Note: Descriptions are shown in the official language in which they were submitted.
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Compositions for Inactivating Pathogenic Microorganisms, Methods
of Making the Compositions, and Methods of Use Thereof
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application claims the benefit under 35 USC ~ 119(e) of U.S.
Application No. 60!457,633, filed June 4, 2003, incorporated herein by
reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present disclosure relates to compositions and methods for
decreasing
the infectivity, morbidity, and/or rate of mortality associated with a variety
of
pathogenic microorganisms.
BACKGROUND OF THE INVENTION
[0003] Pathogenic microorganisms such as bacteria, fungi, viruses, and
bacterial
spores are responsible for a plethora of human and animal ailments. In
addition to
vegetatively growing bacteria, bacteria of the Bacillus genus and others form
stable
spores that resist harsh conditions and extreme temperatures. For example,
contamination of farmlands with B. anthracis can lead to a fatal disease in
domestic,
agricultural, and wild animals, as well as in humans in contact with infected
animals
or animal products. B. a~cth~acis infection in humans is no longer common due
to
effective animal controls that include vaccines, antibiotics, and appropriate
disposal of
infected livestock. However, animal anthrax infection still represents a
significant
problem due to the difficulty of decontaminating land and farms. Moreover, B.
ahth~acis spores can be used as a biological weapon. While an anthrax vaccine
is
available and can be used for the prevention of classic anthrax, genetic
mixing of
different bacterial strains can render it ineffective.
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[0004] Other members of the Bacillus genus are also reported to be etiological
agents for many human diseases. B. ce~eus is a common pathogen involved in
food
borne diseases due to the ability of the spores to survive cooking procedures.
It is
also associated with local sepsis, wound and systemic infection.
[0005] Although antibiotic and antimicrobial therapy is very effective and a
mainstay of modern medicine, these therapies suffer from several
disadvantages. For
example, bacterial strains can develop antibiotic resistance. A person
infected with an
antibiotic resistant strain of bacteria faces serious and potentially life-
threatening
consequences because antibiotics cannot eliminate the infection. Pneumococci,
which
cause pneumonia and meningitis, Salmonella and E. coli which cause diarrhea,
and
enterococci which cause blood stream, surgical wound, and urinary tract
infections
can all develop antibiotic resistance resulting in fatal infections.
[0006] Moreover, antibiotics are not effective in eliminating or inactivating
bacterial
spores and viruses. Disinfectants and biocides, such as sodium hypochlorite,
formaldehyde and phenols can be effective against bacterial spores, but are
not well
suited for decontamination of the environment, equipment, or casualties. The
toxicity
of these compounds can result in tissue necrosis and severe pulmonary injury
following contact or inhalation of volatile fumes. Furthermore, the corrosive
nature
of commonly used disinfectants and biocides renders them unsuitable for
decontamination of sensitive equipment.
[0007] Viruses are additional pathogens that infect human and animals which
currently lack effective means of inactivation. For example, influenza A virus
is a
common respiratory pathogen widely used as a model system to test anti-viral
agents
in vitro and ih vivo. The envelope glycoproteins of influenza A, hemagglutinin
(HA)
and neuraminidase (NA), which determine the antigenic specificity of viral
subtypes,
mutate readily, rendering antibodies incapable of neutralizing the virus.
Current anti-
viral compounds and neuraminidase inhibitors are minimally effective and viral
resistance is common.
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SUMMARY OF THE INVENTION
[0008] Accordingly, there remains a need in the art for anti-pathogenic
compositions and methods that decrease the infectivity, morbidity, and/or
mortality
associated with pathogenic exposure while minimizing microbial resistance,
toxicity
to the recipient, and deleterious effects to equipment and the environment.
[0009] To address these and other needs, the present invention provides
emulsions
comprising an aqueous phase, an oil phase comprising an oil and an organic
solvent,
and at least one surfactant. The emulsion comprises particles preferably
having an
average diameter of less than or equal to about 250 nm.
[0010] In one embodiment, the invention provides a method of reducing the
average
nanoemulsion particle size of a composition comprising a nanoemulsion,
comprising
treating a nanoemulsion comprising an aqueous phase, an oil phase comprising
an oil
and an organic solvent, and a surfactant, and having nanoemulsion particles of
an
average diameter of greater than or equal to about 250 nm, so as to reduce the
average
diameter of the nanoemulsion particles to less than or equal to about 250 nm.
[0011] In another embodiment, the invention provides a method of making a
nanoemulsion, comprising passing a first nanoemulsion through a high pressure
homogenizer or a microfluidizer under conditions effective to reduce the
average
diameter of the nanoemulsion particles less than or equal to about 250 nm. The
nanoemulsion comprises an aqueous phase, an oil phase comprising an oil and an
organic solvent, and one or more surfactants. The nanoemulsion particles have
an
average diameter of greater than or equal to about 250 nm.
[0012] The invention also provides a method of inactivating a microorganism,
comprising contacting the microorganism with a composition comprising a
nanoemulsion for a time effective to inactivate the microorganism. The
nanoemulsion
comprises an aqueous phase; an oil phase comprising an oil and an organic
solvent
and one or more surfactants. The nanoemulsion particles have an average
diameter of
less than or equal to about 250 nm.
[0013] The invention further provides a method of inactivating a pathogenic
microorganism comprising contacting a subject infected with the microorganism
with
a composition comprising a nanoemulsion. The nanoemulsion comprises an aqueous
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phase, an oil phase comprising an oil and an organic solvent, and one or more
surfactants, wherein the nanoemulsion comprises particles having an average
diameter
of less than or equal to about 250 nm.
[0014] In an additional embodiment, the invention provides an immunogenic
composition comprising a nanoemulsion, wherein the nanoemulsion comprises an
aqueous phase, an oil phase comprising an oil and an organic solvent, and a
surfactant, wherein the nanoemulsion comprises nanoemulsion particles having
an
average diameter of less than or equal to about 250 nm and a microorganism or
a
portion thereof.
[0015] In a further embodiment, the invention provides method of vaccinating
against a microorganism comprising administering to a subject the a
composition
comprising a nanoemulsion, wherein the nanoemulsion comprises an aqueous
phase,
an oil phase comprising an oil and an organic solvent, and one or more
surfactants,
wherein the nanoemulsion comprises particles having an average diameter of
less than
or equal to about 250 nm. The microorganism is inactivated by the composition
and
an immunological response by the subject is elicited.
[0016] In another embodiment, the invention provides a method of preventing an
infected state caused by a microorganism, comprising administering to a
subject,
either before or after exposure to a microorganism, a composition comprising a
nanoemulsion. The nanoemulsion comprises an aqueous phase, an oil phase
comprising an oil and an organic solvent, and one or more surfactants, wherein
the
nanoemulsion comprises particles having an average diameter of less than or
equal to
about 25 0 nm.
[0017] The invention further provides a kit comprising a composition
comprising a
nanoemulsion, wherein the composition is provided in a single formulation or a
binary formulation, wherein the binary formulation is mixed prior to using the
composition.
[0018] The above described and other features are exemplified by the following
figures and detailed description.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0019]Figure Average separation of neat (100%) emulsions
1. stored at 55 C.
[0020]Figure Average settling of 10% emulsions stored at
2. 55 C.
[0021]Figure Average settling of 2.5% emulsions stored
3. at 55 C.
[0022]Figure Change in pH after accelerated stability testing.
4. pH of neat and
diluted s measured on day 0 and after 31 days incubation
emulsions at 55 C.
i
[0023]Figure Dependence of nanoemulsion particle size of
5. passage number and
pressure
in
Avestin
EmulsiFlex~
C3.
[0024]Figure Log reduction of Mycobacterium fortuitum by
6. X8PC.
DETAILED DESCRIPTION OF THE PREFERRED EMBQDIMENTS
[0025] Unless otherwise specified, "a" or "an" means "one or more". The
present
inventors discovered that compositions having emulsion particles with an
average
particle diameter of less than or equal to about 250 nm ("small particle size
nanoemulsion") have improved stability and/or activity. These small particle
size
nanoemulsions are useful in a wide range of applications for decreasing the
infectivity, morbidity, and/or rate of mortality associated with a variety of
pathogenic
microorganisms. As used herein, the term "pathogenic microorganism" refers to
a
biological microorganism that is capable of producing an undesirable effect
upon a
host animal, and includes, for example, without limitation, bacteria, viruses,
bacterial
spores, molds, mildews, fungi, and the lilce. This includes all such
biological
microorganisms, regardless of their origin or of their method of production,
and
regardless of whether they exist in facilities, in munitions, weapons, or
elsewhere.
[0026] Small particle size nanoemulsion compositions are useful, for example,
as
therapeutics for humans or animals, for decontaminating surfaces, individuals
or
locations colonized or otherwise infected by pathogenic microorganisms, for
prophylaxis, treatment, and vaccine compositions, for decreasing the
infectivity of
pathogenic microorganisms in foodstuffs, and the like. The inactivation of a
broad
range of pathogenic microrganisms, including, for example, vegetative bacteria
and
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enveloped viruses and bacterial spores, combined with low toxicity, make small
particle size nanoemulsions well-suited for use as a general decontamination
agent
before a specific pathogen is identified.
A. Nanoemulsion Compositions
[0027] Particle size reduction to produce a small particle size nanoemulsion
from a
standard emulsion is efficiently and economically accomplished by high-
pressure
homogenizer or microfluidizer. Small particle size nanoemulsions can be
rapidly
produced in large quantities and are stable for many months at a broad range
of
temperatures.
[0028] An emulsion is a composition containing an aqueous phase and an oil
phase.
The term "emulsion" refers to, without limitation, any oil-in-water
dispersions or
droplets, including lipid structures that can form as a result of hydrophobic
forces that
drive apolar residues (e.g., long hydrocarbon chains) away from water and
polar head
groups toward water, when a water immiscible phase is mixed with an aqueous
phase.
These other lipid structures include, but are not limited to, unilamellar,
paucilamellar,
and multilamellar lipid vesicles, micelles, and lamellar phases. Classical or
standard
emulsions comprise lipid structures having an average particle size of greater
than
about 5 ~.m in diameter. Standard nanomulsions having smaller particle sizes
are
known, and comprise lipid structures having an average particle diameter of
about
500 nm to about 5 ~.m. In one embodiment, a standard nanoemulsion has an
average
particle size of about As used herein, "small particle size nanoemulsions"
refers to
emulsions having an average particle diameters of less than or equal to about
250 iun.
In one embodiment, average particle diameter is less than or equal to about
200 nm,
less than or equal to about 150 nm, less than or equal to about 100 rim, or
less than or
equal to about 50 nm. As used herein, the term "nanoemulsion" can encompass
both
standard and small particle size nanoemulsions.
[0029] Emulsion particle size can be determined using any means known in the
art,
such as, for example, using laser light scattering.
[0030] A nanoemulsion composition contains about 5 to about 50 percent by
volume (vol °fo) of aqueous phase. As used herein, percent by volume
(vol %) is
based on the total volmne of an emulsion or small particle size nanoemulsion.
In one
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embodiment, the aqueous phase is about 10 to about 40 vol %. In another
embodiment, the aqueous phase is about 15 to about 30 vol %. The aqueous phase
ranges from a pH of about 4 to about 10. In one embodiment the pH of the
aqueous
phase ranges from about 6 to about 8. The pH of the aqueous phase can be
adjusted
by addition of an acid or a base such as, for example, hydrochloric acid or
sodium
hydroxide. In one embodiment, the aqueous phase is deionized water
(hereinafter
"diH20") or distilled water.
[0031] The oil phase of a nanoemulsion contains an oil and an organic solvent.
The
oil phase of a nanoemulsion contains about 30 to about 90 vol % oil, based on
the
total volume of the nanoemulsion. In one embodiment, the nanoemulsion contains
about 60 to about 80 vol % oil. In another embodiment, the nanoemulsion
contains
about 60 to about 70 vol % oil. The oil phase also contains from about 3 to
about 15
vol % of an organic solvent based on the total volume of the nanoemulsion. In
one
embodiment, the nanoemulsion contains about 5 to about 10 vol % of an organic
solvent.
[0032] Suitable oils include, but are not limited to, soybean oil, avocado
oil,
squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil,
sunflower oil,
fish oils, cinnamon bark, coconut oil, cottonseed oil, flaxseed oil, pine
needle oil,
silicon oil, mineral oil, essential oil, flavor oils, water insoluble
vitamins, and
combinations comprising one or more of the foregoing oils. In one embodiment,
the
oil is soybean oil.
