U.S. patent application number 12/663904 was filed with the patent office on 2010-07-29 for biomimetic particles and films for pathogen capture and other uses.
Invention is credited to Richard H. Spedden.
Application Number | 20100190690 12/663904 |
Document ID | / |
Family ID | 40096156 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100190690 |
Kind Code |
A1 |
Spedden; Richard H. |
July 29, 2010 |
BIOMIMETIC PARTICLES AND FILMS FOR PATHOGEN CAPTURE AND OTHER
USES
Abstract
The identification and immobilization of glycosylated molecules
having biomimetic properties, more particularly
naturally-occurring, tissue-derived, non-immunological 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 is described herein. The biomimetic
glycosylated films and particles constructed therefrom have
industrial, environmental, diagnostic and/or therapeutic utility 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.
Inventors: |
Spedden; Richard H.;
(Clarksville, MD) |
Correspondence
Address: |
SMITH PATENT CONSULTING, LLC
515 East Braddock Road, Suite B
ALEXANDRIA
VA
22314
US
|
Family ID: |
40096156 |
Appl. No.: |
12/663904 |
Filed: |
June 11, 2008 |
PCT Filed: |
June 11, 2008 |
PCT NO: |
PCT/US2008/066605 |
371 Date: |
December 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11761045 |
Jun 11, 2007 |
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12663904 |
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61027375 |
Feb 8, 2008 |
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61044710 |
Apr 14, 2008 |
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Current U.S.
Class: |
514/1.1 ;
435/7.1; 436/518 |
Current CPC
Class: |
Y10T 428/31504 20150401;
C08L 5/04 20130101; C08L 1/02 20130101; C08L 91/06 20130101; A61L
2300/232 20130101; C08L 91/06 20130101; C08L 91/06 20130101; Y10T
428/31844 20150401; G01N 33/5432 20130101; A61L 31/16 20130101;
A61L 29/16 20130101; A61L 27/54 20130101; C08L 89/00 20130101; C08L
91/06 20130101; A61P 31/00 20180101; C08L 5/04 20130101; C08L 1/02
20130101; C08L 89/00 20130101 |
Class at
Publication: |
514/8 ; 436/518;
435/7.1 |
International
Class: |
A61K 38/14 20060101
A61K038/14; G01N 33/543 20060101 G01N033/543; G01N 33/53 20060101
G01N033/53; A61P 31/00 20060101 A61P031/00 |
Claims
1-97. (canceled)
98. A solid state film with biomimetic properties having at least
one functionally active surface, said film comprising a substrate
having a plurality of glycosylated molecules immobilized therein,
wherein said glycosylated molecules are either harvested or derived
from biological fluid or tissue, or comprise a functional mimic of
a component of said biological fluid or tissue.
99. The biomimetic film of claim 98, wherein the glycosylated
molecules comprise endogenous cell-surface glycan signatures
harvested or derived from biological fluid or tissue or functional
mimics thereof.
100. The biomimetic film of claim 99, wherein said glycan signature
are selected from the group consisting of pathogen binding moieties
and self antigens that masks the foreign nature of the film and
thereby reducing its immunogenic potential.
101. The biomimetic film of claim 98, wherein the glycosylated
molecules are selected from the group consisting of glycoproteins,
glycolipids, glycosides, glycosylated transmembrane proteins,
glycodendrimers, glycodendriproteins, and non-immunological
sialated glycosylated molecules.
102. The biomimetic film of claim 98, wherein the glycosylated
molecules include surface moieties of attaching and effacing (A/E)
lesions or functional mimics thereof.
103. The biomimetic film of claim 99, wherein said glycan
signatures are harvested or derived from the inner or outer walls
or membranes of the organs of the gastrointestinal tract, urinary
tract, pulmonary tract, blood vessels, amniotic sac, ocular sac,
nervous system, muscoskeletal system.
104. The biomimetic film of claim 99, wherein said glycan
signatures are isolated from body fluids selected from the group
consisting of mucous, blood, blood plasma, saliva, urine, synovial
fluid, breast milk, tears, fluids of the reproductive system,
aqueous or vitreous humor, bone marrow, and cerebrospinal
fluids.
105. The biomimetic film of claim 98, wherein said substrate is
non-polar or exhibits hydrophobic surface properties and is
selected from the group consisting of resin, natural wax, synthetic
wax, natural and synthetic plastics, polymers, hydrocarbon
mixtures, natural and synthetic fats, ceramics, glass, diatomaceous
earth, and metal.
106. The biomimetic film of claim 98, wherein said substrate
further comprises a detectable label selected from the group
consisting of a fluorescent label, a radioactive label, a dye, and
a compound that enhances magnetic resonance imaging.
107. The solid state biomimetic film of claim 98, wherein said film
comprises a relatively non-polar substrate and said plurality of
glycosylated molecules are immobilized to the upper surface of said
substrate, further wherein one or more amphiphilic molecules having
hydrophobic tail ends and hydrophilic head ends are embedded in or
chemically, electrically or mechanically linked via said
hydrophobic tail ends to the relatively non-polar substrate,
further wherein the amphiphilic molecules either themselves
comprise biomimetic moieties or have such biomimetic moieties
attached or conjugated to their hydrophilic head ends such that the
biomimetic moieties project from said upper surface so as to
provide at least one surface of said film with functional
activity.
108. The solid state biomimetic film of claim 107, wherein 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.
109. A therapeutic biocompatible particle formed from the solid
state biomimetic film of claim 1, said particle of a size or
geometric configuration that prevents its absorption by the
biological tissue of an organism.
110. The therapeutic particle of claim 109, wherein said biological
tissue is selected from the group consisting of 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 a recombinant version of human, animal or plant
tissue or a human, animal or plant tissue biological surrogate,
further wherein said biological tissues are selected from the group
consisting of epithelium, connective tissue, muscle tissue, nerve
tissue, material associated with or contained in amniotic fluid
surrounding a fetus, aqueous humour, blood, blood plasma,
interstitial fluid, breast milk, mucus, pus, saliva, serum, tears,
urine, cerebrospinal fluid, synovial fluid, intracellular fluid,
aqueous humour, vitreous humour and other bodily fluids.
111. The therapeutic particle of claim 109, wherein the substrate
comprises a nonbiodegradable material capable of maintaining the
immobilization of said glycosylated molecules when ingested by a
living organism.
112. The therapeutic particle of claim 109, wherein the particle is
in the form of a wax micelle.
113. The therapeutic particle of claim 109, wherein the particle is
in the form of a worm micelle.
114. A pharmaceutical composition comprising the therapeutic
particle of claim 109 formulated for introduction into a living
organism.
115. The pharmaceutical composition of claim 114, wherein said
composition further comprises one or more additional agents
selected from the group consisting of antibiotic agents,
probiotics, vitamin supplements such as iron, or therapeutic agents
for oral rehydration therapy such as oral rehydration salts,
solution, and electrolytes.
116. A method for making the biomimetic film of claim 98 comprising
the following steps, with the resultant films being disposed 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 c
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. 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.
117. The method of claim 116, further comprising the step of
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.
118. A method of forming the biomimetic film of claim 98 from
biological cell- or membrane-bound glycosylated molecules
comprising 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; c. optionally filtering or otherwise selectively
separating a fraction of interest containing glycosylated molecules
with biomimetic properties, said fraction may or may not comprise a
liquid or other molecules; d. immobilizing said biomimetic
molecules as part of a film on an appropriate substrate; and e.
optionally removing said film from any surrounding liquid to form a
dry construct.
119. The method of claim 118, wherein the step of selectively
separating a fraction of interest containing glycosylated molecules
with biomimetic properties comprises the following steps for
molecules which are amphiphilic in nature: 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. conducting any desired procedures to affix the
molecules to the less-polar substrate beyond the hydrophobic forces
inherent in the film; and e. removing, or replacing with another
liquid, some or all of said solution.
120. The method of claim 118, wherein the transformation is
achieved through cooling of the non-polar liquid.
121. A method of using of biomimetic film of claim 98 comprising
the steps of: a. exposing said surface to a liquid sample which may
or may not contain pathogens or toxins; b. allowing sufficient time
for said surface to bind to target pathogens or toxins present in
said sample; c. optionally separating said surface, to which said
target pathogens or toxins may be bound, from said sample; and d.
optionally, analyzing said surface 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 surface and
subjecting said pathogen or toxins, or media containing such, to
analysis techniques to detect presence or type.
122. A method of using the biomimetic film of claim 98, comprising
the steps of: a. extracting of a bodily fluid of an organism,
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.
inducing contact of said bodily fluid to one or more surfaces with
the pathogen and toxin binding moieties of the present invention;
c. optionally, reintroducing of the bodily fluid into the organism;
and d. optionally, subjecting said surfaces of the present
invention to analysis techniques to determine presence and/or type
of pathogens or toxins that may have bound to the surface.
123. A method of using the therapeutic particle of claim 109,
comprising the steps of: a. administering one or multiple oral
doses of a plurality of particles of constructs of the present
invention to the host; b. allowing the particles of the present
invention to contact areas of the alimentary system of the host
where pathogens may be present; and c. allowing the host organism
to discharge the particles through defecation, vomiting,
expectoration or other means of natural or induced discharge.
124. A method of using the therapeutic particle of claim 109,
comprising the steps of: a. administering one or multiple doses of
a plurality of particles of the present invention to the host
through a catheter or similar device or through other means of
physically placement into a region of suspected infection or a
point in the host where the particles will migrate to a region
where pathogens may be present; b. allowing the particles of the
present invention to contact those areas of the host where
pathogens may be present; and c. allowing the host organism to
discharge the particles through defecation, urination, vomiting,
mucosal flow, expectoration or other means of natural or induced
discharge or removing the particles from the host through a
catheter or other device.
125. A medical device with enhanced surface biocompatibility formed
from or coated with the biomimetic film of claim 98.
126. The device of claim 125, wherein said biomimetic glycosylated
molecules are harvested or derived from tissue of an intended host
organism of said medical device or an antigen-matched donor thereof
or from a culture of tissue from said recipient or donor.
Description
PRIORITY
[0001] This application claims the benefit of U.S. Utility patent
application Ser. No. 11/761,045 filed Jun. 11, 2007, referred to
hereinafter as the '045 Application, the contents of which are
incorporated by reference herein in its entirety. This application
further claims the benefit of U.S. Provisional Patent Application
Nos. 61/027,375 filed Feb. 8, 2008 and 61/044,710 filed Apr. 14,
2008, referred to hereinafter as the '375 and '710 applications
respectively, the entire contents of which are incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] 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-immunological 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.
BACKGROUND OF THE INVENTION
[0003] Most 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.
[0004] 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.
In contrast, the surface features of the pathogen (often referred
to as surface antigens or antigenic fragments) 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.
[0005] 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. Consequently, immunological techniques constitute
powerful tool for the capture and identification of pathogens.
However, these techniques are limited in their ability to capture
unexpected, uncharacterized, rapidly evolving or engineered
pathogens. Accordingly, one aspect of the present invention
addresses this need, filling an important gap in the field of
capture, concentration and detection of pathogens. More
particularly, 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. By harnessing the host and tissue
specificity of the glycan signature, 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.
[0006] The alimentary tract of biological organisms (humans,
animals, birds, fish, etc.) 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.
[0007] In certain instances, antibiotics may be recommended.
However, 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. In addition,
antibiotics are not always readily available, particularly in
economically disadvantaged locations in the world. Moreover,
antibiotic therapy is not always advisable, even in the treatment
of bacterial infections. For example, certain types of bacterial
pathogens, such as Shiga toxin-producing Escherichia Coli (STEC),
particularly 0157:H7 and its variations, exert their deleterious
effects through the production and release of toxic chemicals.
