U.S. patent application number 15/421918 was filed with the patent office on 2017-07-20 for materials for disinfection produced by non-thermal plasma.
The applicant listed for this patent is DREXEL UNIVERSITY. Invention is credited to ARI D. BROOKS, MOOGEGA COOPER, DANIL V. DOBRYNIN, UTKU K. ERCAN, ALEXANDER FRIDMAN, GREGORY FRIDMAN, GENNADY FRIEDMAN, MARK INGERMAN, SURESH G. JOSHI, SIN PARK, ALEXANDER E. POOR, ALEXANDER RABINOVICH, NATALIE SHAINSKY.
Application Number | 20170202218 15/421918 |
Document ID | / |
Family ID | 45560047 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170202218 |
Kind Code |
A1 |
FRIDMAN; GREGORY ; et
al. |
July 20, 2017 |
Materials for Disinfection Produced by Non-Thermal Plasma
Abstract
Aspects of the present subject matter are directed to a method
comprising contacting an fluid, optionally containing an added
organic material, with a non-thermal plasma to form a disinfection
composition, wherein the disinfection composition is a liquid, and
contacting a surface with the disinfection composition, wherein the
surface is at least partially disinfected upon contact with the
disinfection composition. Additional aspects of the present subject
matter are directed to a method comprising forming a disinfection
composition by contacting an organic material with a non-thermal
plasma, wherein the disinfection composition is a liquid. A further
aspect of the present subject matter is directed to a disinfection
composition comprising an organic material contacted by a
non-thermal plasma, wherein the disinfection composition is a
liquid.
Inventors: |
FRIDMAN; GREGORY;
(PHILADELPHIA, PA) ; PARK; SIN; (PHILADELPHIA,
PA) ; SHAINSKY; NATALIE; (PHILADELPHIA, PA) ;
DOBRYNIN; DANIL V.; (PHILADELPHIA, PA) ; RABINOVICH;
ALEXANDER; (CHERRY HILL, NJ) ; FRIEDMAN; GENNADY;
(RICHBORO, PA) ; FRIDMAN; ALEXANDER;
(PHILADELPHIA, PA) ; COOPER; MOOGEGA; (PASADENA,
CA) ; BROOKS; ARI D.; (CHERRY HILL, NJ) ;
JOSHI; SURESH G.; (SECANE, PA) ; POOR; ALEXANDER
E.; (PHILADELPHIA, PA) ; ERCAN; UTKU K.;
(PHILADELPHIA, PA) ; INGERMAN; MARK; (WYNNEWOOD,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DREXEL UNIVERSITY |
Philadelphia |
PA |
US |
|
|
Family ID: |
45560047 |
Appl. No.: |
15/421918 |
Filed: |
February 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13813750 |
Sep 16, 2014 |
9585390 |
|
|
PCT/US11/46382 |
Aug 3, 2011 |
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15421918 |
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61370392 |
Aug 3, 2010 |
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61370409 |
Aug 3, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2/22 20130101; A01N
37/46 20130101; A61L 2/0011 20130101; A61L 2/14 20130101; A01N
43/16 20130101; A61L 2202/24 20130101; A01N 61/00 20130101; A01N
37/30 20130101; A61L 2/16 20130101; A01N 61/00 20130101; A01N 37/46
20130101; A01N 25/34 20130101; A01N 25/02 20130101; A01N 25/34
20130101; A01N 25/02 20130101 |
International
Class: |
A01N 37/46 20060101
A01N037/46; A61L 2/00 20060101 A61L002/00; A01N 43/16 20060101
A01N043/16; A61L 2/22 20060101 A61L002/22; A61L 2/14 20060101
A61L002/14 |
Claims
1. A device for at least partially disinfecting a human body part,
comprising: a power supply for generating a plasma; a
plasma-treating chamber in which a fluid and plasma can be
introduced to form a disinfection composition; a disinfection
chamber for contacting a human body part or medical device with the
disinfection composition, wherein the human body part is at least
partially disinfected upon contact with the disinfection
composition.
2. The device of claim 1, wherein said plasma is a dielectric
barrier discharge, a corona discharge, or a pulsed corona
discharge, ark, spark, gliding arc, radio frequency discharge,
microwave discharge or any combination thereof.
3. The device of claim 1, wherein said plasma may be of different
type applied at different times and in different locations.
