STABLE BIOCIDAL DELIVERY SYSTEMS AND TREATMENT AGAINST BIOFOULING
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority benefit under 35 USC § 119 of
U.S. Provisional Patent Application Serial No. 61/297,026 filed January 21, 2010.
FIELD OF INVENTION
[0002] The field of the invention generally relates to biocidal delivery systems
for providing products or compounds, such as chemicals, to industrial systems. The
invention also relates to compositions for use in a targeted delivery of said compositions
to bacterial bio-films in various environments.
BACKGROUND OF THE INVENTION
[0003] Bacterial bio-films exist in natural, medical, and industrial environments.
The bio-films offer a selective advantage to microorganisms to ensure the
microorganisms' survival or to allow them a certain time to exist in a dormant state
until suitable growth conditions arise. Unfortunately, this selective advantage poses
serious threats to health, or to the efficiency and lifetime of industrial systems. The
bio-films must be minimized or destroyed to improve the efficiency of industrial
systems, or remove the potential health threats.
[0004] Many industrial or commercial operations rely on large quantities of
water for various reasons, such as for cooling systems, or said systems may produce
large quantities of wastewater, which result in the creation of bio-films that need to be
treated. These industries include, but are not limited to, agriculture, petroleum, oil
drilling, oil pipelines, oil storage, gas drilling, gas pipelines, gas storage, chemical,
pharmaceutical, mining, metal plating, textile, papermaking, brewing, food and
beverage processing, and semiconductor industries. In these operations, naturally
occurring bio-films are continuously produced and often accumulate on numerous
structural or equipment surfaces or on natural or biological surfaces. In industrial
settings, the presence of these bio-films causes a decrease in the efficiency of industrial
machinery, requires increased maintenance and presents potential health hazards. An
example is the surfaces of water cooling towers which become increasingly coated with
bio-film slimes produced by a wide variety of microorganisms which constrict water
flow and reduce heat exchange capacity. Specifically, in flowing or stagnant water,
bio-films can cause serious problems, including pipeline blockages and the corrosion of
equipment by the growth of micro-organisms and microbes the thrive beneath the biofilm
as well as the growth of potentially harmful pathogenic bacteria. Water cooling
tower bio-films may form a harbor or reservoir that perpetuates growth of pathogenic
microorganisms such as Legionella pneumophila.
[0005] Another example of industrial systems are those systems that are found
in the food and beverage industries. Food preparation lines are routinely plagued by
bio-film build-up both on the machinery and on the food product where bio-films often
include potential pathogens. Industrial bio-films, such as those found in the food
industry, are complex assemblages of insoluble polysaccharide-rich biopolymers, which
are produced and elaborated by surface dwelling microorganisms. More particularly,
bio-films or microbial slimes are composed of polysaccharides, proteins and
lipopolysaccharides extruded from certain microbes that allow them to adhere to solid
surfaces in contact with water environments and form persistent colonies of sessile
bacteria that thrive within a protective film. The film may allow anaerobic species to
grow, producing acidic or corrosive conditions. To control these problems, processes
and antimicrobial products are needed to control the formation and growth of bio-films.
Control of bio-films involves the prevention of microbial attachment and/or the removal
of existing bio-films from surfaces. While removal in many contexts is accomplished
by short cleansing treatments with highly caustic or oxidizing agents, the most
commonly used materials to control bio-films are biocides and dispersants.
[0006] In U.S. Patent 5,41 1,666 to Hollis et al., a method of removing a biofilm
or preventing buildup of a bio-film on a solid substrate is taught, that comprises a
combination of at least two biologically produced enzymes, such as an acidic or alkaline
protease and a glucoamylase or alpha amylase and at least one surfactant. U.S. Patent
6,759,040 to Manyak et al. teaches a method for preparing bio-film degrading, multiple
specificity, hydrolytic enzyme mixtures that are targeted to remove specific bio-films
while U.S. Patent 5,512,213 to Paterson et al. teaches a method for stabilizing an
aqueous solution containing an isothiazolin compound against chemical decomposition
through the incorporation of a stabilizing amount of a metal salt. The cation of said
metal salt is an alkali metal while the anion is selected from the group consisting of
acetate, citrate, phosphate and borate.
[0007] Finally, U.S. Patent 6,267,897 to Robertson et al., relates to a method of
inhibiting bio-film formation in commercial and industrial water systems by adding one
or more plant oils to the system. However, although the biocides are effective in
controlling dispersed microorganism suspensions, i . e . , planktonic microbes, biocides
do not work well against sessile microbes, the basis of bio-films. This is due to the fact
that biocides have difficulty penetrating the polysaccharide/protein slime layers
surrounding the microbial cells. Thicker bio-films see little penetration of biocides and
poor biocide efficacy is the result. One known method of trying to better control biofilms
has been the addition of dispersants and wetting agents to biocide compositions to
enhance biocide efficacy. Bio-dispersants may operate to keep planktonic microbes
sufficiently dispersed so that they do not agglomerate or achieve the local densities
necessary to initiate the extracellular processes responsible for anchoring to a surface,
or initiating film- or colony-forming mechanisms. As components in biocidal treatment
formulations, these bio-dispersants have helped in opening channels in the bio-film to
allow better permeability of the toxic agents and to better disperse the microbial
aggregates and clumps that have been weakened and released from the surfaces.
However, bio-dispersants have proven to be more effective in preventing initial bio-film
formation than in removing existing bio-films. In many cases, the activity of biodispersants
has been responsible for only 25 to 30% biomass removal from bio-fouled
surfaces, even when used in conjunction with a biocidal agent.
