porous particles, which are also within the scope of the present invention, have a more complex relationship between particle size and effective surface area), and accordingly, smaller particle sizes may be advantageous.
  • the therapeutic biocompatible particles of the present invention may take the form of fibers or worm micelles (also nanowires) with or without embedded substrate materials. Worm micelles can have diameters as small as the length of two molecules as long as the effective length is sufficient to prevent absorption into the surrounding tissue.
  • the fibers or worm micelles can be part of a construct of entangled fiber-like particles which in concert provide an effective diameter or configuration to impede absorption into surrounding tissue.
  • the particles can be in the form of a free-standing film, so long as at least one dimension is sufficient to prevent absorption of a majority of the particles by the surrounding tissue.
  • the substrate for the construction of the therapeutic biocompatible particle can be of any material which will not substantially degrade in form or ability to immobilize the surface moieties when subjected to the environment present in the host. It is further an object of the present invention that the substrate can consist of natural or synthetic polymers, natural or synthetic waxes, ceramics, metals, materials of biological origin or combinations thereof.
  • the substrate may also be a vesicle or porous in nature, so long as the overall dimension of the particle remains such that the majority of particles will not be absorbed by the surrounding tissue.
  • a porous substrate or vesicle allows for inclusion of other materials in the substrate which are intended to be released in the host which have therapeutic benefit in concert with or independent of the substrate and substrate surface molecules of the present invention.
  • the present invention is not limited to a particular construction method. However, the following methods are suitable for constructing biocompatible particles of the present invention and are therefore provided for illustration purposes.
  • An illustrative method for making a biomimetic films and biocompatible particles of the present invention suitable for therapeutic use comprises the following steps: a. provide a relatively polar solvent having sufficient quantities dissolved therein of one or more amphiphilic molecules which include glycosylated molecules, or conjugates of such, or molecules which can be conjugated with glycosylated molecules, or molecules with other pathogen or toxin binding moieties; b. expose the polar solvent to a relatively non-polar liquid, the non-polar liquid being immiscible in the polar solvent; the relatively non-polar liquid can optionally comprise a material which can reversibly experience a state change from solid to a relatively non-polar liquid under stimulus; c.
  • the amphiphilic molecules can align so as to form a membrane which separates the polar solvent from the non-polar liquid; d. induce or allow transformation of the non-polar liquid to a corresponding non- polar solid having upper and lower surfaces, wherein the hydrophobic tail ends of the amphiphilic molecule are embedded in or chemically, electrically or mechanically linked to the upper surface of the non-polar substrate and the hydrophilic tail ends project from the upper surface into the polar solvent so as to yield a film having functional activity; optionally, prior to or during transition of the non-polar liquid to a solid state the substrate and film can be manipulated in order to yield a film having a geometric form, said manipulation can include, but not be limited to agitation so as to form micelles, worm micelles or micelle-like constructs; the transformation of the non-polar liquid to a corresponding non-polar solid can be performed in a manner so as to maintain the approximate geometric form achieved by the non-polar liquid prior to or during the transition to a solid form, for example, said transformation can
  • adsorb or conjugate molecules with pathogen and/or toxin binding moieties or other properties to the functional surface of the film if the desired functionality is not present from the preceding steps.
  • An alternate method for making the biomimetic films, particles, and constructs thereof comprising the following steps: a. provide a liquid having sufficient quantities dissolved therein of one or more glycosylated or other molecules with pathogen or toxin binding properties; b. expose said liquid to a solid with surface moieties appropriate for conjugation with said molecules; c. allow or induce the conjugation of said molecules to said surface moieties in a manner which results in the pathogen or toxin binding moieties bound on the surface of the substrate, d. Optionally, remove said film from any surrounding liquid to form a dry construct.
  • An illustrative method for using the biomimetic films, particles, and constructs thereof may comprise the following steps: a.
  • Another method of use for the biomimetic films, particles, and constructs thereof comprising the following steps: a. Extract a portion of bodily fluid of a suspected host to a pathogen or toxin infection, including, but not limited to amniotic fluid, aqueous or vitreous humour, blood and blood plasma, bone marrow fluids, cerebrospinal fluid, interstitial fluid, lymph fluids and pleural fluid b. Induce contact of said bodily fluid to one or more surfaces with the pathogen and toxin binding moieties of the present invention. c. Remove said surfaces from contact with said bodily fluid. d. Optionally, reintroduce said bodily fluid with pathogens/toxins removed into the organism. e.
  • the present invention relates to the creation of therapeutic materials which bind to pathogens and toxins and facilitate their removal and expulsion from an infected body.
  • therapeutic materials which bind to pathogens and toxins and facilitate their removal and expulsion from an infected body.
  • particles of a biocompatible nature which are large enough (or have appropriate geometric configuration) so as not to be absorbed into surrounding tissue at the point of therapeutic application.
  • the minimum particle size to avoid adsorption is dependent in part on the properties of the surrounding tissues. It is the object of the present invention that the minimum particle size of constructs of the present invention for therapeutic use be such that a majority of particles are not absorbed into surrounding tissue.
  • constructs such as nano-thickness films and worm micelles 29 (also nanofibers) are of use in the constructs of the present invention. Worm micelle constructs which are interwoven or entangled in a random manner can form aggregate structures (also hydrogels) with properties of larger constructs than the individual worm micelles.
  • aggregate structures can also have the appearance of tangled balls of yarn and in some cases these aggregate structures can appear similar to some depictions of casein micelles, but this analogy is not meant to be limiting.
