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| United States Patent Application |
20070184120
|
| Kind Code
|
A1
|
|
Stutzenberger; Fred J.
; et al.
|
August 9, 2007
|
Adhesin-specific nanoparticles and process for using same
Abstract
The present invention is generally directed to compositions useful in
preventing and/or treating disease due to infection by any of a variety
of biologically active pathogenic microorganisms. The compositions
include nanoparticles formed of a hydrophobic polymeric core, hydrophilic
linking agents bound to the core, and biofunctional materials bound to
the linking agents. The biofunctional materials are functionally
identical to receptors on host cell surfaces that can be recognized and
bound by adhesins on the surface of the targeted pathogenic
adhesin-bearing microorganisms. In one embodiment, the binding action
between the nanoparticles and the microorganisms can lead to the
formation of large agglomerated complexes, which can then be easily
removed from an area, including the digestive tract of an infected
individual. The compositions of the present invention can also be
utilized in preventing enteric infections via the ability to purge
animals of enteropathogens prior to transport and processing for human
consumption.
| Inventors: |
Stutzenberger; Fred J.; (Clemson, SC)
; Latour; Robert A. JR.; (Clemson, SC)
; Sun; Ya-Ping; (Clemson, SC)
; Tzeng; Tzuen R.; (US)
|
| Correspondence Name and Address:
|
DORITY & MANNING, P.A.
POST OFFICE BOX 1449
GREENVILLE
SC
29602-1449
US
|
| Assignee Name and Adress: |
Clemson University
|
| Serial No.:
|
677132 |
| Series Code:
|
10
|
| Filed:
|
October 1, 2003 |
| U.S. Current Class: |
424/499; 424/489; 435/235.1; 435/243; 435/252.1 |
| U.S. Class at Publication: |
424/499; 424/489; 435/235.1; 435/243; 435/252.1 |
| Intern'l Class: |
A61K 9/16 20060101 A61K009/16 |
Claims
1. A composition capable of binding to a biologically active microorganism
comprising: a nanoparticle, said nanoparticle comprising a hydrophobic
polymeric core, a hydrophilic linking agent bound to said polymeric core,
and a biofunctional material bound to said linking agent, wherein said
biofunctional material comprises a binding site for adhesins present on
the surface of the biologically active microorganism.
2. The composition of claim 1, wherein the hydrophobic polymeric core
comprises styrene monomer.
3. The composition of claim 1, wherein the hydrophobic polymeric core
comprises methyl methacrylate monomer.
4. The composition of claim 1, wherein the hydrophobic polymeric core is a
crosslinked polystyrene polymeric core.
5. The composition of claim 1, wherein the nanoparticle is between about
30 nm and about 5 .mu.m in average diameter.
6. The composition of claim 1, wherein the nanoparticle is between about
50 nm and about 1 .mu.m in average diameter.
7. The composition of claim 1, wherein the linking agent comprises a
hydrophilic polymer selected from the group consisting of a polypeptide
and a polyethylene glycol.
8. The composition of claim 1, wherein the linking agent is a derivatized
polyethylene glycol.
9. The composition of claim 1, wherein the linking agent has a degree of
polymerization between about 10 and about 200.
10. The composition of claim 1, wherein the linking agent has a degree of
polymerization between about 20 and about 100.
11. The composition of claim 1, wherein the biofunctional material is
selected from the group consisting of glycolipids, glycoproteins,
peptides, polypeptides, lipids, monosaccharides, polysaccharides, and
derivatives thereof.
12. The composition of claim 1, wherein the biofunctional material is a
carbohydrate moiety.
13. The composition of claim 1, wherein the nanoparticle comprises more
than one different biofunctional material.
14. The composition of claim 1, wherein the composition is an aqueous
suspension.
15. The composition of claim 1, wherein the biologically active
microorganism is a pathogenic bacterial organism.
16. The composition of claim 1, in which the biologically active
microorganism is an enteropathogen.
17. The composition of claim 16, wherein the enteropathogen is C. jejuni.
18. The composition of claim 1, wherein the biologically active
microorganism is selected from the group consisting of a bacterial
pathogen, a viral pathogen, and a parasitic pathogen.
19. The composition of claim 1, wherein the biofunctional material is an
antigentic epitope.
20. The composition of claim 1, wherein the nanoparticle further comprises
a fluorescent label.
21. A process for forming a composition capable of binding to a
biologically active microorganism comprising: providing a hydrophobic
monomer; providing a hydrophilic linking agent; binding the hydrophilic
linking agent to the hydrophobic monomer so as to form a macromonomer
comprising the hydrophobic monomer and the hydrophilic linking agent;
providing additional hydrophobic monomer; polymerizing the additional
hydrophobic monomer with the macromonomer to form a nanoparticle
comprising a core of the polymerized hydrophobic monomer and the
hydrophilic linking agent bound to the core; and binding a biofunctional
material to the hydrophilic linking agent, wherein the biofunctional
material is functionally identical to a receptor recognized by an adhesin
on the surface of the biologically active microorganism.
22. The process of claim 21, wherein the biofunctional material is bound
to the hydrophilic linking agent after the polymerization of the
additional hydrophobic monomer with the macromonomer.
23. The process of claim 21, wherein the biofunctional material is bound
to the hydrophilic linking agent prior to the polymerization of the
additional hydrophobic monomer with the macromonomer, the macromonomer
further comprising the biofunctional material.
24. The process of claim 21, further comprising forming an aqueous
suspension comprising the nanoparticle.
25. A process for agglomerating biologically active microorganisms
comprising: providing a composition comprising multiple nanoparticles,
the nanoparticles comprising a hydrophobic polymeric core, multiple
hydrophilic linking agents bound to the polymeric core, and a
biofunctional material bound to the linking agents, wherein the
biofunctional material bound to the linking agents comprises a binding
site for an adhesin present on the surface of a biologically active
microorganism; and contacting the composition with the biologically
active microorganisms, wherein upon contact, the nanoparticles bind with
one or more microorganisms and the microorganisms bind to one or more
nanoparticles such that an agglomerated complex of nanoparticles and
microorganisms is formed.
26. The process of claim 25 wherein the biologically active microorganism
is an enteropathogenic bacteria.
27. The process of claim 25, wherein the biologically active microorganism
is an enteropathogenic eukaryote.
28. The process of claim 25, wherein the agglomerated complex of
nanoparticles and microorganisms is formed within the digestive system of
an animal.
29. The process of claim 25, wherein the biologically active
microorganisms are contaminants that pose a danger to the general public.
30. A process for labeling microorganisms comprising: providing a
composition comprising a nanoparticle, said nanoparticle comprising a
hydrophobic polymeric core, a hydrophilic linking agent bound to said
polymeric core, a biofunctional material bound to said linking agent, and
a label bound to the polymeric core, wherein said biofunctional material
is functionally identical to a receptor for an adhesin present on the
surface of a biologically active microorganism; and contacting said
biologically active microorganism with said nanoparticle, said
biofunctional material binding with said microorganism at said adhesin.
31. The process of claim 30, wherein the label is a fluorescent label.
32. The process of claim 31, wherein the fluorescent label is a
crosslinking agent within the polymeric core.
33. The process of claim 31, wherein the fluorescent label is bound to the
surface of the polymeric core.
34. The process of claim 30, wherein the biologically active microorganism
a pathogenic bacteria.
35. The process of claim 30, wherein the biologically active microorganism
is a eucaryotic organism.
36. A process for removing a pathogenic microorganism from the digestive
tract of an animal comprising: introducing a composition to the digestive
tract of an animal, the composition comprising a suspension of
nanoparticles, the individual nanoparticles comprising a hydrophobic
polymeric core, a hydrophilic linking agent bound to the polymeric core,
and a biofunctional material bound to the linking agent, wherein the
biofunctional material bound to the linking agent is functionally
identical to a receptor for an adhesin present on the surface of the
pathogenic microorganism; forming agglomerated complexes comprising the
nanoparticles and the pathogenic microorganisms within the digestive
tract of the animal; and removing the agglomerated complexes from the
digestive tract of the animal.
37. The process of claim 36, wherein the animal is poultry.
38. The process of claim 37, wherein the animal is a chicken.
39. The process of claim 36, wherein the animal is a cow.
40. The process of claim 36, wherein the pathogenic microorganism is C.
jejuni.
41. The process of claim 36, wherein the pathogenic microorganism is E.
coli.
42. The process of claim 36, wherein the biofunctional material is a
carbohydrate moiety.
43. The process of claim 42, wherein the carbohydrate moiety is D-mannose
or mannan.
44. The process of claim 36, wherein the hydrophobic polymeric core is a
polystyrene polymer and the linking agent is a polyethylene glycol
polymer.
45. The process of claim 36, wherein the composition is introduced to the
digestive tract of the animal before the animal is transported to a
slaughterhouse.
Description
BACKGROUND OF THE INVENTION
[0001] The biological sources of infection to humans are wide and varied.
For instance, bacteria in the genera Campylobacter, Listeria, Salmonella,
Shigell and Vibrio, as well as various protozoa such as Giardia
intestinalis, and Entamoeba histolytica have long been known to cause
enteric infections. More recently, bacteria in the genera Aeromonas,
Escherichia and Helicobacter as well as rotaviruses and Norwalk-like
viruses have been identified as causative agents of enteric infections.
Recently, concern has been raised that biological pathogens, including
viruses and spores, may be utilized by bioterrorists to cause widespread
infection through introduction into food, water, or air.
[0002] Enteropathogens are spread via contaminated food or water, and are
some of the most virulent and easily communicated pathogens. Symptoms of
enteropathogenic infections can range in severity from mild transient
diarrhea, cramping and nausea to life-threatening dehydration, toxemia
and circulatory collapse. Improved food sanitation, municipal water
purification and personal hygiene have greatly reduced the incidence of
enteric infection in the developed world. These diseases still pose a
dangerous threat, however, especially in the developing world,
particularly for children, as infection often leads not only to disease,
but often to death.