[0033] Suitable organic solvents include, but are not limited to, organic
phosphate
solvents, alcohols, and combinations comprising one or more of the foregoing
solvents. Suitable organic phosphate solvents include, but are not limited to,
dialkyl
and trialkyl phosphates having one to ten carbon atoms, more preferably two to
eight
carbon atoms. The alkyl groups of the di- or trialkyl phosphate can all the
same or the
alkyl groups can be different. In one embodiment, the trialkyl phosphate is
tri-n-butyl
phosphate. Without being held to theory, it is believed that organic solvents
used in
the small particle size nanoemulsions serve to stabilize the nanoemulsion and
remove
or disrupt the lipids in the membranes of pathogens.
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[0034] Suitable alcohols include, for example, C1-C12 alcohols, diols, and
triols, for
example glycerol, methanol, ethanol, propanol, octanol, and combinations
comprising
one or more of the foregoing alcohols. In one embodiment, the alcohol is
ethanol or
glycerol, or a combinations thereof.
[0035] Small particle size nanoemulsion compositions can also contain one or
more
surfactants, present in the aqueous phase, the oil phase, or both phases of a
nanoemulsion. While not limited to any particular proposed mechanism, a
nanoemulsion composition may function to remove proteins from bacterial
membranes, such that a surfactant that will "strip" a membrane of its proteins
may be
useful. A nanoemulsion can contain about 3 to about 15 vol % of surfactant,
based on
the total volume of nanoemulsion. In one embodiment, the nanoemulsion contains
about 5 to about 10 vol % of surfactant.
[0036] Suitable surfactants include, but are not limited to, a variety of
ionic and
nonionic surfactants, as well as other emulsifiers capable of promoting the
formation
of nanoemulsions. Surfactants that allow the oil phase to remain suspended in
the
water phase can be used. In one embodiment, the nanoemulsion comprises a non-
ionic surfactant such as a polysorbate surfactant, i. e., polyoxyethylene
ether. Other
useful surfactants include, but are not limited to, the polysorbate detergents
sold under
the tradenames TWEEN° 20, TWEEN° 40, TWEEN° 60,
TWEEN° 80,
phenoxypolyethoxyethanols and polymers thereof, such as Triton° (i.e.,
X-100, X-
301, X-165, X-102, X-200), Poloxamer° 407 , Spans (20, 40, 60, and 80),
tyloxapol,
and combinations comprising one or more of the foregoing surfactants.
Additional
appropriate surfactants include Brij°30, Brij°35,
Brij°52, Brij°56, Brij°58, Brij°72,
Brij°76, Brij°78, Srij°92, Brij°97,
Brij°98, and Brij° 700. Anionic surfactants
include, but are not limited to sodium dodecyl sulfate (SDS). Mixtures of
surfactants
are also contemplated. In one embodiment, the surfactant is TWEEN° 20
or Triton°
X-100 or a combination thereof. Triton X-100 is a strong non-ionic detergent
and
dispersing agent widely used to extract lipids and proteins from biological
structures.
It also has virucidal effect against a broad spectrum of enveloped viruses. In
another
embodiment, the surfactant is nonoxynol-9.
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[0037] Nanoemulsion compositions can further contain various additives.
Exemplary additives include, for example, activity modulators, gelling agents,
thickeners, auxiliary surfactants, other agents that augment cleaning and
aesthetics,
and combinations comprising at least one of the foregoing, so long as they do
not
significantly adversely affect the activity and/or stability of the emulsions.
Additives
can be incorporated into the nanoemulsion or formulated separately from the
nanoemulsion, i. e., as a part of a composition containing a nanoemulsion.
[0038] "Activity modulators" are additives that affect the activity of a
nanoemulsion
against the target microorganism. Exemplary activity modulators are
interaction
enhancers such as germination enhancers, therapeutic agents, buffers, and the
like,
which are described below.
[0039] One class of activity modulators thus includes "interaction enhancers,"
compounds, or compositions that increase the interaction of the nanoemulsion
with
the cell wall of a bacterium (e.g., a Gram positive or a Gram negative
bacteria) or a
fungus, or with a virus envelope. Again, without being bound by theory, it is
proposed that the activity of the emulsions is due, in part, to the
interaction of a
nanoemulsion with a microorganism membrane or envelope. Suitable interaction
enhancers include compounds that increase the interaction of the nanoemulsion
with
the cell wall of Gram negative bacteria such as T~ibrio, Salmonella, Shigella,
Pseudomouas, Escherichia, Klebsiella, P~oteus, Ehterobacte~, Sen~atia,
Mof°axella,
Legiohella, Bo~detella, Helicobacter, Haemophilus, Neisse~ia, B~ucella,
Yersinia,
Pasteu~ella,Bacteiods, and the like.
[0040] One exemplary interaction enhancer is a chelating agent. Suitable
chelating
agents include ethylenediaminetetraacetic acid (EDTA),
ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and combinations
thereof.
Chelating agents can be prepared in water or in a buffer, such as, for
example, TRIS
buffer. Chelating agents can be premixed with the aqueous phase or can be
added to a
diluent. Chelating agents can be used at a concentration of about 1 ~.M to
about 50
mM, based on the total volume of the nanoemulsion composition. In one
embodiment, the concentration of the chelating agent is between about 100 ~.M
to
about 50 mM. In a further embodiment, the concentration of chelating agent can
be
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greater than or equal to about 25 ~,M, greater than or equal to about 50 ~,M,
greater
than or equal to about 70 wM greater than or equal to about 80 ~,M, greater
than or
equal to about 100 ~M, greater than or equal to about 1 mM, or greater than or
equal
to about 2 mM. In an additional embodiment, the concentration of chelating
agent
can be less than or equal to about 40 mM, less than or equal to about 27 mM,
less than
or equal to about 25 mM, less than or equal to about 10 mM, or less than or
equal to
about 5 mM.
[0041] Another exemplary interaction enhancer is a cationic halogen-containing
compound. A cationic halogen-containing compound can be premixed with the
aqueous phase, or it may can be provided in combination with a nanoemulsion in
a
distinct formulation. A cationic halogen-containing compound can be used at a
concentration of about 0.5 to about 7 vol. %, based on the total volume of the
nanoemulsion. In one embodiment, a cationic halogen-containing compound can be
used at a concentration of about 0.5 to about 3 vol. %, based on the total
volume of
the nanoemulsion.
[0042] Suitable cationic halogen-containing compounds include, but are not
limited
to, cetylpyridinium halides, cetyltrimethylammonium halides,
cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,
cetyltributylphosphoniurn halides, dodecyltrimethylammonium halides,
tetradecyltrimethylammonium halides, alkylbenzyldimethylammonium salts and
combinations comprising one or more of the foregoing compounds. Suitable
halides
in the cationic halogen-containing compounds include chloride, fluoride,
bromide and
iodide. In one embodiment, the halide is chloride or bromide. In another
embodiment the cationic halogen-containing compound is cetylpyridinium
chloride or
benzalkonium chloride or a combination thereof.
[0043] A "germination enhancer" enhances the germination of, for example,
spores.
Suitable germination enhancing agents include nucleosides, a-amino acids,
salts and
combinations thereof. Useful nucleosides include inosine. Useful a-amino acids
include, for example, glycine and the L-enantiomers of alanine, valine,
leucine,
isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the allcyl
esters
thereof. Suitable salts, include, for example, sodium chloride, ammonium
chloride,
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magnesium chloride, calcium chloride, phosphate buffered saline (PBS), and
potassium chloride. In one embodiment, the germination enhancer is a mixture
of
glucose, fructose, asparagine, sodium chloride, ammonium chloride, calcium
chloride,
and potassium chloride. In another embodiment, the germination enhancer is a
combination containing L-alanine, inosine, PBS, and ammonium chloride.
[0044] Certain growth media contain germination enhancers and buffers. Thus,
when testing nanoemulsions for their ability to inactivate spores, addition of
germination enhancers may not be required, if the tests are conducted using
media
containing such germination enhancers. Similarly, the addition of certain
growth
media to emulsions can enhance sporicidal activity.
[0045] An effective amount of germination enhancer can be readily determined
by
one of ordinary skill in the art. Nucleosides and amino acids can be used in
amounts
of about 0.5 mM to about 100 mM. In one embodiment, nucleosides and amino
acids
are used at a concentration of about 1 mM to about 50 mM. In another
embodiment,
nucleosides and amino acids are used at a concentration of about 0.5 mM to
about 5
mM. Salts can be present in amounts of about 0.5 mM to about 100 mM and PBS
can be used at concentrations of about O.OSx to about lx.
[0046] A germination enhancer can be incorporated into the aqueous phase prior
to
formation of the nanoemulsion. In one embodiment a germination enhancer is
active
at approximately neutral pH. In another embodiment, a germination enhancer can
be
active between pH of about 6 to about 8. Adjustment of pH of a nanoemulsion
composition containing a germination enhancer can be achieved by any suitable
means, such as, for example, dilution of a nanoemulsions in PBS or by
preparations of
a neutral nanoemulsion or by the addition of hydrochloric acid or sodium
hydroxide.
(0047] "Therapeutic agent" refers to an agent that decreases the infectivity,
morbidity, and/or rate of mortality associated with a pathogenic microorganism
when
administered to a subject affected by a pathogenic microorganism. Suitable
therapeutic agents include, for example, antimicrobial agents, antiviral
agents,
antifungal agents, and the like, and combinations comprising one or more of
the
foregoing agents. There are many antimicrobial agents currently available for
use in
treating bacterial, fungal and viral infections. Generally, these agents
include agents
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that inhibit cell wall synthesis (e.g., penicillins, cephalosporins,
cycloserine,
vancomycin, bacitracin), imidazole antifungal agents (e.g., miconazole,
ketoconazole
and clotrimazole), agents that act directly to disrupt the cell membrane of
the
microorganism (e.g., polymyxW and colistimethate and the antifungals nystatin
and
amphotericin B), agents that affect the ribosomal subunits to inhibit protein
synthesis
(e.g. chloramphenicol, the tetracyclines, erythromycin and clindamycin),
agents that
alter protein synthesis and lead to cell death (e.g. aminoglycosides), agents
that affect
nucleic acid metabolism (e.g. the rifamycins and the quinolones),
antimetabolites
(e.g., trimethoprim and sulfonamides), and the nucleic acid analogues (e.g.
zidovudine, gangcyclovir, vidarabine, and acyclovir) which act to inhibit
viral
enzymes essential for DNA synthesis. Other useful therapeutic agents include,
but are
not limited to antimicrobials such as phenylphenol, propyl paraben and
poly(hexamethylene biguanide) hydrochloride (PHMB).
[0048] Optionally, nanoemulsion compositions can be formed into gels by adding
a
gelling agent. Suitable gelling agents include, for example, hydrogels such
as, for
example, Natrosol~ 250H NF (Hercules, Inc. Wilmington, DE). A hydrogel can be
added at concentration of about 0.5 wt % to about 5 wt %, based on the total
volume
of the gel. Other suitable gelling agents include, but are not limited to,
about 0.05 wt
to about 3 wt % cellulose polymer, such as cellulose gum or cationic guar
derivatives, and up to about 10 wt % petrolatum, glycerin, polyethylene
glycol,
incroquat behenyl TMS, cetyl palmitate, glycerol stearate, and the like.
[0049] A variety of auxiliary surfactants can optionally be used to enhance
the
properties of a nanoemulsion composition. The choice of auxiliary surfactant
depends
on the desire of the user with regard to the intended purpose of the
composition and
the commercial availability of the surfactant. In one embodiment, the
auxiliary
surfactant is an organic, water-soluble surfactant.
[0050] Other optional additives such as perfumes, brighteners, enzymes,
colorants,
detergent builders, suds suppressors, and the like can also be used in the
compositions
to enhance aesthetics and/or cleaning performance. Detergent builders
sequester
calcium and magnesium ions that might otherwise bind with and render less
effective
the auxiliary surfactants or co-surfactants. Detergent builders are
particularly useful
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when auxiliary surfactants are used, and when the compositions are diluted
prior to
use with hard tap water, especially water having a hardness of , above about
12
grains/gallon.
[0051] A nanoemulsion composition can contain a suds suppressor. A suds
suppressor is a low-foaming co-surfactant that prevents excessive sudsing
during
employment of the compositions on hard surfaces. Suds suppressors are also
useful in
formulations for no-rinse application of the composition. Concentrations of
about 0.5
vol % to about 5 vol % are generally effective. Selection of a suds suppressor
depends on its ability to formulate in a nanoemulsion composition and the
residue as
well as the cleaning profile of the composition. The suds suppressor should be
chemically compatible with the components in a nanoemulsion composition and
functional at the pH of a given composition. In one embodiment the suds
suppressor
or composition containing a suds suppressor does not leave a visible residue
on
surfaces on which a composition is applied.