4. The device of claim 1, wherein the plasma is a non-thermal
plasma having an intensity of at least about 0.1 J/cm.sup.2.
5. The device of claim 1, wherein the said fluid is in the form of
an aerosol or mist.
6. The device of claim 1, wherein the fluid comprises water,
saline, phosphate buffer composition, or a combination thereof.
7. The device of claim 1, wherein the fluid comprises ethanol or
isopropanol.
8. The device of claim 1, wherein the fluid contains an organic
material.
9. The device of claim 8, wherein the organic material comprises an
amino acid, anti-oxidant, and/or an alginate gel.
10. The device of claim 8, wherein the organic material comprises
N-Acetyl Cysteine.
11. The device of claim 8, wherein the organic material is
dissolved in the fluid.
12. The device of claim 8, wherein the organic material in the
disinfection composition is at a concentration of at least about
2.5 mM.
13. The device of claim 1, wherein the medical device comprises
intravenous tubing, heplocks, intravenous catheters, a nebulizer,
and urinary catheters.
14. The device of claim 1, wherein the human body part is a hand,
arm, or portion thereof.
15. The device of claim 1, wherein the surface is contacted with
the disinfection composition remote from the plasma.
16. The device of claim 1, wherein the plasma-treating chamber is
spatially separate from the disinfection chamber, and the two
chambers are in fluid communication with one another.
17. The device of claim 1, wherein the plasma-treating chamber and
the disinfection chamber are the same chamber, and the device is
configured to form the disinfection composition before allowing
contact of the disinfection with the human body part or medical
device.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 13/813,750, filed Sep. 16, 2014, which is the
National Stage of International Application No. PCT/US2011/046382,
filed Aug. 3, 2011, which claims the benefit of U.S. Provisional
Patent Application No. 61/370,392, filed Aug. 3, 2010, and U.S.
Provisional Patent Application No. 61/370,409, filed Aug. 3, 2010,
the disclosures of which are incorporated herein by reference in
their entireties.
TECHNICAL FIELD
[0002] Aspects of the disclosed subject matter are in the field of
disinfection using plasma.
BACKGROUND
[0003] Plasma is an ionized gas. However, in addition to ions and
electrons, plasma typically contains chemically active molecules as
well as electronically excited atoms and molecules all of which do
not remain in their active state for a long time (less than several
seconds in most cases at atmospheric pressure) outside plasma.
Recently, plasmas have been shown useful for disinfection and
sterilization of materials, water, air, and living tissues. Current
technologies include applying plasma itself or blowing active
agents produced in plasma directly to the surface being sterilized
to inactivate micro-organisms. In order to place the active agents
in the plasma in direct contact with the surface, the device
generating the plasma or a flow transporting mechanism (such as a
tube) is placed in an appropriate position.
SUMMARY
[0004] The present invention is directed toward the use of
plasma-treated materials as disinfecting agents.
[0005] Various embodiments of the present invention are directed to
methods comprising contacting a fluid with plasma to form a
disinfection composition; and contacting the disinfection
composition with a surface, wherein said surface is at least
partially disinfected upon contact with the disinfection
composition.
[0006] Other embodiments provide methods comprising forming a
disinfection composition by contacting fluid with a plasma, wherein
the disinfection composition comprises a liquid and is capable of
at least partially disinfecting a surface.
[0007] Still other embodiments provide disinfection compositions
prepared by contacting a fluid with plasma, wherein the
disinfection composition comprises a liquid and is capable of at
least partially disinfecting a surface. In certain embodiments,
these disinfection composition are provided on a woven or non-woven
fabric.
[0008] Other embodiments provide the methods for drying these
disinfection compositions to form disinfecting powders, as well as
the disinfecting powders. Other embodiments provide for methods and
compositions wherein the disinfecting powder compositions are
reconstituted to form a liquid, aerosol, or both. In certain
embodiments, these disinfecting powders are provided in the form of
a kit, with instructions as to how to reconstitute the dry
powders.
[0009] Some embodiments are directed to plasma-treated alginate
gels, as wound bandages or dressings.
[0010] Some embodiments provide methods comprising contacting
liquid with a non-thermal plasma to form a disinfection
composition; and contacting a surface with the disinfection
composition, wherein the surface is at least partially disinfected
upon contact with the disinfection composition.