[0003] Attempts have been made to develop antibiotics and vaccinations
against some of the pathogens that cause enteric infections, but these
attempts have met with limited success. Such agents tend to be quite
expensive, both in development and production costs. In addition, the
sheer variety of pathogenic microorganisms, combined with the ability of
the pathogens to quickly develop resistance to antibiotics and vaccines,
makes the prospect of developing long lasting, inexpensive preventatives
and/or treatments for these diseases by such methods dim. Added to these
problems, even in those instances when the immunity gained from an
inoculation is accurate for the actual pathogen encountered by the
individual, the mucosal immunity gained is short lived (only six months
in the case of the cholera vaccine).
[0004] As such, what is needed in the art is a method of preventing such
infections before they begin. What are needed in the art are products and
methods that can prevent the initial attack of a host by a biological
pathogen. In addition, what are needed in the art are products that can
be utilized to label pathogens in, for example, identification
procedures.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the present invention is directed to a
nanoparticle that includes a hydrophobic polymeric core, a hydrophilic
linking agent bound to the polymeric core, and a biofunctional material
that is capable of binding to a biologically active microorganism bound
to the linking agent. For instance, the biofunctional material bound to
the nanoparticle can include a binding site for adhesins present on the
surface of a targeted microorganism.
[0006] In another embodiment, the present invention is directed to a
composition comprising the nanoparticle. In one embodiment, the
composition can include an aqueous suspension of the nanoparticles.
[0007] The nanoparticles of the invention can be formed in a variety of
sizes and can include many different core, linking, and biofunctional
materials. For example, the nanoparticles can have an average diameter
between about 30 nm and about 5 .mu.m. In one embodiment, the
nanoparticles can have an average diameter between about 50 nm and about
1 .mu.m.
[0008] The hydrophobic polymeric core can be, for example, a styrene
polymer or a methyl methacrylate polymer. In one embodiment, the
hydrophobic polymeric core can be a crosslinked polystyrene core.
[0009] Exemplary hydrophilic linking agents can include polyethylene
glycol and polypeptides. In certain embodiments of the invention, the
linking agents may be derivatized, for example to bind to both the core
material and the biofunctional material as desired. The linking agent can
generally extend off of the surface of the core for a distance. For
instance, the linking agent can have a degree of polymerization between
about 10 and about 200. In one embodiment, the linking agent can have a
degree of polymerization between about 20 and about 100.
[0010] The biofunctional material can include glycolipids, glycoproteins,
peptides, polypeptides, lipids, monosaccharides, polysaccharides, and
derivatives thereof. In one embodiment, the biofunctional material can be
a simple carbohydrate moiety, such as mannose or mannan, for example.
Optionally, the nanoparticles can include more than one type of
biofunctional material on the surface.
[0011] The nanoparticles can be specifically designed to target a
biologically active microorganism such as, for example, pathogenic
bacterial, eucaryotic, and/or viral microorganisms. In one embodiment,
the composition can target specific enteropathogenic microorganisms such
as C. jejuni or E. coli.
[0012] The nanoparticles of the invention can optionally carry additional
materials, such as antigenic epitopes to be used in immunization or
labeling agents, for delivery to the targeted microorganisms.
[0013] The nanoparticles can be formed by any suitable polymerization
method, and may be formed utilizing either a converging or a diverging
polymerization approach. In a diverging approach to polymerization, a
macromolecule can be formed including the linking agent and a monomer of
the core material. The macromolecule can then be polymerized with
monomers of the core material to form a non-biofunctionalized
nanoparticle. Finally, the biofunctional material can be bound to the
surface of the nanoparticle following polymerization. In a converging
approach, the macromolecule can include a monomer of the core material,
the linking agent, and the biofunctional material. The macromolecule thus
obtained can then be polymerized with monomers of the core material and
the biofunctionalized nanoparticles can be formed in a single
polymerization step.
[0014] The compositions of the present invention can be utilized to
agglomerate targeted microorganisms. The large, agglomerated complexes
formed of nanoparticles bound to multiple microorganisms can be removed
from an area and the composition can thus be utilized to decontaminate an
area. In one embodiment, the agglomerated complexes can be formed within
the digestive tract of an animal after the biofunctional nanoparticles
that are biofunctionalized to compete with the native host cell receptors
for the microbial adhesin sites have been ingested. In this embodiment,
the formation of the agglomerated complexes can be used to purge the
animal of the microorganisms. For example, the large agglomerated
complexes can be removed from the digestive tract via the natural
peristaltic action of the animal. For instance, animals can be purged of
pathogenic microorganisms prior to transport and processing. In one
particular embodiment, chickens can be purged of C. jejuni prior to
transport. In another particular embodiment, cattle can be purged of E.
coli prior to being sent to a slaughterhouse. This can help to prevent
the spread of enteric pathogens from an infected group of animals to
later groups of animals processed on the same equipment as well as help
to prevent diseases due to pathogen infection of consumers.
[0015] The compositions of the present invention can also be utilized to
label targeted microorganisms. For instance, the nanoparticles can
include a label, such as a fluorescent label, which can then be detected
following the binding of the nanoparticles to the targeted
microorganisms.
BRIEF DESCRIPTION OF THE FIGURES
[0016] A full and enabling disclosure of the present invention, including
the best mode thereof, to one of ordinary skill in the art, is set forth
more particularly in the remainder of the specification, including
reference to the accompanying figures, in which:
[0017] FIG. 1 is a formation mechanism for a carboxylic acid
terminated-styrene-polyethylene glycol (PEG) macromonomer that can be
utilized in forming biofunctionalized nanoparticles of the present
invention;
[0018] FIG. 2 illustrates a formation mechanism for
2-aminoethyl-.alpha.-D-pyranmannoside;
[0019] FIG. 3 illustrates a formation mechanism for D-Mannose-containing
styrene-PEG macromonomer as may be used in forming mannose
biofunctionalized nanoparticles of the present invention;
[0020] FIG. 4 illustrates a convergent method for forming
biofunctionalized nanoparticles including a polystyrene core, a PEG
linking agent, and a mannose biofunctional component;
[0021] FIG. 5 is a formation mechanism for a carboxylic acid terminated
PEG macromonomer that can be utilized in forming biofunctionalized
nanoparticles of the present invention;
[0022] FIG. 6 illustrates two divergent approach formation mechanisms for
biofunctionalized nanoparticles, one mechanism from the carboxylic acid
terminated styrene-PEG macromonomer of FIG. 1 (nanoparticles 6A) and the
other from the carboxylic acid terminated PEG macromonomer of FIG. 5
(nanoparticles 6B);
[0023] FIG. 7 is a transmission electron microscopy (TEM) image of the
polystyrene core of a nanoparticle with PEG linking agents attached,
prior to biofunctionalization;
[0024] FIG. 8 is .sup.1H NMR spectra in the aromatic region for enkaphalin
in D.sub.2O solution (top) and for enkaphalin functionalized
nanoparticles 6A in concentrated aqueous suspension (bottom);
[0025] FIGS. 9a-d are TEM images of E. coil ORN178 agglutinated with
D-mannose biofunctionalized nanoparticles;
[0026] FIG. 9e is a TEM image of E. coil ORN178 mixed with
non-biofunctionalized nanoparticles;
[0027] FIG. 9f is a TEM image of adhesin deficient E. coli ORN208 mixed
with biofunctionalized nanoparticles of the present invention; and
[0028] FIGS. 10A and 10B graphically compare the number of E. coli ORN178
colony forming units (CFU) obtained when mixed with biofunctionalized
nanoparticles (right bar on the graph) and when mixed with a
nonfunctionalized control substance (left bar on the graph).
[0029] Repeat use of reference characters in the present specification and
drawings is intended to represent the same or analogous features or
elements of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Reference will now be made in detail to various embodiments of the
invention, one or more examples of which are set forth below. Each
embodiment is provided by way of explanation of the invention, not
limitation of the invention. In fact, it will be apparent to those
skilled in the art that various modifications and variations may be made
in the present invention without departing from the scope or spirit of
the invention. For instance, features illustrated or described as part of
one embodiment, may be used in another embodiment to yield a still
further embodiment. Thus, it is intended that the present invention
should cover such modifications and variations as come within the scope
of the appended claims and their equivalents.
[0031] The present invention is generally directed to materials and
compositions useful in preventing and/or treating disease due to
infection by any of a variety of biologically active pathogenic
microorganisms. More specifically, the compositions of the present
invention include a component that can securely bind to a targeted
biologically active microorganism, such as a pathogenic microorganism.
[0032] The invention is also directed to methods for utilizing the
disclosed materials. For example, the disclosed materials can be utilized
for prevention and/or treatment of infections caused by pathogenic
microorganisms. The materials can also be utilized for identifying
biological microorganisms or for decontaminating areas that may contain
pathogenic microorganisms. Moreover, the disclosed materials can be very
safe and non-toxic to hosts. As such, the materials may be safely used
with humans or animals.
[0033] The compositions of the present invention include biofunctionalized
nanoparticles that can bind to targeted microorganisms. The
biofunctionalized nanoparticles include a hydrophobic polymeric core,
hydrophilic linking agents attached to the polymeric core, and
biofunctional moieties attached to the linking components. The
biofunctional moieties can be functionally identical to host cell
receptors that can be recognized and bound by the targeted microorganisms
in nature. As such, when the compositions of the present invention are in
the vicinity of the targeted microorganisms, the microorganism will bind
to the biofunctional moiety of the nanoparticle. In one embodiment, this
binding can prevent initial infection of a host by the microorganism. In
another embodiment, this binding can occur after initial attack of a
pathogenic microorganism, but can lead to the detachment of the pathogen
from the cell of an already infected host or the inactivation of the
pathogenic agent.
[0034] In another embodiment, the compositions of the present invention
can be utilized to decontaminate or purge an area of biologically active
microorganisms via formation of agglomerated complexes of one or more
nanoparticles bound to multiple pathogens. The agglomerated complexes can
be easily collected or removed from a contaminated area. The
nanoparticles of the present invention can also be labeled and used to
tag or otherwise identify targeted biological materials.