(0052] Low-foaming co-surfactants can be used as a suds suppressor to mediate
the
suds profile in a nanoemulsion composition. Examples of suitable suds
suppressors
include block copolymers, alkylated primary and secondary alcohols, and
silicone-
based materials. Exemplary block co-polymers include, e.g., Pluronic°
and Tetronic°
(BASF Company). Alkylated alcohols include those which are ethoxylated and
propoxylated, such as, tergitol (Union Carbide) or poly-tergent° (Olin
Corp.).
Silicone-based materials include DSE (Dow Corning). The suds suppressors can
be
incorporated into the composition by any means known in the art.
B. Method of Making Small Particle Size Nanoemulsions
[0053] Small particle size nanoemulsions and compositions containing small
particle size nanoemulsions can be produced by any suitable means. A small
particle
size nanoemulsion can be formed in the first instance or can be formed from a
nanoemulsion having larger particles. For example, a small particle size
nanoemulsion can be produced by reducing the particle size of a classical or
standard
nanoemulsion (hereinafter "standard nanoemulsion"), to produce a small
particle size
nanoemulsion wherein the average nanoemulsion particle size is less than about
250
nm. In other words, a nanoemulsion having an average particle diameter of
greater
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than about 250 rim is treated in a manner effective to produce particles
having an
average diameter of less than or equal to about 250 nm. In one embodiment,
small
particle size nanoemulsion particles have an average diameter of less than or
equal to
about 200 nm, less than or equal to about 150 nm, less than or equal to about
100 nm,
and less than or equal to about 50 nm.
[0054] Methods for the production of a standard nanoemulsion by mixing an oil
phase with an aqueous phase are well-known. A nanoemulsion can be formed by
blending an oil phase with an aqueous phase on a volume-to-volume basis
ranging
from about 1:9 to about 5:1, about 5:1 to about 3:1, or about 4:1, oil phase
to aqueous
phase. The oil and aqueous phases can be blended using an apparatus capable of
producing shear forces sufficient to form a nanoemulsion such as, for example,
a
French press or a commercial low shear or high shear mixer. In one embodiment,
the
standard emulsions are prepared under conditions of high shear to produce a
nanoemulsion having a substantially uniform particle size distribution. In one
embodiment, a standard nanoemulsion for use in preparing a nanoemulsion
composition is comprised of particles having an average diameter of about 500
nm to
about 5 ~.m, about 500 nm to about 1 ~.m, 400 nm to about 5 ~,m, 400 nm to
about 1
~,m, from about 250 nm to about 5 ~.m, and from about 250 nm to about 1 ~,m.
To
obtain the desired pH, the pH of the aqueous phase can be adjusted using
hydrochloric
acid or sodium hydroxide.
[0055] Forming a small particle size nanoemulsion from a standard nanoemulsion
can be accomplished, for example, by passing the standard nanoemulsion though
a
microfluidizer (Microfluidics Corp., Newton, MA) several times at a pressure
sufficient to produce a desired particle size. A microfluidizer is a
homogenizer that
operates by pumping a fluid stream into an interaction chamber. The
interaction
chamber contains fixed-geometry microchannels that accelerate the fluid
stream,
resulting in high turbulence, shear, and cavitation. A H230Z (chamber 400 ~m
upstream of H21 OZ chamber (200 wm) can be used. Other chamber size and
configurations (Y or Z) can be used in forming a nanoemulsion using a
microfluidizer. During homogenization, a nanoemulsion can be circulated
through a
heat exchanger coil or otherwise cooled to keep the temperature of the
nanoemulsion
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from increasing significantly. In one embodiment, a standard nanoemulsion is
passed
though the microfluidizer for two to five passes at a pressure of about 2,000
to about
10,000 psi. In another embodiment, the pressure is from 3,000 to about 4,000
pounds
per square inch. These conditions can vary depending on factors such as
standard
nanoemulsion particle size, nanoemulsion composition, and desired final
particle size
[0056] Another means of forming a small particle size nanoemulsion is passage
of a
standard nanoemulsion through a high pressure homogenizes, like an EmulsiFlex~
high pressure homogenizes (Avestin, Inc., Ottawa, Canada). The number of
passages
through the homogenizes as well as the flow rate will depend on the particle
size of
the standard nanoemulsion, nanoemulsion composition, and the desired particle
size
of the resulting small particle size nanoemulsion. Operating pressure is
independent
from flow rate and will remain at the set value over the process time. In one
embodiment, the operating pressure is from about 2,500 to about 20,000 psi. As
with
the microfluidizing method discussed above, a nanoemulsion can be cooled using
a
heat exchanger or other method and the nanoemulsion can be passed though the
homogenizes from about two to about five times. The particle size depends
inversely
on both the number of passages and on the operating pressure. See Figure 5.
[0057] In addition to the above described methods, one can produce a small
particle
size nanoemulsion directly, without premixing. The direct use of, for example,
either
a microfluidizer or a high pressure homogenizes, as described above, can
result in a
small particle size nanoemulsion with the properties discussed above for a
small
particle size nanoemulsion produced from a premixed standard nanoemulsion.
[0058] Small particle size nanoemulsions can have a consistency ranging from a
semi-solid cream to a watery liquid similar to skim milk. Creamy emulsions can
be
used as-is or mixed with water.
[0059] A nanoemulsion can be prepared in a diluted or an undiluted form. In
one
embodiment a nanoemulsion shows suitable stability in both diluted and
undiluted
forms. By suitable stability, it is meant that the emulsions do not show any
signs of
separation (oil phase from aqueous phase) for at least 6 months. In another
embodiment a nanoemulsion does not show any sign of separation up to about 2
years. In a fi~ther embodiment, a nanoemulsion does not show any sign of
separation
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for up to about 3 years. Settling of the diluted emulsions is an acceptable
characteristic and does not indicate separation of an oil phase from an
aqueous phase.
Settling is due to separation of emulsions from its diluent, not an oil phase
separating
from an aqueous phase. Such settling is readily reversed by simple shaking of
the
nanoemulsion, while separation of the concentrated emulsions are not reversed
by
simple mixing, requiring instead re-emulsification.
[0060] The emulsions can also contain a first nanoemulsion emulsified within a
second nanoemulsion, wherein the first and second emulsions can each contain
an
aqueous phase, an oil phase, and a surfactant. The oil phase of each of the
first and
second nanoemulsion can contain an oil and an organic solvent. The first and
second
nanoemulsion can be the same or different. A nanoemulsion can also contain a
first
nanoemulsion re-emulsified to form a second nanoemulsion.
[0061] One useful parameter for characterizing a nanoemulsion is "zeta
potential."
Zeta potential is the electrical potential of a shear plane (an imaginary
surface
separating a thin layer of liquid that shows elastic behavior) bound to a
solid surface
that shows normal viscous behavior. The stability of hydrophobic colloids
depends,
in part, on the zeta potential. Zeta potential of a nanoemulsion can be about -
50 mV
to about +50. In one embodiment, the zeta potential of the emulsions can be
greater
than or equal to about +10 mV. In another embodiment, the zeta potential is
greater
than or equal to about +20 mV. In a further embodiment, the zeta potential of
the
emulsions can be less than or equal to about +45 mV, less than or equal to
about +40
mV or less than or equal to about +30 mV.
[0062] In one embodiment a nanoemulsion, comprising optional therapeutic
agents,
can be provided in the form of pharmaceutically acceptable compositions. The
terms
"pharmaceutically acceptable" or "pharmacologically acceptable" refer to
compositions that do not produce significant adverse, allergic, or other
untowaxd
reactions when administered to an animal or a human
[0063] Compositions for pharmaceutical use typically comprise a
pharmaceutically
acceptable carrier, for example, solvents, dispersion media, coatings,
isotonic and
absorption delaying agents and the like, and combinations comprising one or
more of
the foregoing carriers as described, for instance, in IZEMINGTON' S
PHARMACEUTICAL
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SCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and 1461-1487
(1975), arid THE NATIONAL FORMULARY XIV 14th Ed., Washington: American
Pharmaceutical Association (1975). Suitable carriers include, but are not
limited to,
calcium carbonate, carboxymethylcellulose, cellulose, citric acid, dextrate,
dextrose,
ethyl alcohol, glucose, hydroxymethylcellulose, lactose, magnesium stearate,
maltodextrin, mannitol, microcrystalline cellulose, oleate, polyethylene
glycols,
potassium diphosphate, potassium phosphate, saccharose, sodium diphosphate,
sodium phosphate, sorbitol, starch, stearic acid and its salts, sucrose, talc,
vegetable
oils, water, and combinations comprising one or more of the foregoing
carriers. The
use of such media and agents for pharmaceutically active substances is well
known in
the art. Except insofar as any conventional media or agent is incompatible
with the
emulsions of the present invention, their use in therapeutic compositions is
contemplated. Supplementary active ingredients also can be incorporated into
the
compositions.
[0064] For topical applications, the pharmaceutically acceptable carriers can
take
the form of a liquid, cream, foam, lotion, or gel, and may additionally
comprise
organic solvents, emulsifiers, gelling agents, moisturizers, stabilizers,
surfactants,
wetting agents, preservatives, time release agents, and minor amounts of
humectants,
sequestering agents, dyes, perfumes, and other components commonly used in
pharmaceutical compositions for topical administration.
C. Methods of Using Nanoemulsion Compositions to Inactivate a Pathogenic
Microorganism
[0065] Nanoemulsion compositions are particularly useful in applications where
inactivation of pathogenic microorganisms is desired. The term inactivating
means
killing, eliminating, neutralizing, or reducing the capacity of a pathogenic
microorganism to infect a host on contact. Nanoemulsion compositions are
useful for
decreasing the infectivity, morbidity, and/or rate of mortality associated
with a variety
of pathogenic microorganisms.
[0066] A method of inactivating a pathogenic microorganism comprises
contacting
the pathogenic microorganism with an amount a nanoemulsion composition which
is
effective to inactivate the microorganism. The step of contacting can involve
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contacting any substrate which may be or is suspected to be contaminated with
a
nanoemulsion composition. By substrate it is meant, without limitation any
subject,
such as a human or an animal (contact can be in vivo or ex vivo, any article,
any
surface, or any enclosure. A pathogenic microorgansm can be, without
limitation, a
bacteria, a virus, a fungus, a protozoan or a combination thereof.
[0067] The step of contacting can be performed for any amount of time
sufficient to
inactivate a microorganism. In one embodiment, inactivation occurs within
about 5
minutes to about 10 minutes after initial contact. However, it is understood
that when
the emulsions are used in a therapeutic context and applied topically or
systemically,
the inactivation may occur over a longer period of time, for example, 5, 10,
15, 20, 25
30, 60 minutes or longer after administration.
[0068] The step of contacting can be performed using any appropriate means of
application. For example, compositions can be administered by spraying,
fogging,
misting, exposure to aerosols, wiping with a wet or saturated cloth or
towlette,
drenching, immersing.
[0069] Nanoemulsion compositions can be used to inactivate vegetative bacteria
and bacterial spores upon contact. Bacteria inactivated by nanoemulsion
compositions can be Gram negative or Gram positive bacteria. Gram negative
bacteria include, for example and without limitation, Tlibrio, Salmonella,
Shigella,
Pseudomohas, Escherichia, Klebsiella, P~oteus, Ehterobacte~, Se~~atia,
Mo~axella,
Legionella, Bordetella, Gay°d~erella, Haemophilus, Neisseria, Brucella,
Ye~sinia,
Pasteurella, Baete~oids, and Helicobacte~. Gram positive bacteria include, for
example, and without limitation, Bacillus, Clostridium, A~throbacte~,
Micrococcus,
Staphyloeoccus, Streptococcus, Listeria, Co~yhebacte~ia, Planococcus,
Mycobactey~ium, Noca~dia, Rhodococcus, aid acid fast Bacilli such as
Mycobacterium. In one embodiment, nanoemulsion compositions can be used to
inactivate Bacillus, including, without limitation B. ahthy~acis, B. cereus,
B. ci~culans,
B. subtilis, and B. megate~iu~. Nanoemulsion compositions can also be used to
inactivate Clostf~idium, e.g., C. botulihum, C. pe~friagens, and C. tetani.