[0008] Therefore, a clear need still exists for an efficient and effective means
for delivering antimicrobial compounds that are better able to penetrate existing biofilms
and bio-film matrices, and more effective in killing microorganisms contained
within a bio-film matrix, thus killing and eliminating bio-film, as well as preventing
future formation nor buildup of bio-film, in systems, such as industrial systems.
Decreasing the fouling of microfiltration systems, and providing less frequent cleaning
and/or replacement which would enhance the overall filtration process, are also needs
which should be addressed.
SUMMARY OF THE INVENTION
[0009] In one exemplary embodiment, a biocidal-delivery system has been
found which increases the efficiency and effectiveness of introducing antimicrobial
compounds into complex bio-film matrices, through the use of liposome carriers, which
can be used in natural, medical and industrial applications. In industrial applications,
the delivery system can minimize or eliminate fouling in industrial systems, including,
but not limited to, aqueous systems, such as piping, heat exchangers, condensers,
filtration systems and media, and fluid storage tanks.
[0010] According to one embodiment of the invention, liposomes containing an
antimicrobial agent, such as a hydrophilic biocide, are added to a water system prone to
bio-fouling and bio-film formation. The liposomes, being similar in composition to the
outer surface of the microbial cell wall structure or to the material on which the
microbes feed, are readily incorporated into the microbes present in the existing biofilm.
Once the liposomes become entrained with the bio-film matrix, digestion,
decomposition or degradation of the liposome proceeds, releasing the antimicrobial
agent, or biocidal aqueous core reacts locally with the bio-film- encased
microorganisms. Upon the death of the organisms, the polysaccharide/protein matrix
cannot be replenished and decomposes and thereby results in reduced bio fouling of the
water bearing system. Depending on the particular system involved, this bio-film
removal or destruction therefore results in increased heat transfer (industrial heat
exchanger), increased flux (filter or filtration membrane), less deposit of colloidal and
particulate solids and dissolved organics on the surface of the microfiltration
membrane, thereby reducing the frequency and duration of the membrane cleaning and
ultimate replacement, or general reduction of corrosive surface conditions in pipelines,
tanks, vessels or other industrial equipment.
[0011] An alternate embodiment of the invention provides for a delivery system
of actives into a natural, medical or industrial system, which can be chosen from the
group consisting of anti-corrosion treatments, pesticides for agriculture and commercial
home uses, food additives and preservatives, chemical and biological detection, color
and flavor enhancement, odor control and aquatic pest management.
[0012] More specifically, the present invention is an improvement of the
delivery system described in the Published PCT Application WO 2009/020694 Al
wherein the liposome biocidal delivery system is formulated about a stabilized anti
microbial system comprised of a non-oxidizing biocide such as the group consisting of
the isothiazolins. It has been found that isothiazolins undergo chemical decomposition
in presence of high temperature, high pH, reducing agents, and aggressive
nucleophiles. When liposome is added to isothiazolin solutions, the reducing property
of lipids is detrimental to isothiazolin stability. The oxidizing properties and acidic salt
solution (pHl~3) of the isothiazolin anti-microbial compounds also causes liposome
degradation and eventually physical separation. At elevated temperature, these
degradation and separation processes accelerate resulting in unsatisfactory product not
suitable for commercial use. Individually, these biocides generally exhibit a greater
than 50% degradation after one week at 50 °C for both materials. One aspect of the
present invention then comprises the use of a stabilizing oxidizer composition such as
sodium chlorate, and more particularly, a stabilized blend of a buffer selected from the
group consisting of a citrate salt, a chlorate salt, an acetate salt and mixtures thereof.
Even more preferred are stabilizer compositions comprised of a sodium citrate buffer, a
sodium acetate buffer, a sodium chlorate buffer, a sodium citrate/sodium chlorate
buffer mixture, and a sodium acetate/sodium chlorate buffer mixture.
[0013] The various features of novelty that characterize the invention are
pointed out with particularity in the claims annexed to and forming a part of this
disclosure. For a better understanding of the invention, its operating advantages and
benefits obtained by its uses, reference is made to the accompanying drawings and
descriptive matter. Changes to and substitutions of the various components of the
invention can of course be made. The invention resides as well in sub-combinations
and sub-systems of the elements described, and in methods of using them.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Approximating language, as used herein throughout the specification and
claims, may be applied to modify any quantitative representation that could permissibly
vary without resulting in a change in the basic function to which it is related.
Accordingly, a value modified by a term or terms, such as "about", is not limited to the
precise value specified. In at least some instances, the approximating language may
correspond to the precision of an instrument for measuring the value. Range limitations
may be combined and/or interchanged, and such ranges are identified and include all
the sub-ranges included herein unless context or language indicates otherwise. Other
than in the operating examples or where otherwise indicated, all numbers or
expressions referring to quantities of ingredients, reaction conditions and the like, used
in the specification and the claims, are to be understood as modified in all instances by
the term "about".
[0015] As used herein, the terms "comprises," "comprising," "includes,"
"including," "has," "having" or any other variation thereof, are intended to cover a
non-exclusive inclusion. For example, a process, method, article or apparatus that
comprises a list of elements is not necessarily limited to only those elements, but may
include other elements not expressly listed or inherent to such process, method article
or apparatus.
[0016] A delivery system has been found which increases the efficiency and
effectiveness of introducing antimicrobial compounds into complex bio-film matrices
through the use of liposome carriers, which can be used in natural, medical and
industrial applications. In industrial applications, the delivery system can minimize or
eliminate fouling in industrial systems, including, but not limited to, aqueous systems,
such as cooling towers, piping, heat exchangers, condensers, filtration systems and
media, and fluid storage tanks.