Studies have shown that 5-10% of individuals infected by STEC will
develop hemolytic-uremic syndrome (HUS), and, of those, half will
have renal damage and one in ten will die or have renal
failure..sup.1 However, in vivo treatment of shiga toxin-producing
bacteria using antibiotics can be dangerous because the bacteria
have been shown to produce more toxin when stressed..sup.23 .sup.1
Cheleste M Thorpe, Shiga toxin-producing Escherichia coli
infection, Clin Infect Dis. 2004 May 1; 38 (9):1298-303.sup.2 Wong,
C, M. D., et al. "The Risk of the Hemolytic-Uremic Syndrome after
Antibiotic Treatment of Escherichia Coli O157:H7 Infection", The
New England Journal of Medicine, Jun. 29, 2000, Vol. 342, No. 26,
pp. 1930-1936..sup.3Mulvey G.; Rafter D. J.; Armstrong G. D.,
Potential for using antibiotics combined with a Shiga
toxin-absorbing agent for treating O157:H7 Escherichia coli
infections, Canadian Journal of Chemistry, Volume 80, Number 8,
August 2002, pp. 871-874(4)
[0008] Accordingly, since antibiotics are not always advisable,
available or even effective, alternative therapeutic treatments for
pathogenic infections are heartily sought. 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
removing both the pathogen and its associated toxin, without
producing the undesirable side effect of inducing additional toxin
production associated with antibiotic therapy. The technology of
the present invention extends to the treatment of infection
associated with numerous pathogens, including both natural and
engineered disease producing agents, and thus constitutes a marked
improvement in the treatment of infection by toxin-producing
pathogens.
[0009] In the current art of immunologically based pathogen capture
and/or detection in diagnostic samples, 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.
[0010] 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. Current antibody immobilization
procedures utilize conventional molecular conjugating techniques,
which are principally based on conjugation of molecules using
chemical binding techniques, such as with biotintylation or binding
with various polymers, often block copolymers. Immobilization of
antibodies can also be achieved by means of adsorption on a
properly charged surface. Common techniques for conjugation
molecules of biologic origin and other molecules, natural and
synthetic, include, but are not limited to reduction amination,
diazo coupling, use of isothiocyanates, amidation, use of
hom-bifunctional reagents, cycloadditions, maleimide addition
thioether linkages, oxime conjugation, stauding ligation olefin
metathesis, biotintylation and PEGylation. 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.
[0011] 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.
[0012] 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. 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. When 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.
[0013] These types of structures, often referred to interchangeably
as micelles, or vesicles, are well known in science and nature,
with liposomes being a specific subset of these structures.
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.
[0014] A useful rendition of these structures in conjunction with
non-polar liquids has been achieved through the use of biological
amphiphilic compounds to form micelles and micelle-like structures.
In particular, lipo-glycoprotein membranes and micelles formed
therefrom are described in U.S. Pat. Nos. 5,824,337, 6,528,092 and
7,148,031, and U.S. Patent Publication US2007/0141694, all to
Elaine Mullen, the entire contents of which are incorporated herein
by reference. In these patents, the unique and important
contribution of Mullen in the creation of the lipo-glycoprotein
membranes over other micelle-like constructs is the presentation of
important glycans and other biological structures at the surface of
the membrane in a manner that can be beneficially used. The
presence of these glycans allows for the transport of substances
that can be dissolved or suspended in lipids as well as the capture
and concentration of biological and inorganic entities that
naturally bind to the selected glycan structures.
[0015] 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.
[0016] The immobilization of amphiphilic molecules on latex based
polymer particles is described in U.S. Pat. No. 4,929,662, to
Hogenmuller, et. al., and in U.S. Pat. Nos. 4,952,622 and
5,109,038, both to Chauvel, et. al., the entire contents of which
are incorporated herein by reference. The techniques described in
these patents are applicable to one aspect of the present invention
in the creation and construction of biomimetic films and particles
comprising amphiphilic moieties, particularly glycolsylated
molecules, harvested from tissue, wherein the hydrophobic tails of
said molecules are bound to the surface of a latex film or particle
through hydrophobic interaction. 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.
[0017] In U.S. Pat. No. 5,919,408, the entire contents of which are
incorporated herein by reference, Muller, et. al., teach a process
for the production of pseudolatices and micro- or nanoparticles
with embedded pharmaceutical preparations. The techniques of
forming said particles are also of use in creation and construction
of amphiphilic biomimetic surfaces, films, and particles disclosed
herein.
[0018] Previous work done in the field combining polymer substrata
with biological amphiphilic compounds at the surface has been
focused on production of micelle-like structures, without attention
to preserving or utilizing biological surface properties of any
surfactants used to form biomimetic constructs with utility in
pathogen capture. Accordingly, 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.
SUMMARY OF THE INVENTION
[0019] Thus, in view of the foregoing, it is an object of the
present invention to provide novel biomimetic films and particles
suitable for pathogen capture and other uses, as well as methods of
making and using same.
[0020] It will be understood by those skilled in the art that one
or more aspects of this invention can meet certain objectives,
while one or more other aspects can meet certain other objectives.
Each objective may not apply equally, in all its respects, to every
aspect of this invention. As such, the following objects can be
viewed in the alternative with respect to any one aspect of this
invention.
[0021] A primary objective of the present invention is to provide
novel biomimetic films and particles for pathogen capture. In
particular, 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. Of particular interest are 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.
[0022] Accordingly, it is an object of the present invention to
construct 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.
[0023] It is a further object of the present invention to utilize
the 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.
[0024] It is yet another object of the present invention to produce
the biomimetic films and particles in an appropriate particulate
form and use it in the context of an agglutination assay.
[0025] It is yet another object of the present invention to affix
non-immunological, glycosylated molecules, particularly molecules
from biological tissue, to magnetic beads, and expose such magnetic
beads with these moieties to a solution which may or may not
contain pathogens which exhibit the appropriate tissue tropism. 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.
[0026] It is yet another object of the present invention to provide
the biomimetic films and particles of the present invention with
one or more type of non-immunological, 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.
[0027] It is yet another object of the present invention that the
non-immunological, 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.
[0028] It is yet another object of the present invention to
construct the 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.
[0029] It is yet another object of the present invention to
immobilize the non-immunological, glycosylated molecules to a
substrate which further includes a semi-conductor device, examples
of which include, but not limited to, lab-on-a-chip type
devices.
[0030] It is yet another object of the present invention to provide
various methods for constructing embodiments of the present
invention.
[0031] 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. In this manner, 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.
[0032] Accordingly, it is an object of the present invention to
provide therapeutic biocompatible particles with a minimum nominal
diameter sufficient to inhibit absorption of a majority of the
particles by the surrounding tissue, the diameter being greater
than 20 nanometers, preferably greater than 50 nanometers, more
preferably greater than 500 nanometers.
[0033] It is further an object of the present invention to provide
therapeutic biocompatible particles in the form of micelles, such
as wax micelles, or fibers or worm micelles (also nanowires), with
or without embedded substrate materials. 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.
Alternatively, 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.
[0034] It is an object of the present invention to provide
therapeutic biocompatible particles expressing surface molecules
comprising pathogen binding glycosylated molecules. In a preferred
embodiment, 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] It is a further object of the present to formulate the
therapeutic particle as a pharmaceutical composition suitable for
ingestion or administration by other means. Such pharmaceutical
compositions may optionally include additional
[0039] It is a further object of the present invention to provide a
therapeutic dose that consists of a plurality of particles of the
present invention and that those particles can include a range of
sizes, shapes, substrate materials, and types of surface
molecules.
[0040] It is yet another object to provide unique methods of making
and using the therapeutic biocompatible particles and films of the
present invention.
[0041] A final objective of the present invention is to provide
functionally active solid state films of glycosylated amphiphilic
molecules. In particular, it is an objective of the present
invention to provide a non-polar solid surface or film, for example
of a polymer, plastic or wax, that has embedded in it or conjugated
or chemically bound to it the hydrophobic end(s) of a glycosylated
amphiphilic molecule, such that its opposing hydrophilic end(s)
having useful functional properties are expressed at or above the
surface of the solid. These properties may be viable either in air
or when a polar solvent, such as water, is present.
[0042] 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.
[0043] 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. In conventional membranes between a polar
solvent and a non-polar liquid, if the polar solvent is removed,
the alignment of the hydrophobic and hydrophilic ends of the
amphiphilic compounds fails and the membrane is destroyed. However,
with the solid state films of the present invention, 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.
[0044] Accordingly, it is an object of the present invention to
provide 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.
[0045] It is a further object of the present invention to provide a
solid state micelle or micelle-like structure with a surface film
of the present invention, 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.
[0046] It is yet a further object of the present invention to
provide a method for making a film of the present invention,
including the following steps: [0047] (a) providing a polar solvent
having sufficient quantities of one or more glycosylated
amphiphilic molecules dissolved therein; [0048] (b) exposing the
polar solvent to a non-polar liquid, the non-polar liquid being
immiscible in the polar solvent; [0049] (c) allowing the
glycosylated amphiphilic molecules to align so as to form a
membrane that separates the polar solvent from the non-polar
liquid; and [0050] (d) inducing 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 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.
[0051] It is a further object of the present invention to provide a
method of extracting a target molecule of interest from a sample,
including the following steps: [0052] (a) exposing the sample to a
solid state membrane of the instant invention, wherein the
functional groups present at the hydrophilic tail end of the
glycosylated amphiphilic molecules have a binding affinity for a
target molecule; [0053] (b) allowing sufficient time for the
functional groups to bind target molecules present in the sample;
and [0054] (c) removing or separating the membrane, to which the
target molecules are bound, from the sample.
[0055] It is yet another object of the present invention to provide
a method of enhancing the biocompatibility of a medical device
including the step of coating a solid state membrane of the present
invention onto one or more exposed surfaces of the device. Medical
devices having such membrane coatings are also provided herein.
[0056] It is a further object of the present invention to provide
economical solid surfaces which can be imbued with useful
properties of glycosylated amphiphilic compounds such as
glycoproteins. 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. Accordingly, it is an object of the present
invention to utilize the glycosylated amphiphilic membranes of the
present invention, in the form of a solid surface film or a film
coating, to isolate and extract target compounds from a particular
environment or sample.
[0057] It is a further object of the present invention to provide
compositions and methods for preventing an immune response in a
mammal. In that vein, it is an object of the present invention to
utilize the glycosylated amphiphilic membranes of the present
invention in the fabrication of or as a film coating for artificial
organs, implants, and transplant materials, including both living
and nonliving tissue. It is well-accepted that a material that
mimics the surface properties of a biological cell or tissue is
less likely to generate an immune response. Accordingly, through
the selection of 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.
[0058] Within the context of biocompatibility, the use of lipids
and waxes from a biocompatible entity as the non-polar substrate is
anticipated as a means to further insure compatibility or achieve
synergistic benefits. For example, the non-polar substrate may
comprise a natural wax or fat isolated from an antigen-matched
human donor.
[0059] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying figures and examples.
However, it is to be understood that both the foregoing summary of
the invention and the following detailed description are of a
preferred embodiment and not restrictive of the invention or other
alternate embodiments of the invention. In particular, while the
invention is described herein with reference to a number of
specific embodiments, it will be appreciated that the description
is illustrative of the invention and is not constructed as limiting
of the invention. Various modifications and applications may occur
to those who are skilled in the art, without departing from the
spirit and the scope of the invention, as described by the appended
claims. Likewise, other objects, features, benefits and advantages
of the present invention will be apparent from this summary and
certain embodiments described below, and will be readily apparent
to those skilled in the art having knowledge of various pathogen
binding glycan motifs, amphiphilic compounds, self-assembly
techniques and peptide synthesis. Such objects, features, benefits
and advantages will be apparent from the above in conjunction with
the accompanying examples, data, figures and all reasonable
inferences to be drawn therefrom, alone or with consideration of
the references incorporated herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] Various aspects and applications of the present invention
will become apparent to the skilled artisan upon consideration of
the brief description of the figures and the detailed description
of the present invention and its preferred embodiments which
follows:
[0061] FIG. 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.