  • Aggregate structures of worm micelles are of value in the constructs of the present invention since the resultant structure can have the equivalent shape and size of a particulate construct of sufficient size so as not to be absorbed into surrounding tissue even though the individual components might not be that large.
  • One preferred embodiment of the therapeutic biocompatible particle is the micelle, more particularly a wax or worm micelle as discussed above.
  • a micelle of the present invention is the micelle, more particularly a wax or worm micelle as discussed above.
  • glycosylated films may be formed from the glycosylated films previously described, preferably having a sufficient size and/or a geometric configuration so as not to be readily absorbed into biological tissue of an organism, including but not limited to the walls or membranes (either inside or outside) of the organs of a body, alimentary tract (also gastrointestinal tract), urinary tract, pulmonary tract, blood vessels, amniotic sac, ocular sac, nervous system (for example, but not limited to, the brain or spine), membranes of the musculoskeletal system or cell walls;
  • Suitable glycosylated pathogen binding moieties include, but are not limited to lactosylceramide structures, which are thought to be important in the binding of EHEC to human tissues; Gal ⁇ l-4Gal ⁇ l-4Glc, which has been shown to be important in binding of Shiga-like toxin 2 (Stx2); GaIa(I -4)Gal ⁇ (l-4)GlcNAc, which has been shown to
  • the glycosylated molecules are themselves amphiphilic in nature or conjugated to hydrophobic moieties to form an amphiphilic molecule. In this manner, these molecules can self-align through hydrophobic interaction to form films and structures of the present invention, particularly in conjunction with non-polar or less-polar surfaces.
  • the conjugates can include synthetic moieties, such as the products of chemical synthesis to produce combinations of molecules in a predetermined structure. Said conjugates can also include moieties known for their binding properties, such as block copolymers, or more specifically polyethylene glycol (PEG), or natural binding molecules, such as heparin or its derivatives.
  • substrates that are relatively non-polar material, with particular value in materials that can transition from a relatively non-polar liquid or gel to a solid, often crystalline structure.
  • substrate materials of value in the context of the present invention include, but are not limited to natural wax and synthetic wax, natural and synthetic plastics, polymers or hydrocarbon mixtures.
  • substrate materials comprising natural and synthetic fats and resins are also of interest as substrate materials in the present invention.
  • such constructs of film and substrate can form a micelle or micelle-like structure, wherein the non- polar substrate and hydrophobic tail ends are sequestered in the interior of the micelle while the hydrophilic head ends are present on the outer surface of the micelle.
  • the glycosylated molecules presented by the wax micelle may consist of a heterogeneous population of amphiphilic molecules or conjugates such that the combination of different amphiphilic molecules or conjugates in a surface provides functionality beyond what can be achieved with a homogeneous population. Further, said heterogeneous population of amphiphilic molecules: a.
  • b. can contain a mixture of amphiphilic molecules in a manner which effectively controls the surface density of a specific glycosylated molecule due to the presence of other displacing material in the surface film
  • c. can contain a heterogeneous mixture of amphiphilic molecules or conjugates with pathogen and/or toxin binding properties present in the film in a manner which provides for multiple pathogen and/or toxin binding mechanisms or moieties.
  • Another preferred embodiment of the therapeutic biomimetic film is the worm micelle.
  • the design and construction as well as the delivery the worm micelles of the present invention is analogous to that set forth above.
  • biocompatible pathogen binding particles and associated pharmaceutical formulations thereof be designed for use, recommended for use, or administered to the intended host in a manner consistent with the first dose being provided to the host organism before the pathogen or toxin has been identified.
  • This use is in contrast with the therapeutic use of antibiotics in, for example, the case of a gastro-intestinal infection, wherein it is recommended that the bacteria be identified before antibiotics are administered, to avoid the chance of increased bacterial release of toxins.
  • the particles and formulations of the present invention can be provided to a host organism at any point in the cycle of an infection.
  • the particles may be formulated as a pharmaceutical composition suitable for administration to human or animal subjects in need thereof.
  • Preferred subjects are those having a complex gastro-intestinal system, including but not limited to humans, livestock, for example hoofed animals, poultry and fish, domestic pets and animals at zoos and game parks, and wild species.
  • a suitable therapeutic dose may include a plurality of biocompatible particles, those particles can include a range of sizes, shapes, substrate materials, and types of surface molecules.
  • a pharmaceutical composition of the present invention can be formulated in a liquid or gel matrix, which may optionally include other molecules of therapeutic, nutritional or psychological (such as those which provide taste or texture properties) benefit.
  • Other molecules of therapeutic benefit include materials designed to facilitate ingestion of the matrix, including such that might reduce the possibility of vomiting.
  • It may also be desirable to include as well as agents known to be of value in oral rehydration therapy (also ORT), such as oral rehydration salts or solutions and electrolytes, to synergistically treat the dehydration that is often a component of gastrointestinal infections.
  • ORT oral rehydration therapy
  • sources of iron with the formulation, as the production of toxin in enterohemorrhagic E.coli is thought to be enhanced under iron deficient conditions.
  • the formulation may also include antibiotics or probiotics.
  • the additional therapeutic agents noted above can either be formulated with the therapeutic particles of the present invention, administered in a delivery matrix or administered as a separate dose.
  • the benefit of simultaneous treatment both with the present invention and with antibiotics is that the present invention has the potential to capture some of the additional toxins which might be expelled by the pathogens in response to the antibiotic treatment. This may allow destruction of some of the bacteria by antibiotics without the resulting short-term rise in toxin production causing unacceptable stress on the host's tissues.