[0035] In general, the nanoparticles of the present invention can target
and bind to any biologically active microorganism that initiates
attachment to a host via an attaching/effacing (A/E) mechanism.
Microorganisms have evolved a number of mechanisms to facilitate the A/E
mechanism of attachment. For purposes of this disclosure, the A/E
mechanism is defined as the utilization by the microorganism of one or
more surface adhesins to recognize and bind with receptors that are
either on the surface of a host cell or a component of the extracellular
matrix (ECM). Adhesins are usually proteins or polypeptides located on
the surface of microorganisms that can display various specificities for
a wide range of host cell receptor molecules. Adhesins can be present on
pathogens as components of filamentous, nonflagellar structures, known as
pili or fimbriae, or alternatively as afimbrial monomeric or multimeric
proteins anchored within the surface membrane of the microorganism.
Adhesins need not be proteins or polypeptides, however. Other nonprotein
components of membranes, including lipopolysaccharides (LPS), and
lipteichoic acid, for example, can function as adhesins as well.
Biologically active microorganisms that utilize the A/E mechanism and can
be targeted by the nanoparticles of the present invention can include
prokaryotic microorganisms and eucaryotic microorganisms as well as
viruses and spores.
[0036] The nanoparticles of the present invention have at their center a
polymeric, hydrophobic core. Polymeric materials are generally preferred
for the cores of the nanoparticles as they can allow for wide variation
in size of the nanoparticles, which can desirably vary depending upon the
application. In addition, polymeric materials can be used to form the
core relatively inexpensively when compared to other materials utilized
for drug delivery mechanisms in the past (e.g. gold). They can also be
quite safe to use, and will not exhibit toxicity found with other
materials, such as heavy metals.
[0037] In one embodiment, the core can be polystyrene. Other materials may
also be utilized as the polymeric core material, however. For example, in
one embodiment, the polymeric core may be a polymethyl methacrylate
material. Copolymers of styrene and methyl methacrylate may also form the
hydrophobic polymeric core. Other materials that can be utilized as the
core material can include polymers or copolymers of methyl acrylate,
vinyl acetate, (.alpha.-methylstyrene, lactic acid, and the like. In one
embodiment, the core can be formed of biodegradable polymers.
[0038] Generally, between about 50% and about 80% of the weight of the
nanostructure can be the core material with the remainder of the weight
split between the linking agents, the biofunctional materials, and any
other material that can be part of the nanoparticle. Variations will
obviously occur depending upon chain length and make-up of the
macromonomer forming the core as well as depending upon the make-up of
the other materials.
[0039] Bound to the surface of the polymeric core of the nanoparticles are
hydrophilic linking agents. In one particular embodiment, the linking
agents can be covalently bound to the surface of the polymeric core. The
linking agents of the nanoparticles can generally serve two purposes,
they can provide a level of hydrophilicity to the nanoparticles and
encourage the nanoparticles to remain in suspension in an aqueous
environment, and they can tether a biofunctional moiety to the exterior
surface of the nanoparticles. To provide the desired amount of
hydrophilicity to the nanostructures, the relative amount of the
hydrophilic linking agent material, while it can be a very high
percentage of the weight of the nanoparticle, if desired, can be quite
small. For example, the relative amount of the hydrophilic linking agent
material can be less than about 5% of the weight of the nanoparticle, and
can provide a suitable level of hydrophilicity to the nanoparticles. In
one embodiment, the relative amount of the hydrophilic linking agent need
be only from about 3% to about 5% of the molecular mass of the
nanoparticle to provide enough hydrophilicity to the nanoparticle.
[0040] The linking agents generally extend off of the surface of the core
for a distance so as to allow better access to the target microorganisms
by the biofunctional materials that are bound to the nanostructures via
the linking agents. For example, the linking agents can, in one
embodiment, comprise a polymer having a degree of polymerization between
about 10 and about 200. In one embodiment, the linking agent can have a
degree of polymerization between about 20 and 100, in one embodiment
between about 25 and 40, for example between about 29 and about 36.
[0041] Suitable inking agents can include hydrophilic polymeric materials
that can be bound to both the core material and one or more biofunctional
moieties. In certain embodiments, the linking agent can be derivatized in
order to promote the formation of the bonds, e.g. covalent bonds. In one
embodiment, the linking agent can be a diol or a glycol. For example, in
one embodiment, the linking agent can by polyethylene glycol or a
derivative thereof. Other materials that can be utilized as the linking
agent, either alone or in combination with each other, can include
peptides, polypeptides, oligosaccharides, polysaccharides, polyacrylic
acid, polyacrylamides, and the like.
[0042] The outer surface of the nanoparticles of the present invention
includes one or more biofunctional moieties bound to the linking agents.
The biofunctional moieties of the present invention can be functionally
identical to receptors recognized and bound by the targeted
microorganisms, as will be further discussed herein. In general, however,
the biofunctional moieties of the present invention can include
glycolipids, glycoproteins, polypeptides, lipids, monosaccharides,
polysaccharides, and derivatives of any of the above.
[0043] Various methods can be utilized to synthesize the nanoparticles of
the present invention, depending primarily on the individual constituents
of the nanoparticles. In one particular embodiment, the linking agents
can be covalently attached to both the polymeric core material as well as
to the biofunctional material. The covalent attachments of this
embodiment can prevent the desorption of the biofunctional material and
allow the active sites of the nanoparticles to be more accessible to the
targeted microorganisms. In addition, the formation of covalent
attachments between the different sections of the nanoparticles can
result in materials that can remain stable essentially indefinitely.
[0044] In general, an emulsion polymerization or a dispersion
polymerization method can be used to form the nanoparticles. An emulsion
polymerization is herein defined as a polymerization reaction whereby
monomer(s), initiator, dispersion medium (and possibly colloid
stabilizer) are mixed forming initially an inhomogeneous system conducive
to the polymerization of the monomer(s) resulting in particles of
colloidal dimensions. A dispersion polymerization is herein defined as a
precipitation polymerization in which monomer(s), initiator(s), and
colloid stabilizer(s) are mixed in a solvent forming initially a
homogeneous system conducive to the polymerization of the monomer(s) and
producing polymer that is insoluble in the solvent, resulting in
formation of polymer particles.
[0045] Other suitable polymerization method may alternatively be used in
forming the nanoparticles of the present invention, however. For example,
microemulsion polymerization, precipitation polymerization, precipitation
polycondensation, precipitation polyaddition, or suspension
polymerization methods may be used as are generally known in the art.
[0046] In general, the biofunctionalized nanoparticles of the present
invention can be formed through either a converging or a diverging
polymerization approach. For instance, in those embodiments in which the
biofunctional moiety to be attached to the surface of the nanoparticle is
not susceptible to damage under the conditions of polymerization, a
converging approach may be preferred. For example, in those embodiments
wherein a carbohydrate moiety such as a monosaccharide, a polysaccharide,
or a derivative thereof is the biofunctional material, a converging
polymerization process can be utilized. In other embodiments, such as
when the biofunctional moieties may be sensitive to the polymerization
conditions, a divergent polymerization process may be preferred.
[0047] No matter which formation process is utilized, the formed polymeric
core can be a very stable material that can remain stable essentially
indefinitely. In some embodiments, following polymerization of the core
material, the core can be cured or crosslinked to stabilize the
nanoparticle. For example, the core can be crosslinked with a divinyl
compound such as, for example, divinyl benzene, ethylene glycol
dimethacrylate (EGDMA), or 1,6-hexanedio diacrylate (HDODA).
[0048] In general, a converging polymerization process is one in which a
macromonomer is first formed including the biofunctional moiety, the
linking agent, and a monomer of the core material. The macromonomer is
then polymerized with other monomers of the core material to form the
biofunctionalized nanoparticles of the present invention. For example, in
one embodiment, the nanoparticles of the present invention can include a
polystyrene core, a polyethylene glycol linking agent, and a
monosaccharide such as, for example, a D-mannose biofunctional moiety. In
one particular embodiment, this nanoparticle can be formed according to
the following converging approach: [0049] 1) Preparation of
.omega.-Carboxylic acid-styrene-polyethylene glycol macromonomer 6A as
illustrated in FIG. 1. [0050] 2) Preparation of
2-aminoethyl-.alpha.-D-pyranmannoside as illustrated in FIG. 2. [0051]
3) Preparation of D-Mannose-containing styrene-PEG macromonomer from
.omega.-carboxylic acid-styrene-PEG macromonomer and
2-aminoethyl-.alpha.-D-pyranmannoside as shown in FIG. 3. [0052] 4)
Polymerization of styrene with the D-Mannose-containing styrene-PEG
macromonomer according to a dispersion copolymerization protocol as shown
in FIG. 4.
[0053] In another embodiment, a diverging approach may be utilized to
prepare the nanoparticles. In a diverging approach, the polymeric core
with the attached linking agents may be formed prior to the attachment of
the biofunctional moiety to the surface of the nanoparticle. For example,
FIG. 6 illustrates two possible mechanisms for polymerizing a polystyrene
core with attached carboxylic acid-terminated PEG linking agents. FIG. 6A
uses the .omega.-Carboxylic acid-styrene-polyethylene glycol macromonomer
which can be prepared according to the process illustrated in FIG. 1.
FIG. 6B uses a shorter carboxylic acid-terminated PEG macromonomer which
can be prepared according to the mechanism illustrated in FIG. 5. In
either case, polystyrene-PEG nanoparticles can be prepared via, for
example, dispersion polymerization of the macromonomer with styrene, to
form the corresponding non-biofunctionalized nanoparticles, as shown in
FIG. 6.