[0011] Other embodiments are directed to devices for at least
partially disinfecting a human body part, comprising a power supply
for generating a plasma; a plasma-treating chamber in which a fluid
and plasma can be introduced to form a disinfection composition; a
disinfection chamber for contacting a human body part or medical
device with the disinfection composition, wherein the human body
part is at least partially disinfected upon contact with the
disinfection composition.
[0012] Some aspects of the present subject matter are directed to
methods comprising placing organic molecules in a carrier material,
such as but not limited to, liquid, gel, or powder, generating
plasma, contacting the carrier material with the plasma itself or
with plasma generated chemically active and electronically excited
molecules and atoms to create a disinfection compound which
includes the organic molecules modified as a result of exposure to
plasma or plasma generated active species and placing a surface in
contact with the disinfection compound to at least partially
disinfect at least a portion of the surface or using the
disinfecting compound inside another material to cause at least
disinfection of the latter. The contact between gas phase plasma
and the carrier material can occur in various ways including
through generation of plasma within voids or bubbles inside the
carrier material, through generation of plasma around aerosolized
carrier material, through generating plasma on a boundary between
gas and bulk carrier material, or through passing plasma generated
active species past the carrier material which could be in bulk or
aerosolized form.
[0013] The disinfection compound may be of various types of
materials, including water and organic materials, as well as being
in a gaseous, gelatinous or liquid state. Further, the disinfection
compound may be an antioxidant, such as N-Acetyl Cysteine. It may
be an amino acid such as Cysteine. In certain embodiments, the
disinfection compound is dissolved in a liquid. Suitable liquids
include saline, deionized water, phosphate buffered saline, or a
combination thereof. The amount of organic material in the
disinfection compound may vary. In certain embodiments, the organic
material in the disinfection compound is at a concentration of at
least about 2.5 mM. In other embodiments, the organic material in
the disinfection compound is at a concentration of at least about 5
mM. In still other embodiments, the organic material in the
disinfection compound, or composition, is at a concentration of at
least about 10 mM.
[0014] In some examples, the gelatinous substance may be an
alginate. Alginates are gelatinous substances obtained from certain
seaweeds and used as stabilizers and water retainers in beverages,
ice cream, ices, frozen custard, emulsions, desserts, baked goods,
and confectionery ingredients.
[0015] Plasma employed to create the disinfection compound may be
non-thermal plasma such as an atmospheric pressure dielectric
barrier discharge, corona discharge or pulsed corona discharge. The
plasma may have different intensities depending on the embodiment.
In applying plasma to a surface of bulk carrier material the
non-thermal plasma may have surface power density of at least about
0.1 J/cm.sup.2, or at least about 0.5 J/cm.sup.2, or at least about
1 J/cm.sup.2, or at least about 5 J/cm.sup.2.
[0016] Plasma may also be in the form of thermal plasma such as an
arc. Mixed forms of plasma (plasma whose gas temperature varies
dramatically over the extent of the ionized gas), such as a gliding
arc discharge, may also be employed in some embodiments. Plasma
intensity in such cases may also depend on the specific embodiment
or specific end point application. Different types of plasma or
active species from different forms of plasma can be applied
sequentially to the carrier material to generate disinfection
compound.
[0017] In certain embodiments, the surface contacted with the
disinfection composition is a medical device. In other embodiments,
the surface is a living tissue. Advantageously, the surface may be
contacted with the disinfection composition remote from the
non-thermal plasma. In some embodiments, the surface is contacted
with the disinfection composition immediately after the
disinfection composition is formed. For example, the surface may
contact the disinfection composition in less than a minute after
the disinfection composition is formed. The surface may be
contacted with the disinfection composition in less than 10 seconds
after the disinfection composition is formed. The surface may be
contacted with the disinfection composition in less than 1 second
after the disinfection composition is formed. The surface may be
contacted with the disinfection composition in less than 0.1 second
after the disinfection composition is formed.
[0018] In other embodiments, the surface is contacted with the
disinfection composition a longer period of time after the
disinfection composition is formed. Suitable periods of time
include at least about 20 minutes, at least about 60 minutes, at
least about 90 minutes, or at least about 120 minutes. The surface
may be in contact with the disinfection composition for varying
amounts of time. Suitable amounts of time include, for example, at
least about 600 seconds, at least about 60 seconds, at least about
30 seconds, at least about 15 seconds, or at least about 5 seconds.