  • the biocompatible particles and subsequent pharmaceutical formulations thereof may be administered in any conventional manner, including oral delivery, percutaneous administration, intravenous administration, intranasal administration and the like.
  • the particles are formulated for ingestion, although introduction through a medical device, such as a catheter or hypodermic needle, or introduction during a surgical procedure, or as an irrigation therapy procedure such as might be used to flush pathogens or toxins from an organ or bodily system, is also contemplated.
  • the therapeutic pathogen binding particles of the present invention can be introduced into a biological organism, with or without other molecules and in a liquid or solid form, and subsequently removed or expelled from said biological organism.
  • the particles of the present invention can be introduced into a biological organism, with or without other molecules and in a liquid or solid form, and subsequently removed or expelled from said biological organism.
  • inventions can be optionally be recovered after expulsion or removal from the host and subsequently subjected to diagnostic techniques known by those skilled in the art to detect the presence and optionally the type of pathogen bound to the particle as part of a medical diagnostic procedure.
  • diagnostic techniques known by those skilled in the art to detect the presence and optionally the type of pathogen bound to the particle as part of a medical diagnostic procedure.
  • indicator molecules detectable by medical imaging techniques, to provide indication of presence of particles of the present invention in a treated the host.
  • Such indicator molecules may be provided on the substrate itself or conjugated to the therapeutic particle.
  • Indicator molecules suitable for indicating presence, location or density of constructs of the present invention within a biological organism include, but are not limited to, a fluorescent label, a radioactive label, a dye and a compound which enhances magnetic resonance imaging. Labels which are detectable by external instruments, such as a radioactive label, could permit identification of areas in the host organism where constructs of the present invention are bound to pathogens which are securely bound to tissue in the host.
  • the particles and formulations of the present invention can be produced and stored in a dried form for later rehydration.
  • the particles may further be provided with a solid matrix which provides benefits for transport and storage, further wherein the matrix may include other molecules of therapeutic, nutritional or psychological benefit (similar to those projected for liquid matrices).
  • the particles of the present invention can also be provided with an exterior coating layer that protects the particles of the present invention from the environment for a particular period of time or until a specific area of the host is encountered, for example as would be the case if particles of the present invention are enclosed in a capsule, tablet or other matrix to be ingested.
  • an exterior coating layer that protects the particles of the present invention from the environment for a particular period of time or until a specific area of the host is encountered, for example as would be the case if particles of the present invention are enclosed in a capsule, tablet or other matrix to be ingested.
  • Formulation and time release delivery of materials of therapeutic value through means of a protective capsule is well known by those skilled in the art, and the utilization of such techniques are an object here.
  • An illustrative treatment method suitable for use with the therapeutic particles and formulations of the present invention may include the following steps:
  • An alternative therapeutic treatment comprises the steps of:
  • Another alternative method for therapeutic treatment or diagnostic using the particles of the present invention is as follows: 1. Extraction of a bodily fluid of an organism, including, but not limited to bone marrow fluids, amniotic fluid, aqueous or vitreous humour, blood and blood plasma, interstitial fluid, lymph fluids and pleural fluid 2. Inducing contact of said bodily fluid to one or more surfaces with the pathogen and toxin binding moieties of the present invention. 3. Optionally, reintroducing of the bodily fluid into the organism.
  • the present invention further relates to are glycosylated amphiphilic molecules composed of hydrophobic "tails” and hydrophilic "heads” that self align to form a membrane at the interface of a polar solvent and a non-polar liquid or a solid.
  • the present invention is directed to a solid state membrane, typically a thin film, composed of a non-polar solid material having the hydrophobic "tail” of a glycosylated amphiphilic molecule embedded in or linked to its surface such that the hydrophilic "head” protrudes from the solid surface and presents useful properties to the surrounding environment.
  • a membrane or film in accordance with the present invention is produced when a non-polar liquid, in the presence of a polar solvent and an amphiphilic biological compound, undergoes a transformation from liquid to solid, through thermal, chemical or radiative means, with the resultant effect that the amphiphilic molecule is affixed or "locked" to the surface, more particularly the hydrophobic ends of the amphiphilic compounds are mechanically or chemically linked to or embedded in the non-polar solid.
  • the membrane and micelles produced therefrom remain stable even in the absence of the polar solvent, thereby allowing the hydrophilic components of the amphiphilic compounds to present useful properties at the surface thereof.
  • This aspect of the present invention arose with the discovery that many amphiphilic molecules, particularly glycolipids and glycoproteins endogenous to plant and animal tissue fluids, spontaneously aggregate at the interface between certain aqueous solutions and certain non-polar liquids to form a flexible membrane.
  • the lipophilic components of the molecules adhere, through non-covalent hydrophobic interactions, to molecules of oil or other non-polar liquids and present their hydrophilic moieties toward the aqueous phase to produce a membrane, which, when agitated (e.g., rolled, shaken, or forced through a filter), stretches and breaks into closed vesicles or micelles.
  • the glycoprotein membrane and the micelles produced by association of non-polar liquids with glycoproteins are mechanically and chemically more stable than those formed from phospholipids.
  • the polar surfaces of the glycoprotein micelles do not fuse easily and are quite stable in aqueous media. They are able to retain their shape and hold their contents and are very resistant to destruction. For example, they can be selectively extracted using physical means such as separatory funnel or syringe which, in turn, enables the creation of micelle populations that are relatively uniform in size.