[0017] According to one embodiment of the invention, liposomes containing a
biocidal or antimicrobial agent or compound are added to an industrial system prone to
bio-fouling and bio-film formation. The liposomes, being similar in composition to
microbial membranes or cells, are readily incorporated into the existing bio-film. Once
the antimicrobial compound-containing liposomes diffuse into, adsorb or otherwise
become entrained with the bio-film matrix, the microorganisms existing within the biofilm
matrix will ingest the liposome structure, resulting in the decomposition or
disintegration of the liposome inside the intracellular matrix of the microorganism,
thereby releasing the antimicrobial compound into the intracellular matrix of the
microorganism, ultimately resulting in the death of the microorganism. That is lipid
decomposition and biocide release can be programmed to occur by making the lipid
matrix sensitive to pH, redox potential, Ca + concentration, or other changes.
Thereafter the biocidal component that may be concentrated in the aqueous core of the
liposome or in the lipid membrane portion of the liposome, is released to react directly
with the bio-film-encased microorganisms. Thus, rather than adding a biocide at high
levels to the bulk water system, a small quantity of liposome-encased biocide is taken
up by the bio-film or by free (planktonic) organisms, and degradation of the liposome
releases the biocide locally in or at the target organisms or their film matrix niche. The
biocide thus attains a high concentration locally to kill the target organisms, and upon
the death of the organisms, the polysaccharide/protein matrix that forms the bio-film
cannot be maintained or regenerated and decomposes, and thereby results in reduced
fouling of the water bearing system, resulting in increased heat transfer, increased flux,
less deposit of colloidal and particulate solids and dissolved organics on the surface of
the micro-filtration membrane, thereby reducing the frequency and duration of the
membrane cleaning and ultimate replacement or other benefits.
[0018] Liposomes, or lipid bodies, are systems in which lipids are added to an
aqueous buffer to form vesicles, structures that enclose a volume. The liposomes may
be comprised of lipids selected from the group consisting of phospholipids, lecithin,
phosphatidyl choline, glycolipid, triglyceride, sterol, fatty acid, sphingolipid, or
combinations thereof.
[0019] More specifically, liposomes are microscopic vesicles, most commonly
composed of phospholipids and water. The liposomes may be made from phospholipids
derived from various sources, including, but not limited to soybeans and eggs. When
properly mixed, the phospholipids arrange themselves into a bi-layer or multi-layers,
very similar to a cell membrane, surrounding an aqueous volume core. Liposomes can
be produced to carry various compounds or chemicals within the aqueous core, or the
desired compounds can be formulated in a suitable carrier to enter the lipid layer(s).
Liposomes can be produced in various sizes and may be manufactured in submicron to
multiple micron diameters. The liposomes may be manufactured by several known
processes. Such processes include, but are not limited to, controlled evaporation,
extrusion, injection, micro-fluid processors and rotor-stator mixers. Liposomes can be
produced in diameters ranging from about 10 nanometers to greater than about 15
micrometers. When produced in sizes from about 100 nanometers to about 20
micrometer sizes the liposomes are very similar in size and composition to most
microbial cells. The biocide or antimicrobial compound containing-liposomes should
be produced in sizes that mimic bacterial cells, for example, from about 0.05 to about
15 m, or alternately, about 0. 1 to 10.0 m. Details pertaining to liposome production
processes may be gleaned, for example, in U.S. Patents 5,807,572 and 7,491,409.
Both of these patents are incorporated by reference herein.
[0020] In one embodiment, effective amounts of the biocide containing liposome
is introduced into an industrial system which is prone to bio-fouling and bio-film
formation, or can be introduced into systems that already exhibit signs of bio-fouling or
bio-film formation. The effective amount will vary according to the antimicrobial
compound or biocide, and the aqueous system to which it is added, but one embodiment
provides from about 0.01 ppm to about 100 ppm, with an alternative of from about
0.05 to about 50 ppm, alternately from about 0.05 to about 5.0. The liposomes, being
similar in composition to microbial membranes, or cell walls, are readily incorporated
into the existing bio-film and become entrained within the bio-film matrix. The
liposomes containing biocides have improved penetration of the bio-film matrix, due to
similarity in composition and structure with the bio-film. Once the liposome is
incorporated or entrained within the existing bio-film matrix, the liposome will begin to
disintegrate. Upon the decomposition or programmed disintegration of the liposome,
the biocidal compound contained within the aqueous core of the liposome is released to
react directly with the bio-film encased microorganisms, resulting in their demise.
Upon the death of the organisms, the polysaccharide/protein matrix will rapidly
decompose, freeing the surface from contaminating microbes.
[0021] More specifically, one aspect of the present invention is directed to a
liposomal-encapsulated biocidal delivery system wherein the non-oxidizing biocidal
compound is stabilized by a citrate/ chlorate buffer composition in which the mixture
buffer provides a stability to the biocide active that is much greater than either of the
buffers alone. A principal feature of one embodiment of the present invention is that
the liposomes constitute extremely small hydrophobic bodies that may readily survive in
and disperse in systems, such as for example, aqueous or natural systems, and yet will
adsorb to or penetrate a bio-film and preferentially target or be targeted by the microbes
that inhabit, constitute or sustain the bio-film. As such, the liposomes deliver a
biocidal agent directly to the microbes or bio-film, resulting in effective locally biocidal
level of activity, without requiring that the industrial system as a whole sustain a high
dose. Thus, where conventional bio-film treatment may require dosing with a bulk
biocidal chemical at a certain level, delivery via liposome may be dosed at levels an
order of magnitude or more lower in the aqueous system, yet still achieve, or build up
to a level that effectively controls or removes bio-film. This lower level of biocide
concentration has positive effects on the environment due to the efficacy resulting from
the delivery system. Additionally, depending upon the particular system that is being
treated, an embodiment provides for flexibility in where the liposomes are actually
delivered into the system. If there is one particular area in a system that is prone to
bio-film creation, the delivery of the liposomes may be delivered to that particular
portion or point of the system, such that the delivery of the biocidal delivery
composition is to a targeted location, and not necessarily privy to or exposed to the
entire system. As smaller doses of the liposome containing biocides are needed due to
the efficacy of the biocides in this format, an entire system or process need not be
flooded with or treated with biocides.