[0062] FIG. 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.
[0063] FIG. 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.
[0064] FIG. 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0065] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
embodiments of the present invention, the preferred methods,
devices, and materials are now described. However, before the
present materials and methods are described, it is to be understood
that this invention is not limited to the particular molecules,
compositions, methodologies or protocols herein described, as these
may vary in accordance with routine experimentation and
optimization. It is also to be understood that the terminology used
in the description is for the purpose of describing the particular
versions or embodiments only, and is not intended to limit the
scope of the present invention which will be limited only by the
appended claims.
[0066] I. Elements of the Present Invention:
[0067] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. However,
in case of conflict, the present specification, including
definitions, will control. Accordingly, in the context of the
present invention, the following definitions apply:
[0068] As used herein and in the appended claims, the singular
forms "a", "an" and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
a "molecule" is a reference to one or more molecules and
equivalents thereof known to those skilled in the art, and so
forth.
[0069] In the context of the present invention, the term
"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. The term 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.
[0070] The present invention makes reference to glycan signatures
and glycosylated molecules, and in certain instances
"non-immunological, sialated, glycosylated molecules". As noted
above, examples of suitable glycosylated molecules of interest in
this invention 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.
[0071] As used herein, the term "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. In the former context, glycosylation frequently
arises as a result of co-translational or post-translational
modification. In the latter context, 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.
[0072] In the context of the present invention, a glycosylated
lipid (or "glycolipid") is a biological molecule composed of a
lipid and a carbohydrate, typically an oligosaccharide, whereas a
glycosylated protein (or "glycoprotein") is a biological molecule
composed of a protein and an oligosaccharide. In the context of
glycoproteins, the addition of sugar chains to a protein occurs
either at an asparagine moiety (referred to herein and elsewhere in
the art as "N-glycosylation") or at a hydroxylysine,
hydroxyproline, serine or threonine moiety (referred to herein and
elsewhere in the art as "O-glycosylation"). In the context of the
self-assembled films of present invention, glycoproteins with
N-linked glycoyslations may provide advantages in forming
self-assembled films without the assistance of other amphiphilic
molecules.
[0073] 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. Furthermore, 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. For example, the
glycoproteins "ovotransferrin", "ovalbumin" and "ovomucoid" are
found in certain egg whites (albumen) and the glycoprotein
"Glyocophorin A" is commonly found in blood plasma, Similarly,
kappa-Casein is found in cow's milk, and 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.
Other glycoproteins suitable for use in the context of the present
invention include, but are not limited to, human immunoglobulins,
such as IgG1; hormones, such as pituitary hormones lutropin (LH),
thyrotropin, and pro-opiomelanocortin; proteoglycans, and
derivatives thereof. Further examples of 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).
[0074] Proteoglycans are a special class of heavily glycosylated
glycoproteins comprised of a core protein with one or more
glycosaminoglycan (GAG) chains. The 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. Examples of 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 (SLRPs) include
decorin, biglycan, fibromodulin and lumican.
[0075] In the context of the present invention, the term
glycosylated transmembrane molecule, or 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.
[0076] In the context of the present invention, 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.
[0077] In addition to being isolated from natural sources,
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.
[0078] As used herein, the term "glycan" is synonymous with the
term "polysaccharide" as referring to compounds composed of a large
number of glycosidically linked monosaccharide units (typically ten
or more).
[0079] As used herein, the term "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". When the
anomeric carbon is attached via a nitrogen atom, the resulting
molecule is preferably designated as a "glycosylamine" rather than
an "N-glycoside". Illustrative examples of 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.
[0080] The present invention makes reference to the immobilization
of non-immunological, 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. As used herein and in the appended
claims, the term "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. As such,
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.
[0081] As used herein and in the appended claims, 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.
[0082] The present invention makes reference to biomimetic surfaces
or films composed of solid substrates that find utility in the
field pathogen capture. As discussed in greater detail below, 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.
[0083] As used herein and in the appended claims, the term
"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.
[0084] As discussed in greater detail below, 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.
[0085] As used herein and in the appended claims, the term "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. In certain instances, the
presence and/or level of target molecule in a sample will correlate
with a particular disease or disorder (e.g., a bacterial infection,
heavy metal poisoning, cancer, etc.).
[0086] As discussed in greater detail below, the biomimetic films
and particles of the present invention find particular utility in
the context of pathogen capture. As used herein and in the appended
claims, the term "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).
[0087] 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. As used
herein and in the appended claims, the term "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. Examples of heavy metals
associated with serious illness (e.g., heavy metal poisoning)
include, but are not limited to, lead, mercury, copper, cadmium,
manganese, aluminum, beryllium, molybdenum, vanadium, strontium,
zinc, and iron.
[0088] 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.
[0089] In the context of the present invention, the terms "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. Within the concept of this invention, 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.
[0090] As noted above, the term "non-polar" refers to a substance
that is relatively uncharged, typically water insoluble
(hydrophobic). In the context of the self-assembled amphiphilic
films of the present invention, the non-polar substrate must be
capable of stable expression both as a liquid and a solid. In
preferred embodiments, the non-polar substrate exists in solid
state at a conventional room temperature (typically between 15 and
25.degree. C.). However, bearing in mind that thermal degradation
of many amphiphilic compounds is a function of time at elevated
temperature and that the non-polar substrate needs to be in a
liquid state for only a very short period of time to permit the
hydrophobic tails of the amphiphilic compound to become bound in
the surface, relatively high processing temperatures can indeed be
used without causing substantial degradation of the amphiphilic
compound.
[0091] Furthermore, in that pressure and temperature are inversely
related, 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. Examples of 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.
[0092] As used herein and in the appended claims, the term
"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.
[0093] The hydrophobic component of an amphiphilic molecule is
typically a large hydrocarbon moiety, such as a long chain of the
form CH.sub.3(CH.sub.2).sub.n, 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. Often, 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. Pat. No.
7,109,280 (Kulkarni, et. al.), the entire contents of which are
incorporated by reference herein. Block copolymers can be used to
construct a hydrophobic tail with the same functionality as the
hydrophobic amine based tails of glycoproteins.
[0094] Surfactants such as sodium dodecyl sulphate, benzalkonium
chloride, octanol, and cocaminopropyl betaine are examples of
amphiphilic compounds with known industrial uses. However, as noted
above, 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.
[0095] As discussed in greater detail below, the solid state
biomimetic films of the present invention may be used to create
useful micelles of novel construction. As used herein and in the
appended claims, 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. In a
typical micelle, 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.
[0096] As discussed in greater detail below, 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. As noted above, the
term "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.
[0097] As discussed in greater detail below, 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.
[0098] As used herein and in the appended claims, the term "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. In certain instances, the
presence and/or level of target molecule in a sample will correlate
with a particular disease or disorder (e.g., a bacterial infection,
heavy metal poisoning, cancer, etc.).
[0099] As discussed in greater detail below, 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. As used herein and in the appended claims,
the term "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.
Examples of heavy metals associated with serious illness (e.g.,
heavy metal poisoning) include, but are not limited to, lead,
mercury, copper, cadmium, manganese, aluminum, beryllium,
molybdenum, vanadium, strontium, zinc, and iron.
[0100] As discussed in greater detail below, the biomimetic films
and particles of the present invention find therapeutic utility, as
pharmaceutical formulations suitable for in vivo administration. In
a preferred embodiment, the biomimetic particle takes the form of
an ingestible micelle, suitable for oral delivery. However, 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.
[0101] As discussed in greater detail below, the biomimetic films
and particles of the present invention find utility as coatings for
medical devices. In the context of the instant invention, 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.
[0102] II. Non-Immunological Biomimetic Films for Pathogen
Capture:
[0103] As noted previously, it is an object of the present
invention to immobilize non-immunological glycosylated molecules
with biomimetic properties on the surface of solid substrates for
the purpose of pathogen, capture, concentration and/or detection.
Central to the instant invention is the discovery that 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.
[0104] A. Identifying and Isolating Suitable Biomimetic
Molecules:
[0105] 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. 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.
[0106] 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. For example, a
gastro-intestinal infection may exhibit adhesins to glycoproteins
(particularly glycomucins) present in a specific section of the
small intestine. In the context of the methods of the present
invention, 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.
[0107] In addition to extracting molecules of interest from tissue
obtained directly from the host organism, 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. For
example, porcine tissue is often used as a biological surrogate for
human tissue. Avian eggs have been demonstrated to produce
glycosylated molecules of interest. There are a wide range of other
potential surrogates and biological sources.
[0108] 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. .sup.4 .sup.4
Hamilton et al., Humanization of Yeast to Produce Complex
Terminally Sialylated Glycoproteins Science 8 Sep. 2006: 1441-1443
DOI: 10.1126/science.1130256
[0109] 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). For
example, 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).
[0110] Glycosylated molecules in bodily fluids may be present as
individual molecules and readily available for extraction and
immobilization on a surface. In many other situations, 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). In those
instances, the cell must be lysed or ruptured in a manner whereby
the molecules of interest can be captured and immobilized. Methods
for cell lysis are known to those skilled in the art and 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).
[0111] 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. In the context of the present
invention, this trait can be used to extract molecules of interest
from a solution of bodily fluids or solutions containing lysed cell
material. In certain aspects of the present invention, 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. In other aspects of the present invention, 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.
[0112] B. Immobilizing Biomimetic Glycosylated Molecules:
[0113] 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. In addition, in that many of
the 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.
[0114] 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).
[0115] 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.
[0116] Alignment and immobilization through the use of surfactant
forces is also of interest in the present invention. The creation
and use micelle-like films and structures using amphiphilic
molecules is known to those skilled in the art. The present
invention expands on that art and provides novel constructs and
methods based on immobilization of glycosylated amphiphiles on
solid substrates. Techniques of relevance to the creation of
constructs of interest in the present invention have also been
taught in the use of surfactants to create solid lipid
nanoparticles (SLN). The solid lipid nanoparticles tend to be
micelle-like particles between 10 nanometers and 1 micrometer
(sometimes 10 micrometers) in size.
[0117] In Published U.S. Patent Application No. 2006/0083781, the
entire contents of which are included herein by reference, 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.
[0118] Many of the techniques of value in the immobilization of
glycosylated molecules of interest in the present invention are
suggested in connection with the immobilization of other types of
molecules, for use in other applications. The techniques are
generally based on conjugation of proteins with polymers
(particularly copolymers), other proteins or carbohydrates.
[0119] Techniques taught by others for the immobilization of
bioactive peptides can also be utilized to immobilize biomimetic
molecules derived from biological tissue. Heparin and heparin-like
molecules can be usefully conjugated to the biomimetic molecules of
the present invention and these conjugated constructs can then be
further conjugated to other molecules to create an immobilization
structure. Heparin binding of bioactive peptides and synthetic
molecules has been taught by Zamora in U.S. Pat. No. 6,921,811 and
U.S. Pat. No. 7,297,343 and patent application US 2006/0024347, the
entire contents of which are included herein by reference. The use
of heparin binding of bioactive peptides has also been taught by
Stupp, et. al. in patent application US 2007/0277250, the entire
contents of which are included herein by reference.
[0120] 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 nanofibers, also worm
micelles. The nanofiber structures consist solely of amphiphilic
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.
[0121] Yet others have taught the immobilization of bioactive
species on cross-linked block copolymer surfactants, and as above
it is the object of the present invention to use the same
techniques to immobilize biomimetic molecules harvested from
biological tissue. See, for example, U.S. Pat. No. 5,897,955 to
Paul D. Drumheller, the entire contents of which are incorporated
herein by reference for relevant techniques applied to bioactive
peptides. In particular, Drumheller describes the attachment of
bioactive species using a cross-linking compound to copolymer
surfactants which are bound to a substrate.