  • the glycoprotein micelles may be used as carriers for substances that are not, when used alone, capable of forming relatively stable micelles (e.g., lipids, lipophilic, and lipid-like moieties).
  • the Mullen micelles can be kept for months at room temperature in sterile aqueous solution and indeed in some instances can, under very restrictive conditions, be dried and subsequently rehydrated.
  • the dried membranes tend to lose their elasticity and are quite fragile. If fact, when the aqueous solvent is evaporated, only a diaphanous, brittle monolayer of glycoprotein remains. It is accordingly an object of the present invention to provide a means for stabilizing the micelle membrane, so as to facilitate the long term storage and convenient transport of both membranes and micelles formed therefrom.
  • the present invention utilizes a non- polar material, first in a liquid form to first initiate membrane formation, then, in a solid form in which the hydrophobic "tails" of the amphiphilic molecule are embedded in or linked to its surface while the hydrophilic "heads” protrude therefrom so as to present useful properties to the surrounding environment.
  • the transformation from liquid to solid either through thermal, chemical or radiative means, results in "locking" of the amphiphilic molecule to non-polar material.
  • the hydrophobic ends of the amphiphilic compounds are affixed to the surface of the solid, either through mechanical, chemical or physical means. The resulting membrane or film remains stable even if the polar solvent is removed, thereby allowing the hydrophilic components of the amphiphilic compounds to present useful properties at the surface of the solid material.
  • the amphiphilic molecules that form the surface of the membranes and micelles of the present invention can vary in form and source.
  • the amphiphilic molecules need only to act as surfactants in the presence of the selected polar solvent and the fluid state of the non- polar substrate, be it wax, plastic, fat, polymer or other hydrocarbon.
  • the selected polar solvent and the fluid state of the non- polar substrate be it wax, plastic, fat, polymer or other hydrocarbon.
  • This concentration is known as the critical micelle concentrations (CMC).
  • the substrate material can also vary greatly.
  • the substrate needs to be non-polar (or sufficiently non-polar) in the fluid form to permit formation of micelle or micelle-like alignment of the amphiphilic compounds at the interface with the polar solvent.
  • the solid material Upon solidification, the solid material must provide a mechanical or chemical bond such that the amphiphilic material remains oriented with the hydrophilic end presented to the surrounding environment and thus exhibiting useful properties.
  • the solid or solid- film material may be allowed to solidify without agitation, resulting in a relatively flat or contour conforming surface.
  • the non-polar material for example a wax or plastic polymer
  • the non-polar material when in liquid form is agitated in the polar solvent in the presence of an amphiphilic compound at the proper concentration and temperature, then micelle structures spontaneously will form. These structures can be preserved in the transition of the non-polar materials from liquid to a solid state.
  • the size of the solid micelle-like structures can be influenced during formation through the degree of agitation, concentration of amphiphilic compound and selection of amphiphilic compound.
  • a typical method for a making a glycosylated amphiphilic membrane or film of the present invention generally includes the following steps:
  • the polar solvent may optionally be removed so as to yield a dry solid state film.
  • the structures of the present invention have been shown to have a level of stability after removal of the non-polar solvent. This is considered a very useful property in allowing preservation of the material of the invention. Lack of moisture will decrease the likelihood of biological attack during storage. Additionally, removal of moisture allows for storage of the material at reduced temperature without the risk of ice crystals damaging the biological compounds.
  • Fluid micelles and micelle-like structures have an inherent problem of reduced dimensional stability when exposed to high-shear conditions such as in a turbulent non-polar solvent flow. The solid micelles and micelle-like structures of this invention are more stable in those conditions and thus are more suited to applications involving turbulent flow, for example in the context of environmental filters.
  • the biomimetic films and particles, especially the micelle particles, of the present invention have a wide range of utilities, ranging from environmental sensing to pathogen capture and therapeutic applications.
  • the membranes of the present invention are particularly useful for extracting target molecules, for example, contaminating substances such as pathogens, toxins, and the like, from a particular sample based on tissue tropism.
  • Selectivity based on tissue tropism provides a significant advantage over current selectivity filters which are either very specific to a particular serotype of bacteria, where only bacteria which are anticipated can be found, or very general as with a sized-based filter, where potentially harmful bacteria could get lost in the biological clutter.
  • Selectivity based on tissue tropism permits capture of bacteria and toxins which have the potential to bind to the host tissue and thus at least have the potential to represent an infectious agent. This selectivity permits the present invention to potentially capture unexpected, emerging or engineered pathogens, providing a powerful tool in the early detection which is so critical in containing the spread of communicable disease.
  • constructs of the present invention can be utilized for therapeutic treatment in the early stages of an infection. This is particularly beneficial in the case of toxin producing gastrointestinal infections in which the use of antibiotics in contraindicated.
  • Constructs of the present invention can be administered as an oral dose, bind to free pathogens or toxins in the intestines and subsequently be expelled by the body. When a substrate such as wax is utilized, this material is generally regarded as safe and is usually considered environmentally benign.
  • a typical method for extracting a target molecule of interest from a particular sample using a glycosylated amphiphilic membrane or film of the present invention generally includes the following steps:
  • a force which promotes contact between the target molecule and the membrane include, but are not limited to, mechanical acceleration, centrifugal force, electrical force, magnetic force, hydraulic force and various other means for agitation.
  • the membrane may then be analyzed for the presence of target molecule using any number of commercially available detection and measurement technologies (e.g., PCR, immunoassays, DNA microarrays, protein microarrays, etc.).