It should be understood that the surface contact time may exceed
600 seconds. For example, an exemplary and non-limiting example may
be that the disinfection composition is left on the surface for
several days.
[0019] The extent to which the surface is disinfected may vary. In
certain embodiments, the surface is at least about 50% disinfected
upon contact with the disinfection composition. In other
embodiments, the surface is at least 75% disinfected upon contact
with the disinfection composition. Preferably, the surface is at
least 90% disinfected upon contact with the disinfection
composition. More preferably, the surface is at least 95%
disinfected upon contact with the disinfection composition.
[0020] An additional aspect of the present subject matter is
directed to a method comprising forming a disinfection composition
by contacting water and/or an organic material with a non-thermal
plasma. The disinfection composition may be a liquid. In certain
embodiments, the disinfection composition may be an aerosol. In
certain embodiments, the organic material is dissolved in a liquid.
Suitable liquids include saline, deionized water, phosphate
buffered saline, or a combination thereof. The amount of organic
material in the disinfection composition may vary. In certain
embodiments, the organic material in the disinfection composition
is at a concentration of at least about 2.5 mM. In other
embodiments, the organic material in the disinfection composition
is at a concentration of at least about 5 mM. In still other
embodiments, the organic material in the disinfection composition
is at a concentration of at least about 10 mM.
[0021] An additional aspect of the present subject matter is
directed to a disinfection composition comprising an organic
material contacted by a non-thermal plasma. The disinfection
composition may be a liquid. In certain embodiments, the
disinfection composition is an aerosol. The organic material may be
an antioxidant, preferably N-Acetyl Cysteine. The organic material
may be dissolved in a liquid. Suitable liquids include, for
example, saline, deionized water, phosphate buffered saline, or a
combination thereof.
[0022] A further aspect of the present subject matter is directed
to a product formed by the process of contacting an organic
material with a non-thermal plasma. The organic material may be an
antioxidant, preferably N-Acetyl Cysteine. In certain embodiments,
the organic material is dissolved in a liquid. Suitable liquids
include, for example, saline, deionized water, phosphate buffered
saline, or a combination thereof.
[0023] In certain other embodiments the present subject matter is
directed to a disinfection composition comprising an aqueous liquid
contacted by a non-thermal plasma. This aqueous liquid may be water
and may contain organic material or may be free of added organic
material. The aqueous liquid may be water which optionally
contained inorganic salts, for example a phosphate buffer.
[0024] In certain embodiments, the non-thermal plasma is an
atmospheric pressure dielectric barrier discharge. The plasma
intensity may be at least about 0.1 J/cm.sup.2, or at least about
0.5 J/cm.sup.2, or at least about 1 J/cm.sup.2, or at least about 5
J/cm.sup.2.
[0025] This summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This summary is not intended to identify
key features or essential features of the claimed subject matter,
nor is it intended to be used as an aid in determining the scope of
the claimed subject matter.
[0026] Other features of the subject matter are described
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The foregoing and other aspects of the present subject
matter will become apparent from the following detailed description
of the subject matter when considered in conjunction with the
accompanying drawings. For the purpose of illustrating the subject
matter, there is shown in the drawings embodiments that are
presently preferred, it being understood, however, that the subject
matter is not limited to the specific instrumentalities disclosed.