[0122] Bhaskaran, et. al. in U.S. Pat. Appl. 2004/0136952, the
entire contents of which are incorporated herein by reference,
teaches methods of synthesizing polymer conjugates of growth factor
proteins and other compounds while maintaining a high level of
functionality of these biological compounds. These techniques also
find utility in the context of the present invention in the context
of using similar techniques to form conjugates with biomimetic
molecules harvested from biological tissue.
[0123] Techniques known in the art for immobilization of bioactive
molecules on metallic and polymeric surfaces by the process of
adsorption can be applied in the immobilization the biomimetic
molecules harvested from biological tissue of the present
invention, and such application is an object of the present
invention..sup.5 6 .sup.5 Jennissen, H. P., Chatzinikolaidou, M,
Rumpf, H. M., (2000) Modification of metal surfaces and biocoating
of implants with bone-morphogenetic protein 2 (BMP-2), DVM Bericht
313:127-140..sup.6 Schrier, J. A., DeLuca, P. P., Porous Bone
Morphogenetic Protein-2 Microsheres: Polymer Binding and In Vitro
Release, AAPS PharmsciTech 2001; 2 (3) Article 17.
[0124] Methods currently in use for the immobilization of
antibodies on solid substrates are also of particular use in
connection with the constructs of this 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.
[0125] 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. Many of these materials and techniques
are of applicable to the creation of the constructs of the present
invention, as in the binding of to solid substrates of various
biomimetic molecules harvested from biological tissue, and also in
the binding of antibodies. A typical example involves the use of
biotintylation to conjugate a glycoprotein to a magnetic bead with
activin present on the surface.
[0126] Block copolymers have also been used to immobilize
antibodies in solid surfaces. In work done at Purdue University,
antibodies were immobilized on polycarbonate surfaces using
Poly-L-Lysine and the resultant surfaces were used for pathogen
capture to facilitate detection..sup.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. .sup.7 Chen, W.-T. (Speaker), M. R. Ladisch, T.
Geng, and A. K. Bhunia, Pathogen Capture and Concentration on
Functionalized Polycarbonate Membrane Detection and Sample
Preparation Based on Immuno-Filters, BIOT Division Paper 140, 227th
ACS National Meeting, Section: High Throughput Screening/Genomics
and Proteomics, Anaheim, Calif. (Mar. 30, 2004).
[0127] 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. al., the
entire contents of which are incorporated herein by reference. The
patents describe the use of such constructs in the formation of
vesicles that enclose materials of biological interest which may
then embedded or conjugated with carrier material. The vesicle
walls are semi-permeable to permit the material of biological
interest to diffuse into the surrounding environment. It is an
object of the present invention that these same constructs and
surface block copolymers can be used to immobilize the biomimetic
glycosylated molecules harvested from biological tissue to form
constructs of the present invention. Polyethylene glycol (PEG) is
an example of a polymer that is applicable to forming conjugations
with biomimetic molecules to create constructs of the present
invention.
[0128] In sum, the science of forming conjugates of proteins, and
other functional biological materials relevant to this patent, and
other molecules, specifically other biological and synthetic
compounds, is well understood by those skilled in the art. In fact,
numerous methods for forming protein to protein conjugates are
available and readily known to those skilled in the art..sup.89
These techniques are of use in the context of the present invention
in the immobilization of biomimetic molecules harvested from
biological tissue to substrates in the formation of constructs of
the present invention. To the extent that some of these techniques
can also be used to form amphiphilic conjugates they are also
applicable to other embodiments of the present invention. .sup.8
Stowell, C. P. and Lee, Y. C., Neoglycoproteins: The Preparation
and Application of Synthetic Glycoproteins, ADVANCES IN
CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY (Vol 37), 1980, Academic
Press, Inc., pp. 225-281.sup.9 Pozsgay, V., Kubler-Kielb, J.,
Conjugation methods towards synthetic vaccines, Carbohydrate-Based
Vaccines, ACS Symposium Sieries, R. Roy, Editor, In press,
2006.
[0129] In U.S. Pat. No. 6,322,810, the entire contents of which are
included herein by reference, 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 large enough to not be readily absorbed by
surrounding tissue this generally places the constructs of the
present invention in a size range greater than the 20 nanometer
diameter limit taught by Alkan-Onyuksel
[0130] C. Constructing Biomimetic Films for Pathogen Capture:
[0131] 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.
[0132] An illustrative method for making biomimetic films and
particles of the present invention comprises the following steps,
with resultant films on solid substrates: [0133] 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; [0134] 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; [0135] c. allowing the amphiphilic molecules to align so
as to form a membrane which separates the polar solvent from the
non-polar liquid; and [0136] 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 [0137] 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. [0138] f. Optionally, remove said
film from any surrounding liquid to form a dry construct.
[0139] 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.
[0140] In the context of the above-described construction method,
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.
[0141] An alternate method for making biomimetic films and
particles of the present invention comprises the following steps,
with resultant films on solid substrates: [0142] a. providing a
liquid having sufficient quantities dissolved therein of one or
more molecules with biomimetic properties or pathogen or toxin
binding properties; [0143] b. exposing said liquid to a solid with
surface moieties appropriate for conjugation with said molecules;
[0144] 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. [0145] d. Optionally, removing said film
from any surrounding liquid to form a dry construct.
[0146] Biomimetic films from biological cell- or membrane-bound
glycosylated molecules may be constructed according to the
following steps: [0147] 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.
[0148] 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. [0149] c. Optionally, filtering or otherwise selectively
separating a fraction of interest containing glycosylated molecules
with biomimetic properties, said fraction may or may not comprise a
liquid or other molecules [0150] d. Immobilizing said biomimetic
molecules as part of a film on an appropriate substrate. [0151] e.
Optionally, removing said film from any surrounding liquid to form
a dry construct.
[0152] The methods above are equally applicable when the biological
tissue includes attaching and effacing (A/E) lesions or their
functional analogues.
[0153] Alternatively, the biomimetic films from biological cell- or
membrane-bound glycosylated molecules may be constructed as
follows: [0154] 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.
Examples of 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. [0155]
b. In the case where molecules of interest are bound in cell
membranes of other constructs, 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.
[0156] c. 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.
[0157] In the context of the above method, non-immunological,
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: [0158] 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; [0159] b. If
necessary, adding amphiphilic molecules to achieve critical micelle
concentration, before or after exposing said solution to said
less-polar material; [0160] c. Allowing the amphiphilic compounds
to form a film at the interface between said solution and said
less-polar material; [0161] d. Conducting any desired procedures to
affix the molecules to the less-polar substrate beyond the
hydrophobic forces inherent in the film; and [0162] e. Removing or
replacing some or all of said solution with another liquid.
[0163] The resulting 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. In that context, 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.
[0164] The present invention further provides a method for using
the biomimetic films for pathogen capture, the method comprising
the steps of [0165] a. exposing said film to a liquid sample which
may or may not contain pathogens or toxins, [0166] b. allowing
sufficient time for said film to bind to target pathogens or toxins
present in said sample; [0167] c. optionally separating said film,
to which said target pathogens or toxins may be bound, from said
sample. [0168] d. optionally, 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.
[0169] 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.
[0170] In the context of the above method of use, 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.
[0171] In the context of the above method of use, 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.
[0172] In the context of the above method of use, 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.
[0173] In the context of the above method of use, 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.
[0174] In the context of the above method of use, 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.
[0175] In the context of the above method of use, 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: [0176] a. Enrichment of the
sample with a nutrient compound, [0177] b. Incubation of the sample
at a temperature between 35 degrees and 40 degrees centigrade for a
period of time, [0178] c. Incubation in an anaerobic
environment.
[0179] In the context of the above method of use, 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.
[0180] In the context of the above method of use, 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.
[0181] III. Treatment for Infection by Toxin Producing
Pathogens:
[0182] The 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. In particular,
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. For roughly spherical
particles, 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.
[0183] Thus, 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. 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. In the context of the present
invention, 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.
[0184] 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. Additionally, to blind all the adhesins
on a specific pathogen (i.e., to completely coat the pathogen), 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.
[0185] In the case of the present invention, where 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.
[0186] In the context of therapeutic treatment for infection with a
bacteria associated with toxin production, such as E. coli 0157:H7,
the present invention also represents an improvement over the use
of antibiotics which can trigger an immediate release of high
concentrations of toxin. As mentioned in the New England Journal of
Medicine article referenced above, the use of antibiotic treatment
is not recommended in the case of E. coli 0157:H7 infection.
Similar hazards may be present with infection by other
toxin-producing bacteria, including strains of Vibrio cholerae,
Salmonella, Shigella, Clostridium, Helicobacter, enteropathogenic
E. coli (EPEC). and enteroaggregative E. coli (EAEC). 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.
[0187] Additionally, since in many instances, 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. There is also
the potential that 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.
[0188] Of particular interest in the context of the present
invention are the patents of Dr. G. D. Armstrong and various
co-inventors, listed below, the entire contents of which are
included herein by reference: [0189] 5,484,773--Treatment of
Antibiotic-Associated Diarrhea. [0190] 5,620,858--Method of
Removing Shiga-like Toxins from Biological Samples. [0191]
5,627,163--Treatment of Traveller's Diarrhea (claims to binding E.
coli). [0192] U.S. Pat. No. 5,635,606--A Method of Removing Toxin
A. [0193] U.S. Pat. No. 5,637,576--Treatment of Traveller's
Diarrhea. [0194] U.S. Pat. No. 5,661,131--Treatment of Cholera.
[0195] U.S. Pat. No. 5,679,653--Diagnosis and treatment of
bacterial dysentery. [0196] U.S. Pat. No. 5,811,409--Treatment of
Cholera. [0197] U.S. Pat. No. 5,817,633--Treatment of Cholera.
[0198] U.S. Pat. No. 5,849,714--Treatment of bacterial dystentery.
[0199] U.S. Pat. No. 5,858,698--Methods for detection of
enteropathogenic e. coli. [0200] U.S. Pat. No. 5,891,860--Treatment
of traveller's diarrhea. [0201] U.S. Pat. No. 5,939,397--Treatment
of cholera. [0202] U.S. Pat. No. 5,955,449--Diagnosis and treatment
of bacterial dysentery. [0203] U.S. Pat. No. 5,962,423--Treatment
of bacterial dysentery. [0204] U.S. Pat. No. 6,013,635--Treatment
of C. difficile toxin B associated conditions. [0205] U.S. Pat. No.
6,069,137--Treatment of traveller's diarrhea. [0206] U.S. Pat. No.
6,107,282--Treatment of C. difficile toxin B associated conditions.
[0207] U.S. Pat. No. 6,121,242--Treatment of bacterial dysentery.
[0208] U.S. Pat. No. 6,224,891--Compounds and methods for the
treatment of bacterial dysentery using antibiotics and toxin
binding oligosaccharide compositions. [0209] U.S. Pat. No.
6,262,037--Pharmaceutical compositions for the amelioration of
enteropathogenic E. coli infection. [0210] U.S. Pat. No.
6,291,435--Treatment of diarrhea caused by enteropathogenic
Escherichia coli. [0211] U.S. Pat. No. 6,310,043--Treatment of
bacterial infections. [0212] U.S. Pat. No. 6,358,930--Treatment of
C. difficile toxin B associated conditions. [0213] U.S. Pat. No.
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. However 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.
Among those unique aspects of the present invention in the field of
therapeutic use for treatment of infection in comparison to said
patents are: [0214] 1. In certain aspects, 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. [0215] 2. In other aspects of the
present invention, 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. In certain instances, certain elements of the patents
referenced above can be utilized in conjunction with the present
invention to provide unique constructs with additional value.
[0216] 3. In certain aspects, 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. [0217] 4. 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.
[0218] A. Identifying Suitable Pathogen-Binding Molecules:
[0219] As noted previously, it is an objective of the present
invention to provide 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.