Other
bacteria that can be inactivated by a nanoemulsion include, but are not
limited to, H.
influehzae, N. gonorrhoeae, S. agalactiae, S. pneumonia, S pyogenes and Tl
chole~ae
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(classical and Eltor), and Yersinia , including, Y. pestis, Y. ehte~ocolitica,
and F
pseudotubef culosis. In another embodiment, the bacteria is B. a~thracis. In
another
embodiment, the bacteria is Mycobate~iuu~ tuberculosis.
[0070] Contacting a bacterial spore with a nanoemulsion inactivates the spore.
Without being bound to any theory, it is proposed that the sporicidal ability
of the
nanoemulsions is by initiation of germination without complete reversion to
the
vegetative form, leaving the spore susceptible to disruption by the emulsions.
Induction of germination using germination enhancers such as inosine and L-
alanine
can result in acceleration of the sporicidal activity of the nanoemulsion,
while
inhibition of iutiation of germination with I?-alanine can delay sporicidal
activity.
This unique action of a nanoemulsion, which can be better in efficiency than 1
bleach, is interesting because Bacillus spores are generally resistant to most
disinfectants including many commonly used detergents. The sporicidal effect
can
start almost immediately. In one embodiment the sporicidal effect occurs
within 30
minutes of contact with a nanoemulsion.
[0071] Contacting a nanoemulsion composition with a virus can inactivate a
virus.
The effect of nanoemulsion compositions on viral agents can be monitored using
any
suitable means, such as, for example, plaque reduction assay (PRA), cellular
enzyme-
linked immunosorbent assay (ELISA), P-galactosidase assay, and electron
microscopy (EM).. Viruses which can be inactivated by contact with a
nanoemulsion
composition include, without limitation, and virus of the families
Baculoviridae,
Herpesviridae, Iridoviridae, Poxviridae, "African Swine Fever Viruses,"
Adenoviridae, Caulimoviridae, Myoviridae, Phycodnaviridae, Tectiviridae,
Papovaviridae, Circoviridae, Parvoviridae, Iiepadnaviridae, Gystoviridae,
Birnaviridae, Reoviridae, Coronaviridae, Flaviviridae, Togaviridae,
"Arterivirus,"
Astroviridae, Caliciviridae, Picornaviridae, Potyviridae, Retroviridae,
Orthomyxoviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Arenaviridae,
and
Bunyaviridae. In one embodiment, the virus is herpes, pox, papilloma, corona,
influenza, hepatitis, sendai, sindbis and vaccinia viruses, west nile, hanta,
and viruses
which cause the common cold.
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(0072] In yet another embodiment, contacting a nanoemulsion with a fungus
inactivates the fungus. In one embodiment, the fungus is a yeast, such as, for
example
various species of Candida (e.g., Candida albicerhs) or filamentous yeast
including
but not limited to Aspe~gillus species or dermatophytes such as T~ichophytoh
~°ubrum,
Trichophyto~ mehtag~ophytes, Mic~ospo~um cahis, Mic~osporum gypseum, and
Epide~ophytoh floccosum, and types thereof, as well as others.
[0073] The methods and compositions, or components of the methods and
compositions can be formulated in a single formulation, or can be separated
into
binary formulations for later mixing during use, as may be desired for a
particular
application. Such components can advantageously be placed in kits for use
against
microbial infections, decontaminating instruments and the like. Such kits may
contain all of the essential materials and reagents required for the delivery
of the
formulations to the site of their intended action as well as any desired
instructions.
[0074] For i~c vivo use, the methods and compositions may be formulated into a
single or separate pharmaceutically acceptable syringeable composition. In
this case,
the container means may itself be an inhalant, syringe, pipette, eye dropper,
or other
like apparatus, from which the formulation may be applied to an infected area
of the
body, such as the lungs, injected into an animal, or even applied to and mixed
with the
other components of the kit.
[0075] A kit also can include a means for containing the vials in close
confinement
for commercial sale (e.g., injection or blow-molded plastic containers into
which the
desired vials are retained). Irrespective of the number or type of containers,
the kits
also may comprise, or be packaged with, an instrument for assisting with the
injection/administration or placement of the ultimate complex composition
within the
body of an animal. Such an instrument may be an inhalant, syringe, pipette,
forceps,
measured spoon, eyedropper, or any such medically approved delivery vehicle.
[0076] Actual amounts of nanoemulsions and additives in the compositions can
be
varied so as to provide amounts effective to inactivate vegetative as well as
sporular
microorganisms and pathogens. Accordingly, the selected amounts will depend on
the nature and site for treatment, the desired response, the desired duration
of biocidal
action, the condition of the subject being treated, and other factors. A
nanoemulsion
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composition can comprise, for example, about 0.001% to about 100% nanoemulsion
per milliliter of liquid composition. In one embodiment, a nanoemulsion
composition
can contain about 0.01 % to about 90% nanoemulsion per milliliter of liquid.
These
are merely exemplary ranges. A nanoemulsion composition can also comprise
greater
than about 0.25%, about 1.0%, about 5%, about 10%, about 20%, about 35%, about
50%, about 65%, about 80%, about 90%, or about 95% of nanoemulsion per
milliliter
of liquid composition.
[0077] The small particle size nanoemulsions as described herein are more
stable
than standard emulsions under a variety of conditions, showing substantially
no
observable separation or settling for up to one month, preferably up to four
months,
more preferably up to or more than one year, up to about 21 °C,
preferably up to 40
°C,. Such stability is at no dilution, up to 2.5% dilution, up to 10%
dilution, more
preferably up to 50% dilution or higher.
[0078] The small particle size nanoemulsions perform equal to or better than
standard emulsions in inactivating a pathogenic microorganism, exhibiting a
less than
10% failure rate, preferably a less than 5% failure rate, more preferably a
less than 1%
failure rate, and most preferably a 0% failure rate against pathogens. The
invention is
further illustrated by the following non-limiting examples.
1. Prevention and Treatment of Infection
[0079] Nanoemulsion compositions are useful for the prevention and treatment
of
infection. A method of inactivating a pathogenic microorganism comprises
contacting
a subject infected with or suspected to be infected with the microorganism
with a
nanoemulsion composition comprising an aqueous phase, an oil phase, and one or
more surfactants. The oil phase comprises an oil and an organic solvent, as
discussed
above. The nanoemulsion particles have an average diameter of less than or
equal to
about 250 nm. In one embodiment, the particles have an average diameter of
less
than or equal to about 200 run, less than or equal to about 150 nm, less than
or equal
to about 100 nm, or less than or equal to about 50 nm.
[0080] The pathogenic microorganism may have systemically infected the subject
or on the surface of the subject. Where the microorganism is not on the
subject, the
is delivered to the site of infection by any suitable method, for example
injection, oral
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administration, suppositories, and the like. In one embodiment the subject is
an
animal. In a further embodiment, the animal is a human.
[0081] Exemplary infected states that can be treated or prevented with
nanoemulsions include, but are not limited to, bacterial, fungal, protozoal,
and/or viral
vaginal infection, sexually transmitted diseases (STDs), skin infections such
as, acne,
impetigo, athlete's foot, onychomycosis, candidiasis and other acute fungal
infections,
herpes simplex and zoster and infections associated with psoriasis or other
skin
inflammatory diseases. In one embodiment, an infected state is particularly
susceptible to topical treatment. As used herein, "infected states" is
inclusive of
contamination with pathogenic microorganisms, and treatment and prevention of
such
infected states includes, but is not limited to, wound decontamination,
decontamination of skin, airways, and/or mucosal surfaces (e.g., with anthrax
spores,
viruses, bacteria, and/or fungi); and the like. Nanoemulsion compositions can
also be
used as a surgical irrigant. The emulsions can be used in the personal health
care
industry in deodorants, soaps, body wash, acne/dermatophyte treatment agents,
treatments for halitosis, and skin disinfecting.
[0082] Nanoemulsions can be used in a variety of combination therapies,
particularly those directed to microorganisms. This approach is often
advantageous in
avoiding the problems encountered as a result of multidrug resistance, for
example.
[0083] In one embodiment, a nanoemulsion can be used in the prevention or
treatment of genital infections. Such sexually transmitted genital infections
include,
but are not limited to genital herpes, human papilloma virus (HPV), human
immunodeficiency virus (HIV), trichomoniasis, gonorrhea, syphilis, and
chlamydia.
A nanoemulsion can be applied to the genitals either before or after sexual
intercourse
or both before and after sexual intercourse. In one embodiment, a nanoemulsion
is
introduced into the vagina of a female, at about the time of sexual. In
another
embodiment, a nanoemulsion is introduced into the vagina of a female prior to
intercourse. A nanoemulsion can also be administered to other mucous
membranes.
Application of a nanoemulsion composition to genitalia can be accomplished
using
any appropriate means including, for example, ointments, jellies, inserts
(suppositories, sponges, and the like), foams, and douches.
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[0084] A nanoemulsion can also be used in the treatment of nonsexually
transmitted genital infections, such as fungal, protozoan, bacterial
infections. Fungal
infections treatable with a nanoernulsion include, but are not limited, to
tinea, candida
(e.g., Cahdida albicans). Nonsexually treated bacterial infections treatable
with a
nanoemulation include, but are not limited, nonspecific vaginitis and
bacterial
vaginitis caused by, for example, Ga~d~ze~ella vagihalis, Ga~d~ey°alla
mobiluncus,
and Mycoplasma hominis.
[0085] Nanoemulsion compositions can also be used for the prevention and
treatment of respiratory infection. Nanoemulsion compositions can be used to
prevent
infection by, without limitation, the common cold, influenza, tuberculosis,
legionnaire's disease, and acute respiratory syndrome (SARS). In one
embodiment, a
nanoemulsion composition is applied to the respiratory passages using, for
example, a
nasal spray, such that the spray coats the respiratory passages before
exposure to these
pathogens. In another embodiment, this use can substantially inactivate or
eliminate a
respiratory pathogen preventing the pathogen from inducing a pathogenic
response.
The use of a nanoemulsion in the prevention and treatment of a respiratory
infection
can also stimulate an immunological response against a specific pathogen which
can
protect from further exposure to the same pathogen.
2. Immunogenic Compositions and Vaccine Applications:
[0086] A nanoemulsion can be mixed with a microorganism, a recombinant
antigen,
or a combination thereof to yield an immunogenic composition. The
concentration of
microorganism can range from approximately 102 to approximately 101°.
The
concentration of antigen can range from approximately 1 ~,g to approximately 1
mg,
either alone or mixed with other adjuvants which include but are not limited
to CpG
oligonucleotides. As used herein "ilnmunogenic composition" refers to any
composition capable of eliciting an immune response. In one embodiment, the
immune response results in production of protective antibodies.
[0087] An immunogenic nanoemulsion composition can be administered topically
on the skin or on mucosal membranes of a subj ect. Administration of an
immunogenic composition can be performed using any appropriate means and using
any appropriate formulation knov~m in the art.
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[0088] In one embodiment, a nanoemulsion composition contains substantially
inactivated B. ahth~acis. In one embodiment, a nanoemulsion composition
contains a
peptide comprising at least a portion of anthrax protective antigen (PA). PA
is known
to provide protection against infection in conventional vaccines. A vaccine
can also
contain an attenuated strain of B. auth~acis. In one embodiment, PA is
isolated from
B. avrthy~acis extract. In another embodiment, PA is recombinant PA. B.
ahtlz~aeis PA
concentration can range from about 1 ~.g to about 1 mg. In one embodiment , B.
ahthT°acis PA concentration ranged from between about 2.3 ~,g to about
30 ~,g.
[0089] In another embodiment, a nanoemulsion contains substantially
inactivated
vaccinia for use as can be used as a small pox vaccination. In a fiu ther
embodiment, a
nanoemulsion. In a further embodiment, a nanoemulsion can be used to create a
vaccine against influenza virus. In an additional embodiment, a vaccine
composition
can contain an inactivated influenza virus or a portion thereof
[0090] A nanoemulsion composition can contain substantially inactivated
Mycobateria tuberculosis or a portion thereof. In one embodiment, the
Mycobate~ia
tuberculosis-containing nanoemulsion composition is efficacious as a vaccine
against
tuberculosis.
[0091] A nanoemulsion composition can contain substantially inactivated
hepatitis
virus or an portion thereof. In one embodiment, the hepatitis-containing
nanoemulsion is efficacious as a vaccine against hepatitis infection. In
another
embodiment, the hepatitis virus is hepatitis A, hepatitis B, or hepatitis C,
or a mixture
thereof. In a further embodiment, the hepatitis virus is hepatitis B.