[0022] Indeed, while the terms "antimicrobial" or "biocide" or "biocidal" have
been employed to describe the agent carried by the liposome, these agents need not be
the highly bioactive materials normally understood by those terms, but may include a
number of relatively harmless materials that become highly effective simply by virtue
of their highly localized release. Thus, for example, surfactants or harmless
ammonium or phosphonium halide salts, when released locally, may affect the normal
action of extracellular colony-forming secretions, and are to be included as
antimicrobial or biocidal agents for purposes of the invention, and the same mechanism
may be employed to deliver other treatment chemicals to the targeted bio-film sites.
[0023] Aqueous systems that can be treated by this method include, but are not
limited to, potable and non-potable water distribution systems, cooling towers, boiler
systems, showers, aquaria, sprinklers, spas, cleaning baths, air washers, pasteurizers,
air conditioners, fluid transporting pipelines, storage tanks, ion exchange resins, food
and beverage processing lines, metalworking fluid baths, coal and mineral slurries,
metal leaching fluids, wastewater treatment facilities, mollusk control, pulp and
papermaking operations, acid mine drainage, or any application prone to bio-fouling by
microbial species. Application such as oil drilling, oil storage tanks or oil pipelines,
where bio-films form in stagnant or pooled aqueous sumps or lenses along the conduit
system, may also be effectively treated.
[0024] Additional applications for liposome delivery of a treatment chemical
comprise natural, medical and industrial systems, such as, but not limited to anticorrosion
treatments for equipment generally, delivery of hormone, vitamin or
antioxidant treatments or antibiotic and gene therapies for medical or veterinary
purposes, delivery of pesticides for agriculture and commercial home uses, effective
formulations of food additives and preservatives, targeted delivery for chemical and
biological detection systems, color and flavor enhancement, odor control, fungicides,
rodenticides, insecticides, mildew control and aquatic pest management.
[0025] Anti-microbial liposomes are systems in which lipids are added to an
aqueous anti-microbial compound solution to form vesicles, structures that enclose a
portion of the anti-microbial solution. Liposomes maybe consist of lipids selected from
the group consisting of phospholipids, lecithin, phosphatidyl choline, glycolipids,
triglycerides, sterol, fatty acid, sphingolipid, or combinations thereof.
[0026] As briefly mentioned above, it is well documented that isothiazolins
undergo chemical decomposition in presence of high temperature, high pH, reducing
agents, and aggressive nucleophiles. When liposome is added to isothiazolin solutions,
the reducing property of lipids is detrimental to isothiazolin stability. Moreover, the
oxidizing properties and acidic salt solution (pHl~3) of the isothiazolin anti-microbial
compounds also cause liposome degradation and eventually physical separation. At
elevated temperature, these degradation and separation processes accelerate resulting in
unsatisfactory product not suitable for commercial use.
[0027] One aspect of the present invention comprises the addition of a
combination of a citrate salt, acetate salt, or chlorate salt buffer composition to the
isothiazolin liposome composition to regulate pH and redox potential in the solution.
The result is a stabilized micro-biocidal composition that is resistant to chemical
decomposition and a homogenous liposome solution free of physical phase separation to
a degree that is surprisingly and unexpectedly enhanced over the inclusion of either
compound alone. Whereas theoretically any salt form may be used, the sodium salt
form is preferable for any one of a number of reasons.
[0028] In order to prepare the isothiazolin anti-microbial liposome, lipids are
added to an isothiazolin solution to form liposome vesicles, which encapsulate a portion
of the isothiazolin compounds dissolved in solution. Individually, isothiazolins and
liposomes are stable. Commercial isothiazolin products such as R&H Kathon®886F is
stabilized by magnesium nitrate. Commercial phospholipids and lecithin such as
Cargill Lecigran®6000G is stabilized by tocopherols. But when blended together,
isothiazolin compounds and liposome are incompatible. Magnesium nitrate and
tocopherols cannot provide sufficient stabilizing effect, resulting in chemical
degradation of 3-isothiazolin and irreversible phase separation of the liposome lipids
from the isothiazolin solution when the pH drops below 1. and temperature rises
above 35 °C. Additional stabilizers are needed to address these issues. In one
embodiment, the present invention employs citrate buffers and chlorate salts as
additional stabilizers to ensure product compatibility and extend shelf life. Suitable
stabilizer buffer systems include sodium citrate, sodium chlorate, sodium acetate and
mixtures thereof.
[0029] Various types of biocides, for example non-oxidizing biocides, can be
incorporated into the liposome and are effective. Preferably, the non-oxidizing biocide
useful in the practice of the present invention is an isothiazolin, most preferably, 3-
isothiazolin. These isothiazolin-3-one liposome formulations are more effective at
killing and removing bio-films when compared to the same isothiazolin-3-one
compounds at the same active concentrations, which are introduced into systems, but
not incorporated in liposomes, as the liposome containing biocides readily penetrate the
microbial bio-films and are highly effective at destroying the bio-film matrix. This
liposome delivery method may comprise 5-chloro-2-methyl-4-isothizolin-3-one and 2-
methyl-4-isothiazolin-3-one, but any substituted isothiazolin-3-one based biocide can be
made significantly more effective when delivered in a liposome biocidal delivery system
or composition.