  • the amount of target molecule bound to the membrane may be used to estimate the amount of target molecule present in the sample.
  • Such levels can also be used to diagnose the presence of a disease condition (e.g., a bacterial infection, cancer, etc.)
  • the instant technology also finds utility as an alternative to Hemagglutination, wherein pathogens are detected through their ability to bind to multiple red blood cells at once, resulting in visible agglutination of the cells.
  • the films and particles of the instant invention find application in a number of divergent environments, the structure and form of the inventive film is not particularly limited.
  • the films of the present invention may take the form of a biological or environmental filter or sensor, such as a microarray or biochip sensor having specificity (via the functional ends of the embedded glycosylated amphiphile) for one or more proteins, antibodies, tissues, or chemical substances.
  • biomimetic film of the present invention having binding specificity for one or more target molecules, for example glycoproteins exhibiting sugars specific to a particular class of pathogens, with the intent of placing a liquid sample of interest in the pipe in contact with said membranes.
  • target molecules for example glycoproteins exhibiting sugars specific to a particular class of pathogens
  • the biomimetic films of the present invention surface-embedded or associated with glycosylated amphiphiles, find utility both as singular solid state materials and as film coatings on other materials or for other devices.
  • the "other" material on which the biomimetic film is disposed may serve as structural or geometric support or, alternatively, may address thermal or density issues.
  • the "other" material may comprise a useful device or implement, for example, a sample containing vessel, pipe, tube, or the like.
  • a glycosylated amphiphilic membrane of the present invention may be anchored or adhered to a stream contacting surface of a vessel through which the fluid stream sample is passed.
  • the useful implement may comprise a medical device that would benefit from biological properties displayed by the glycosylated amphiphilic membrane of the present invention.
  • Medical devices particularly implantable medical devices, often fail as a result of a biological organism's reaction to its introduction.
  • the reaction arises from the identification of the introduced device as “foreign” and involves subsequent protective attempts by the organism's immune system to remove, sequester or destroy the perceived injurious stimuli and initiate the healing process.
  • An example of such a reaction is transplant rejection, which occurs when the immune system of a recipient of a transplant attacks the transplanted organ or tissue.
  • non-specific binding (NSB) of proteins at medical implant surfaces is believed to be at least partially responsible for triggering the foreign body response, which in turn can lead to device failure or rejection. This "biofouling" is also blamed for device infection incidence, thrombosis, and sensor deterioration over time in vivo.
  • solid state membranes expressing such endogenous glycosylated amphiphiles will mimic endogenous tissues and thus be recognized by the immune system as “self rather than “foreign”.
  • Such "biomimetic" membranes are particularly useful as antigenicity reducing coatings for medical devices.
  • a biomimetic film of the present invention using a glycosylated amphiphile isolated, extracted, harvested or otherwise derived from an intended recipient or, alternatively, an antigen-matched donor.
  • an ideal donor would be an HLA-identical sibling
  • alternative donors include an HLA-phenotypically matched unrelated donor (MUD), a partially mismatched related donor (PMRD) or a cord blood donor (CBD), who can be a phenotypically matched or mismatched related or unrelated donor.
  • Medical devices that would benefit from such antigenicity reducing coatings include both temporary implants (i.e., devices intended for limited introduction, for example angioplasty catheters) and more permanent implants (i.e., devices intended for long term insertion, for example cardiac pacemakers).
  • medical devices contemplated by the instant invention include, but are not limited to, needles, catheters (e.g., intravenous, urinary, and vascular catheters), stents, shunts (e.g., hydrocephalus shunts, dialysis grafts), tubes (e.g., myringotomy tubes, tympanostomy tubes), implants (e.g., breast implants, intraocular lens), prosthetics, and artificial organs, as well as cables, leads, wires, and electrodes associated therewith (e.g., leads for pace makers and implantable defibrillators, bipolar and monopolar RF electrodes, vascular guidewires).
  • Also contemplated are devices such as wound dressings, sutures, staples, anastomosis devices, vertebral disks, bone pins, suture anchors, hemostatic barriers, clamps, screws, plates, clips, vascular implants, tissue adhesives and sealants, tissue scaffolds, various types of dressings, bone substitutes, intraluminal devices, vascular supports, and other body contacting devices that may benefit from enhanced biocompatibility.
  • the solid state films and membranes of the instant invention may possess analytic, diagnostic and/or therapeutic utility.
  • the glycosylated amphiphilic molecule selected may have a binding specificity for a target molecule of biological or medical interest.
  • Glycosylated amphiphilic membranes so fabricated can be used to capture and extract such materials, either in vivo or ex vivo.
  • target molecules of biological or medical interest include, but are not limited to, biological entities such as hormones, proteins, nucleic acid molecules, (e.g., RNA and/or DNA), circulating cells, and particulate analytes of biological origin that serve as diagnostic indicia or biomarkers of disease, for example cancer.
  • biological entities such as hormones, proteins, nucleic acid molecules, (e.g., RNA and/or DNA), circulating cells, and particulate analytes of biological origin that serve as diagnostic indicia or biomarkers of disease, for example cancer.
  • foreign materials particularly biohazardous materials, such as contaminants, drugs, toxins, heavy metals, pathogens and the like.
  • the glycosylated amphiphilic membranes of the present invention can serve not only to detect the presence of the target molecule but also effectively remove it from (i.e., detoxify) the sample.
  • the target molecule of interest is a heavy metal, such as lead, mercury, copper, or the like
  • the selected glycosylated amphiphile may act as a chelating agent that binds the heavy metal and effectively eliminates it from circulation.