[0220] In a preferred embodiment, 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 (including a tissue culture), produced by organisms
developed through recombinant techniques, or alternatively produced
from phage display techniques..sup.10 .sup.10 Smith, George P.,
Petrenko, Valery A., Phage Display, Chem. Rev. 1997, 97,
391-410
[0221] Examples of 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. 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. Examples of suitable biological tissues
include, but are not limited to epithelium, connective tissue,
muscle tissue, nerve tissue, mucous or serous cells, 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.
[0222] Examples of suitable glycosylated molecules include but are
not limited to those expressing the following moieties: [0223]
Galabiose structure Gal.alpha.1-4Gal which has been shown to be
important in binding of uropathogenic Escherichia coli, Pseudomonas
aeruginosa (PA-I lectin), and 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
(Stx1), both from E. coli strains, and enterotoxin B from
Staphylococcus aureus;.sup.11 .sup.11 Noriko Suzuki, Kay-Hooi Khoo,
Hao-Chia Chen, James R. Johnson, and Yuan C. Lee, Isolation and
Characterization of Major Glycoproteins of Pigeon Egg White, J.
Biol. Chem., Vol. 276, Issue 26, 23221-23229, Jun. 29, 2001 [0224]
Lactosylceramide structures which are thought to be important in
the binding of EHEC to human tissues;.sup.12 .sup.12 Teneberg,
Susann, Jonas Angstrom, Asa Ljungh, Carbohydrate recognition by
enterohemorrhagic Escherichia coli: characterization of a novel
glycosphingolipid from cat small intestine, Glycobiology vol. 14
no. 2 pp. 187.+-.196, 2004 [0225] Gal.alpha.1-4Gal.beta.1-4Glc,
which has been shown to be important in binding of Shiga-like toxin
2 (Stx2).sup.13; and .sup.13 Tomoda H.; Arai M. 1; Koyama N.;
Matsui H.; O mura S.; Obata R.; Lee Y. C., Purification of
Shiga-like toxin 1 by pigeon egg white glycoproteins immobilized on
Sepharose gels, Analytical Biochemistry, Volume 311, Number 1,
December 2002, pp. 50-56(7) [0226]
Gal.alpha.(1-4)Gal.beta.(1-4)GlcNAc which has been shown to be
important in binding of Shiga-like toxin 1 (Stx1),.sup.Ibid
[0227] 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.
[0228] As noted above, 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. However, in the
context of therapeutic constructs of the present invention, more
particularly ingestible biocompatible glycosylated particles, the
pathogen binding glycosylated molecule is not limited to those that
mimic endogenous pathogen binding structures. In fact, any number
of glycosylated molecules may be utilized as the pathogen binding
moiety, including antibodies and products of a phage display
process. Moreover, the therapeutic biocompatible 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.
[0229] Accordingly, additional examples of 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.
[0230] In addition to tagging a pathogen for an immune system
response, antibodies are also known to interfere with the
pathogen's ability to bind to and infect host tissues. As noted
previously, binding and infection of pathogen with host tissue
arises from the specific interaction between pathogen expressed
adhesins (e.g., lectins) and glycan signatures exhibited by the
host tissue. 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..sup.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. .sup.14 Sharon N.,
Carbohydrates as future anti-adhesion drugs for infectious
diseases, Biochim Biophys Acta. Apr. 17, 2006 (4):527-37.
[0231] For example, 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..sup.15
16 Krippner, et. al., in International Publication No.
WO/2007/048190, the entire contents of which are incorporated
herein by reference, describe the formation of glycodendrimer and
protein 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
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..sup.17 Accordingly, both
glycodendrimers and glycodendriproteins may be used in the
construction of the ingestible biomimetic films and particles of
the present invention. .sup.15 Touaibia, Mohamed; Roy, Rene,
Glycodendrimers as Anti-Adhesion Drugs Against Type 1 Fimbriated E.
coli Uropathogenic Infections, Mini Reviews in Medicinal Chemistry,
Volume 7, Number 12, December 2007, pp. 1270-1283.sup.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.sup.17 Rendle, P. M.,
A. P. Seger, J. Rodrigues, N. J. Oldham, R. R. Bott, J. B. Jones,
M. M. Cowan, B. G. Davis Glycodendriproteins: a Synthetic
Glycoprotein Mimic Enzyme with Branched Sugar-display Potently
Inhibits Bacterial Aggregation, J. Am. Chem. Soc. 2004, 126,
4750-4751
[0232] 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. Likewise, 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 U.S. Pat. No. 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.
[0233] 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 Ser. Nos. 11/413,826, 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.
[0234] Lactoferrin (also known as lactotransferrin) also has
pathogen binding properties of interest. .sup.18192021 Studies have
shown that some pathogens exhibit host tropism based on lactoferrin
properties specific to a single host species..sup.22 A method for
immobilization of lactoferrin on a substrate is described by
Satyanarayan A. Naidu in U.S. Pat. No. 6,172,040, the contents of
which is incorporated herein by reference in its entirety. Naidu
describes the use of immobilized lactoferrin as a coating for beef
carcasses during processing as a means of preventing bacterial
adhesion to the beef tissue. The present invention contemplates a
unique use of lactoferrin in the context of biomimetic films and
particles of the present invention, in both the fields of pathogen
capture for concentration and detection and pathogen capture in
therapeutic uses. .sup.18 van der Strate B W, Beljaars L, Molema G,
et al. (2002). "Antiviral activities of lactoferrin.". Antiviral
Res. 52 (3): 225-39..sup.19 Weinberg E D (2002). "Human
lactoferrin: a novel therapeutic with broad spectrum potential.".
J. Pharm. Pharmacol. 53 (10): 1303-10..sup.20 Valenti P, Antonini G
(2006). "Lactoferrin: an important host defence against microbial
and viral attack.". Cell. Mol. Life. Sci. 62 (22): 2576-87..sup.21
Ward P P, Paz E, Conneely O M (2006). "Multifunctional roles of
lactoferrin: a critical overview.". Cell. Mol. Life. Sci. 62 (22):
2540-8..sup.22 DHAENENS, L., F. SZCZEBARA, M. O. HUSSON,
Characterization, and Immunogenicity of the Lactoferrin-Binding
Protein from Helicobacter pylori, INFECTION AND IMMUNITY, Vol. 65,
No. 2, February 1997, p. 514-518
[0235] In binding to the intestinal epithelium, Enteropathogenic
Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli
(EHEC) exhibit a broad range of binding motifs depending on the
stage of the infection. 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..sup.23 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. Specifically, 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..sup.24 Such 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). Thus, the surface moieties
of attaching and effacing (A/E) lesions may be harvested, for
example by rupturing the cell membranes. Alternatively, synthetic
equivalents of such surface expressing moieties may be
synthetically derived, for example by means of phage display to
develop an intimin recognition product. In either event, 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. .sup.23 Teneberg, Susann, Jonas Angstrom, Asa Ljungh,
Carbohydrate recognition by enterohemorrhagic Escherichia coli:
characterization of a novel glycosphingolipid from cat small
intestine, Glycobiology vol. 14 no. 2 pp. 187.+-.196, 2004.sup.24
Donnenberg M S, Escherichia coli: Virulence Mechanisms of a
Versatile Pathogen. ed. San Diego: Academic Press (2002).
[0236] Trans-membrane molecules, as well as other glycan signature
sequences, 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.
[0237] Finally, 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. In this manner, the resulting construct can provide
functionality beyond what can be achieved with a homogeneous
population. In instances wherein 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. Likewise, in instances wherein the
pathogen binding film results from a mixture of amphiphilic
molecules which self-align, the other amphiphilic molecules can be
selected so as the mixture forms a more stable amphiphilic film
than one of just biomimetic molecules.
[0238] B. Constructing Therapeutic Biocompatible Particles:
[0239] In the context of the present invention, 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.
[0240] 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. Alternatively, 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.
[0241] 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.
[0242] 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. Use of 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.
[0243] 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.
[0244] An illustrative method for making a biomimetic films and
biocompatible particles of the present invention suitable for
therapeutic use, comprises the following steps: [0245] 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; [0246] 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; [0247] c. allow the amphiphilic
molecules to align so as to form a membrane which separates the
polar solvent from the non-polar liquid; [0248] 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 be achieved through rapid cooling of or removal
of pressure force on the non-polar liquid. [0249] e. if additional
properties are required: 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. [0250] f. Optionally, remove said
film from any surrounding liquid to form a dry construct.
[0251] An alternate method for making the biomimetic films,
particles, and constructs thereof comprising the following steps:
[0252] a. provide a liquid having sufficient quantities dissolved
therein of one or more glycosylated or other molecules with
pathogen or toxin binding properties; [0253] b. expose said liquid
to a solid with surface moieties appropriate for conjugation with
said molecules; [0254] 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. [0255] d. Optionally, remove said film from any
surrounding liquid to form a dry construct.
[0256] An illustrative method for using the biomimetic films,
particles, and constructs thereof may comprise the following steps:
[0257] a. Administer one or multiple doses of a plurality of
particles of said constructs to a suspected host of a pathogen or
toxin infection through a catheter or similar device or through
other means of physical placement into a region of suspected
infection or a point in the host where the particles will migrate
to a region where pathogens and or toxins may be present, or one or
multiple oral doses of a plurality of particles of said constructs
to a suspected host to a pathogen or toxin infection. [0258] b.
Allow the particles of the present invention to contact those areas
of the host where pathogens and/or toxins may be present. [0259] c.
Allow the host organism to discharge the particles through
defecation, urination, vomiting, mucosal flow, expectoration or
other means of natural or induced discharge or remove the particles
from the host through a catheter or other device.
[0260] Another method of use for the biomimetic films, particles,
and constructs thereof comprising the following steps: [0261] 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 [0262] b. Induce contact of said bodily fluid to one
or more surfaces with the pathogen and toxin binding moieties of
the present invention. [0263] c. Remove said surfaces from contact
with said bodily fluid. [0264] d. Optionally, reintroduce said
bodily fluid with pathogens/toxins removed into the organism.
[0265] e. Optionally, subject said surfaces of the present
invention to analysis techniques to determine presence and/or type
of pathogens or toxins which may have bound to the surface.
[0266] As noted previously, 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. Of value in the therapeutic aspects of the present invention
are 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. As
a general rule, for roughly spherical particles, this may require
particles with nominal diameters greater than 20 nanometers. To
produce particles which can pass through the gastro-intestinal
tract without being absorbed through the intestinal wall, studies
suggest that at a greater than 50 nanometer particle size, less
than 50% are absorbed through the intestinal wall, and as a general
rule particles of 500 nanometers and larger do not pass into
epithelia of the gastrointestinal tract..sup.25262728 This presumes
a roughly spherical shape, though a cylindrical shape with a length
longer than that threshold might be absorbed if oriented properly.
Additionally, the properties of the intestinal wall will vary from
host to host and also vary based on condition of health of the
intestinal wall tissue. .sup.25 Jani P, Halbert G W, Langridge J,
Florence A T. Nanoparticle uptake by the rat gastrointestinal
mucosa: quantitation and particle size dependency. J Pharm
Pharmacol. 1990 December; 42(12):821-6..sup.26 Desai M P,
Labhasetwar V, Amidon G L, Levy R J. Gastrointestinal uptake of
biodegradable microparticles: effect of particle size. Pharm Res.
1996 December; 13(12):1838-45..sup.27 Hussain N, Jaitley V,
Florence A T. Recent advances in the understanding of uptake of
microparticulates across the gastrointestinal lymphatics. Adv Drug
Deliv Rev. 2001 Aug. 23; 50(1-2):107-42..sup.28 Francis M, Cristea
M, Winnik F M. Polymeric micelles for oral drug delivery: Why and
how Pure Appl. Chem. 2004; 76:1321-1335.
[0267] The probability that a cylinder with a length greater than
50 nanometers is absorbed decreases with increasing length. Even
film or thread-like constructs with diameters less than 50
nanometers will not easily be absorbed if the length is
significantly greater than that. Very thin constructs, having
diameters far smaller than 50 nanometers, are also of value in the
present invention so long as the length is sufficient to impair
absorption. Accordingly, constructs such as nano-thickness films
and worm micelles.sup.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. These 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.