The drawings are not necessarily drawn to scale. In the
drawings:
[0028] FIG. 1 shows the percentage of surviving Staphylococcus
aureus cells after being contacted with plasma at different plasma
energies from a dielectric barrier discharge generated at 500 Hz
(0.13 W/cm.sup.2);
[0029] FIG. 2 shows the percentage of surviving Escherichia coli
cells after being contacted with plasma at different energies;
[0030] FIG. 3 shows the response of NAC alone on Escherichia coli
cells upon incubation for 30 minutes at room temperature wherein
the cells were washed twice with phosphate buffered saline;
[0031] FIG. 4 shows the response of NAC alone on Escherichia coli
cells upon incubation for 30 minutes at room temperature wherein
the cells were not washed;
[0032] FIG. 5A shows the response of NAC alone on Staphylococcus
aureus cells upon incubation for 30 minutes at room temperature
wherein the cells were washed twice with phosphate buffered
saline;
[0033] FIG. 5B shows a comparison of membrane potential between
cells contacted with traditional direct plasma sterilization and
plasma contacted NAC composition at varying plasma energies;
[0034] FIG. 6 shows a gel electrophoresis comparison of
disinfection with traditional direct plasma sterilization, the
application of NAC only, and the application of an exemplary
disinfection composition;
[0035] FIG. 7 shows the percentage of surviving Escherichia coli
cells after being contacted with exemplary disinfection
compositions comprising different concentrations of NAC contacted
with a plasma at an energy of 0.78 J/cm.sup.2;
[0036] FIG. 8 shows the percentage of surviving Escherichia coli
cells after being contacted with exemplary disinfection
compositions comprising 5 mM NAC contacted with a plasma at varied
energies compared to traditional direct plasma sterilization;
[0037] FIG. 9 shows the percentage of surviving Escherichia coli
cells after being contacted with an exemplary disinfection
composition comprising 5 mM NAC and phosphate buffered saline
remotely contacted with plasma at different energies compared to
traditional direct plasma sterilization;
[0038] FIG. 10 shows the percentage of surviving Escherichia coli
cells after being contacted with an exemplary disinfection
composition comprising 5 mM NAC and phosphate buffered saline
remotely contacted with plasma at different energies;
[0039] FIG. 11 shows the percentage of surviving Escherichia coli
cells after initially being contacted with plasma, followed by
being contacted with 5 mM NAC compared to traditional direct plasma
sterilization;
[0040] FIG. 12 is a schematic of experimental conditions for
exemplary disinfection compositions;
[0041] FIG. 13 shows a comparison of percentage of surviving cells
of S. aureus and E. coli (7 log) allowed to dry on the surface of
glass, immediately contacted with Plasma treated-NAC solution (5
mM) and mixed with no delay time, and holding time of 3
minutes;
[0042] FIG. 14 shows the effect of holding time on cell survival
percentage. A 5 mM NAC solution was treated with plasma over time,
and applied almost immediately (no delay time) to E. coli
suspension PBS (7 Log), and the mixture was held for variable time
period before plated for colony counts;
[0043] FIG. 15 shows the effect of holding time on cell survival
percentage. A 5 mM NAC solution was treated with plasma over time,
and applied almost immediately (no delay time) to E. coli
suspension PBS (9 Log), and the mixture was held for variable time
period before plated for colony counts; and
[0044] FIG. 16A shows the effect of delay time on cell survival;
NAC (5 mM final) in PBS treated with Plasma over the time, and was
kept a side (delayed mixing with bacteria) for variable times
(Delay Times) before mixing with E. coli (7 log) cultures. The
mixture was held for 1 min (Holding Time) before transfer for
colony assay. Bar represents the amount of plasma energy
(c/cm.sup.2) applied to NAC solution.
[0045] FIG. 16B shows the data associated with Experiments With
Added Organic Materials--Series 2, below, showing that
fluid-mediated plasma inactivates E. coli (1.times.10.sup.7 CFU/ml)
(sterilizes) in a time-dependent manner. Plasma-treated NAC
solution has stronger antimicrobial properties and a significantly
greater biocidal effect than treated de-ionized water (DIW) or
phosphate-buffered saline (PBS).
[0046] FIG. 16C shows that treated NAC solution causes cell
density-dependent inactivation of E. coli. Within 3 min holding
(contact) time, it inactivated up to 1.times.10.sup.8 CFU/ml. (*, P
value<0.05, against comparable conditions). See Experiments With
Added Organic Materials--Series 2, below.
[0047] FIG. 16D shows data that plasma-treated antimicrobial NAC
solution inactivates a wide array of pathogens in planktonic form.
Three minutes of plasma treatment produced sufficient antimicrobial
properties in solution, inactivating almost all pathogens in their
planktonic and biofilm forms, except C. albicans and C. glabrata
(which required 3.2 min; data not shown). (Non-treated [0 min]
samples were considered 100%). See Experiments With Added Organic
Materials--Series 2, below.
[0048] FIG. 16E shows data that plasma-treated antimicrobial NAC
solution inactivates a wide array of pathogens in biofilm-embedded
form.
[0049] FIG. 16F shows the holding (contact) time required by
plasma-treated NAC solution to complete inactivation of E. coli
bacteria. The graph shows a significant, rapid decline in surviving
E. coli as holding time increases. (*, P<0.05; against control,
C).
[0050] FIG. 16G shows that plasma-treated NAC solution retains
antimicrobial effects up to 90 days at room temperature. See
Experiments With Added Organic Materials--Series 2, below.