[0054] According to the diverging approach, following formation of the
non-biofunctionalized nanoparticles, the desired biofunctional moiety can
be attached to the PEG linking compound at a functional group either at
the terminus or elsewhere along the PEG chain. For example, in one
embodiment, the biofunctional moiety can be a polypeptide sequence, such
as, for instance, the tripeptide enkephalin (1-3 Tyr-Gly-Gly). In this
embodiment, covalent attachment of the bioactive species can be attained
via a carbodiimide-activated coupling reaction. In general, a carboxylic
acid group on the linking agent can be an excellent platform for the
attachment of the biofunctional moieties, though other functionalities on
the linking agent could alternatively be utilized. For instance, in other
embodiments, functionalities can include epoxy functionalities, amine
functionalities, aldehyde functionalities, thiol functionalities, and the
like.
[0055] The biofunctionalized nanoparticles of the invention can generally
be anywhere from about 30 nm in average diameter up to several microns in
average diameter. For instance, the nanoparticles can be between about 50
nm and about 1 micron in average diameter. For example, in one
embodiment, nanoparticles can have an average diameter of about 250 nm.
In another embodiment, the nanoparticles can have an average diameter of
about 750 nm. The size of the nanoparticles can generally be controlled
by standard methods as are known in the art including, for example,
controlling the ratio between the individual components during
polymerization, controlling the length of the polymeric chain component
of the macromonomer, or through solvent selection.
[0056] The biofunctionalized material bound to the surface of the
nanoparticles can include a material that is functionally identical to
receptors recognized by the adhesins present on the target microorganism.
As such, when the nanoparticles are in the presence of the target
microorganism, the microorganism can recognize and bind to the
biofunctionalized material on the nanoparticle. Many adhesins function as
lectins, mediating microbial interactions with carbohydrate moieties of
glycoproteins or glycolipids on host cell surfaces. Thus, in one
embodiment, the biofunctional material of the disclosed nanoparticles can
be a simple carbohydrate moiety or a functionalized derivative thereof,
such as fucose, mannose, dextrose, mannan, galactose, or galactosamine,
for example. Carbohydrate biofunctional moieties, among other advantages,
are relatively inexpensive, non-toxic, and readily available. Other
possible biofunctional materials that can be utilized in the present
invention include specific amino acid moieties present in receptor
proteins. Glycoproteins, glycolipids, or other components that can
potentially serve as cellular surface receptors for adhesins can be
utilized as the biofunctional materials of the present invention. Protein
constituents of the ECM can also be utilized as biofunctional materials
in those instances wherein the target microorganism communicates with the
host via the ECM.
[0057] The biofunctional materials of the disclosed nanoparticles can be
identically functional to receptors identified and bound by the surface
adhesins of microorganisms that utilize an A/E attachment mechanism. This
can include prokaryotic microorganisms, including both bacteria and
cyanobacteria; eucaryotic microorganisms, including individual eucaryotic
cells within an organism as well as parasites and the like, such as
various amoebas, ciliates, flagellates, or sporozoans; viruses; or
spores.
[0058] Many bacterial adhesins and their corresponding receptors are known
in the art and may be targeted according to the present invention. See,
for example, M. A. Mulvey & S. J. Hultgren, Adhesin Bacterial,
Encyclopedia of Microbiology, Vol. 1, Academic Press, 42-47 (2000). For
exemplary purposes only, the following table (Table 1) lists some of the
possible biofunctional materials that may be utilized on the
biofunctionalized nanoparticles of the present invention along with the
corresponding adhesins and the bacterial pathogen which carries these
specific adhesins. It should be noted that in certain instances, the
receptor for a given adhesin may be an entire protein. In the present
invention, the biofunctional material can generally include a polypeptide
sequence of the protein that can be recognized and bound by the adhesin,
and need not include the entire protein structure, though this is not
prohibited in the disclosed nanoparticles.
TABLE-US-00001
TABLE 1
Biofunctional
Organism Adhesin Material
Escherichia coli P pili (PapG) Gal.alpha.(1-4)Gal
Type 1 pili D-mannose
(FimH) (uroplakin 1a &
1b, CD11, CD18,
uromodulin)
Curli (CsgA) Fibronectin/
laminin/plasminogen
Prs pili Gal.alpha.(1-4)Gal
S pili .alpha.-sialyl-2,3-
.beta.-galactose
K88 pili (K88ad) IGLad (nLc.sub.4Cer)
K99 pili (FanC) NeuGc(.alpha.(2-3)Gal.beta.4Glc
DR family Decay
DR Accelerating
DR-II Factor
AFA-I (SCR-3 domain)
AFA-II
F1845
Nonfimbrial Glycophorin A
adhesins 1-6
M hemagglutinin A.sup.M determinant
of glycophorin A
Intimin Tir (EPEC encoded
phosphoprotein)
Neisseria Type-4a pili CD46
Opa proteins CD66 receptor
family/HSPG
Opa.sub.50 Vitonectin/fibronectin
Opc HSPG/Vitronectin
LOS ASGP-R
Inducible adhesin Lutropin receptor
Listeria Internalin E-cadherin
monocytogenes
Haemophilus Hemagglutinating AnWj antigen/
influenzae pili lactosylceramide
Hsp-70-related Sulfoglycolipids
proteins
HMW1, HMW2 Negatively charged
Glycoconjugates
Campylobacter jejuni CadF Fibronectin
Yersinia Invasin .beta.1 integrins
YadA Cellular
fibronectin/
collagen/laminin
Bordetella pertussis FHA CR3 integrin
Pertactin, BrkA Integrins
Pertussin toxin Lactosylceramides/
gangliosides
Mycobacterium BCG85 complex, FAP Fibronectin
proteins
Streptococcus Protein F family Fibronectin
Polysaccharide CD44
capsule
ZOP, FBP4, GAPDH Fibronectin
Lipoteichoic acid Fibronectin/macrophage
(LTA) scavenger receptor
M protein CD46/fucosylated
glycoconjugates/
fibronectin
Staphylococcus FnbA, FnbB Fibronectin
Can Collagen
Protein A (Spa) von Willebrand factor
C1fA Fibrinogen
EbpS Elastin
[0059] This exemplary table of biofunctional materials is non-limiting,
and microorganisms that can be targeted by the biofunctionalized
nanoparticles of the present invention can include these as well as many
other bacteria including, for example, bacteria in the genera
Campylobacter, Listeria, Salmonella, Shigella, Vibrio, Aeromonas,
Moraxella, Escherichia and Helicobacter. Major enteropathogenic species
that can be specifically targeted by the present invention can include
Salmonella typhi, Enterobacter aerogenes, Streptococcus faecalis,
Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomanas aeruginose,
Dichelobacter nodus, Vibrio parahaemolyticus, Yersinia enterocolitica,
Vibrio cholerae 01, Vibrio cholerae 0139, non-01 Vibrio cholerae, Vibrio
fluvialis, Aeromonas hydrophila, and Plesiomonas shigelloides.
[0060] In one embodiment, the nanoparticles of the present invention can
target commensal microorganisms such as yeast or other fungi. For
example, the yeast Candida albicans is a human commensal. The ability of
Candida to adhere to the host is a fungal virulence factor similar to
that of other microbial systems, and is considered a significant step in
the development of candidiasis. The present invention can also target
viral pathogens. In particular, the biofunctional materials on the
surface of the nanoparticles can include those which can be recognized
and bound by the pathogenic capsid surfaces of a virus. For example, the
nanoparticles can be biofunctionalized so as to target various
rotaviruses, Norwalk-like viruses, adenoviruses, astroviruses,
coronaviruses, enteroviruses, or other viral agents. For example, in one
embodiment, nanoparticles can be biofunctionalized with the GP120 protein
of HIV to provide a particulate immunizing preparation.
[0061] Exemplary eucaryotic enteropathogens that can be targeted by the
present inventions include the parasites Giardia intestinalis, Entamoeba
histolytica, Cryptosporidium parvum, Cycolsporocayetanensis, Dientamoeba
fragilis, Isospora belli, Balantidium coli, and Strongyloides
stercoralis.
[0062] In one embodiment, the compositions of the present invention can
prevent infection of a host by a pathogenic microorganism by eliminating
the bioactivity of the pathogen through binding the pathogen to the
nanoparticle. For example, the nanoparticles of the invention can
irreversibly bind to the targeted pathogens at the adhesins and mask
those particular adhesins, preventing the pathogen from binding to host
cells and infection of the host. The nanoparticles of the present
invention can, in some embodiments, be quite large in relation to many of
the pathogenic microorganisms to which they can bind. Therefore, the
presence of the nanoparticles irreversibly bound to the surface of the
pathogenic microorganism can, in these embodiments, sterically hinder
binding and subsequent invasion of a host cell by the microorganism.
[0063] In one embodiment, the compositions of the present invention can be
utilized to agglomerate microorganisms. The biofunctionalized
nanoparticles will generally have multiple biofunctional material
components across the surface of the nanoparticles, with estimates of
numbers of individual biofunctional moieties attached to the surface of
the nanoparticles in the tens of thousands. In addition, the
microorganisms themselves have multiple adhesins across their surfaces.
The natural ligand-receptor systems in nature are high-affinity binding
systems. Thus, when the compositions of the invention are in the presence
of multiple microorganisms, as would be expected in an area contaminated
with the microorganism, a single nanoparticle can bind to more than one
microorganism. In addition, a microorganism that is already bound to a
nanoparticle at one adhesin location can bind to another nanoparticle at
another adhesin site should the two come into proximity. In this manner,
as the nanoparticles and microorganisms continue to contact each other
and bind to one another, large aggregate complexes of the two can be
formed. Formation of aggregates of the nanoparticles with the
microorganisms can eliminate the bioactivity of the microorganism through
both steric hindrance and adhesin masking. In addition, experimental
results indicate that the formation of large aggregates can lead to the
death of the microorganisms.
[0064] The formation of the large agglomerated complexes can not only
prevent infection of a host by the microorganisms, as described above,
but can also provide a means for gathering, identifying, or removing the
microorganisms from a location.
[0065] The nanoparticles of the present invention can have many different
practical applications, a few of which are discussed in more detail
below.