.sup.29Vijayan, Kandaswamy, Discher, Dennis E., Block Copolymer
Worm Micelles in Dilution: Mechanochemical Metrics of Robustness as
a Basis for Novel Linear Assemblies Journal of Polymer Science Part
B: Polymer Physics, Volume 44, Issue 24 (p 3431-3433)
[0268] 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 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.alpha.1-4Gal.beta.1-4Glc,
which has been shown to be important in binding of Shiga-like toxin
2 (Stx2); Gal.alpha.(1-4)Gal.beta.(1-4)GlcNAc, which has been shown
to be important in binding binding of Shiga-like toxin 1 (Stx1); or
any one or more of the pathogen binding sequence set forth in
Appendix A included herewith. Synthetic equivalents thereof are
also contemplated.
[0269] In a preferred embodiment, 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.
[0270] In the context of the micelles with self-assembled
amphiphilic films, it is preferable to utilize 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.
[0271] As noted above, it is an object that the amphiphilic
molecules self-align due to hydrophobic forces to form the film. It
is a further object of the present invention that 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: [0272] a. can contain a
mixture of glycosylated amphiphilic molecules with pathogen and/or
toxin binding properties and other amphiphilic molecules which
together form a more stable amphiphilic film than one of just said
glycosylated molecules, [0273] 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, and/or
[0274] 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.
[0275] 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.
[0276] C. Formulation and Delivery of Biocompatible Pathogen
Binding Particles:
[0277] It is an object of the present invention that the
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. However, it is also noted that the particles and
formulations of the present invention can be provided to a host
organism at any point in the cycle of an infection.
[0278] 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. In the
context of the present invention, 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.
[0279] 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. It may also be
desirable to include sources of iron with the formulation, as the
production of toxin in enterohemorrhagic E. coli is thought to be
enhanced under iron deficient conditions..sup.30 31 The formulation
may also include antibiotics or probiotics. .sup.30 Todar, Kenneth,
Todar's Online Textbook of Bacteriology, 2008,
http://www.textbookofbacteriology.net/e.coli.html.sup.31 Roth, R.
I., Panter, S. S., Zegna, A. I., Arellano, F. A., Levin, J.,
Effects of iron on bacterial endotoxin. Journal of Endotoxin
Research 4:44, 273-278, 1997.
[0280] 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.
[0281] 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.
In a preferred embodiment, 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.
[0282] 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 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.
[0283] Also contemplated is the inclusion of 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.
[0284] 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).
[0285] 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.
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.
[0286] An illustrative treatment method suitable for use with the
therapeutic particles and formulations of the present invention may
include the following steps: [0287] 1. Administering one or
multiple oral doses of a plurality of particles of the present
invention to the host. [0288] 2. Allowing the particles of the
present invention to contact areas of the alimentary system of the
host where pathogens may be present. [0289] 3. Allowing the host
organism to discharge the particles through defecation, vomiting,
expectoration or other means of natural or induced discharge.
[0290] An alternative therapeutic treatment comprises the steps of:
[0291] 1. Administering one or multiple doses of a plurality of
particles of the present invention to the host through a catheter
or similar device or through other means of physically placement
into a region of suspected infection or a point in the host where
the particles will migrate to a region where pathogens may be
present. [0292] 2. Allowing the particles of the present invention
to contact those areas of the host where pathogens may be present.
[0293] 3. Allowing the host organism to discharge the particles
through defecation, urination, vomiting, mucosal flow,
expectoration or other means of natural or induced discharge or
removing the particles from the host through a catheter or other
device.
[0294] Another alternative method for therapeutic treatment or
diagnostic using the particles of the present invention is as
follows: [0295] 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 [0296] 2. Inducing contact of
said bodily fluid to one or more surfaces with the pathogen and
toxin binding moieties of the present invention. [0297] 3.
Optionally, reintroducing of the bodily fluid into the organism.
[0298] 4. Optionally, subjecting said surfaces of the present
invention to analysis techniques to determine presence and/or type
of pathogens or toxins which may have bound to the surface.
[0299] IV. Solid State Films from Glycosylated Amphiphilic
Molecules:
[0300] 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. In
this aspect, 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.
[0301] 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. This
technology is discussed in detail in U.S. Pat. Nos. 5,824,337
(Mullen), 6,528,092 (Mullen), and 7,148,031 (Mullen), the contents
of which are incorporated by reference herein in their entirety.
Accordingly, micelles that embody this technology are often
referred to herein as "Mullen micelles".
[0302] In many instances 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. For example, unlike phospholipids based liposomes
and micelles, 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 reparatory funnel or syringe
which, in turn, enables the creation of micelle populations that
are relatively uniform in size. In addition, 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).
[0303] Depending upon the amphiphilic compound utilized, 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.
However, 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. To that end, 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. More
particularly, 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.
[0304] 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. In the context of stable films of
amphiphilic molecules that self-assembly as a result of on
hydrophobic forces, in many cases it is necessary to achieve a
threshold density of molecules with amphiphilic properties in the
polar solution. This concentration is known as the critical micelle
concentrations (CMC). In a situation where biological molecules of
interest are not present in a solution at or above the critical
micelle concentration, it may be necessary to have present in the
solution (through nature or design) other amphiphilic molecules
which may or may not be glycosylated in order to achieve a
sufficient concentration of amphiphilic molecules to form a stable
self-assembled film which includes the biological molecules of
interest.
[0305] 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. 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.
[0306] In preparation of the constructs of this invention, the
solid or solid-film material may be allowed to solidify without
agitation, resulting in a relatively flat or contour conforming
surface. If the non-polar material, for example a wax or plastic
polymer, 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.
[0307] As with fluid micelles, 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.
[0308] Accordingly, a typical method for a making a glycosylated
amphiphilic membrane or film of the present invention generally
includes the following steps: [0309] (a) providing a polar solvent
having sufficient quantities of one or more glycosylated
amphiphilic molecules dissolved therein; [0310] (b) exposing the
polar solvent to a non-polar liquid, the non-polar liquid being
immiscible in said polar solvent; [0311] (c) allowing the
glycosylated amphiphilic molecules to align so as to form a
membrane that separates the polar solvent from the non-polar
liquid; and [0312] (d) inducing transformation of the non-polar
liquid to a corresponding non-polar solid, for example, through the
application of thermal, chemical or radiative stimulus, such that
the hydrophobic tail ends of the amphiphilic molecule become
embedded in or chemically or mechanically linked to the non-polar
substrate while the hydrophilic tail ends project from the
substrate to provide the membrane with a functionally active
surface.
[0313] The polar solvent may optionally be removed so as to yield a
dry solid state film. As mentioned above, depending upon the
desired membrane form, shape and structure, it may be further
desirable to manipulate the reactants prior to, during or
subsequent to transition. For example, by controlling the temporal
component of the transition process (for example, speeding up the
transition process through rapid cooling of the non-polar liquid),
one can drive the production of micelle constructs having
particular size or geometry. Alternatively, agitating the film
prior to or during transition will result in the formation of
micelle-like constructs.
[0314] 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.
[0315] 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.
[0316] V. Utilities of the Films, Particles, and Constructs of the
Present Invention:
[0317] 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.
[0318] Because the pathogen and toxin binding membranes can be
immobilized on relatively stable, benign, and economical surfaces
of a size and configuration which will generally not be absorbed by
surrounding tissue, the 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.
[0319] 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: [0320] (a) exposing the sample to a solid state
membrane or membrane coated implement or device; [0321] (b)
allowing a sufficient time for the functional groups present on the
hydrophilic tail end of the glycosylated amphiphile to bind target
molecules present in the sample; and [0322] (c) removing or
separating the membrane, having target molecules bound thereto,
from the sample.
[0323] In certain instances, it may be desirable to subject the
sample to a force which promotes contact between the target
molecule and the membrane. Illustrative examples of such forces
include, but are not limited to, mechanical acceleration,
centrifugal force, electrical force, magnetic force, hydraulic
force and various other means for agitation.
[0324] Upon removal from the sample, 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.). In certain instances, 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.)
[0325] Since the technology can exhibit both the properties of
binding to pathogens and physical manifestation in a microscopic
form (micelle construct), 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.
[0326] In that 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. For example, 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.
Alternatively, in the context of pathogen capture and filtration,
it may be beneficial to coat a vessel or pipe or filter media
contained within with a 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. Accordingly,
in addition to serving as a source for micelle construction, 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.
[0327] In the context of the instant invention, 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. Alternatively, the "other" material may
comprise a useful device or implement, for example, a sample
containing vessel, pipe, tube, or the like. For example, in those
instances where the sample is in the form of a continuous fluid
stream, 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. In
other embodiments, it may be desirable to adhere a membrane coating
of the instant invention to the walls of a centrifuge, such that
contaminating substances present in the sample are accelerated
towards and then captured by the hydrophilic moieties present on
the surface of the film, the particular hydrophilic moieties having
binding specificity for target contaminants of interest. In further
embodiments, the useful implement may comprise a medical device
that would benefit from biological properties displayed by the
glycosylated amphiphilic membrane of the present invention.
[0328] Medical devices, particularly implantable medical devices,
often fail as a result of a biological organism's reaction to its
introduction. In many instances, 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.
In addition, 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.
[0329] Accordingly, in the context of medical devices, it is highly
advantageous to be able to mask the foreign nature of the device so
as to prevent the rejection process. One means to achieve this goal
involves coating the medical device with a biocompatible material
that minimizes or substantially eliminates such negative reactions.
In addition, functionally inert surface coatings for medical
implant devices can effectively limit host rejection of the medical
implant device through attached ligands, incorporated drugs, and
reduced NSB responsible for biofouling of such devices in or ex
vivo. The glycosylated amphiphilic membranes of the instant
invention find particular utility as such biocompatible coatings.
In that many glycosylated moieties are native to living systems,
they are less likely to trigger immune response. Accordingly, solid
state membranes expressing such endogenous glycosylated amphiphiles
(or functionally active fragments or derivatives thereof) 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.
[0330] In certain instances, in the interest of further reducing
potential immune response, it may be desirable to construct 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.
While 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.
[0331] 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). Examples of
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.
[0332] In addition to enhancing the biocompatibility of medical
devices, the solid state films and membranes of the instant
invention, more particularly the functionally active glycosylated
moieties provided on the surface thereof, may possess analytic,
diagnostic and/or therapeutic utility. For example, 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. Examples of 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. Also contemplated are foreign materials,
particularly biohazardous materials, such as contaminants, drugs,
toxins, heavy metals, pathogens and the like.
[0333] In certain instances, the glycosylated amphiphilic membranes
of the present invention (as well as medical devices formed
therewith) can serve not only to detect the presence of the target
molecule but also effectively remove it from (i.e., detoxify) the
sample. For example, when 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.
[0334] The glycosylated amphiphilic membranes of the instant
invention also find utility in the areas of environmental detection
and detoxification. For example, 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). By positioning a filter with a functionally active surface
in contact with a stationary or flowing water source, 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. Alternatively, 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.
[0335] Depending upon the detection methodology utilized, it may be
desirable to transport the biomimetic films to a separate location,
such as a laboratory setting, for analysis. In that the target
molecule is firmly bound to the membrane, it no longer constitutes
a danger to either the surrounding environment or to the analyst
(i.e., safe handling through conjugation). Accordingly, 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. In the context of hazardous materials, such as pathogens,
the lysing solution is likely to effectively kill the pathogen,
thereby eliminating the dangers associated with conventional
detection methods that utilize live pathogens.
[0336] Hereinafter, the present invention is described in more
detail by reference to the Examples. However, the following
materials, methods and examples only illustrate aspects of the
invention and in no way are intended to limit the scope of the
present invention. As such, methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention.