[0092] A nanoemulsion composition can contain substantially inactivated HIV or
a
portion thereof. In one embodiment, the HIV-containing nanoemulsion is
efficacious
as a vaccine against HIV infection.
[0093] A nanoemulsion composition can be applied to mucosa to prevent
infection
by a microorganism both as a prophylaxis and as a broad spectrum immunogenic
composition. This administration results in a broad spectrum prophylactic
immunogenic composition which can both prevent infection by a pathogenic
microorganism and also cause an immune response against a pathogenic
microorganism which comes in contact with the nanoemulsion-coated mucosa of a
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subject. In one embodiment, this immunogenic response can provide protection
or
future protection to the subject against a microorganism. In another
embodiment, the
microorganism causes influenza, tuberculosis, the common cold, SARS or other
respiratory diseases.
[0094] In one embodiment, a nanoemulsion composition can be administered prior
to contact with microorganisms. Upon contact with a pathogenic microorganism,
the
microorganism is inactivated. The nanoemulsion-inactivated microorganism can
stimulate an immune response in a subject. In other words, a nanoemulsion
composition can inactivate a microorganism and also function an as an adjuvant
to aid
in stimulating an immune response against the microorganism or its antigens.
In one
embodiment, the immune response results in antibodies capable of neutralizing
a
microorganism and thus providing immunological protection of the subject
against the
microorganism. Examples of microorganisms which can be used in conjunction
with
a nanomemulsion include, but are not limited to bacteria, bacterial spores ,
viruses,
protozoa, and fungi.
3. Decontamination of Medical Devices
[0095] Nanoemulsion compositions axe useful for decontaminating surfaces
colonized or otherwise infected by pathogenic microorganisms. These
applications
include, for example, disinfecting or sterilizing medical devices, contact
lenses antd
the like, particularly when the devices or lenses are intended to be used in
contact
with a patient or wearer. As used herein "medical devices" includes any
material or
device that is used on, in, or through a patient's body in the course of
medical
treatment, whether prophylactic or therapeutic treatment. Medical devices
include,
but are not limited to, such items as implants, for example urinary catheters,
intravasculax catheters, dialysis shunts, wound drain tubes, slcin sutures,
vascular
grafts, implantable meshes, intraocular devices, heart valves, and the like;
wound care
devices, for example wound dressings, surgical sutures, biologic graft
materials, tape
closures and dressings, surgical incise drapes, and the like; drug delivery
devices, for
example skin patches, mucosal patches and medical sponges; and body cavity and
personal protection devices, for example tampons, sponges, surgical and
examination
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gloves, toothbrushes, birth control devices such as IUD's and IUD strings,
diaphragms
and condoms; and the like.
[0096] For applications of this type, the compositions may be conveniently
provided
in the form of a liquid or foam, and may be provided with emulsifiers,
surfactants,
buffering agents, wetting agents, preservatives, and other components commonly
found in compositions of this type. The nanoemulsion compositions can be
impregnated into absorptive materials, such as sutures, bandages, and gauze,
or coated
onto the surface of solid phase materials, such as staples, zippers, and
catheters to
deliver the compositions to a site for the prevention or therapy.
4. Sterilization and Disinfectant Applications
[0097] The present invention is also useful for disinfection and sterilization
for
medical, hospital, ambulance, institutional, educational, agricultural, food
processing,
and industrial applications.
[0098] In one embodiment, a nanoemulsion composition can be used to prevent
contamination, disinfect or sterilize other surfaces, including surfaces used
in the food
industry, for example equipment and areas where food is processed, packaged
and
stored; vehicles; machinery; household surfaces, and other surfaces. For
example, a
nanoemulsion composition can be used to eliminate contamination in meat
processing
plants, particularly of microorganisms such as Liste~ia mo~ocytogenes,
Salmonellae
species and Eschey~ichia species by cleaning slaughterhouses or food packaging
facilities on a continual basis with the composition. In addition,
nanoemulsion
compositions can be formulated into sprays for hospital, food processing and
serving
facilities, and household uses such as cleaning and disinfecting patient
rooms,
household appliances, kitchen and bath surfaces, and the like.
[0099] When used in liquid form to decontaminate surfaces, the emulsions can
be
admixed with an aqueous carrier liquid. The aqueous carrier liquid is
preferably not
toxic and is chemically compatible with the inventive emulsions. The aqueous
carrier
liquid can comprise solvents commonly used in hard surface cleaning
compositions.
Such solvents are preferably chemically stable at the pH of the emulsions,
have good
filming/residue properties, and are miscible with water. Preferred carrier
liquids
comprise water or a miscible mixture of a C2-C4 alcohol and water. The alcohol
or
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glycerol can be used to adjust the viscosity of the compositions Preferably,
the
aqueous carrier liquid is water or a water-ethanol mixture containing from
about 0 to
about 50% ethanol or other solvents. Alternatively, when used to clean hard
surfaces,
the emulsions may be in the form of a gel, foam, or cream, preferably a gel,
and may
be provided with emulsifiers, surfactants, buffering agents, wetting agents,
preservatives, and other components commonly found in compositions of this
type.
[0100] A nanoemulsion can also be used for mold remediation for building,
equipment, and facilities. Examples of molds include, but are not limited to
Cladosporium, Fusa~ium, Alte~haf°ia, Curvula~ia, Aspe~gillus, and
Penicillium.
[0101] A nanoemulsion composition can also be used in the food industry in
preventing and treating food contaminated with pathogens. Thus, such
compositions
may be used to reduce or inhibit microbial growth or otherwise abrogate the
deleterious effects of microbial contamination of food. For example, a
nanoemulsion
composition can be used to kill bacteria and fungus on poultry eggs, fruit,
vegetables,
and meat. Also, the inclusion of a nanoemulsion compositions within the food
product itself would be effective in killing bacteria that may have been
accidentally
contaminated meat or poultry. A nanoemulsion composition can be included in
juice
products to prevent growth of certain fungi, which cause contamination and
lead to
production of mycotoxins. For these applications, the nanoemulsion
compositions are
applied in food industry acceptable forms such as washes, dips, additives,
preservatives, or seasonings. The use of media and agents for additives,
preservatives, and seasonings that are acceptable in food industry is well
known in the
art. Except insofar as any conventional additives, preservatives and
seasonings are
incompatible with the emulsions, their use in preventing or treating food born
microbes and their toxic products is contemplated. Supplementary active
ingredients
may also be incorporated into the compositions.
5. Siodefense Applications
[0102] Nanoemulsion compositions are also useful for biodefense applications,
such
as, for example, decontamination of a building, surface, garment, and
personnel, and
disinfection or sterilization of soil and/or waterways contaminated with a
pathogenic
microorganism, for example as a result of a biological warfare attack.
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[0103] Nanoemulsion compositions can be delivered and applied for
detoxification
and decontamination using any appropriate means. Such decontamination
procedures
are well known to those of skill in the art and may involve simple application
of the
formulation in the form of a liquid spray or may require a more rigorous
regimen. For
example, nanoemulsion compositions can be applied by, without limitation,
spraying,
fogging, misting, exposure to aerosols, wiping with a wet or saturated cloth
or
towlette for personal skin decontamination; drenching, immersing, spraying
with a
hand-held spray bottle or backpack-mounted spray apparatus, showering,
spraying
with a curtain spray, pouring, dripping, and bathing in the liquid
formulation.
Additionally, a nanoemulsion can be deployed in a semi-solid carrier, such as
in gels,
lotions, creams, and pastes. Deployment can be accomplished by people,
deployed
from aircraft, helicopters, trucks, tanks, railroad, boats, bicycle, or by
automated
systems, including mobile robots.
[0104] Deployment can include applying the formulation to a surface inside of
an
industrial setting selected from, for example, a food processing plant, a
hospital, an
agricultural facility, an institutional building, an ambulance, and a cooking
area.
[0105] A fog (e.g., aerosols with particulate sizes ranging from 1-30 ~.m) can
be
used to achieve effective decontamination in areas where decontamination by a
foam
would be difficult, if not impossible. One example is the interior of air
conditioning
ducts. A fog can be generated at registers and other openings in the duct and
travel a
significant distance inside of the duct to decontaminate hard to reach places.
A
relatively automated fog-based decontamination system can be set-up at the
scene of
an attack. Remotely activated foggers can be placed inside of a facility and
turned on
at periodic intervals (from a remote location) to completely decontaminate the
facility.
This method greatly decreases the potential for decontamination personnel to
be
exposed to a biological waxfare agent.
[0106] A nanoemulsion can be used to decontaminate wounds contaminated with or
suspected to be contaminated with bacteria, bacterial spores, virus, fungus,
protazoa
or combinations thereof. In one embodiment the bacteria is B. a~thf~acis. In
another
embodiment, the virus is smallpox. In a further embodiment, the bacteria is a
Yersihia species..
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[0107] A nanoemulsion can also be used to decontaminate skin contaminated with
or suspected to be contaminated with bacteria, bacterial spores, virus,
fungus,
protazoa or combinations thereof. In one embodiment the bacteria is B.
ahth~acis. In
another embodiment, the virus is smallpox. In a fi~rther embodiment the
bacteria is
Ye~siv~ia species.
[0108] A nanoemulsion is also useful for prophylaxis treatment of skin against
bacteria, bacterial spores, virus, fungus, protazoa, or combinations thereof.
In one
embodiment the bacteria is B. anthracis. In a further embodiment, the
bacterial spore
is cutaneous B. ahthracis spore. In another embodiment, the virus is smallpox.
In a
further embodiment, the bacteria is Yersihia species.
[0109] A nanoemulsion is also useful for battlefield prophylaxis treatment of
mucosa against bacteria, bacterial spores, virus, fungus, protazoa or
combinations
thereof. In one embodiment the bacteria is B. anth~acis. In a further
embodiment, the
bacterial spore is B. anthracis spore. In another embodiment, the virus is
smallpox.
In a further embodiment, the bacteria is Ye~sihia species. In one embodiment,
nanoemuslions can be applied intranasally prior to and/or immediately after
suspected
contamination by bacteria, bacterial spores, virus, fungus, or combinations
thereof.
[0110] A nanoemulsion composition is also useful for decontamination of
surfaces
contaminated by or suspected to be contaminated by bacteria, bacterial spores,
virus,
fungus, protazoa or combinations thereof as the result of, for example, a
biological
warfare attack. In one embodiment the bacteria is B. a~th~acis. In a further
embodiment, the bacterial spore is B. ahth~°acis spore. In another
embodiment, the
virus is smallpox. In a further embodiment, the bacteria is Yef°sihia
species. In one
embodiment, a nanoemulsion composition can be applied prior to and/or
immediately
after suspected contamination by bacteria, bacterial spores, virus, fungus, or
combinations thereof. In a further embodiment, the nanoemulsion composition is
applied intranasally. Nanoemulsion can be applied to surfaces using any
appropriate
means. In one embodiment, a nanoemulsion is delivered as a spray, liquid, fog,
foam,
or aerosol to contaminated or suspected contaminated surfaces.
[0111] A nanoemulsion composition is also useful for decontamination buildings
contaminated by or suspected to be contaminated by bacteria, bacterial spores,
virus,
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fungus, or combinations thereof. In one embodiment the bacteria is B.
ahth~acis. In a
further embodiment, the bacterial spore is B. anthy~acis spore. In another
embodiment,
the virus is smallpox. In a further embodiment, the virus is Yersi~ia species.
In one
embodiment, nanoemuslions can be applied intranasally prior to and/or
immediately
after suspected contamination by bacteria, bacterial spores, virus, fungus, or
combinations thereof. Nanoemulsion can be applied to surfaces using any
appropriate
means. In one embodiment, a nanoemulsion is delivered as a spray, liquid, fog,
foam,
or aerosol to contaminated or suspected contaminated surfaces.
Example 1. Comparison of Standard Emulsions and Small pParticle Size
Nanoemulsions.
[0112] The nanoemulsions are described by the components of the nanoemulsion
according to Table 1. Unless otherwise noted, the oil is soybean oil. In the
formulations, the detergent is listed first, followed by the volume percentage
of the
detergent (e.g., W2o5 refers to 5 vol % of Tween 20). In the formulations, the
designation L2 refers to a small particle size nanoemulsion produced by a
microfluidizer, while the absence of the L2 designation refers to a standard
nanoemulsion (i.e., average particle sizes of 250 nm to about 1 micrometer).
The
designation L3 refers to nanoemulsions produced using an Avesting high
pressure
homogenizer.