Purging
[0066] In one embodiment, the process of the present invention can be
utilized to purge a pathogenic microorganism from a person or an animal
that is already infected by or a carrier of a pathogenic microorganism.
In this embodiment, a composition including the nanoparticles can be
ingested by the individual to form agglomerated complexes with the
microorganisms in the digestive tract.
[0067] For example, the composition can be an aqueous composition
including the suspended nanoparticles that is simply swallowed by the
individual. Alternatively, the composition can include encapsulated
nanoparticles or nanoparticles otherwise prepared for release following
ingestion within the digestive system at a time or location where the
targeted microorganisms can be expected to be encountered. Upon contact
between the two, the microorganisms and the nanoparticles can form
agglomerated complexes within the digestive tract. The large agglomerated
complexes can be removed from the digestive tract via the natural
peristalsis of the individual at a rate dependent on intestinal transit
time.
[0068] In some embodiments, infection of the individual by a pathogenic
microorganism may have already begun prior to ingestion of the disclosed
compositions. For instance, individual microorganisms may already be
bound to the surface of the digestive tract at the time the composition
including the biofunctionalized nanoparticles comes into contact with the
microorganisms. In this embodiment, agglomerated complexes can form at
the surfaces where the pathogens have attached themselves to the walls of
the digestive tract. The formation of the agglomerated complexes can
still remove the microorganisms from the digestive tract, even in this
embodiment, as the shear forces of intestinal contents acting on the
agglomerated complexes during peristalsis can physically wrench the
infecting agents from the walls of the digestive tract.
[0069] In one preferred embodiment, the compositions of the present
invention can be utilized to prevent enteric disease in humans brought
about by infection by any of a variety of enteropathogens that can be
carried by consumable livestock. In general, in this embodiment,
infection can be prevented by decontaminating or purging the source or
carrier of the pathogens prior to processing and human ingestion.
[0070] For example, in one embodiment, the present invention is directed
to the prevention of gastroenteritis cause by bacterial infection by C.
jejuni. C. jejuni is the leading cause of bacterial enteric infection in
the United States and Europe. While C. jejuni is not carried by healthy
individuals, it is often isolated from healthy cattle, poultry, and even
flies. It is also sometimes present in non-chlorinated water sources. The
most common source of C. jejuni infection in the United States is
chicken. Human feeding studies in the past have suggested that only about
400 to 500 bacteria can initiate clinical infection in some individuals,
though this can obviously vary from host to host.
[0071] In modern chicken processing plants, a single plant may process
several thousand chickens a day. Loads of chickens are delivered to a
plant from many different sources over the course of a single day. Though
the entire plant is often decontaminated on a daily basis to prevent the
spread of bacterial infection, unfortunately, if a single load of C.
jejuni infected animals passes through the plant, it is practically
impossible to prevent the spread of the bacteria throughout the
processing line over the course of the day. Food products delivered from
the contaminated processing line can then carry the pathogen and can lead
to human infection.
[0072] In one preferred embodiment, the present invention is directed to a
process for purging infected animals of pathogenic microorganisms before
they reach a processing plant. For instance, the compositions of the
present invention can be utilized to purge the C. jejuni bacteria from
chickens prior to transport to a processing plant. In this embodiment,
compositions of the invention can be fed to the chickens prior to
transport. For instance, chickens can be gavaged prior to transport to a
processing plan with a composition including a suspension of
nanoparticles as herein described. The nanoparticles in this case could
incorporate biofunctional materials specifically targeted for the C.
jejuni bacteria. For example, biofunctionalization of the nanoparticles
with a D-mannose moiety can be utilized to agglomerate certain strains of
C. jejuni within the digestive tract of an animal such as chickens
carrying the pathogen.
[0073] In general, a composition comprising between about 50 mg and 200 mg
D-mannose biofunctionalized nanoparticles per kg of body weight may be
ingested by an animal. In one embodiment, the composition can comprise
about 100 mg D-mannose biofunctionalized nanoparticles per kg body
weight.
[0074] Following ingestion, the D-mannose biofunctionalized nanoparticles
of the present invention can bind to C. jejuni within the digestive tract
of the chicken, and large agglomerated complexes of bacteria and
nanoparticles can form which can pass through the chicken via natural
peristalsis. This can be done so as to purge C. jejuni from the animals
prior to the arrival of the chickens at the processing plant.
[0075] In general, a chicken has a digestive retention time of about 200
minutes (see, for example, J. L. Grimes, et al., Journal of Poultry
Research, Vol. 6, No. 4, p. 339, 1997). Thus, targeted pathogens within
the digestive tract could be largely purged from the animals within about
3-4 hours of ingestion, depending on individual digestive retention time.
Following this period, and providing the animals are kept away from any
source of recontamination of the pathogens, levels of the pathogens
within the digestive tract of the animals could remain greatly diminished
until the time of processing.
[0076] Though described here in some detail for the enteropathogen C.
jejuni, it should be understood that the purging processes of the present
invention may be equally applicable to other pathogens and other carriers
to prevent human infection by the pathogens via human consumption. For
instance, similar to C. jejuni, certain pathogenic E. coli has exhibited
strong adhesin-specific interactions with D-mannose molecules. As such,
in certain embodiments of the present invention, D-mannose
biofunctionalized nanoparticles can be utilized to aggregate E. coli
bacteria in the prevention of E. coli enteroinfection similar to the
process described above for the prevention of C. jejuni enteroinfection.
Decontamination
[0077] In another embodiment, the formation of large agglomerated
complexes of the nanoparticles of the present invention with
microorganisms can be utilized to decontaminate an area. For instance, in
one embodiment, the nanoparticles can be biofunctionalized to target
microorganisms which can be considered a danger to the general public
such as through contamination of personnel, buildings (interior or
exterior), equipment, soil, water, or vegetation. In this particular
embodiment, the nanoparticles can be spread over the contaminated area,
such as in a powder or spray form, so as to contact the microorganisms
and form large, agglomerated complexes. These complexes can be large
enough so as to be simply swept, vacuumed, or otherwise removed from the
contaminated area.
Treatment through Increased Specificity
[0078] In one embodiment, the nanoparticles of the present invention can
be biofunctionalized with a combination of different biofunctional
materials. This can provide increasing levels of specificity of the
nanoparticles to a specific microorganism. A single microorganism can
often include multiple adhesins on the surface, with varying receptor
specificities. In addition, the different adhesins on the surface of a
single microorganism can often function synergistically. For example,
many different microorganisms could be expected to bind to nanoparticles
biofunctionalized with a monosaccharide such as mannose or galactose, for
example, as these are fairly common receptors for a wide variety of
adhesins. Greater binding specificity could be obtained by the
nanoparticles by including combinations of receptor structures on the
surface, where the combination of structures can be recognized only by
the targeted microorganism.
[0079] For example, in one embodiment, a polyethylene based linking agent
can be synthesized with multiple residue-like side groups placed
precisely along the backbone chain. Similarly, different biofunctional
materials can be placed on different linking agents across the surface of
the nanoparticle. Either or both of these approaches in concert can
provide a protein-like synthetic macromolecular structure on the surface
of the nanoparticle that is similar in structure to natural host proteins
to which the microorganism can adhere. The nanostructure obtained can
have structural and functional specificity to a specific target
microorganism. This design can maximize the probability for the
nanoparticles to bind to protein structures on the targeted
microorganisms with very specific high-affinity binding.
[0080] In either case, a more natural protein-like surface can be formed
on the nanoparticle which can be more specifically recognized and bound
by the targeted microorganisms, while avoiding binding to other,
non-targeted microorganisms. For example, in one embodiment, the
nanoparticles of the present invention may be utilized to treat an
existing infection, such as an existing enteroinfection, for example. In
this embodiment, the biofunctionalized nanoparticles can be specifically
designed so as to target and bind the infective agents, but avoid binding
to other microorganisms in the alimentary canal, microorganisms which may
be necessary for the health of the individual.
Identification
[0081] In another embodiment, the nanoparticles can be labeled with a
fluorescent label such as fluorescein isothiocyanate. The labeled
nanoparticles can then be used to identify microorganisms bearing
adhesins to which they bind. For example, fluorescent-labeled
nanoparticles can be utilized to identify pathogens in biological
samples. In this embodiment, the nanoparticles can bind to and thus label
pathogens in biological samples such as autopsies, biopsies, urine
samples, oral swabs, fecal smears or sputum samples. The targeted
pathogens can be infectious agents, such as enteropathogenic bacteria or
parasites. Alternatively, the labeled nanoparticles can be targeted
toward endogenous entities that are indicative of disease, such as cancer
cells, for example, or agents which can be found in a biological sample
that may be a byproduct of a disease process. Thus, the compositions of
the present invention can in this embodiment be used for diagnoses of
disease or recognition and identification of the presence of
disease-causing agents or their products.
[0082] In another embodiment, the labeled nanoparticles can be utilized to
identify places or areas that are contaminated by a suspected pathogenic
microorganism. In this embodiment, the labeled nanoparticles can also be
utilized to estimate the level of contamination by the number and size of
agglomerated complexes in a given area. For example, samples can be taken
from an industrial site suspected of being contaminated by a known
pathogen. Water, plant, and soil samples can be exposed to nanoparticles
of the invention that have been biofunctionalized to be recognized and
specifically bound by adhesins on the surface of the suspected pathogen.
[0083] In another embodiment, the labeled nanoparticles of the present
invention can be used to identify unknown adhesins on the surface of a
microorganism. For example, nanoparticles including a label and a known
biofunctional moiety can be exposed to a microorganism. Adhesins on the
microorganism can be identified by observing binding affinity for the
microorganism to the labeled nanoparticles.
[0084] Fluorescent labeling of compounds is generally known in the art and
thus is not described in detail herein. Fluorescent labels that can be
incorporated into the biofunctionalized nanoparticles include
cross-linking agents in the polymeric core of the nanoparticles or
fluorescent probes attached to the surface of the nanoparticles. For
example, anthracene, which is a strongly fluorescent molecule, can be
employed as a cross-linking agent in a polymerization process. Similarly,
divinyl porphyrins, which are strongly fluorescent in the extreme red to
near-infrared wavelength regions (650-850 nm) can be used as crosslinking
agents in the nanoparticles.