[0051] FIG. 16H shows accelerated aging (maturation) data of
treated NAC solution at elevated temperature of 50.degree. C.,
demonstrating that for .about.2 years of equivalent time, the
treated solution retains strong biocidal activity, a quality
required by an ideal antimicrobial solution. See Experiments With
Added Organic Materials--Series 2, below.
[0052] FIG. 16I shows the effects of hold time on biocidal effect,
on exposure of plasma-treated NAC on E. coli. For 0 minute hold,
bacteria were exposed to treated NAC, then immediately plated. The
other times indicate hold times prior to plating.
[0053] FIG. 16J shows the effects of killing persistence of plasma
treated NAC solutions on 10.sup.7 CFU/mL colonies of E. coli, 15
minute hold times treated 1 minute;
[0054] FIG. 16K shows the effects of killing persistence of plasma
treated NAC solutions on 10.sup.7 CFU/mL colonies of E. coli, 15
minute hold times treated 3 minutes;
[0055] FIG. 17 shows complete inhibition of 10.sup.7 CFU/mL E. coli
by alginate gels exposed to 15 seconds of plasma treatment (see
Experiments with Alginate Gels--Series 2 below). Note that the
treatment duration is the time the alginate gels were subjected to
plasma before inoculation with the E. Coli.
[0056] FIG. 18 shows complete inhibition of 10.sup.9 CFU/mL E. coli
by alginate gels exposed to 60 seconds of plasma treatment (see
Experiments with Alginate Gels--Series 2 below). Note that data for
15 second (left) and 60 second (right) treatments are presented for
each E. Coli concentration.
[0057] FIG. 19 shows complete inhibition of 10.sup.7 CFU/mL of
various pathogens by gels exposed to 15 seconds of plasma treatment
(see Experiments with Alginate Gels--Series 2 below). Note that the
data for each bacteria strain is presented for each treatment
duration (top to bottom corresponds to left to right; e.g., data
for E. Coli is leftmost and for Candida glabrata is rightmost
within each treatment duration).
[0058] FIG. 20 shows the results of alginate gels exposed to 3
minutes of plasma treatment result in total inhibition of 10.sup.7
CFU/mL E. coli after 10 minutes of contact (see Experiments with
Alginate Gels--Series 2 below). Note that the data for treatment is
presented for hold time (i.e., top to bottom of legend corresponds
to left to right within each hold time.
[0059] FIG. 21 shows that the effect of gels exposed to 3 minutes
of plasma treatment lasts 3 weeks (see Experiments with Alginate
Gels--Series 2 below). Note that Tx refers to calcium alginate gels
treated with non-thermal non-equilibrium dielectric-barrier
discharge plasma (power density, 0.29 W/cm.sup.2), and delays are
given in days (1, 7, 14, 21, and 28). For each day delay, data for
No Tx is on left and 3 min Tx is on right.
[0060] FIG. 22 shows the results of E. coli inactivation by plasma
treated water, showing that 60 sec treatment was sufficient to
inactivate 2.times.10.sup.3 CFU/mL E. Coli. Times of 30 s, 60 s,
and 90 s correspond to plasma energies of 3.9, 7.8, and 11.9
J/cm.sup.2, respectively
[0061] FIG. 23 shows the results of colony assays demonstrating a
rapid inactivation of E. coli and S. aureus (at 10.sup.7 CFU/ml),
by application of plasma-treated PBS (see Experiments Without
Organic Materials Added--Series 3, below). The killing effect was
plasma-exposure time dependent. The left bar within each plasma
energy set indicates E. coli; the right bar indicates S. aureus,
Bar, standard deviation; *, indicates statistically significant
(p<0.05) inactivation as compare to the corresponding plasma
non-treated cells
[0062] FIG. 24 shows the results of XTT assays demonstrating
bacterial biomass-dependent responses towards applications of
plasma-treated PBS (see Experiments Without Organic Materials
Added--Series 3, below). The planktonic cultures (10.sup.6 to
10.sup.9 CFU/ml) were exposed to 3.9 J/cm.sup.2 (30 sec) of plasma
energy. Bar, standard deviation; *, P<0.05 for given cell
concentration as compare to plasma untreated cells; first bar from
left within each CFU represents data for E. coli; second for S.
aureus; and third for MRSA95
[0063] FIG. 25 shows the results of XTT assays showing a rapid
inactivation of MRSA-USA300 and -USA400 when embedded in biofilms,
upon applications of plasma-treated PBS (see Experiments Without
Organic Materials Added--Series 3, below). All biofilm-embedded
pathogens were inactivated in less than 150 seconds (required 19.5
J/cm.sup.2 plasma energy). Bar, standard deviation; *, significant
inactivation (p value<0.05) as compared to plasma non-treated
bacteria or biofilms; first bar from left within each plasma energy
field represents data for E. coli; second for S. aureus; third for
MRSA95; fourth for USA300; and rightmost bar for USA400.