[0030] An example of an isothiazolin-3-one compound is
lsothiazolin-3-one
Where:
R = H, CI, Br, I , CnH
X = H, CI, Br, I , CnH
Y = H, CI, Br, l , CnH
[0031] In preparing the biocidal liposomes of the present invention comprising
isothiazolin, the active isothiazolin compound is incorporated into the liposome in an
amount of from about 1.0 wt% to about 12.0 wt% and preferably in an amount of from
about 10.0 wt% to about 12.0 wt%. The amount of stabilizing buffer composition
added to the liposome formulation is from about 0.02 wt% to about 10.0% wt% and,
preferably, in an amount of from about 0.03 wt% to about 5.5 wt%. The liposome
formulations are usually mixtures of particles of various sizes. Whereas the liposome
particle sizes may be formulated up to 200 microns, preferably the liposome size useful
in the practice of the present invention will range from about 100 nanometers to about
10 microns in diameter.
[0032] Liposomes of the present invention may be created as multi-layer bodies,
in which one or more additional layers are provided to enhance the stability of the
liposomes or to effectuate a programmed release of the underlying lipid body and
contents. Thus, this technology may be used to encapsulate medicines for intracorporal
delivery, such that the additional layers may include a protective layer that is
hydrolyzed or otherwise breaks down over time to provide a sustained release or longer
lifetime of the underlying liposome. This additional layer may also include an
encapsulating polymer that selectively breaks down when the multi-layer liposome
encounters a low-pH environment, like the corrosive high acidity environment that may
develop beneath a bio-film.
[0033] A layer may also be compounded to be vulnerable to sulfur-fixing
bacteria, causing the liposome to specifically release its biocide in proximity to these
corrosive organisms often present in a waste or pipeline system. Furthermore, several
such layers may be employed to assure a sufficient lifetime of the liposome, preferably
on the order of several days as well as an ability to target a specific niche or
environment in the bio-film. This assures that the liposomes will effectively encounter
the target organisms or bio-film colonies and deliver their biocides thereto. The lipid
material itself may be treated to provide enhanced resistance to hydrolysis or decay, or
the added layers may be formed of various hardened or cross-linkable oils or polymers.
[0034] An alternate embodiment of the invention provides for a biocidal
delivery composition for delivering at least one antimicrobial composition into a biofilm
present in an industrial system, wherein the bio-film comprises at least one
microorganism species; b) the biocidal delivery composition comprises a liposome
structure containing at least one lipid or phospholipid type component; and c) the
liposome structure encapsulates at least one non-oxidizing antimicrobial composition in
combination with a stabilizer composition.
[0035] A further embodiment provides for the targeted delivery of biocide
actives into an industrial system, such as an industrial aqueous system, by introducing
into said system an effective amount of said biocides in a critical area of said system.
By targeting an area, and entry at a specific point in a process, the efficacy of the
liposome system provides for a noteworthy impact on the environment as well as the
cost of maintaining a system, as the entire system does not need to be flooded with
biocides, only the specific area of interest.
[0036] The present invention will now be more specifically described and
detailed in the following examples to better show one skilled in the art how to best
carry out and practice the metes and bounds of the present invention. It is to be
emphasized that they are for illustrative purposes only, and should not be construed as
limiting the spirit and scope of the invention as recited in the claims that follow.
EXAMPLE
[0037] Three batches of liposomes (150 nanometers average diameter) were
created that incorporated an isothiazolin biocide, KathonT ^available from Rohm &
Haas, Philadelphia, PA) as the active ingredient. The liposomes were then placed in
microtiter plates that had microbial bio-films coating them. The microbe inhibiting
efficacy of the isothiazolin liposomes was then compared with non-liposomal
isothiazolin biocide when used at the same isothiazolin concentrations. The liposomes
containing isothiazolin penetrated the bio-film and inhibited the bio-film organisms
much more effectively than the non-liposomal isothiazolin solution. The biocide -
containing liposomes were comprised of the following components in their respective
percent ranges.
Component Percentage (% by wt)
a) KATHON®886F (14.0% isothiazolin) 78.67
b) DEIONIZED WATER 0.67
c) LECIGRAN®6000G 10.0
d) SODIUM CHLORATE (50% sol.) 8.0
e) SODIUM CITRATE DIHYDRATE 2.33
f) CITRIC ACID MONOHYDRATE 0.33
[0038] The degradation of the 3-isothiazolin liposomes can be qualitatively
observed by the formation of an insoluble precipitate. Quantitatively, gas
chromatography (GC) and high pressure liquid chromatography (HPLC) analysis were
used to determine actives concentrations for samples stored under accelerated storage
conditions (50° C). The stability of isothiazolin liposomes known in the art are as
follows.
[0039] Various isothiazolin formulations with stabilizers were tested at 38 °C
and 50 °C. As can be seen from Table 1 below, the citrate buffer and chlorate salt
stabilizer combination extended shelf life from 29 days to 85 days during 38 °C/100 °F
storage, while either the citrate buffer or chlorate salt alone only extends shelf life to 49
and 42 days, respectively. Thirteen (13) stabilized anti-microbial liposomal
compositions were prepared in the following component ratios as set forth in Table 1.
The isothiazolin-encapsulated liposomes with the different buffer stabilizer compounds
incorporated therein were compared for stability over time at the four (4) different
temperatures and the days to irreversible separation are set forth below:
Table 1
[0040] It is evident from the table that the sodium citrate/sodium chlorate
buffer composition provided unexpectedly high levels of stability for the liposomebiocide
composition than either buffer added alone. Stability of the anti-microbial
liposomal compounds was the measured as a function of pH over time. Whereas the
liposomes containing the isothiazolin/sodium acetate buffer and the
isothiazolin/sodium citrate buffers alone showed good biocidal stability at 38 °C/100
°F for forty-two (42) and forty-nine (49) days respectively, liposomes containing the
isothiazolin biocide with combinations of the sodium acetate/sodium chlorate and
sodium citrate/sodium chlorate buffers exhibited surprisingly superior biocidal
stability at the same elevated temperatures for fifty-one (51) and eighty-five (85) days,
respectively.