[0085] Luminescent probes attached to the surface of the nanoparticles can
be attached similar to the biofunctional materials in formation of the
nanoparticles. Though not required, in some embodiments the biofunctional
materials can be loaded onto the nanoparticles in relatively high
concentrations prior to attachment of the labels. The fluorescent probes
can then be incorporated in lower concentration so as to avoid
interference with the affinity of the nanoparticles for the microorganism
adhesin sites. For example, the fluorescent probes can be in a ratio to
the biofunctional moieties of between about 1:50 to about 1:150.
[0086] The selection of specific luminescent probes can depend at least in
part upon the desired application of the nanoparticles. For example, in
tracing and analysis of enteropathogens in biological samples (such as
biopsies or fecal smears, for instance), light transmission through the
biological environment is a major consideration. Possible luminescent
probes that can be incorporated into the nanoparticles can include, for
example, porphyrins, FITC, and anthracene.
[0087] In one embodiment, derivatized phathlocyanines and
metellophathlocyanines can be utilized as fluorescent probes. For
example, diimide-catalyzed amidation and esterification chemistries can
be used to link derivatized fluorescent probes to the nanoparticles, as
is generally known in the art.
Drug Delivery
[0088] In another embodiment, the nanoparticles can be utilized as a
delivery vehicle for an agent such as a vaccination agent to a biological
system.
[0089] There are three basic requirements for a successful vaccination
process: long biological half-life of the vaccine, the ability to induce
an antibody response to a native challenge, and the establishment of a
long-term response. In one embodiment of the present invention, an
antigenic epitope such as, for example, a purified viral capsid
component, can be attached to the biofunctionalized nanoparticles to
create a preparation that includes immunogenic particles similar in size
to the native virus. Particulate antigens in general have the potential
for much longer biological half-life than those of soluble antigens.
Thus, the particulate vaccine can be retained within the recipient's
system for a longer period of time. In this embodiment, the particulate
vaccine can be prevented from rapidly diffusing from the site of entry
and the process can diminish the rate of excretion for the vaccine. This
extended biological half-life of the vaccine agent within the host can
increase the likelihood of a strong antibody response by the host, and
subsequently can lengthen the protective term in the host. This is one
manifestation of the adjuvant effect (an example is the precipitation of
bacterial toxins by alum to create an insoluble antigenic form that is
released slowly over months). In addition, in this embodiment, the
nanoparticle-derived antigen could have a stronger opsonizing
(macrophage-stimulating) potential than that of the same antigen in the
soluble state, since macrophages respond more avidly to particulate
antigens.
[0090] In yet another embodiment, nanoparticles could serve as carriers
for peptides too small to be immunogenic in themselves. This strategy is
analogous to earlier demonstrations of the hapten-carrier phenomenon.
Haptens are small organic molecules that are antigenic, but not
immunogenic. When haptens are complexed to large molecules such as
proteins, they function as the immunodominant epitope of the
hapten-carrier conjugate. Many biologically important substances,
including drugs, peptide hormones, and steroid hormones can function as
haptens. Use of this phenomenon has been widespread in radioimmunoassay
testing for prohibited drug use in athletes and racehorses.
[0091] The compositions of the present invention can also include other
biologically active agents, in addition to the biofunctionalized
nanoparticles as herein described. For instance, in one embodiment, the
nanoparticles can be utilized in conjunction with antibiotic agents to
improve the efficacy of the antibiotic.
[0092] The use of antibiotic agents can sometimes lead to undesired side
effects. For example, in meningococcal infections, when the bacterial
pathogen is lysed by an antibiotic, the bacteria release endotoxins into
the circulatory system of the infected host. The free endotoxins can then
cause undesired effects as they spread through the host and exacerbate
the inflammatory response. In this embodiment, biofunctionalized
nanoparticles that target the released endotoxins can be used in
conjunction with the antibiotics. Thus, the nanoparticles can bind the
endotoxins that are released as the bacteria lyse and thus can prevent
spread of the endotoxins through agglomeration, masking, and/or steric
hindrance. As the biofunctionalized nanoparticles can be designed with
high affinity for the specific endotoxins, they can bind to and hold the
disease causing agents until the agents are either degraded or destroyed
with the nanoparticles by the body's natural defense systems or excreted
from the system.
[0093] The present invention may be better understood with reference to
the following examples.
EXAMPLE 1
[0094] The macromonomer labeled 6A in FIG. 1 was prepared according to the
formation process illustrated therein.
[0095] Materials. p-Chloromethylstyrene (90%), 4-dimethylamino pyridine
(DMAP, 99%), hydrochloric acid, N-hydroxysuccinimide (93+%), MES hydrate
(99%), nitrobenzene (99%), pyridine (99%), sodium hydride (NaH, 60%
dispersed in mineral oil), sodium bicarbonate (powder), and succinic
anhydride (99%) were purchased from Acros, azobisisobutyronitrile (AIBN,
98%) and polyethylene glycol methacrylate (M.sub.n.about.526) from
Aldrich, 1-ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride
(EDAC), triphenylphosphine (99%), sodium azide (99%), and
p-toluenesulfonyl chloride (98%) from Alfa Aesar, styrene from Baker,
polyethylene glycol (M.sub.w.about.2000) from Fluka, and
tetrabutylammonium bromide (98%) from Lancaster. Ethanol, chloroform, and
diethyl ether were purchased from Fisher and used without further
purification. THF was distilled over sodium, and methylene chloride was
distilled over calcium hydride before use. DMF was distilled over calcium
hydride under reduced pressure before use. Deuterated water (99.9%) and
chloroform for NMR measurements were obtained from Cambridge Isotope
Laboratories.
[0096] Preparation of Hydroxy-St-PEG Macromonomer (Macromonomer A on FIG.
1). NaH (0.6 g, 15 mmol) was added to a mixture of polyethylene glycol
(M.sub.w.about.2000, 20 g, 10 mmol) and THF (100 mL). After being kept at
40.degree. C. for 4 h, the reaction mixture was cooled to 34.degree. C.,
added with p-chloromethylstyrene (1.83 g, 12 mmol), kept at 30.degree. C.
for 24 h, and then added with HCl until neutral pH. The resulting
solution was concentrated and precipitated into cold ether, followed by
filtration for the solids and drying in a vacuum oven at room temperature
to obtain A (20.1 g, 95% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): 7.38
(d, 2H), 7.30 (d, 2H), 6.72 (m, 1H), 5.74 (d, 1H), 5.24, (d, 1H), 4.65
(s, 2H), 3.4-4.0 (m, 180H) ppm. C NMR (125 MHz, CDCl.sub.3): 137.95,
136.99, 136.60, 128.01, 126.26, 113.82, 73.00, 72.60, 70.41, 69.45, 61.78
ppm.
[0097] Preparation of Tosylate-St-PEG Macromonomer (Macromonomer B on FIG.
1). Macromonomer A (10 g, 4.3 mmol) was dissolved in dry
CH.sub.2Cl.sub.2, followed by the addition of pyridine (5.2 g, 65 mmol).
The mixture was cooled to 0.degree. C., and then p-toluenesulfonyl
chloride (12.65 g, 65 mmol) was added. The resulting mixture was stirred
at room temperature for 12 h. After CH.sub.2Cl.sub.2 was removed on a
rotary evaporator, to the resulting oil were added CHCl.sub.3 (20 mL) and
H.sub.2O (10 mL). The organic layer was washed with HCl (2 M), aqueous
NaHCO.sub.3 (5%), and then H.sub.2O. It was dried with MgSO.sub.4 powder,
filtered, and precipitated into cold ether. The filtration for the solids
was followed by washing with cold ether several times and drying in a
vacuum oven at room temperature to obtain B (9.5 g, 92% yield). .sup.1H
NMR (500 MHz, CDCl.sub.3): 7.78 (d, 2H), 7.30 (m, 6H), 6.69 (m, 1H), 5.72
(d, 1H), 5.22 (d,1 H), 4.54 (s, 2H), 4.13 (t, 2H), 3.48-3.40 (m, 180 H),
2.44 (s, 3H) ppm. C NMR (125 MHz, CDCl.sub.3): 144.83, 137.95, 136.96,
136.59, 133.04, 129.89, 128.03, 127.99, 126.25, 113.81, 70.78, 70.69,
70.61, 69.44, 68.90, 68.50, 21.71 ppm.
[0098] Preparation of Azide-St-PEG Macromonomer (Macromonomer C on FIG.
1). Macromonomer B (8.9 g, 4.2 mmol) was dissolved in DMF (60 mL), and to
the solution was added NaN.sub.3 (2.76 g, 42 mmol) and tetrabutylammonium
bromide (0.2 g, 0.54 mmol). After being kept at 40.degree. C. for 4 h,
the mixture was precipitated into cold ether to remove DMF. The resulting
solids were re-dissolved in CHCl.sub.3, and the solution was dried with
MgSO.sub.4. Upon filtration to remove MgSO.sub.4, the CHCl.sub.3 solution
was concentrated and again precipitated into cold ether, followed by the
filtration again and drying in a vacuum oven at room temperature to
obtain C (8.3 g, 95% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): 7.78 (d,
2H), 7.30 (d, 2H), 6.69 (m, 1H), 5.72 (d, 1H), 5.22 (d, 1H), 4.54 (s,
2H), 3.80-3.54 (m, 180 H), 3.38 (t, 2H) ppm. C NMR (125 MHz, CDCl.sub.3):
137.95, 137.00, 136.60, 128.02, 126.28, 113.84, 73.00, 70.63, 68.90,
68.50, 50.74 ppm.
[0099] Preparation of Amine-St-PEG Macromonomer (Macromonomer D on FIG.