EXAMPLES
Example 1
[0337] 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 50 nm to 1000
nm 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.
[0338] The porcine small intestine wax micelles were demonstrated
for research and analytical use in this case. However, 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
[0339] 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 Coli strain which is always fimbirated.
Example 3
[0340] Shaw, et. al., .sup.32 demonstrated that Tir can be
translocated into red blood cell (RBC) membranes, and that the
resulting transmembrane molecules (described by Race, et.
al..sup.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). 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. In this
example, 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. It is also envisioned that 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 50 nm to 1000 nm 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 individual are scanned at regular intervals
to detect location and accumulation levels of radioactively tagged
paraffin particles, to determine points and gross quantities of
intimin expressing bacteria. .sup.32 Shaw, Robert K., Sarah
Daniell, Gad Frankel and Stuart Knutton, Enteropathogenic
Escherichia coli translocate Tir and form an intimin-Tir intimate
attachment to red blood cell membranes, Microbiology (2002), 148,
1355-1365..sup.33 Race, Paul R., Jeremy H. Lakey, and Mark J.
Banfield Insertion of the Enteropathogenic Escherichia coli Tir
Virulence Protein into Membranes in Vitro, J. Biol. Chem., Vol.
281, Issue 12, 7842-7849, Mar. 24, 2006
Example 4
[0341] A hydrogel is prepared using techniques taught by Stupp, et.
al. in US patent application 2005/0209145. In this application
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.
Example 5
[0342] Paraffin wax (100 ml) was melted at a temperature of
70.degree. C. Pigeon egg white (50 ml) was dissolved in distilled
water (400 ml) and the resultant mixture was filtered and then
heated to 70.degree. C. The melted wax was added to the egg white
mixture, which was then agitated and quickly cooled to a
temperature below 40.degree. 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) were
dried, rewetted, and exposed to fluorescent lectins. The lectins
were found to successfully bind to the sugars on the embedded
glycoproteins. The glycoproteins of pigeon egg white express
Gal(1-4)Gal.beta.(1-4)GlcNAc, which has been shown to bind to
Shiga-like Toxin 1..sup.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..sup.35 Accordingly, the material can be a component in an
orally administered therapeutic treatment for these two types of
infections. .sup.34 Tomoda, Hiroshi; Masayoshi Arai, Nobuhiro
Koyama, Hidenori Matsui, Satoshi mura, Rika Obata and Yuan C. Lee,
Purification of Shiga-like toxin 1 by pigeon egg white
glycoproteins immobilized on Sepharose gels, Analytical
Biochemistry Vol 311, Issue 1, 1 Dec. 2002, pp 50-56.sup.35 Haataja
S, Tikkanen K, Liukkonen J, Francois-Gerard C, Finne J.,
Characterization of a novel bacterial adhesion specificity of
Streptococcus suis recognizing blood group P receptor
oligosaccharides, J Biol. Chem. 1993 Feb. 25; 268(6):4311-7.
[0343] 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.
Example 6
[0344] 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.degree. 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..sup.3637 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. .sup.36 Hamilton, Stephen R., et. al., Humanization of Yeast
to Produce Complex Terminally Sialylated Glycoproteins, Science 8
Sep. 2006: Vol. 313. no. 5792, pp. 1441-1443.sup.37 Amano, Koh, et.
al., Engineering of mucin-type human glycoproteins in yeast cells,
PNAS | Mar. 4, 2008, vol. 105, no. 9, pp. 3232-3237
Example 7
[0345] A solution containing Tamm-Horsfall glycoproteins (also
uromodulin) and microcrystalline wax was heated to 98.degree. 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 50 nm to 1000 nm 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.
Example 8
[0346] 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 Coli 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.
Example 9
[0347] 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
[0348] 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)..sup.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 solution
under agitation to form micelles. The solution is then cooled to
form semi-solid, but pliable micelles with glycosylation sites
expressed on the surface. These micelles are used as an
agglutination assay in the study of potential brain pathogens.
.sup.38 Liu H W, Chen C H, Tsai C L, Lin I H, Hsiue G H,
Heterobifunctional poly(ethylene glycol)-tethered bone
morphogenetic protein-2-stimulated bone marrow mesenchymal stromal
cell differentiation and osteogenesis, Tissue Eng. 2007 May;
13(5):1113-24.
Example 11
[0349] 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.
Example 12
[0350] 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
Neisseria meningitidis in the spinal fluid for diagnostic, research
or therapeutic purposes..sup.39 .sup.39 Evans & Oakhill
Biochem. Soc. Trans. 30 (4): 705-7, 2002
INDUSTRIAL APPLICABILITY
[0351] The 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. As such, 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 personnel) subject
to intestinal illnesses in visited locations, and researchers doing
work on pathogen and toxin tissue tropism. 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.
[0352] In addition, 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. In that 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.
[0353] All patents and publications mentioned herein are
incorporated by reference in their entirety. Nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
[0354] While the invention has been described in detail and with
reference to specific embodiments thereof, it is to be understood
that the foregoing description is exemplary and explanatory in
nature and is intended to illustrate the invention and its
preferred embodiments. Through routine experimentation, one skilled
in the art will readily recognize that various changes and
modifications can be made therein without departing from the spirit
and scope of the invention.
[0355] Other advantages and features will become apparent from the
claims filed hereafter, with the scope of such claims to be
determined by their reasonable equivalents, as would be understood
by those skilled in the art. Thus, the invention is intended to be
defined not by the above description, but by the following claims
and their equivalents.
TABLE-US-00001 APPENDIX A Known Pathogen Target Sequences (b1-3)Gal
(b1-4)Gal Fuc Fuc(a1-2)[Gal(a1-3)]Gal(b1-3)GalNAc(b1-4)[Gal
(b1-4)Glc(b1-1)Cer]NeuAc(a1-3)
Fuc(a1-2)[Gal(a1-3)]Gal(b1-3)GalNAc(b1-4)[NeuAc
(a1-3)]Gal(b1-4)Glc(b1-1)Cer
Fuc(a1-2)[Gal(a1-3)]Gal(b1-4)GlcNAc(b1-6)[Gal
(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer]NeuAc
(a1-3)Gal(b1-4)GlcNAc(b1-3)
Fuc(a1-2)[Gal(a1-3)]Gal(b1-4)GlcNAc(b1-6)[NeuAc
(a1-3)Gal(b1-4)GlcNAc(b1-3)]Gal(b1-4)GlcNAc(b1-3)
Gal(b1-4)Glc(b1-1)Cer
Fuc(a1-2)[Gal(a1-3)Gal(b1-3)]GlcNAc[Fuc(a1-4)]
Fuc(a1-2)[GalNAc(a1-3)Gal(b1-3)]Fuc(a1-4)[GlcNAc]
Fuc(a1-2)[GalNAc(a1-3)Gal(b1-3)]GlcNAc
Fuc(a1-2)[GalNAc(a1-3)Gal(b1-3)]GlcNAc[Fuc(a1-4)]
Fuc(a1-2)Gal(b1-3)[Fuc(a1-4)]Gal
Fuc(a1-2)Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1-3)Gal(b1-4) Glc(b1-1)Cer
Fuc(a1-2)Gal(b1-3)Fuc(a1-4)[GlcNAc]
Fuc(a1-2)Gal(b1-3)GalNAc(b1-4)Gal(b1-4)Glc(b1-1) Cer
Fuc(a1-2)Gal(b1-3)GlcNAc Fuc(a1-2)Gal(b1-4)GlcNac
Fuc(a1-3)[Gal(b1-4)]GlcNAc(b1-6)[Gal(b1-3)]GalNAc
(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)Cer
Fuc(a1-3)[Gal(b1-4)]GlcNAc(b1-6)[GalNAc(b1-3)Gal
(a1-4)Gal(b1-4)Glc(b1-1)Cer]Gal(b1-3)
Fuc(a1-3)[NeuAc(a1-3)Gal(b1-4)]GlcNAc(b1-3)Gal
(b1-4)[Fuc(a1-3)]GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer
Fuc(a1-3)[NeuAc(a1-3)Gal(b1-4)]GlcNAc(b1-3)Gal
(b1-4)[GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer]Fuc(a1-3)
Fuc(a1-3)[NeuAc(a1-3)Gal(b1-4)]GlcNAc(b1-3)Gal (b1-4)Glc(b1-1)Cer
Fuc(a1-3)[NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal
(b1-4)]GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer
Fuc(a1-4)[Fuc(a1-2)Gal(b1-3)]Gal
Fuc(a1-4)[Fuc(a1-2)Gal(b1-3)]GlcNAc(b1-3)Gal(b1-4) Glc(b1-1)Cer
Fuc(a1-4)[Gal(b1-3)]GlcNAc(b1-3)Gal(b1-4)Glc
Fuc(a1-4)[Gal(b1-3)]GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer Gal
Gal(a1-3)[Fuc(a1-2)]Gal(b1-3)GalNAc(b1-4)[Gal
(b1-4)Glc(b1-1)Cer]NeuAc(a1-3)
Gal(a1-3)[Fuc(a1-2)]Gal(b1-3)GalNAc(b1-4)[NeuAc
(a1-3)]Gal(b1-4)Glc(b1-1)Cer
Gal(a1-3)[Fuc(a1-2)]Gal(b1-4)GlcNAc(b1-6)[Gal
(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer]NeuAc
(a1-3)Gal(b1-4)GlcNAc(b1-3)
Gal(a1-3)[Fuc(a1-2)]Gal(b1-4)GlcNAc(b1-6)[NeuAc
(a1-3)Gal(b1-4)GlcNAc(b1-3)]Gal(b1-4)GlcNAc(b1-3)
Gal(b1-4)Glc(b1-1)Cer Gal(a1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)Cer
Gal(a1-3)Gal(b1-3)[Fuc(a1-2)]Fuc(a1-4)[GlcNAc]
Gal(a1-3)Gal(b1-3)[Fuc(a1-2)]GlcNAc[Fuc(a1-4)]
Gal(a1-3)Gal(b1-4)Glc Gal(a1-3)Gal(b1-4)Glc(b1-1)