Table 1
Component Symbol
Tween 20 wao
Ethanol E
Cetylpyridinium chloride C
EDTA ED
Triton X-100 X
Tributyl phosphate P
Glycerol G
Benzallconium chloride BA
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[0113] A first nanoemulsion is produced from a mixture containing 548
milliliters
of water, 2.24 grams of EDTA, 25 grams of cetylpyridiunium chloride, 125
milliliters
of Tween 20, 200 milliliters of ethanol and 1600 milliliters of soybean oil.
The first
nanoemulsion is pre-mixed with a Silverson L4RT mixer and a fine emulsifier
screen
for 10 minutes at 10,000500 revolutions per minute.
[0114] The first nanoemulsion is then processed in a Microfluidics M-11 OEH
microfluidizer processor using an H210Z (200 ~.m) chamber downstream of an
H230Z (400 ~.m) chamber. The first nanoemulsion is passed through the
microfluidizer 3 to 4 times at a pressure of 3,500500 pounds per square inch
(psi)
using cooling ice in the tray surrounding the chambers. The small particle
size
nanoemulsion produced is referred to as W2oEC ED L2.
[0115] The second nanoemulsion is then diluted with distilled water to produce
a
series of diluted nanoemulsions. The water and the nanoemulsion can be mixed
by
shaking, for example, until the nanoemulsion is incorporated into the water.
Exemplary diluted nanoemulsions are as shown in Table 2. The percentage shown
refers to the volume percentage of the nanoemulsion in the dilution.
Table 2
Formulation water W2o5EC ED
L2
50% W2o5EC ED 500 500 mL
L2 mL
20% WZOSEC ED 800 200 mL
L2 mL
10% W2o5EC ED 900 100 mL
L2 mL
5% W2o5EC ED 950 50 mL
L2 mL
2.5% W2o5EC 975 25 mL
ED L2 mL
Example 2. Method of Making a Small Particle Size Nanoemulsion
[0116] A standard nanoemulsion (i.e., particles sizes of 250 nm to 5
micrometers) is
fornzed as follows. A mixture of 22 vol% distilled water, 1 wt/vol%
cetylpyridinium
chloride, 5 vol% Tween 20, 64 vol% soybean oil, and 8 vol% ethanol based on
the
total volume of the mixture is formed. The nanoemulsion is formed by mixing
for 5
minutes at 10,000500 revolutions per minute with a Silverson L4RT mixer with a
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standard mixing assembly and a fine emulsion screen. The standard nanoemulsion
is
denoted as WZOSEC.
[0117] A small particle size nanoemulsion is formed by passing the WZOSEC
nanoemulsion 4 times through a Microfluidics M-1 lOEH rnicrofluidizer
processor
using an H21 OZ (200 ~,m) chamber downstream of an H23 OZ (400 ~,m) chamber.
The
small particle size nanoemulsion is denoted as WZOSEC L2.
[0118] After formation, the W2o5EC and W2o5EC L2 emulsions are diluted with
water for further testing. Particle sizes are determined by Particle Sizing
Systems
(PSS) Nicomp Model 380. The samples are diluted 1/2000 in distilled water to
measure the particle size. The formulations and data are shown in Table 3.
Table 3
FormulationFormulation Amount of Amount Average Particle
No. nanoemulsionof Size, mn
water
1 W2a5EC - - 421.4
2 50% WaoSEC 90 mL 90 mL 454
3 20% WaoSEC 36 mL 144 mL 437.5
4 10% WZOSEC 18 mL 162 mL 418.8
5% W~nSEC 9 mL 171 mL 427.4
6 2.5% W2o5EC 4.5 mL 175.5 mL 470.3
7 W2o5EC L2 - - 152
8 50% W2o5EC 90 mL 90 mL 99.3, 219.5*
L2
9 20% WZOSEC 36 mL 144 mL 144.2
L2
10% W2o5EC 18 mL 162 mL 153
L2
11 5% W2o5EC L2 9 mL 171 mL 177.8
12 2.5% W2o5EC 4.5 mL 175.5 mL 157.7
L2
fi wnen there is mde range of particle sizes (Nicomp reading), two methods of
calculation are used
[0119] As shown in Table 3, dilution of the emulsions does not appreciably
affect
the particle size of either the standard nanoemulsion or the small particle
size
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nanoemulsion. The average particle size for the W2o5EC emulsions is about 400
to
about 500 nm (samples 1-6) and for the WZOSEC L2 emulsions is about 140 to
about
220 mn (samples 7-12).
Example 3. Effect of Microfluidizer Chamber Size on the Size of Small Particle
Size Nanoemulsion Particles
[0120] A WaoSG BA2 nanoemulsion is passed through different combinations of
microfluidizer chambers as shown in Table 4. The WZOSG BA2 L2 small particle
size
nanoemulsion is made with 1 pass with a Silverson L4RT mixer and 4 passes
through
a microfluidizer. Combinations of chamber having 75, 200, 400 micrometer
microchannels are used to determine the relationship between the size of the
microchannels and the size of the particles produced.
Table 4
~~
Sample First chamber, Second chamber, Particle size,
~,m ~.m nm
1 75 100 174
2 100 75 165
75 200 185
4 200 75 180
75 400 211
6 400 75 199
[0121] As shown in Table 4, the chamber size utilized in the microfluidizer,
when
varied between 75 and 400 Vim, does not significantly affect the particle size
of the
emulsions. In all cases, the paxticle size is less than or equal to about 250
nm.
Example 4. Effect of Number of Passes Through the Microfluidizer on
Emulsion Particle Size
[0122] A W2o5G BA2 nanoemulsion is formed using either a Silverson L4RT mixer
(high shear) or a household hand mixer (low shear). The nanoemulsion is then
passed
through the microfluidizer for 1 to 6 passes and the particle size measured.
The
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relationship between the number of passes in the microfluidizer and the
particle size
of the emulsions are shown in Table 5 and Figure 5.
Table
5'
Sample Type of FirstNumber of PassesNanoemulsion Particle Size
Mixer Through (nm)
Microfluidizer(three independent experiments
with
different emulsion lots)
1 High shear 1 183, 221, 267
2 High shear 2 183, 205, 195
3 High shear 3 210, 202, 201
4 High shear 4 155, 156, 156
High shear 4 220, 157, 180
6 High shear 5 157, 132, 158
7 High shear 6 196, 161, 168
8 Low shear 0 426, 529, 522
9 Low shear 1 275, 210, 205
Low shear 2 218, 168, 218
11 Low shear 3 183, 151, 129
12 Low shear 4 182, 179, 180
[0123] As shown in Table 5 and Figure 5, the number of passes through the
microfluidizer does not have a large effect on the nanoemulsion particle size.
As
shown in Sample 4 and 5, 4 passes through the microfluidizer produces particle
sizes
consistently below 250 run. Regarding high shear versus low shear mixing of
the
starting emulsion, while high shear mixing can produce a more consistent
particle size
distribution than the low shear mixing, high shear mixing of the starting
emulsion is
not required to produce the small particle size nanoemulsions.
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Example 5. Combined Effects of Number of Passes through the Microfluidizer
and Microfluidizer Chamber Size
[0124] The effect of both the number of passes through the microfluidizer and
the
chamber size in the microfluidizer are studied for different formulations. The
starting
emulsions are prepared using either a Silverson L4RT mixer ("Silv") or a Ross
HSM-
410X high shear mixer with a 3 inch X-series rotor/stator pre-set to a 0.010
gap
(Ross) in order to determine the effect of mixing method on the paxticle size
of the
starting nanoemulsion (i,e., prior to passage through the microfluidizer). The
L2
emulsions are produced by passing a standard nanoemulsion produced by
Silverson
mixing through a microfluidizer. The particle sizes are shown in Table 6.
Table
6
Sample FormulationHigh shear InteractiveNumber Particle
Mixer type chamber of size,
used passages mn
1 Nanowash+ Silv - - 410-486
alcohol*
2 W2o5G BA2 Silv, 5 - - 304-371
minutes
mixing
3 W2o5G BA2 Silv, 20 - - 283-340
min
mixing
4 S8G Silv - - 350
W2o5EC Silv - - 3 81
6 W2o5G Silv - - 486
7 W2o5G BA2 Ross - 1 260
8 W2o5G BA2 Ross 2 247
9 W2o5G BA2 Ross 3 281
W2o5G BA2 Ross 4 229-254
11 W2o5G BA2 Microfluidizer400, 200 2 196
12 WaoSG BA2 Microfluidizer400, 200 3 195
13 W2o5G BA2 Microfluidizer200, 200 3 173
14 W2o5G BA2 Microfluidizer75, 200 3 210
W2o5G BA2 Microfluidizer75, 200 3 235
16 W2o5G BA2 Microfluidizer200, 400 3 179
then
diluted
using
75,
200
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17 S8G** Microfluidizer75, 200 3 161
18 W2o5EC Microfluidizer75, 200 3 178
19 W2o5EC Microfluidizer75, 200 3 158
20 WaoSG Microfluidizer75, 200 3 223
21 W~oSGC*** Microfluidizer400, 200 3 189, 200,
225,
226
22 XBGC Microfluidizer400, 200 3 130, 145
23 X8E6G2* * Microfluidizer400, 200 3 249
* *
1 % W 205 U13A2 + 2mM EDTA + 20% ethanol
** 8% SDS, 6% glycerol, 64% soybean oil, 20% water
* * * 5% Tween 20, 8% glycerol, 1 % cetylpyridinium chloride, 64% soybean oil,
22% water
* * * * 8% Triton X 100, 6% ethanol, 2% glycerol, 64% soybean oil, 20% water
[0125] As shown in Table 6, the Silverson high shear mixer (samples 1-6)
produces
particle sizes of about 300 nm to about 500 nm. The Ross high shear mixer
(Samples
7-10), produces particle sizes of 260 nm after 1 pass to about 229 to 254 nm
after 4
passes. The Ross high shear mixer is thus capable of producing smaller
particle sizes
than the Silverson mixer. Also shown in Table 6 is that the samples passed
through
the microfluidizer (samples 11-23) have smaller particle sizes than the
samples mixed
with either high shear mixer (samples 1-10).
[0126] Regaxding the samples passed through the microfluidizer, as shown in
samples 1 l and 12, similar particle sizes are obtained with either 2 or 3
passes through
the microfluidizer. Samples 13-16 show that changing the microchannel size of
the
microfluidizer chamber does not decrease the particle size of the emulsions.
Samples
17-23 illustrate that, independent of the formulation of the emulsions,
emulsions
having particle sizes of less than about 250 nm can be formed by passing the
emulsions through a microfluidizer.
Example 6: Particle Sizes and Zeta Potentials for Different Nanoemulsion
Formulation
(0127] In this experiment, the particle sizes and zeta potentials for
different small
particle size nanoemulsion formulations axe determined. The emulsions axe
formed
by passing a starting nanoemulsion through the microfluidizer for 3 passes
using the
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H230Z + H210Z chambers. The particle size and zeta potential are measured by
Nicomp 380 Particle sizer. The data are shown in Table 7.
Table
7
Sample Formulation ~ Particle SizeZeta
(mV)
1 1% W2o5G BA2 L2 + 2mM EDTA 186 11
2 WaoSG BA2 L2 in water 183 27
3 W2o5GC L2 168-236 30-33
4 WZOSG SA2 OA2 L2* 226 33
W2o5E SA3 L2 154 31
6 W2o5E SA3 L2 + 2 mM EDTA 131 12
7 WZOSG SA3 L2** 215 32
8 WaoSG SA3 L2 + 2 mM EDTA 187, 191 12
9 W2o5E L2 189 -25
WZOSEC L2, premixed 156, 182 31
11 W2o5EC L2 146 41
* 5% Tween 20, 8% glycerol, 2%sterylamine, 2% oleyl alcohol, 61 % soybean
oil, 21 % water
** 5%Tween 20, 8% glycerol, 3% Sterylamine, 61% Soybean oil, 23%
water
[0128] As shown in Table 7, all of the formulations have particle sizes of
less than
or equal to about 250 nm.
Example 7. Stability of Nanoemulsions
[0129] A WaoSEC nanoemulsion was formed containing 5% Tween-20, 8% ethanol,
1% cetylpyridinium chloride, 64% soybean oil, and the balance water. A W2o5EC
L2
nanoemulsion is formed using 2 passes on a microfluidizer. A WZOSGC
nanoemulsion is formed containing 5% Tween-20, 8% glycerol, 1 %
cetylpyridinium
chloride, 64% soybean oil, and the balance water. A WZOSGC L2 nanoemulsion is
formed using 2 passes on a microfluidizer. An X8P nanoemulsion is formed using
8% Triton X-100, 8% tributyl phosphate, and the balance water.