  • the inventive biomimetic films may take the form of a filter for capturing target molecules that may be present in an environmental sample, such an entering or exiting water source (e.g., an agricultural feed trough or runoff ground water).
  • an entering or exiting water source e.g., an agricultural feed trough or runoff ground water.
  • target molecules present in the source become bound to the hydrophilic ends of the glycosylated amphiphiles. In this manner, the target molecules can effectively removed from the source and safely transported to a laboratory setting.
  • the filters may be provided with a means for real-time detection and measurement of target molecule binding.
  • Real-time detection may involve analysis of a change in a readily measurable parameter, for example changes in optical spectra (e.g., fluorescence, color change), electrical activity, electrical field (e.g., conductance), or magnetic field.
  • optical spectra e.g., fluorescence, color change
  • electrical activity e.g., conductance
  • magnetic field e.g., magnetic field
  • a technician may opt to either assess the target molecule in situ, while bound to the membrane or, alternatively, may opt to unbind it from the filter, for example with the help of a lysing solution or the like.
  • the lysing solution is likely to effectively kill the pathogen, thereby eliminating the dangers associated with conventional detection methods that utilize live pathogens.
  • Porcine small intestine was treated in a tissue disruptor and then sonicated to release the membrane bound glycosylated compound.
  • the resultant solution was sterile filtered.
  • the glycoprotein rich filtrate was heated along with food grade paraffin wax to 60 degrees Celsius.
  • the resulting mixture was agitated and then pumped through a zone excited by sonication.
  • the mixture was then sprayed through a small orifice into a continuous stream of cold water.
  • the resulting particles in the 50nm to lOOOnm diameter size range were concentrated and washed.
  • the particles were then added to an LB broth solution with active Salmonella Montevideo present and rotated for 20 minutes at 37 degrees Celsius.
  • the particles were removed from the LB broth solution and then rinsed.
  • One sample was stained and observed under a microscope.
  • Salmonella were observed bound to the surface of the particles.
  • a second sample was processed for PCR analysis techniques known to those skilled in the art. The wax was easily removed early in the process since when it melted, it separated to the top of the vessel, and then upon cooling the wax was easily removed, and the bacterial remnants of lysing remained in the liquid below. The PCR analysis gave positive results for Salmonella.
  • porcine small intestine wax micelles were demonstrated for research and analytical use in this case.
  • the particles were created with material which is generally regarded as safe, and consequently would also be suitable in the context of an ingestible biomimetic particle, for as an oral therapy for pathogens that would bind to the small intestine.
  • Porcine intestine is used as an analogue for human intestine in this case.
  • Example 2 Bovine Serum Albumin (BSA) in solution was heated to 55 degrees Celsius. Food grade paraffin wax, also at 55 degrees was added to the solution. The resulting mixture was agitated at high shear using a food processor and then rapidly quenched in a volume of 25 degree Celsius water. The resultant micelle- like particles in the range of 500 nm to 5000 nm were allowed to rise to the surface and then collected. The particles were then rediluted and separated by rise time in the fluid over three successive dilutions to yield relative size separations. Samples of the various size fractions were successfully demonstrated as agglutination assay in trials on a mutant Escherichia CoIi strain which is always f ⁇ mbirated.
  • BSA Bovine Serum Albumin
  • Shaw, et. al., 32 demonstrated that Tir can be translocated into red blood cell (RBC) membranes, and that the resulting transmembrane molecules (described by Race, et. al. 33 ) which are expressed can result in Tir-intimin binding of other serotypes than those initially secreting the Tir. Accordingly, red blood cells are exposed to Tir producing bacteria per the procedures used by Shaw, et.al. (E.coli strain CVD206 was used in that case, but the technique is applicable to other strains as well).
  • RBC red blood cell
  • the resulting sample of infected red blood cells is then subjected to cell rupture techniques which both destroy the bacteria as well as liberate the transmembrane molecules of Tir, which according to Shaw are in the 78 kDa size range.
  • the transmembrane Tir, with expressed intimin receptors is immobilized directly into a solid substrate based on amphiphilic properties, along with the other transmembrane glycoproteins of RBC using self-aligning film techniques.
  • Tir molecules can also be selectively extracted from the solution using size based fractionation techniques, including but not limited to size-exclusion chromatography and dialysis, and then the selected particles can be added to a crafted solution of selected amphiphilic molecules prior to immobilization.
  • the Tir transmembrane solution is sterile filtered.
  • the glycoprotein rich filtrate is heated to 54 degrees Celsius along with food grade paraffin wax which has a melting point of 52 degrees Celsius.
  • the paraffin includes a radioactive tag.
  • the resulting mixture is agitated and then pumped through a zone excited by sonication.
  • the mixture is then sprayed through a small orifice into a continuous stream of water at 25 degrees Celsius.
  • the resulting particles in the 50nm to lOOOnm diameter size range are allowed to rise to the surface and then collected.
  • the particles are added to an Oral Rehydration Therapy solution of a mixture of sugar and salt and known to those skilled in the art.
  • the solution is provided to an individual with diarrhea and a suspected enteropathogenic Escherichia coli infection. During the course of treatment, the intestinal area of the
  • a hydrogel is prepared using techniques taught by Stupp, et. al. in US patent application 2005/0209145.
  • Stupp teaches the creation of growth factor binding hydrogels based on self-assembling peptide amphiphiles which have been configured through phage display techniques to bind to growth factors.