[0041] In addition to the foregoing, the biocide may be any type of biocide that
is suitable for killing or destroying the desired microbial organism. In one
embodiment, the biocide may be a non-oxidizing or oxidizing compound, or
combinations thereof. In another embodiment, the biocide includes, but is not limited
to, guanidine or biguanidine salts, quaternary ammonium salts, phosphonium salts, 2-
bromo-2-nitropropane-l, 3-diol, 5-chloro-2-methyl-4-isothiazolin-3-one / 2-methyl-4-
isothiazolin-3-one, n-alkyl-dimethylbenzylammonium chloride, 2,2,dibromo-3-
nitrilopropionamidemethylene-bis(thiocyanate), dodecylguanidine hydrochloride,
glutaraldehyde, 2-(tert-butylamino)-4-chloro-6-(ethylamino)-s-triazine, betabromonitrostyrene,
tributyltinoxide, n-tributyltetradecyl phosphonium chloride,
tetrahydroxymethyl phosphonium chloride, 4,5,-dichloro-l,2,-dithiol-3-one, sodium
dimethyldithiocarbamate, disodium ethylenebisdithiocarbamate, Bis(trichloromethyl)
sulfone, 3,5-dimethyl-tetrahydr o-2H- 1,3,5, -thiadiazine-2-thione, 1,2,-
benzisothiazolin-3-one, decylthioethylamine hydrochloride, copper sulfate, silver
nitrate, bromochlorodimethylhydantom, sodium bromide, dichlorodimethylhydantom,
sodium hypochlorite, hydrogen peroxide, chlorine dioxide, sodium chlorite, bromine
chloride, peracetic acid and precursors, sodium trichloroisocyanurate, sodium
trichloroisocyanurate, dibromo, dicyano butane and combinations thereof.
[0042] In one embodiment, the biocide may be guanidine or biguanidine salts,
quaternary ammonium salts and phosphonium salts. Examples of guanidine or
biguanidine salts are of the general formulas:
wherein R, R1, R are independently H, C C2 substituted or non-substituted alkyl
(linear or branched) or aryl, X is an organic or inorganic acid, n is 0-20 and z is 1-12.
[0043] Examples of the general formula of acceptable phosphonium salts
comprises (R P j X wherein R is an alkyl group of from 1 to 8 carbon atoms, Ris
an n-alkyl group giving 8 to 20 carbon atoms, and X is an anion consisting of a
halide, sulfate, nitrate, nitrite, and combinations thereof.
[0044] An alternative formula provides that R is an alkyl group having from
1-8 carbons, R2 is an n-alkyl group having 6-20 carbon groups, and X is an anion
such as halides, sulfates, nitrates, nitrites and mixtures thereof. Preferably, X is
chloride, bromide, iodide, S0 4
= , and N0 3 , N0 2 or mixtures thereof.
[0045] Another embodiment provides and R2 are hydroxyalkyl groups
having from 1-4 carbons and X is an anion such as halides, sulfates, nitrates, nitrites
and mixtures thereof. Preferably, X is chloride, bromide, iodide, S0 4
= , and N0 3 ,
N0 2 or mixtures thereof.
[0046] Quaternary ammonium salts are another example of a biocide or agent
that may be encapsulated or manufactured into a liposome core, and are of the general
formula
3
+-CH —benzyl ring X .
wherein is an n-alkyl group of chain length C - C1 ; R2 and R3 are CH3 or n-alkyl
group of chain length C2- C and X is an anion such as halides, sulfates, nitrates,
nitrites and mixtures thereof.
[0047] The non-biocidal agents may be any type of environmentally friendly
compound or composition that removes or inactivates the protozoa to keep it from
spreading, such as by interfering with its life or reproductive cycle. In one
embodiment, the non-biocidal agent may be used as an adjuvant with a biocide. For
example, non-biocidal agents include, but are not limited to, biodispersants, ethylene
oxide/propylene oxide copolymers, trichlorohexanoic acid, polysiloxanes,
carbosilanes, polyethyleneimine, bacteria, microorganisms, plasmids, phagocytes,
macrophages, toxin-producing microorganisms, amino acids, proteins, peptides,
DNA, RNA, base pairs, antisense RNA pharmaceuticals, antibiotics, chelators,
natural extracts, organic/ inorganic redox agents, organic and inorganic dye
sensitizers, apoptosis signaling reagent, microorganism- and plant-derived extracts and
by-products, metabolic components, preservatives, toxic phytochemicals, microbial
toxins, catalysts that generate free radicals or active oxygen species, L-cystin and
enzymes or combinations thereof.
[0048] The biocide and stabilizer may be incorporated into the vesicle in any
amount sufficient for controlling the microbial organism and will depend on the
specific biocide and stabilizer chosen. In one embodiment, the biocide or nonbiocidal
agent is incorporated into a liposome vesicle in an amount of from about 1.0
wt% - 12 wt%, and the stabilizer is added to the vesicle in an amount of about 0.02-
10.0 wt%.
[0049] In one embodiment, the vesicles are added to the aqueous system in
effective amounts, such that the amount of the biocide is introduced into the aqueous
system from about 0.05 to about 500 micrograms per milliliter. In another
embodiment, the vesicles are added to the aqueous system such that the amount of the
biocide agent is introduced into the aqueous system from about 0. 1 to about 100
micrograms per milliliter. In another embodiment, the vesicle is added to the aqueous
system in an amount of from about 0.01 ppm by volume to about 100 ppm by volume.