1). Macromonomer C (3 g, 1.38 mmol), PPh.sub.3 (4.4 g, 16.5 mmol), and
H.sub.2O (37.3 mg, 2.1 mmol) were mixed with THF (2.5 mL), and the
mixture was stirred at room temperature for 36 h. The reaction mixture
was concentrated and precipitated into cold ether, followed by filtration
for the solids and drying under vacuum at room temperature to obtain D
(2.6 g, 94% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): 7.39 (d, 2H), 7.30
(d, 2H), 6.71 (m, 1H), 5.76 (d, 1H), 5.25 (d, 1H), 4.57 (s, 2H), 3.8-3.4
(m, 180H), 2.88 (t, 2H) ppm. C NMR (125 MHz, CDCl.sub.3): 137.94, 136.97,
136.59, 127.99, 126.25, 113.18, 73.10, 72.98, 70.60, 69.44, 41.83 ppm.
[0100] Preparation of Macromonomer 6A. Macromonomer D (1.96 g, 9.2 mmol),
succinic anhydride (0.18 g, 18.4 mmol), and DMAP (10 mg, 0.08 mmol) were
dissolved in CH.sub.2Cl.sub.2 (20 mL), and the solution was stirred at
room temperature for 36 h. Water (5 mL) was added to quench the reaction.
The solution was washed with water (5 mL.times.3), and the organic layer
was dried with MgSO.sub.4 and filtered. The resulting solution was
concentrated and precipitated into cold ether, followed by filtration for
the solids and drying under vacuum at room temperature to obtain 1 (1.63
g, 80% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): 7.40 (d, 2H), 7.34 (d,
2H), 6.27 (m, 1H), 5.76 (d, 1H), 5.26 (d, 1H), 4.58 (s, 2H), 3.90-3.40
(m, 180H), 2.67 (t, 2H), 2.56 (t, 2H) ppm. C NMR (125 MHz, CDCl.sub.3):
174.34, 172.67, 137.92, 136.58, 128.00, 126.25, 113.83, 72.00-68.00 (m),
31.03, 30.27 ppm.
EXAMPLE 2
[0101] The macromonomer labeled 6B in FIG. 5 was prepared according to the
formation process illustrated therein.
[0102] Materials as described above in Example 1 were used. Succinic
anhydride (5 g, 50 mmol), pyridine (50 mmol), and nitrobenzene (20 drops)
were added to a solution of polyethylene glycol methacrylate (5.26 g, 10
mmol) in CH.sub.2Cl.sub.2 (200 mL). After refluxing at 55.degree. C. for
48 h, the solvent CH.sub.2Cl.sub.2 was removed on a rotary evaporator,
and the solids were dissolved in an aqueous sodium bicarbonate solution
(10%). The resulting solution was filtered to remove solid residues and
then washed with diethyl ether (25 mL.times.3) to collect the organic
layer. The resulting diethyl ether solution was cooled to 0.degree. C.
and acidified with HCl, followed by the extraction with CHCl.sub.3. The
CHCl.sub.3 solution thus obtained was washed with water and then dried
with MgSO.sub.4. Upon the filtration to remove MgSO.sub.4 and then the
evaporation to remove CHCl.sub.3, Macromonomer 6B was obtained as a
yellow oil (5.95 g, 95% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): 6.03
(s, 1H), 5.48 (s, 1H), 4.27 (t, 2H), 4.23 (t, 2H), 3.54 (br, 36H), 2.5
(m, 4H), 1.83 (s, 3H) ppm. C NMR (125 MHz, CDCl.sub.3): 176.8, 172.3,
167.5, 136.4, 125.9, 70.7, 69.2, 63.9, 29.0, 28.9, 18.4 ppm.
EXAMPLE 3
[0103] In this example, nanoparticles including a polystyrene core and
linking agents of carboxylic acid-terminated oligomeric polyethylene
glycols were first synthesized via dispersion polymerization. The
polymeric nanoparticles were then covalently functionalized with
enkephalin peptide molecules in the carbodiimide-activated coupling
reaction to form biofunctionalized nanoparticles according to a diverging
approach.
[0104] Materials: Enkephalin (1-3 Tyr-Gly-Gly) was purchased from Sigma,
Nhydroxysuccinimide (93+%) and MES hydrate
(C.sub.6H.sub.13NO.sub.4SCH.sub.2O, 99%) from Acros,
azobisisobutyronitrile (AIBN, 98%) from Aldrich,
1-ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride (EDAC) from
Alfa Aesar, and ethanol from Fisher. Deuterated water (99.9%) and
chloroform (99.8%) for NMR experiments were supplied by Cambridge Isotope
Laboratories. Biuret reagent and Folin and Ciocalteu's phenol reagent for
the modified Lowry assay were obtained from Sigma.
[0105] Dynamic light scattering measurements were carried out on a Coulter
N4 Plus particle sizer. NMR measurements were performed on a JEOL Eclipse
+500 NMR spectrometer. TEM analyses were conducted on a Hitachi H7000 TEM
system. UV/vis absorption spectra were recorded on a Shimadzu 3100
spectrophotometer.
[0106] Macromonomers 6A and 6B were prepared as described above in
Examples 1 and 2. The macromonomers were then independently utilized to
form polystyrene-PEG nanoparticles via the dispersion polymerization of
each macromonomer with styrene as illustrated in FIG. 6. In a typical
reaction, styrene (1.042 g, 10 mmol) and either macromonomer 6A or 6B
(1.1 g, 0.5 mmol) were added to an ethanol/water mixture (4/1, 25 mL).
The mixture was cooled to 0.degree. C., and to the mixture was added AlBN
(16 mg, 0.1 mmol). Upon degassing, the mixture was stirred at 60.degree.
C. for 24 h, yielding a milky suspension. The suspension was transferred
into a membrane tubing (cut-off molecular weight 12,000) for dialysis
against fresh deionized water for 3 days to yield a purified aqueous
suspension of the nanoparticles formed according to the formation
mechanism illustrated in FIG. 6. The two different nanoparticles
obtained, depending upon the starting macromonomer, are herein referred
to as nanoparticle 6A and nanoparticle 6B.
[0107] The dispersion copolymerizations of the illustrated macromonomers
6A and 6B with styrene yielded polymeric nanoparticles 6A and 6B,
respectively, in aqueous suspensions. The suspensions appeared milky, but
remained stable essentially indefinitely. Each nanoparticle had a
polystyrene core and a shell of hydrophilic PEG chains that were
terminated with carboxylic acids. Such a structural arrangement is
believed to be responsible for the stability of the aqueous suspensions.
[0108] The dynamic light scattering results of the suspensions are shown
in Table 1.
TABLE-US-00002
TABLE 1
Average Diameter Average Average Diameter
Via Light Scattering Diameter ViaTEM Via Light
before before Scattering After
Nano- Functionalization Functionalization Functionalization
particle (nm) (nm) (nm)
6A 175 (25) 160 (18) 174 (21)
6B 180 (20) 190 (60)
Note:
the number in parenthesis is the standard deviation.
[0109] The nanoparticles obtained were of relatively narrow size
distributions. The formation of nanoparticles and their average sizes and
size distributions are confirmed by the results from TEM
characterizations. Shown in FIG. 7 is a typical TEM image of the
nanoparticle 6B. The halo surrounding each particle may be attributed to
the PEG shell. The average sizes determined from the TEM images are
slightly smaller than those obtained from the dynamic light scattering
experiments (Table 1). This seems understandable because the dynamic
light scattering measures hydrodynamic volumes of the nanoparticles,
corresponding to a more expanded surface structure of the nanoparticles
in a suspension. Between the two nanoparticle samples, the difference in
their average sizes has to do with the PEG chain length in the starting
macromonomers. The macromonomer with a longer PEG chain (macromonomer 6A)
corresponds to smaller particles, while the macromonomer of a shorter PEG
chain (macromonomer 6B) corresponds to larger particles.
[0110] To form biofunctionalized nanoparticles 6A, MES hydrate was added
to an aqueous suspension of nanoparticle 6A (10 mL, 5.2 wt %
nanoparticles 6A, 3 mole % --COOH on the nanoparticles) to result in the
buffer condition (0.1 M) of pH equal to 5.6, followed by the addition of
N-hydroxysuccinimide (15 mg) and EDAC (30 mg). The suspension was stirred
at room temperature for 3 h. The mixture was centrifuged to remove the
MES buffer, washed with a phosphate buffer twice, and suspended in the
phosphate buffer (10 mL). To the suspension was added enkephalin (60 mg,
0.2 mmol). The resulting suspension was stirred at room temperature for
24 h, followed by dialysis (membrane tubing cut-off molecular weight
12,000) against fresh deionized water for 3 days.
[0111] The same process was utilized to formed enkaphalin
biofunctionalized nanoparticles 6B from an aqueous suspension of
nanoparticles 6B.
[0112] The functionalization of the polymeric nanoparticles with the
peptide Tyr-Gly-Gly resulted in no meaningful changes in the size
characterizations. The dynamic light scattering and TEM results were
essentially unchanged from those of the nanoparticles before the peptide
functionalization. This also seems reasonable considering the small size
of the peptide.
[0113] Following formation, the nanoparticle samples were dissolved in
organic solvents, resulting in the destruction of the nanoparticles
because of the solubility of the polystyrene copolymers. The resulting
homogeneous solutions in deuterated chloroform were used in NMR
measurements. The solution-phase .sup.1H NMR spectra thus obtained were
not very informative because they were overwhelmed by the extremely broad
signals in the aromatic region arising from polystyrene, as well as broad
peaks resulting from the polymer corona. The relatively low peptide
content overall in the nanoparticle sample also makes the solution-phase
NMR characterization virtually impossible. Thus, the gel-phase NMR
approach was employed to characterize the peptide-functionalized
polymeric nanoparticles in an aqueous suspension.