  • the same phage display techniques are used in this example to develop peptide epitopes with binding affinity for Clostridium difficile spores and to create the associated hydrogels.
  • the hydrogels are further tagged with a radioactive tag.
  • the hydrogels are introduced by catheter into the colon of an individual with suspected residual C. difficile infection in the form of spores.
  • the colon is then irrigated to flush unbound hydrogel from the system.
  • the colon is then scanned for the radioactive tag to identify areas of potential spore infestation.
  • Paraffin wax (100ml) was melted at a temperature of 70° C.
  • Pigeon egg white 50 ml was dissolved in distilled water (400ml) and the resultant mixture was filtered and then heated to 70° C.
  • the melted wax was added to the egg white mixture, which was then agitated and quickly cooled to a temperature below 40° C through the addition of chilled water.
  • Glycoproteins recovered from pigeon egg whites acted as a surfactant and became embedded in the surface of paraffin wax in a manner that the wax formed micelles in the diameter range of 0.001 to 4 mm.
  • the resultant spherical structures i.e., micelles
  • the lectins were found to successfully bind to the sugars on the embedded glycoproteins.
  • the glycoproteins of pigeon egg white express GaI(I -4)Gal ⁇ (l-4)GlcNAc, which has been shown to bind to Shiga-like Toxin I. 34
  • This glycans structure also has potential to bind to Streptococcus suis which can cause meningitis humans and has also been implicated in serious porcine diseases. 35 Accordingly, the material can be a component in an orally administered therapeutic treatment for these two
  • Said spherical structures are mixed with an Oral Rehydration Therapy mixture, which also contains antibiotics known to be effective for E.coli O157:H7.
  • the spherical constructs of the present invention are intended to capture and immobilize some of the increased toxin loading caused by the use of antibiotics.
  • a solution of yeast invertase and low density polyethylene (LDPE) with a melting point of 105 degrees Celsius was heated in an agitated pressure reactor to a temperature of 120° Celsius and a pressure of 15 psig. The solution was then cooled through rapid depressurization of the reactor followed by the addition of chilled water.
  • LDPE micelles having surface embedded glycoproteins recovered from yeast invertase were thus formed, the micelles having a diameter ranging from 500 nm to 2,000 nm.
  • the resultant micelles were dried, rewetted, and exposed to fluorescent lectins. The fluorescent lectins were observed to have successfully bound to the sugars on the embedded glycoproteins.
  • the same procedure is used with genetically modified yeast which produces glycoproteins with sialic acid decorations.
  • the resulting particles provide diagnostic, research and therapeutic particles for application to influenza, particularly potential mutations of the avian influenza virus to human virulence, as well as other pathogens and toxins whose tropism may, in part, be based on sialic acid.
  • Tamm-Horsfall glycoproteins also uromodulin
  • microcrystalline wax was heated to 98° C, until the wax melted the resulting mixture was agitated and then pumped through a zone excited by sonication. The mixture was then sprayed through a small orifice into a continuous stream of cold water. The resulting particles in the 50nm to lOOOnm diameter size range were concentrated and washed. The particles are then suspended in a irrigation solution, which is used to irrigate an infected human bladder.
  • Paraffin wax particles of the present invention having a surface embedded with the glycoproteins of yeast invertase are exposed to two mutant strains of Escherichia CoIi CTF073 in LB broth at 37 degrees Celsius for 20 minutes.
  • the first strain was genetically modified to always express type 1 fimbriae and the second strain was modified to not produce type 1 fimbriae.
  • the particles in solution were then rinsed, stained using crystal violet, and observed under the microscope. In the case of the mutant E.coli which expressed type 1 fimbriae, binding was observed. No binding was observed with in the case of the bacteria with no ability to produce type 1 fimbriae.
  • Wax particles with surface moieties glycosylated molecules of porcine intestine were created using techniques of the present invention.
  • the particles were exposed to Salmonella Kentucky for 30 minutes at 37 degrees Celsius.
  • the particles were then rinsed three times with sterile water.
  • a lysing compound (InstaGene by BioRad) was introduced into the solution with the particles to rupture the bacteria and liberate the DNA.
  • the particle solution was heated to 56 degrees Celsius for 15 minutes, vortexed and then raised to 100 degrees Celsius for eight minutes and then vortexed again.
  • the solution was allowed to cool.
  • the wax separated and formed a solid film at the top of the vial containing the solution.
  • a pipette was used to puncture a hole in the wax and extract DNA containing solution from the vial.
  • a PCR analysis correctly identified the Salmonella. Control samples with no bacteria, properly indicated that case also.
  • Example 10 Glycoproteins are harvested from a culture of porcine brain tissue which has been lysed to disrupt the cell membranes. These glycoproteins are conjugated to a hydrophobic block copolymer utilizing PEG-tethering to form an amphiphilic conjugate. Other glycoproteins been shown to retain bioactivity after covalent bonding with heterobifunctional acrylate-N-hydroxysuccinimide poly(ethylene glycol) (PEG). 38 A polar solution containing this material is agitated. A predominantly non-polar mixture of monounsaturated and saturated fatty acids which are at a temperature above their melting point are added to the
  • An amphiphilic compound is synthesized by PEGylating lactoferrin to a hydrophobic polymeric tail of poly(ethylethylene) utilizing techniques known to those skilled in the art.
  • a polar solution of the PEGylatd lactoferrin amphiphiles is heated to 50 degrees Celsius and then food grade paraffin with a melting point of 48 degrees Celsius is added to the solution.
  • the solution is agitated and then rapidly cooled by dilution to a temperature of 40 degrees Celsius.