In another embodiment, the vesicle is added to the aqueous system in an amount of
from about 0.01 ppm by volume to about 50 ppm by volume. In another
embodiment, the vesicle is added in an amount of from about 0.01 ppm by volume to
about 20 ppm by volume. In another embodiment, the vesicle is added to the aqueous
system in an amount of from about 0.05 ppm by volume to about 5.0 ppm by volume.
[0050] In addition to the exemplary stabilizing agents noted above, additional
stabilizing agents that may be mentioned include:
a) I0 3, HIO 3, periodic, periodate salts
b) metal nitrates - Na, K, Ca, Mg
c) orthoesters - trimethyl orthoformate, triethyl orthoformate, triethyl
orthoacetate, trimethyl orthovalerate, trimethyl orthobenzoate
d) formaldehyde releases
e) phenoxyalkanols - phenoxyethanol, phenoxy isopropanol
f) nitrogen based heterocyclic thiols - 2 mercapto pyridine, MTZ,
2-thiohydantoin, L-cystin
g) EDTA
h) rheological modification agents such as thickeners
i) stearic hindrance agents (long chain repulsive)
j ) yield value modification (carbon as suspending agent)
[0051] In accordance with one embodiment of the invention, a stabilized
biocidal delivery composition is provided for delivering at least one anti-microbial
composition into a bio-film present in an industrial system. The biofilm comprises at
least one micro-organism species therein, and the biocidal delivery composition
comprises a liposome vesicular structure contain at least one lipid or phospho-lipid
component. Further, the liposome structure encapsulates at least one antimicrobial
composition in combination with at least one stabilizer agent. In another aspect of the
invention, the lipid is a member selected from the group consisting of phospholipids,
lecithin, phosphatidyl choline, glycolipid, triglyceride, sterol, fatty acid, sphingolipid,
or combinations thereof. In certain aspects of the invention, the phospholipid may be
derived from soybeans or eggs. Further, the lecithin may be a mixture of lipids.
[0052] In accordance with an exemplary embodiment of the invention, the
antimicrobial composition comprises at least one biocide, such as a nonoxidizing
biocide. The biocide may, for example, be an isothiazolin compound. More
specifically, the isothiazolin biocide may comprise at least one member selected from
the group consisting of 5-chloro-2-methyl-4-isothizolin-3-one, 2-methyl-4-isothiazolin-
3-one, or any combinations thereof.
[0053] In another exemplary embodiment, the stabilizer agent or compound is
a buffer comprised of a mixture of two or more compounds selected from the group
consisting of a citrate salt, a chorate salt buffer, and an acetate salt. The stabilizer
compound buffer may be comprised of a mixture of two or more compounds selected
from the group consisting of the metal salt of a citrate/chorate buffer, a metal salt of
an acetate/ chlorate buffer, and a citrate/ acetate buffer. The buffer stabilizer may be
selected from the group consisting of a sodium citrate buffer, a sodium acetate buffer,
a sodium citrate/sodium chlorate buffer mixture, and a sodium acetate/sodium chlorate
buffer mixture. The buffer stabilizer may be incorporated with the isothiazolin
biocide in an amount from about 0.2% to about 10% of the total biocide liposome
composition, and more preferably, the isothiazolin biocide may be incorporated in an
amount of about 1.0 wt% to about 12.0 wt% of the total biocide liposome
composition. Even more specifically, the isothiazolin biocide may be incorporated in
an amount of about 10.0 wt% to about 12.0 wt% of the total biocide liposome
composition. The liposome structure may be up to about 200 microns in diameter and
preferably, is between about 100 nanometers to about 10 microns in diameter. The
industrial system may be an aqueous system. The industrial system can be chosen
from the group consisting of water distribution systems, cooling towers, boiler
systems, showers, aquaria, sprinklers, spas, cleaning bath systems, air washers,
pasteurizers, air conditioners, fluid transporting pipelines, storage tanks, ion exchange
resins, food and beverage processing lines, paint spray booths, metalworking fluid
baths, coal and mineral slurries, metal leaching fluids, wastewater treatment facilities,
pulping and papermaking suspensions, mollusk control, acid mine drainage, oil
drilling pipes, oil pipelines, oil storage tanks, gas drilling pipes, gas pipelines, or any
industrial application prone to microbial induced bio-film formation or microbial
induced corrosion.
[0054] In another aspect of the invention, methods are disclosed for delivering
an antimicrobial composition into a biofilm in an industrial system comprising the
steps of: a) forming a liposome vesicular structure which encapsulates at least one
isothiazolin antimicrobial composition in combination with a buffer stabilizer
comprised of a mixture of two or more compounds selected from the group consisting
of a citrate salt, a chlorate salt, and an acetate salt, and b) introducing an effective
amount of the liposomes from a) above to the industrial system that is prone to
biofouling or biofilm formation. The liposome structure may be introduced at about
0.01 ppm to about 100 ppm. Further, the liposome structures may be introduced in
the industrial system at certain targeted locations thereof. The liposome structure may
comprise a biocide such as an isothiazolin biocide, and the isothiazolin biocide may be
selected from the group consisting of 5-chloro-2-methyl-4-isothizolin-3-one, 2-methyl-
4-isothiazolin-3-one, and mixtures thereof.
[0055] The buffer stabilizer may be selected from the group consisting of a
sodium citrate buffer, a sodium acetate buffer, a sodium citrate/sodium chlorate buffer
mixture, and a sodium acetate/sodium chlorate buffer mixture. Further, the buffer
stabilizer is incorporated in an amount of 0.2 wt% to about 10 wt% of the total
biocide liposome composition. In another embodiment, the isothiazolin biocide is
incorporated in an amount of 1.0 wt% to about 12.0 wt% of the total biocide liposome
composition.