[0114] The gel-phase NMR measurements were carried out with the same NMR
probe as for solutions, except that highly concentrated nanoparticle
suspensions were used. As shown in FIG. 8 top, the proton NMR spectrum of
the peptide-attached nanoparticles 6A in an aqueous (D.sub.2O) suspension
consisted of broad signals in the aromatic region in addition to the
strong peaks associated with the protons in the PEG corona of the
nanoparticles, but there were no meaningful contributions from the
polystyrene core. The aromatic signals may be attributed to the phenyl
protons in the tyrosine unit of the peptide, in comparison with the sharp
signals observed in the same region for the peptide in solution (FIG. 8
top).
[0115] In the gel-phase .sup.1H NMR spectrum of nanoparticles 6A without
peptide attachment (FIG. 8, bottom), there are the same strong peaks
associated with the protons in the PEG corona of the nanoparticles, but
no aromatic signals. The comparison of .sup.1H NMR results in FIG. 8
provides strong evidence that the peptide species are indeed attached to
the polymeric nanoparticles.
[0116] More quantitatively, the peptide species attached to the polymeric
nanoparticles were analyzed by using the modified Lowry assay. The assay
is commonly used in the determination of peptide and protein contents
through targeting the tyrosine or tryptophan moiety. The testing
procedure involves the mixing of the specimen with the Biuret reagent and
then Folin and Ciocalteu's phenol reagent. The tyrosine or tryptophan
moiety was detected when the colorless solution turned blueish green,
with the extent of the color change (absorbance around 725 nm in the
visible absorption spectrum) corresponding to the tyrosine or tryptophan
concentration. The presence of a tyrosine unit in enkephalin (1-3) makes
the peptide-functionalized polymeric nanoparticles responsive to the
assay. A standard curve was obtained by using solutions of free peptide.
In order to match the effects of light scattering on the absorption
measurements, these solutions for the standard curve were also added with
the same amount of unfunctionalized nanoparticles as the estimated
nanoparticle content in the sample solution for determination. The result
thus obtained for the peptide-functionalized nanoparticles 6A was 0.01 mg
peptide per mg nanoparticles. By using the average diameter obtained from
the TEM analysis (Table 1) for the spherical polystyrene core, with the
same density of 1.047 g/cm.sup.3 as for bulk polystyrene, the average
number of PEG linkers per nanoparticle and the average total mass per
nanoparticle were estimated as 395,000 linkers/particle and
3.6.times.10.sup.12 mg/particle, respectively. Thus, the average number
of peptide species per nanoparticle is 73,000, which corresponds to
.about.20% of the PEG tethers on the nanoparticle.
EXAMPLE 4
[0117] In this example, nanoparticles covalently attached with derivatized
D-mannose molecules were prepared according to the converging approach
illustrated in FIGS. 2-4. 2-aminoethyl-.alpha.-D-pyranmannoside was first
prepared as illustrated in the formation scheme of FIG. 2. A styrene
macromonomer substituted with the derivatized D-mannose was then prepared
according to the formation mechanism of FIG. 3.
[0118] The nanoparticles were then prepared in dispersion copolymerization
of styrene and the styrene macromonomer p-substituted with derivatized
D-mannose according to a converging polymerization approach as herein
described and illustrated in FIG. 4.
[0119] The nanoparticles thus prepared were used to study adhesion
interactions with E. coli ORN178 (provided by Dr. Chu-Cheng Lin,
Department of Zoology, National Taiwan Normal University, Taiwan). E.
coli ORN208, which has abnormal pili that is deficient in the adhesin
sites of the normal pili of E. coli ORN178 was used as a control.
[0120] Early stationary phase cultures of E. coil ORN178 was harvested and
washed twice with phosphate buffer saline (PBS). The bacterial suspension
was mixed with an aqueous suspension of the nanoparticles for 5 min, and
then the mixture was centrifuged at 6,000.times.g for 3 min. The
supernatant containing free nanoparticles was removed, and the pellet was
washed with PBS, centrifuged, and then re-suspended in PBS (1 mL). The
sample for transmission electron microscopy (TEM) imaging was fixed in
cacodylate buffered glutaraldehyde (3.5%, pH.about.7.2) at 4.degree. C.
for 12 h. A droplet of the sample was deposited on a carbon-coated copper
grid, stained with uranyl acetate, and then dried in air for 30 min.
[0121] Early stationary phase cultures of E. coli ORN208 were prepared in
an identical fashion as the E. coli ORN178 described above.
[0122] FIG. 9 shows lower magnification of E. coil ORN178 after mixing
with the D-mannose functional nanoparticles at 9a and 9b, and higher
magnification of E. coli ORN178 after mixing with the D-mannose
functional nanoparticles at 9c and 9d. The individual spheres visible in
9c and 9d are the individual biofunctionalized nanoparticles. As can be
seen, there is significant agglomeration of the E. coli ORN178 cells with
the nanoparticles. For comparison purposes, FIG. 9e shows a TEM image of
the E. coli ORN178 mixed with the non-biofunctionalized nanoparticles 6A
of FIG. 1. As can be seen, there is no agglomeration of the bacteria.
Similarly, FIG. 9f shows the abnormal pili-containing E. coli ORN208
mediated with the D-mannose functional nanoparticles. However, as can be
seen in the image, there is no agglomerated of the abnormal bacteria E.
coli ORN208.
[0123] The cell-nanoparticle bindings were also confirmed to be
irreversible, as was shown by subsequent incubation of the agglomerated
E. coli ORN178 cells with a solution of free D-mannose molecules (10
mg/mL). Following incubation, there was no evidence of dissociation of
the agglomerated complexes.
EXAMPLE 5
[0124] In this example, a Colony Forming Unit (CFU) Reduction Assay was
used to evaluate the agglomeration of adhesin-specific biofunctionalized
nanoparticles to E. coli.
[0125] Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) were used to
cultivate E. coli strain ORN178.
[0126] Exponentially grown E. coli (OD.sub.600=0.5) were diluted to
10.sup.-6, 10.sup.-7 and 10.sup.-8 with phosphate buffered saline (PBS)
in duplicates. 250 .mu.l of the diluted cultures were mixed with equal
volume of mannose biofunctionalized nanoparticles, prepared as described
above in Example 4, mannan biofunctionalized-nanoparticles, which were
prepared in an equivalent protocol as that described for the mannose
biofunctionalized nanoparticles, or PBS control. The mixtures were
incubated at 37.degree. C. for 15 minutes with gentle mixing every 5
minutes to allow agglomeration to occur. At the end of incubation, the
whole content (500 .mu.l) was transferred into a 100.times.15 mm Petri
dish. 20 ml of sterile TSA, previously melted and equilibrated at
45.degree. C., was then poured into the plate and allowed to be mixed
with the culture and solidified. The plates were incubated at 37.degree.
C. for 24 hrs. At the end of incubation time, the number of colonies on
the plates was enumerated.
[0127] FIG. 10A shows approximately 260 CFU of the E. coli mixed with PBS
(control), when no agglomeration of bacteria occurred compared to
approximately 15 CFU obtained when the E. coli agglomerated with the
mannose biofunctionalized nanoparticles (10.sup.-7 dilution). Similarly,
FIG. 10B shows approximately 360 CFU of the E. coli mixed with PBS
(control), when no agglomeration occurred compared to approximately 5 CFU
obtained when the E. coli agglomerated with the mannan biofunctionalized
nanoparticles (10.sup.-7 dilution).
[0128] The mannose biofunctionalized nanoparticles caused a 94% reduction
in the number of colony forming units and mannan, which is a polymer of
mannose, and will include more possible binding sites for the E. coli,
caused a 99% reduction in the number of colony forming units.
EXAMPLE 6
[0129] The ability of the disclosed mannose biofunctionalized
nanoparticles to achieve a marked reduction in C. jejuni CFU was compared
to the CFU-reducing aggregation with a variety of other enteric bacteria.
Strains of Escherichia coli, Salmonella typhi, Enterobacter aerogenes and
Streptococcus faecalis were mixed with mannose biofunctionalized
nanoparticles at nanoparticle:cell ratios of 0.3 to 3 .mu.g:10.sup.6 CFU.
No significant reduction in CFU could be detected.
[0130] Table 2 is a comparison of colony forming units (CFU) obtained when
12.5 micrograms of mannose biiofunctionalized nanoparticles (NP) per ml
of nanoparticle mixture was combined with a variety of enteric bacteria:
Campylobacter jejuni (CJ), Escherichia coli (EC) strain B, Salmonella
typhimurium (ST), Enterobacter aerogenes (EA) and Streptococcus faecalis
(SF).
TABLE-US-00003
TABLE 2
CFU .times. 10.sup.4 CFU .times. 10.sup.5
CJ control 352-383* 31-38
CJ + NP 146-199 7-15
EC control 397-426 40-48
EC + NP 393-430 46-47
ST control TNTC** 283-298
ST + NP TNTC** 278-303
EA control 326-393 27-38
EA + NP 317-323 32-42
SF control 320-380 30-41
SF + NP 367-394 33-41
*Ranges of duplicate platings
**Too numerous to count
[0131] The negative results with other enterics suggest that the
aggregation of C. jejuni by mannose-bearing nanoparticles is not merely a
non-specific binding. Commensal strains of E. coli bind mannose very
weakly while pathogenic strains such as the E. coli ORN178 used in
Examples 4 and 5, above, strongly bind both mannose and mannose
oligosaccharides, so it is not surprising that the nanoparticles could
not cause a reduction in E. coli CFU.
[0132] It will be appreciated that the foregoing examples, given for
purposes of illustration, are not to be construed as limiting the scope
of this invention. Although only a few exemplary embodiments of this
invention have been described in detail above, those skilled in the art
will readily appreciate that many modifications are possible in the
exemplary embodiments without materially departing from the novel
teachings and advantages of this invention. Accordingly, all such
modifications are intended to be included within the scope of this
invention which is defined in the following claims and all equivalents
thereto. Further, it is identified that many embodiments may be conceived
that do not achieve all of the advantages of some embodiments, yet the
absence of a particular advantage shall not be construed to necessarily
mean that such an embodiment is outside the scope of the present
invention.
* * * * *