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[0130] Stability is determined by evaluating the physical appearance of the
emulsions. As used herein, creaming is the presence of a white layer of creamy
material on top of the nanoemulsion that is more opaque than the rest of the
nanoemulsion. Settling is a gradual decrease in opacity of the nanoemulsion
from top
to bottom due to separation of the more dense diluent (water) at the bottom
from the
less dense nanoemulsion at the top. The water appears as transparent layer at
the
bottom of the vial. Settling is classified as follows: Mild settling: the
nanoemulsion
appears cloudy with a gradient of "cloudiness" where it gets more opaque as
you go
upwards. Moderate settling: a partially clear aqueous solution appears on the
bottom
of the sample. The rest of the nanoemulsion appears cloudy with a gradient of
cloudiness getting more opaque as you go up. Some creaming may be on the
surface.
Severe settling: nanoemulsion has the appearance of three distinct layers, a
partially
clear bottom, cloudy middle, and creamy top. Extreme settling: only two
layers, a
thick partially clear bottom and a thin creamy top.
[0131] Separation is the phase separation of the nanoemulsion ingredients.
Separation is classified as follows: Mild separation: the surface of the
nanoemulsion
shows few visible oil droplets. Moderate separation: the surface of the
nanoemulsion
has a film of oil. The bottom of the nanoemulsion may have a clear aqueous
layer.
Severe separation: nanoemulsion has the appearance of three distinct layers, a
clear
aqueous layer on the bottom, a white or cloudy middle layer and a dense oily
layer on
the top. Extreme separation: total separation into an oil layer on top and
water on
bottom.
[0132] The ambient storage stability test includes storing the neat emulsions
in
polypropylene bottles or centrifuge tubes at room temperature (22-
25°C). Containers
may be mixed or opened during the observation period. The emulsions are
observed
for separation or any other changes in appearance. The observation period is
varied
due to different manufacturing dates of the emulsions. The data for W2o5EC
emulsions are shown in Table 8.
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Table 8
Sample Days Bottle Type of
in
Appe~.~ce
stora fullness container
a
1 severe separation
93%:
579 1/4 125m1 PP <7% nanoernulsion
between oil ~ water
2 619 1/4 125m1 PP extreme separation
3 505 2/3 250m1 PP moderate separation-
6%
oil
4 585 2/3 250m1 PP moderate separation-
8%
oil
457 2/3 250m1 PP mild separation-
1% oil
497 2/3 250m1 PP moderate separation-
1.5%
oil
7 184 full 125m1 PP mild-oil drop in
air space
8 224 full 125m1 PP mild-oil drop in
air space
184 3/4 125m1 PP mild separation-
1 % oil
film
224 3/4 125m1 PP moderate separation-
2%
oil film
11 184 2/3 125m1 PP mild separation-
4% oil
12 224 2/3 125m1 PP moderate separation-
6%
oil
13 112 1/4 SOOmI PP intact
14 152 1/4 SOOmI PP moderate separation-
3%
oil
33 full 30m1 PP intact
16 74 full 30m1 PP mild separation-
1 of 4
vials with oil film
17 74 '/2 250m1 PP mild separation
PP= polypropylene
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[0133] The data for WaoSEC L2 emulsions are shown in Table 9.
Table 9
Sample Days Bottle Type of
in
Appearance
storage fullness container
18 116 full 30m1 PP intact
...__............__...._...._...._..._.............._..__._._.___............._
._..._._____....._._.__._.._....._...._..._...._..............___....._....._..
.._._.__..___..........__._._....._.._..._.._...._._..._.._.___..__......._....
...__..
19 157 full 30m1 PP intact
20 74 1/4 60m1 PP intact
.._........._........._.........._....._..__.._.........._..__.._......___.....
._._....__.._._.._..._...._._....._......__..._______....................__....
............................_......_..._........._._......................_....
..........._...._...._.._........_._..._.._._..
21 115 1/4 60m1 PP intact
22 75 full SOOmI PP intact
_......__......._......._._._.....___.....________..........._.._.____......_..
..._......__.___..._....._._....__.__......................_....._............_
..._..._......_...._........_..........._.__......_..._..__....._......_..._._.
..._......._......._.........
23 115 full SOOmI PP intact
24 33 full 30m1 PP intact
_........_........................_...................___..........._..........
......._._.._................._._...._......_......._...__.....................
.........._......___.........._......_._...._...........................___....
......_.._....._...._..._....._..........._.._._...._....._...._............
25 74 full 30m1 PP intact
[0134] As shown in Tables 8 and 9, the small particle size nanoemulsions are
more
stable at room temperature than comparable standard emulsions. Batches of
standard
WaoSEC neat nanoemulsion stored at ambient temperatures longer than 5 months
show oil forming a film or layer on the surface of the nanoemulsion. The
thiclmess of
the oil layer is variable and may be related in part to the amount of air in
the storage
container in addition to the number of times the container has been entered.
[0135] Batches of smaller particle size W205EC L2 neat nanoemulsion are stored
at
ambient temperatures for up to 4 months. No settling or separation is observed
in
these batches.
[0136] Accelerated stability testing is also performed as follows. Glass vials
are
filled with 20 milliliters of neat, 10% diluted and 2.5% diluted nanoemulsion.
The
emulsions are stored at 55 °C and observed 3 times a week for changes
in physical
appearance. One additional set of vials for the WZOSEC L2 emulsions is filled
completely (about 25 milliliters) to eliminate air during storage. These full
vials are
inverted at day 7 to facilitate observation of creaming and separation.
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(0137] Neat emulsions (100%) of standard W2o5EC and small particle size
nanoemulsion WaoSEC L2 under accelerated stability testing at 55°C show
a film of
oil separating after 4 and 5 days, respectively (Fig. l and Table 10).
Table 10
verage verage
Days Days
to Mild to
or Severe
or
oderate Extreme
Separation Separation
anoemulsion eat 10% 2.50% eat 10% 2.50%
8P 3 10
W2o5EC .3
WaoSEC L2 5.3
W2o5EC L2 full*
WZOSGC 5.7
W2o5GC L2 8.7
1~I= No separation
[0138] For comparison, the X8P neat nanoemulsion shows signs of instability
with a
distinct clear aqueous layer on the bottom and a 5% oil layer on the surface.
Neat
emulsions of both WaoSGC and W~oSGC L2 show yellowing of the oil film on the
surface of the nanoemulsion, whereas for W2o5EC and W2o5EC L2, the oil film is
colorless. The neat small particle size nanoemulsions are stable for 1-3 days
longer
than the standard emulsions.
[0139] No diluted nanoemulsion (10% or 2.5%) shows separation of oil after 4
weeks observation at 55°C (Table 10).
[0140] Table 11 shows the settling observed for the nanoemulsions after
accelerated
aging.
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able 11
verage verage
Days Days
to to Severe
Mild or
or
oderate xtreme
Settling Settling
anoemulsion eat 10% 2.50% eat 10% 2.50%
8P 3 3 10 10
W2o5EC 3 3 19 10
W2o5EC L2 10.6 5
W2o5EC L2 fixll* 5
WaoSGC 5 3 26 19
WaoSGC L2 10 3
[0141] On average, the small particle size nanoemulsions exhibit less oil
separation
and less separation of the oil and water layers than the standard emulsions
(Table 10).
The small particle size nanoemulsions exhibit comparable settling and creaming
to the
standard emulsions when undiluted and improved stability when diluted to 10%
or
2.5% (Table 11).
[0142] Settling and creaming are more pronounced in the diluted large particle
size
emulsions compared to the diluted emulsions stored at 55°C (Figs. 2-3,
Table 12).
The 10% W2o5EC nanoemulsion is ~3% settled after 4 weeks, whereas the 10%
WaoSEC L2 nanoemulsion is only 9% settled. The onset of settling occurred
later in
the smaller particle size nanoemulsion, within 10 days for 10% W2o5EC L2
compared
to only 3 days for 10% WZOSEC. Table 12 shows the creaming and settling of the
emulsions.
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[0143] Table 12 shows the separation and settling of emulsions under
accelerated
aging conditions
Table 12
eat ~10% 2.50%
anoemulsion Oil Water Cream SettlingCreamSettling
8P 9 17 13 86 6 94
WaoSEC 2 0 14 83 5 94
WaoSEC L2 3 0 9 42
W2o5EC L2 full*0 0 2 <14 2 28
W2o5GC 0.3 0 13 77 5 93
*
*
W2o5GC L2 0.7** 0 0 11 41
[0144] The W2o SEC L2 nanoemulsion that is stored in vials that are completely
full
show no separation and less settling compared to the same nanoemulsion stored
in
vials containing an air space (Table 12). Interestingly, the bottom breaks off
at the
seam at day 10 and day 21 for 2 of the full vials of diluted nanoemulsion.
[0145] The change in pH after accelerated stability testing is measured. The
pH of
each nanoemulsion is measured at the beginning and at the end of the
accelerated
stability incubation at 55 °C. Diluted emulsions are measured using a 3-
in-1
combination electrode and neat emulsions axe measured with a semi-micro
electrode.
The initial pH of the neat W2o5EC, and W2o5EC L2, WZOSGC, and W2o5GC L2
emulsions is similar for each nanoemulsion, ranging from 4.2-4.4. The pH
increases
with increasing dilution of these nanoemulsion to a pH of 5.6 for the 2.5 %
dilutions.
After 4 weeks at 55 °C, the pH of the neat emulsions remains unchanged,
whereas the
pH of the diluted emulsions decreases to a value similar to that of the neat
nanoemulsion, (4.0 - 4.4). In contrast, W2o5EC L2 incubated in vials that are
filled
completely, slightly increased in pH after 4 weelcs incubation at 55
°C. The difference
between the neat and diluted nanoemulsion is also maintained (Figure 4).
[0146] Additional stress testing is preformed by centrifugation, freezing and
autoclaving. In the centrifugation test, neat (100%) and a 10% dilution of
WaoSEC L2
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nanoemulsion are centrifuged at 1,650xg for 30 minutes at room temperature,
then
stored at room temperature for observation. An additional sample of the 10%
dilution
of W2o5EC L2 is not centrifuged and is stored at room temperature for
comparison.
After storage at room temperature for 6 weeks, no separation of neat or
diluted
emulsions is observed. Only slight creaming is seen in the 10% diluted
emulsions
with no difference between the centrifuged and uncentrifuged sample.
[0147] In the freezing test at-18°C neat nanoemulsion and a 10%
dilution of
WaoSEC L2 are placed at-18°C for 24 hours, and then left at room
temperature for
observation. The neat nanoemulsion WZOSEC L2 is frozen at-18°C for 24
hours then
thawed and observed. After 24 hours at room temperature no separation is
observed
in the neat or 10% diluted nanoemulsion. Creaming is observed in the 10%
diluted
nanoemulsion and no settling were noted.
[0148] In the autoclaving test neat W2o5EC, W2o5EC L2, W2o5GC, and W2o5GC L2
emulsions are placed in a Yamato autoclave for 15 minutes at 121°C, and
then stored
at room temperature for observation. Both emulsions containing ethanol (W2o5EC
and W2o5EC L2) boiled over in the autoclave and severe separation is observed
immediately after autoclaving. The emulsions containing glycerol axe intact
after
autoclaving and displayed no separation up to 3 days when stored at room
temperature.
Example 8 Manufacture of Small Particle Size Nanoemulsions Using a High
Pressure Homogenizer
[0149] This example demonstrates using a high pressure homogenizer (Avestin
Emulsiflex C3) to reduce the particle size of a standard nanoemulsion to
particles
having a diameter of 50-150 nm. The size of the nanoemulsion particles depends
on
the pressure and number of passages.
[0150] First, a standard nanoemulsion containing particles having an average
diameter of 250 nm to 5 micrometers, preferably about 300 nanometer to 1
micrometer is formed. The standard nanoemulsion contains 22 vol % distilled
water,
1 wt/vol % cetylpyridinium chloride, 5 vol % Tween 20, 64 vol % soybean oil, 8
vol
ethanol and 2mM EDTA, based on the total volume of the mixture formed. The
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nanoemulsion is formed by mixing for 5 minutes at 10,0001500 revolutions per
minute with a Silverson L4RT mixer with a standard mixing assembly and a fine
emulsion screen. The standard nanoemulsion is denoted as WZOSEC ED.