  • the resultant wax substrate micelles are washed and then placed in a sterile solution.
  • the mixture is packaged in aseptic packaging as an oral treatment for amoebic dysentery caused by Entamoeba histolytica. These pathogens are known to bind to lactoferrin.
  • Activin is surface immobilized on polymeric filter material. Biotinylated transferrin is then conjugated onto the surface of the filter. The filter is then provided as a means of capture for meningitidis in the spinal fluid for diagnostic, research or therapeutic purposes. 39
  • biomimetic films and particles of the present invention find utility in the capture, concentration and identification of pathogens and toxins in environmental, research and medical contexts, either in vivo, in vitro, or in situ. Constructs of the biomimetic particles also have therapeutic applications, as pharmaceutical formulations, ingestibles, and the like, having particular applicability in the early stages of an infection, before the infecting organism has been identified, when antibiotics tend to be contraindicated yet when therapeutic intervention has the highest potential for success .
  • the present invention has applicability to both civilian and military medical personnel, individuals responsible for public safety in regards to infectious diseases, food supply integrity, both potable and recreational water quality, livestock (including, but not limited to, hoofed animals, poultry, fish) and pet owners, travelers (including leisure, business, military and disaster relief
  • the present invention has particular applicability to disaster relief scenarios where the public is at great risk to waterborne illness; emergency stocks of the present invention can be maintained in a dry form until needed and then administered as part of a hydration therapy to individuals showing symptoms of gastrointestinal illness.
  • the present invention also has applicability to address emerging antibiotic resistant infections.
  • the solid state films and membranes of the present invention formed from the spontaneous aggregation and specific alignment of glycosylated amphiphilic molecules at the interface between a polar solvent and a non-polar liquid, can be used to produce relatively robust and stable micelles having improved long term storage capacity.
  • the biomimetic films and particles of the present invention having surface embedded amphiphilic molecules present useful biological properties to the surrounding environment, they find particular industrial applicability in the areas of environmental sensing, pathogen capture, diagnostic assessment, and therapeutic detoxification.

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Abstract

La présente invention concerne l'identification et l'immobilisation de molécules glycosylées possédant des propriétés biomimétiques, plus particulièrement des séquences de glycane non immunologiques dérivées de tissus et d'origine naturelle ou leurs équivalents fonctionnels, sur des surfaces et des films à semi-conducteurs ou sur des membranes se produisant à l'interface entre des matériaux non polaires et polaires. Les films et les particules glycosylés biomimétiques ainsi produits ont une utilité industrielle, environnementale, diagnostique et/ou thérapeutique lors de la liaison, capture et/ou extraction de pathogènes, toxines et/ou contaminants in vivo, in vitro ou in situ. La présente invention porte aussi sur l'utilisation de tels films et particules biomimétiques lors de l'administration d'autres molécules thérapeutiques ainsi que lors de la construction de dispositifs venant au contact du corps présentant une meilleure biocompatibilité et une immunogenicité réduite.
PCT/US2008/066605 2007-06-11 2008-06-11 Particules et films biomimétiques destinés à la capture de pathogènes et d'autres usages WO2008154603A1 (fr)

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US8518431B2 (en) 2008-08-07 2013-08-27 Bioactive Surgical, Inc. Stem cell capture and immobilization coatings for medical devices and implants
WO2021205077A1 (fr) * 2020-04-09 2021-10-14 Finncure Oy Nanoparticules mimétiques pour prévenir la propagation et diminuer le taux d'infection de nouveaux coronavirus

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US6884842B2 (en) * 1997-10-14 2005-04-26 Alnis Biosciences, Inc. Molecular compounds having complementary surfaces to targets
US20050244425A1 (en) * 1996-04-19 2005-11-03 Henry M. Jackson Foundation For The Advancement Of Military Medicine Histidine-tagged intimin and methods of using intimin to stimulate an immune response and as an antigen carrier with targeting capability
US20060003387A1 (en) * 2004-02-05 2006-01-05 Massachusetts Institute Of Technology Cell display libraries
US20060127950A1 (en) * 2004-04-15 2006-06-15 Massachusetts Institute Of Technology Methods and products related to the improved analysis of carbohydrates

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US6884842B2 (en) * 1997-10-14 2005-04-26 Alnis Biosciences, Inc. Molecular compounds having complementary surfaces to targets
US20050048110A1 (en) * 1999-12-14 2005-03-03 Discher Dennis E. Polymersomes and related encapsulating membranes
US20060003387A1 (en) * 2004-02-05 2006-01-05 Massachusetts Institute Of Technology Cell display libraries
US20060127950A1 (en) * 2004-04-15 2006-06-15 Massachusetts Institute Of Technology Methods and products related to the improved analysis of carbohydrates

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Publication number Priority date Publication date Assignee Title
US8518431B2 (en) 2008-08-07 2013-08-27 Bioactive Surgical, Inc. Stem cell capture and immobilization coatings for medical devices and implants
US8889168B2 (en) 2008-08-07 2014-11-18 Bioactive Surgical Inc. Stem cell capture and immobilization coatings for medical devices and implants
WO2021205077A1 (fr) * 2020-04-09 2021-10-14 Finncure Oy Nanoparticules mimétiques pour prévenir la propagation et diminuer le taux d'infection de nouveaux coronavirus
US11564892B2 (en) 2020-04-09 2023-01-31 Finncure Oy Virus-like particles for preventing the spreading and lowering the infection rate of viruses

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