CLAIMS
1. A stabilized biocidal delivery composition for delivering at least one
anti-microbial composition into a bio-film present in an industrial system, wherein
a) the bio-film comprises at least one micro-organism species;
b) the biocidal delivery composition comprises a vesicular structure;
and
c) the vesicular structure encapsulates at least one antimicrobial
composition in combination with at least one stabilizer composition.
2. The biocidal delivery composition of claim 1 wherein said vesicular
structure is composed of a liposome structure containing at least one lipid or
phospholipid component.
3. The biocidal delivery composition of claim 2 wherein the lipid is one
member selected from the group consisting of phospholipids, lecithin, phosphatidyl
choline, glycolipid, triglyceride, sterol, fatty acid, sphingolipid, or combinations
thereof.
4. The biocidal delivery composition of claim 3 wherein the lipid is a
phospholipid.
5. The biocidal delivery composition of claim 4 wherein the phospholipid
is derived from soybeans or eggs.
6. The biocidal delivery composition of claim 3 wherein the lecithin is a
mixture of lipids.
7. The biocidal delivery composition of claim 3 wherein the antimicrobial
composition comprises at least one biocide.
8. The biocidal delivery composition of claim 7 wherein the antimicrobial
composition comprises a non-oxidizing biocide.
9. The biocidal delivery composition of claim 8 wherein the biocide is an
isothiazolin compound.
10. The biocidal delivery composition of claim 9 wherein the isothiazolin
biocide comprises at least one member chosen from the group consisting of 5-chloro-
2-methyl-4-isothizolin-3-one, 2-methyl-4-isothiazolin-3-one, or any combinations
thereof.
11. The biocidal delivery composition of claim 10 wherein the stabilizer
compound is a buffer comprised of a mixture of two or more compounds selected
from the group consisting of a citrate salt, a chlorate salt buffer, and an acetate salt.
12. The biocidal delivery composition of claim 11 wherein the stabilizer
compound is a buffer comprised of a mixture of two or more compounds selected
from the group consisting of the metal salt of a citrate/ chlorate buffer, a metal salt of
acetate/ chlorate buffer, and a citrate/ acetate buffer.
13. The biocidal delivery system of claim 11 wherein said buffer stabilizer
is selected from the group consisting of a sodium citrate buffer, a sodium acetate
buffer, a sodium citrate/sodium chlorate buffer mixture, and a sodium acetate/sodium
chlorate buffer mixture.
14. The biocidal delivery system of claim 13 wherein said buffer stabilizer
is incorporated with said isothiazolin biocide in an amount of from about 0.2% to
about 10% of the total biocide liposome composition.
15. The biocidal delivery system of claim 14 wherein said isothiazolin
biocide is incorporated in an amount of from about 1.0 wt% to about 12.0 wt% of the
total biocide liposome composition.
16. The biocidal delivery system of claim 15 wherein said isothiazolin
biocide is incorporated in an amount of from about 10.0 wt% to about 12.0 wt% of
the total biocide liposome composition.
17. The biocidal delivery composition of claim 16 wherein the liposome
structure is up to about 200 microns in diameter.
18. The biocidal delivery composition of claim 1 wherein the liposome
structure is between about 100 nanometers to about 10 microns in diameter.
19. The biocidal delivery composition of claim 18 wherein the industrial
system is an aqueous system.
20. The biocidal delivery composition of claim 19 wherein the industrial
system is chosen from the group consisting of water distribution systems, cooling
towers, boiler systems, showers, aquaria, sprinklers, spas, cleaning bath systems, air
washers, pasteurizers, air conditioners, fluid transporting pipelines, storage tanks, ion
exchange resins, food and beverage processing lines, paint spray booths,
metalworking fluid baths, coal and mineral slurries, metal leaching fluids, wastewater
treatment facilities, pulping and papermaking suspensions, mollusk control, acid mine
drainage, oil drilling pipes, oil pipelines, oil storage tanks, gas drilling pipes, gas
pipelines, or any industrial application prone to microbial induced bio-film formation
or microbial induced corrosion.
21. A method for delivering an antimicrobial composition into a bio-film in
an industrial system comprising the steps of: a) forming a liposome structure which
encapsulates at least one isothiazolin antimicrobial composition in combination with a
buffer stabilizer comprised of a mixture of two or more compounds selected from the
group consisting of a citrate salt, a chlorate salt, and an acetate salt; and b)
introducing an effective amount of the liposomes of a) above to an industrial system
that is prone to bio-fouling or bio-film formation.
22. The method of claim 2 1 wherein the liposome structures are introduced
at from about 0.01 ppm to about 100 ppm.
23. The method of claim 22 wherein the liposome structures are introduced
in the industrial system at a targeted location.
24. The method of claim 23 wherein the liposome structure comprises a
biocide.
25. The method of claim 24 wherein the biocide is an isothiazolin biocide.
26. The method of claim 25 wherein the isothiazolin biocide is selected
from the group consisting of 5-chloro-2-methyl-4-isothizolin-3-one, 2-methyl-4-
isothiazolin-3-one, and mixtures thereof.
27. The method of claim 26 wherein said buffer stabilizer is selected from
the group consisting of a sodium citrate buffer, a sodium acetate buffer, a sodium
citrate/ sodium chlorate buffer mixture, and a sodium acetate/sodium chlorate buffer
mixture.
28. The method of claim 27 wherein said buffer stabilizer is incorporated in
an amount of from about 0.2 wt% to about 10 wt% of the total biocide liposome
composition.
29. The biocidal delivery system of claim 14 wherein said isothiazolin
biocide is incorporated in an amount of from about 1.0 wt% to about 12.0 wt% of the
total biocide liposome composition.