[0151] Small particle size nanoemulsions containing particles of various sizes
are
then formed by passing the standard nanoemulsion through an Avestin EmulsiFlex
under different pressures ranging from 3,500 -17,000 psi. The nanoemulsion was
passed between 4-5 times under the same conditions. The machine applies high
pressure to push the nanoemulsion through a dynamic homogenizing valve. Table
13
describes the different nanoemulsion particle size resulting from different
passages
into the emulsifier.
Table 13
Name Passages in Pressure (psi) Particle size
the (nm)
high pressure
emulsifier
WaoSEC ED None -- 277
WZOSEC ED L3 1 17,000 111
WaoSEC ED L3 2 17,000 92
W~oSEC ED L3 3 17,000 91
WzoSEC ED L3 4 17,000 65
W~oSEC ED L3 1 3,500 164
WzoSEC ED L3 2 3,500 123
WaoSEC ED L3 3 3,500 110
WaoSEC ED L3 4 3,500 124
W2o5EC ED L3 5 3,500 130
[0152] Table 13 and Figure # demonstrate that particle size is inversely
dependent
on the amount of pressure applied during homogenization as well as the number
of
passages to which the nanoemulsion is subjected.
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Example 9. Testing of Disinfectants Containing the Nanoemulsions.
[0153] Example 9 compares the efficacy of a standard nanoernulsion versus a
small
particle size nanoemulsion (denoted L2) as a disinfectant.
[0154] The AOAC (Association of Official Analytical Chemist) dilution test is
a
carrier-based test. Carriers (i. e., stainless steel cylinders) are inoculated
with a test
microorganism, dried, exposed to a dilution of a disinfectant product, and
cultured to
assess the survival of the bacteria. A single test involves the evaluation of
60
inoculated carriers contaminated with one microorganism against one product
sample.
In addition to the 60 carriers, 6 carriers are required to estimate carrier
bacterial load
and 6 more are included as extras. Thus, a total of 72 seeded carriers are
required to
perform a single test.
[0155] A contaminated dried cylinder carrier is added to the medication tubes.
Immediately after placing carrier in medication tube, tubes are swirled 3
times before
placing tube into bath. Ten minutes after each carrier is deposited into the
disinfectant, each carrier is removed from the medication tube with a sterile
hook,
tapped against the interior sides of the tube to remove the excess
disinfectant, and
transferred into the primary subculture tube containing the appropriate
neutralizer
(Letheen broth, 10 mL in 20 x 150 mm tubes). The subculture tubes are swirled
for 3-
4 seconds. Transfer into the primary subculture tubes should be within ~ 5
seconds of
the actual time of transfer (10 minutes). The bacterial carrier load on at
least 2
carriers is assayed.
[0156] After a minimum of 30 minutes from when the test carrier was deposited,
each carrier is transferred using a sterile wire hook to a second subculture
tube
containing 10 mL of the appropriate neutralizer. The carriers are transferred
in order,
but the intervals do not have to be timed. The tubes are swirled for 3-4
seconds and
the subcultures incubated at 37 °C for 48 hours. If the broth culture
appears turbid,
the result is positive. A negative result is one in which the broth appears
clear. Each
tube is shaken prior to recording results to determine the presence or absence
of
turbidity. The primary and secondary subculture tubes for each carrier
represent a
"carrier set." A positive result in either the primary or secondary subculture
tube is
considered a positive result for a carrier set.
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[0157] Gram stains are performed on smears taken from the positive culture
tubes.
For additional confirmatory tests, a loop of broth is streaked on the
selective media
appropriate for the test microorganism and incubated for 24 hours at 37
°C.
[0158] Table 14. Gram staining and culture on selective media required to
ensure
the identity of the microorganism.
Table 14
_
S choleraesuis S au~eus P, aerugi~cosa
Gram stain Gram negative Gram positiveGram negative
rods
cocci arrangedrods
in clusters
Selective mediaMacConkey agar Mannitol saltPseudosel
agar
agar
Morphology Pale large colonies,Circular, Circular,
on small, small,
selective mediaagar turning fluorescent initially
light opaque,
color, yellow colonies.turning
fluorescent
green
over time.
Re ular media TSA* TSA TSA
*'1'ryptic soy agar
[0159] Table 15 show the results for a W2o5G BA2 + 2mM EDTA at pH 7.2
nanoemulsion and a W2o5G BA2 L2+ 2mM EDTA at pH 7.2 nanoemulsion with
Staphylococcus aureus.
Table
15
SampleFormulation CarriersTotal Number of Percentage
failed testedexperimentsfailed
1 1% WZOSG BA2 + 16 304 6 5.26%
2 mM EDTA
2 1% W2o5G BA2 L2 2 240 4 0.83%
+
2 mM EDTA
3 1% W2o5G BA2 L2 1 300 6 0.33%
[U16UJ As shown in 'fable 15, a disinfectant made with the small particle size
nanoemulsions has a lower failure ratio than a standard nanoemulsion. The
standard
nanoemulsion has a failure rate of about 5%. The small particle size
nanoemulsions
have a failure rate of less than 1 %.
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[0161] Table 16 also shows results obtained for various formulations exposed
to
Staphylococcus auf~eus.
Table
16
SampleFormulation Number of No. of Percentage
ExperimentsFailed failed
Cylinders
1 1% WZOSG BA2 + 2mM 6 304 5.3%
EDTA pH 7.2
2 1% WZOSG BA2 + 2mM 9 272 11.4%
EDTA pH 8.0
3 1% WaoSG BA2 L2+ 4 240 0.83%
2mM EDTA pH 7.2
4 1% W2o5G BA2 pH 6 300 0.33%
7.2
[0162] Table 16 demonstrates that the small particle size nanoemulsions
(Samples 3
and 4) show greater efficacy against Staphylococcus aureus than the standard
emulsions (Samples 1 and 2).
[0163] Table 17 shows the results obtained for various formulations exposed to
Salmonella choleraesuis.
Table
17
SampleFormulation Number of No. of Percentage
ExperimentsCylindersfailed
tested
1 1% WZOSG BA2 + 2mM EDTA2 120 0%
pH 7.2
2 1% W2o5G BA2 + 2mM EDTA1 30 0%
pH 8.0
3 1% WZOSG BA2 L2+ 2mM 1 60 0%
EDTA pH 7.2
4 1% W2o5G BA2 (LZ) pH 60 240 0%
7.2
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[0164] Table 17 demonstrates that the small particle size nanoemulsions
(Samples 3
and 4) show similar efficacy against Salmonella cholef°aesuis compared
to the
standard emulsions (Samples l and 2). Overall in the disinfectant test, the
small
particle size nanoemulsions perform as well as or better than the standard
emulsions.
Example 10: Bactericidal Properties of the Nanoemulsions Against
Staphylococcus aureus
[0165] The bactericidal activity of the nanoemulsions is tested using a tube
rotation
test. In this test, first a culture is prepared by picking one colony from the
stock
culture plate of Staphylococcus au~eus, streaking fresh TSA and incubating
overnight
at 37 °C. The next morning, one colony is picked from the agar plate
and transferred
into 25 mL of TSB in a 50 mL screw-cap tube and incubated at 37 °C on a
tube
rotator for 4-5 hours until the culture becomes turbid. Bacteria grown for 4-6
hours is
added to 10 mL TSB until the culture media becomes slightly turbid.
[0166] W2o5EC and W2o5EC L2 are used as previously described. The emulsions
are then diluted to 2%, 1 %, 0.2%, 0.1 %, and 0.02% by volume with water.
[0167] Bactericidal testing is performed as follows. In 1.7 mL microfuge
tubes, 0.5
mL cell suspension and 0.5 mL of each of the nanoemulsion dilutions is mixed
and
the tubes capped. A positive control containing 0.5 mL of cell suspension and
0.5 mL
of sterile distilled water is prepared in parallel. The tubes are incubated on
a tube
rotator at 37 °C for 10 minutes. Each of the preparations is serially
diluted (5 log
diluation) in a 96-well plate using PBS. 25~.L from each dilution on is
incubated on
TSA at 37 °C overnight. The colonies on the control and test plates are
counted. The
count on the control plate provides the initial bacterial count. The initial
bacteria
count is provided as:
Initial bacterial count = CFU x 40 x plate dilution
where CFU is the colony forming units per mL. The colonies on each of the test
plates is counted. Plates having between 20 - 50 CFU are counted. The report
log
reduction is provided as:
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Report Log reduction = Log (count on the control treatment) - Log (count on
the treatment).
[0168] The results are shown in Table 18.
Table
18
W2o5EC
Zero Control1% 0.5% 0.1% 0.05% 0.01%
Log 5 5 1 1 3 5 5
Count 193, 215, 0 0 52,77 261, 225,
201 150 236 237
Kill 7.36 100.00 100.0099.67 -26.14 -17.26
Log R. 0.03 6.29 6.29 2.48 -0.10 -0.07
WZOSEC
L2
Zero Control1% 0.5% 0.1% 0.05% 0.01%
Log 5 5 1 1 4 5 5
Count 146, 167, 0, 0 0, 289, 196, 149,
129 184 0 246 206 170
Kill -27.64 100.00 100.0080.55 -46.18 -16.00
Log R. -0.11 6.14 6.14 0.71 -0.16 -0.06
Note: a (-) log killing is considered zero.
[0169] As shown in Table 19, at 0.01% and 0.05% dilution, neither the standard
nanoemulsion nor the small particle size nanoemulsion has a significant effect
on the
viability of the S. au~eus. The 1%, and 0.5% dilutions, however, have similar
effects
on S. au~eus viability, with 100% lcilling at 1% and 0.5% for both particle
sizes. The
0.1 % dilutions show slightly better killing in the nanoemulsion compared with
the
small particle size nanoemulsion.
[0170] Small particle size nanoemulsions have several advantages over standard
emulsions. First, the small particle size nanoemulsions can be more stable
than the
standard emulsions when stored at room temperature or at 55 °C. The
small particle
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size nanoemulsions are capable of resisting separation or settling when stored
at room
temperature for four months. The undiluted small particle size nanoemulsions
can
take about 1 to 3 days longer to exhibit moderate separation than the standard
emulsions. The 2.5 % to 10 % diluted small particle size nanoemulsions can
take
about 2 to 7 days longer to exhibit moderate to extreme settling than the
standard
emulsions. In addition, the onset of phase separation in the small particle
size
nanoemulsions at 55 °C is later than for the standard emulsions.
[0171] Second, the small particle size nanoemulsions perform equal to or
better than
standard emulsions in inactivating bacteria. In a disinfectant test, the small
particle
size nanoemulsions exhibit a less than 1 % failure rate against Staphylococcus
aureus
compared to greater than 5 % for a standard nanoemulsion. In the same test,
both the
and standard nanoemulsions have a 0 % failure rate against Salmonella
cholef~aesuis.
In a tube rotation test, the small particle size nanoemulsions have a slightly
improved
killing compared with the standard emulsions against Staphylococcus aureus
killing
activity.
Example 11: Bactericidal Properties of the Nanoemulsions Against
Mycobacteria fortuitum
[0172] The bactericidal activity of the XBPC nanoemulsion against Mycobacteria
fortuitum is tested using a tube rotation test as described in Example 10.
XBPC
contains 8% Triton X-100, 8% tri-n-butyl phosphate, 1% CPC, 64% soybean oil,
and
19% water.
[0173] Initial bacterial count and report log reduction are calculated as
described in
Example 10. Figure 6 shows the Log Reduction over time of M. fo~tuitunz
treated
with 10%, 1%, and 0.10 % XBPC at both room temperature and 37 °C.
[0174] The results demonstrate that 10 % XBPC produces a 3.5 log reduction in
M.
fortuitum at room temperature within 24 hours. XBPC shows more modest
bactericidal activity at 1 % and 0.10% at room temperature. The results also
demonstrate that at 37 °C 10% XBPC demonstrates the greatest
bactericidal activity
for the shorter time points (1 and 2 hours). However, at greater than or equal
to 3
hours, there is no difference in bactericidal activity between 10% and 1% XBPC
at 37
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°C. Moreover, XBPC continues to provide bactericidal activity M.
fo~tuiturrz against
for at least 48 hours at 37 °C.
***
[0175] While the invention is described with reference to exemplary
embodiments,
it will be understood by those skilled in the art that various changes may be
made and
equivalents may be substituted for elements thereof without departing from the
scope
of the invention. In addition, many modifications may be made to adapt a
particular
situation or material to the teachings of the invention without departing from
the
essential scope thereof. Therefore, it is intended that the invention not be
limited to
the particular embodiment disclosed as the best mode contemplated for carrying
out
this invention. All references and publications cited herein are incorporated
by
reference in their entireties.
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