Gal(a1-3)Gal(b1-4)Glc(b1-1)Cer Gal(a1-3)Gal(b1-4)GlcNAc
Gal(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1) Cer
Gal(a1-3)Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal
(b1-4)Glc(b1-3)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc (b1-1)
Gal(a1-4)Gal Gal(a1-4)Gal(b Gal(a1-4)Gal(b1-1)
Gal(a1-4)Gal(b1-1)Cer Gal(a1-4)Gal(b1-1)Glc Gal(a1-4)Gal(b1-4)Glc
Gal(a1-4)Gal(b1-4)Glc(b1-1) Gal(a1-4)Gal(b1-4)Glc(b1-1)Cer
Gal(a1-4)Gal(b1-4)Glc(b1-1)Me Gal(a1-4)Gal(b1-4)GlcNAc
Gal(a1-4)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1) Cer Gal(a1-4)Glc(b
Gal(b Gal(b1-1)Cer Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1-3)Gal(b1-4)Glc
Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1-3)Gal(b1-4)Glc(b1-1) Cer
Gal(b1-3)[Gal(b1-4)GlcNAc(b1-6)]GalNAc(b1-3)Gal
(a1-4)Gal(b1-4)Glc(b1-1)Cer
Gal(b1-3)[NeuAc(a2-3)]GalNAc(b1-4)Gal(b1-4)[NeuAc
(a2-3)Glc(b1-1)Cer Gal(b1-3)[NeuAc(a2-3)]GalNAc(b1-4)Gal(b1-4)Glc
(b1-1)Cer Gal(b1-3)Gal Gal(b1-3)GalNAc
Gal(b1-3)GalNAc(b1-3)[NeuAc(a2-8)NeuAc(a2-3)]Gal (b1-4)Glc(b1-1)Cer
Gal(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)
Gal(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1) Cer
Gal(b1-3)GalNAc(b1-4)[NeuAc(a1-3)]Gal(b1-3)GalNAc
(b1-4)Gal(b1-4)Glc(b1-1)Cer
Gal(b1-3)GalNAc(b1-4)[NeuAc(a1-3)]Gal(b1-4)Glc (b1-1)Cer
Gal(b1-3)GalNAc(b1-4)[NeuAc(a1-8)NeuAc(a1-3)]Gal (b1-4)Glc(b1-1)Cer
Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-3)]Gal
Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-3)]Gal(b1-4)Glc (b1-1)
Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-3)]Gal(b1-4)Glc (b1-1)Cer
Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-8)NeuAc(a2-3)]Gal (b1-4)Glc(b1-1)
Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-8)NeuAc(a2-3)]Gal (b1-4)Glc(b1-1)Cer
Gal(b1-3)GalNAc(b1-4)[NeuGc(a1-3)]Gal(b1-4)Glc (b1-1)Cer
Gal(b1-3)GalNAc(b1-4)Gal(b1-4)Glc
Gal(b1-3)GalNAc(b1-4)Gal(b1-4)Glc(b1-1)
Gal(b1-3)GalNAc(b1-4)Gal(b1-4)Glc(b1-1)Cer
Gal(b1-3)Glc(b1-3)Gal(b1-4)Glc Gal(b1-3)GlcNAc
Gal(b1-3)GlcNAc(b1-3)Gal(b1-4)Glc
Gal(b1-3)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer
Gal(b1-3)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-3)Gal (b1-4)Glc(b1-1)Cer
Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[Gal(b1-3)]GalNAc
(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)Cer
Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[GalNAc(b1-3)Gal
(a1-4)Gal(b1-4)Glc(b1-1)Cer]Gal(b1-3)
Gal(b1-4)[NeuAc(a2-3)]GlcNAc(b1-3)Gal(b1-4)GlcNAc Gal(b1-4)GalNAc
Gal(b1-4)GalNAc(b1-4)Gal(b1-4)Glc(b1-1)Cer Gal(b1-4)Glc
Gal(b1-4)Glc(b1-1)Cer Gal(b1-4)GlcNAc Gal(b1-4)GlcNAc(b1-3)Gal
Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)
Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer
Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc(b1-3)Gal
(a1-4)Gal(b1-4)Glc(b1-1)Cer GalNAc
GalNAc(a1-3)Gal(b1-3)[Fuc(a1-2)]Fuc(a1-4)[GlcNAc]
GalNAc(a1-3)Gal(b1-3)[Fuc(a1-2)]GlcNAc
GalNAc(a1-3)Gal(b1-3)[Fuc(a1-2)]GlcNAc[Fuc(a1-4)]
GalNAc(a1-3)GalNAc(a1-3)Gal(a1-4)Gal(b1-4)Cer
GalNAc(a1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc (b1-1)Cer
GalNac(b
GalNAc(b1-3)[Gal(a1-3)]Gal(a1-4)Gal(b1-4)Glc(b1-1) Cer
GalNAc(b1-3)Gal GalNAc(b1-3)Gal(a1-3)Gal(b1-4)Glc(b1-1)Cer
GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)
GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)Cer
GalNAc(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc (b1-1)Cer
GalNAc(b1-4)[NeuAc(a1-3)]Gal(b1-4)Glc(b1-1)Cer
GalNAc(b1-4)[NeuAc(a1-8)NeuAc(a1-3)]Gal(b1-4)Glc (b1-1)Cer
GalNAc(b1-4)[NeuAc(a2-3)]Gal(b1-4)Glc(b1-1)
GalNAc(b1-4)[NeuAc(a2-3)]Gal(b1-4)Glc(b1-1)Cer GalNAc(b1-4)Gal
GalNAc(b1-4)Gal(b1-4)Glc(b1-1) GalNAc(b1-4)Gal(b1-4)Glc(b1-1)Cer
Glc(b1-1)Cer GlcNAc GlcNAc(b1-3)Gal
GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer GlcNAc(b1-4)GlcNAc Lac(-)Cer Man
Man(a1-2)Man Man(a1-2)Man(a1-6)Man(a1-6)
Man(a1-3)[Man(a1-6)]Man(a1-6)[Man(a1-2)]Man(a1-3) Man
Man(a1-3)[Man(a1-6)]Man(a1-6)[Man(a1-3)Man]Man (a1-2)
Man(a1-3)[Man(a1-6)]Man(a1-6)Man(a1-4)Man Man(a1-3)Man(a1-4)GalNAc
Man(a1-3)Man(a1-6)Man
Man(a1-6)[Man(a1-3)]Man(a1-6)[Man(a1-2)]Man(a1-3) Man
Man(a1-6)[Man(a1-3)]Man(a1-6)[Man(a1-3)Man]Man (a1-2)
Man(a1-6)[Man(a1-3)]Man(a1-6)Man(a1-4)Man NeuAc
NeuAc(a1-3)[Gal(b1-3)GalNAc(b1-4)]Gal(b1-4)Glc (b1-1)Cer
NeuAc(a1-3)[GalNAc(b1-4)]Gal(b1-4)Glc(b1-1)Cer NeuAc(a1-3)Gal
NeuAc(a1-3)Gal(b1-3)GalNAc(b1-4)[NeuAc(a1-8)NeuAc
(a1-3)]Gal(b1-4)Glc(b1-1)Cer NeuAc(a1-3)Gal(b1-4)
NeuAc(a1-3)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-3)Gal
(b1-4)[Fuc(a1-3)]GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer
NeuAc(a1-3)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-3)Gal
(b1-4)[GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer]Fuc(a1-3)
NeuAc(a1-3)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-3)Gal (b1-4)Glc(b1-1)Cer
NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-3)[NeuAc(a1-3)Gal
(b1-4)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-4) Glc(b1-1)Cer
NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)[Fuc
(a1-3)]GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer
NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1) Cer
NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b
NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc
(b1-3)Gal(b1-4)Glc(b1-1)Cer
NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc
(b1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)Cer
NeuAc(a1-3)Gal(b1-4)GlcNAc(b1-6)[NeuAc(a1-3)Gal
(b1-4)GlcNAc(b1-3)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-4) Glc(b1-1)Cer
NeuAc(a1-6)Gal NeuAc(a1-8)NeuAc(a1-3)[Gal(b1-3)GalNAc(b1-4)]Gal
(b1-4)Glc(b1-1)Cer NeuAc(a1-8)NeuAc(a1-3)[GalNAc(b1-4)]Gal(b1-4)Glc
(b1-1)Cer NeuAc(a1-8)NeuAc(a1-3)[NeuAc(a1-3)Gal(b1-3)GalNAc
(b1-4)]Gal(b1-4)Glc(b1-1)Cer NeuAc(a2-3)
NeuAc(a2-3)[Gal(b1-3)GalNAc(b1-4)]Gal(b1-4)Glc (b1-1)
NeuAc(a2-3)[Gal(b1-3)GalNAc(b1-4)]Gal(b1-4)Glc (b1-1)Cer
NeuAc(a2-3)[Gal(b1-4)]GlcNAc(b1-3)Gal(b1-4)GlcNAc
NeuAc(a2-3)[GalNAc(b1-4)]Gal(b1-4)Glc(b1-1)
NeuAc(a2-3)[GalNAc(b1-4)]Gal(b1-4)Glc(b1-1)Cer
NeuAc(a2-3)[NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)]Gal (b1-3)Glc(b1-1)
NeuAc(a2-3)[NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)]Gal (b1-4)Glc(b1-1)Cer
NeuAc(a2-3)Gal NeuAc(a2-3)Gal(b1-3)[NeuAc(a2-6)]GalNAc
NeuAc(a2-3)Gal(b1-3)[NeuAc(a2-6)]GalNAc(b1-3)Gal
(a1-4)Gal(b1-4)Glc(b1-1)Cer NeuAc(a2-3)Gal(b1-3)GalNAc
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4) Glc(b1-1)
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4) Glc(b1-1)Cer
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-3)]Gal (b1-3)Glc(b1-1)
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-3)]Gal (b1-4)Glc(b1-1)Cer
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-3)NeuAc
(2-8)]Gal(b1-4)Glc(b1-1)Cer
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-8)NeuAc
(a2-3)]Gal(b1-4)Glc(b1-1)
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)[NeuAc(a2-8)NeuAc
(a2-3)]Gal(b1-4)Glc(b1-1)Cer
NeuAc(a2-3)Gal(b1-3)GalNAc(b1-4)Gal(b1-4)Glc(b1-1) Cer
NeuAc(a2-3)Gal(b1-4)GalNAc NeuAc(a2-3)Gal(b1-4)Glc
NeuAc(a2-3)Gal(b1-4)Glc(b1-1) NeuAc(a2-3)Gal(b1-4)Glc(b1-1)Cer
NeuAc(a2-3)Gal(b1-4)Glc(b1-3)[Gal(a1-3)Gal(b1-4)
GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc (b1-1)
NeuAc(a2-3)Gal(b1-4)GlcNAc
NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-3)[NeuAc(a2-3)Gal
(b1-4)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-4) Glc(b1-1)Cer
NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc
(b1-3)Gal(b1-4)Glc(b1-1)Cer
NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-4)Gal(b1-4)Glc(b1-1)
NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-4)Gal(b1-4)GlcNAc
(b1-3)Gal(b1-4)Glc(b1-1)
NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal
(b1-4)GlcNAc(b1-3)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-4) Glc(b1-1)Cer
NeuAc(a2-3)Lac NeuAc(a2-3)NeuAc(2-8)[NeuAc(a2-3)Gal(b1-3)GalNAc
(b1-4)]Gal(b1-4)Glc(b1-1)Cer
NeuAc(a2-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc
NeuAc(a2-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc(b1-3)Gal
(a1-4)Gal(b1-4)Glc(b1-1)Cer NeuAc(a2-6)Gal
NeuAc(a2-6)Gal(b1-3)GlcNAc(b1-4)Gal(b1-4)Glc(b1-1) Cer
NeuAc(a2-6)Gal(b1-4)GlcNAc
NeuAc(a2-6)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1) Cer
NeuAc(a2-6)Gal(b1-4)GlcNAc(b1-4)Gal(b1-4)Glc(b1-1) NeuAc(a2-8)
NeuAc(a2-8)NeuAc(a2-3)[Gal(b1-3)GalNAc(b1-3)]Gal (b1-4)Glc(b1-1)Cer
NeuAc(a2-8)NeuAc(a2-3)[Gal(b1-3)GalNAc(b1-4)]Gal (b1-4)Glc(b1-1)
NeuAc(a2-8)NeuAc(a2-3)[Gal(b1-3)GalNAc(b1-4)]Gal (b1-4)Glc(b1-1)Cer
NeuAc(a2-8)NeuAc(a2-3)[NeuAc(a2-3)Gal(b1-3)GalNAc
(b1-4)]Gal(b1-4)Glc(b1-1) NeuAc(a2-8)NeuAc(a2-3)Gal(b1-4)Glc(b1-1)
NeuAc(a2-8)NeuAc(a2-3)Gal(b1-4)Glc(b1-1)Cer
NeuGc(a1-3)[Gal(b1-3)GalNAc(b1-4)]Gal(b1-4)Glc (b1-1)Cer
NeuGc(a1-3)[GalNAc(b1-4)]Gal(b1-4)Glc(b1-1)Cer
NeuGc(a1-3)Gal(b1-4)Glc(b1-1)Cer
NeuGc(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc
(b1-3)Gal(b1-4)Glc(b1-1)Cer NeuGc(a2-3)Gal(b1-4)Glc
NeuGc(a2-3)Gal(b1-4)Glc(b1-1)
NeuGc(a2-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc
(b1-3)Gal(b1-4)Glc(b1-1)Cer NeuNAc(a2-3)Gal(b
NeuNAc(a2-3)Gal(b1-4)Glc
* * * * *
References