[0064] FIGS. 26A-D show the results of experiments aimed at
determining the efficacy of plasma treating powders or solutions
resulting in powders, as described below in section titled
Preparing and Reconstituting Dry Powder Disinfectants. FIGS. 26A,
C, & D shows the antimicrobial effect of reconstituted NAC
powders derived from plasma-treated solutions. FIG. 26B shows the
antimicrobial effect of plasma-treated NAC powders.
[0065] FIG. 27 is an exemplary device that may be used to sterilize
or disinfect the arm or hand of a person.
[0066] FIG. 28 is an alternate exemplary device that may be used to
sterilize or disinfect the arm or hand of a person.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0067] The present invention is directed to use of plasma as a
disinfecting agent. Various embodiments of the present invention
are directed to methods comprising contacting a fluid with plasma
to form a disinfection composition; and contacting the disinfection
composition with a surface, wherein said surface is at least
partially disinfected upon contact with the disinfection
composition.
[0068] Other embodiments provide methods comprising forming a
disinfection composition by contacting fluid with a plasma, wherein
the disinfection composition comprises a liquid and is capable of
at least partially disinfecting a surface.
[0069] Still other embodiments provide disinfection compositions
prepared by contacting a fluid with a plasma, wherein the
disinfection composition comprises a liquid and is capable of at
least partially disinfecting a surface. In certain embodiments,
these disinfection composition are provided on a woven or non-woven
fabric.
[0070] Other embodiments provide the methods for drying these
disinfection compositions to form disinfecting powders, as well as
the disinfecting powders. Other embodiments provide for methods and
compositions wherein the disinfecting powder compositions are
reconstituted to form a liquid, aerosol, or both. In certain
embodiments, these disinfecting powders are provided in the form of
a kit, with instructions as to how to reconstitute the dry
powders.
[0071] Some embodiments are directed to plasma-treated alginate
gels, as wound bandages or dressings.
[0072] Some embodiments provide methods comprising contacting
liquid with a non-thermal plasma to form a disinfection
composition; and contacting a surface with the disinfection
composition, wherein the surface is at least partially disinfected
upon contact with the disinfection composition.
[0073] Other embodiments are directed to devices for at least
partially disinfecting a human body part, comprising a power supply
for generating a plasma; a plasma-treating chamber in which a fluid
and plasma can be introduced to form a disinfection composition; a
disinfection chamber for contacting a human body part or medical
device with the disinfection composition, wherein the human body
part is at least partially disinfected upon contact with the
disinfection composition.
[0074] The present subject matter may be understood more readily by
reference to the following detailed description taken in connection
with the accompanying figures and examples, which form a part of
this disclosure. It is to be understood that this subject matter is
not limited to the specific devices, methods, applications,
conditions or parameters described and/or shown herein, and that
the terminology used herein is for the purpose of describing
particular embodiments by way of example only and is not intended
to be limiting of the claimed subject matter.
[0075] Similarly, unless specifically otherwise stated, any
description as to a possible mechanism or mode of action or reason
for improvement is meant to be illustrative only, and the invention
herein is not to be constrained by the correctness or incorrectness
of any such suggested mechanism or mode of action or reason for
improvement. Throughout this text, it is recognized that the
descriptions refer to methods of using, the compositions used in
those methods, as well as the methods of manufacturing the
compositions used in those methods.
[0076] When values are expressed as approximations by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. In general, use of the term "about"
indicates approximations that can vary depending on the desired
properties sought to be obtained by the disclosed subject matter
and is to be interpreted in the specific context in which it is
used, based on its function, and the person skilled in the art will
be able to interpret it as such. In some cases, the number of
significant figures used for a particular value may be one
non-limiting method of determining the extent of the word "about."
In other cases, the gradations used in a series of values may be
used to determine the intended range available to the term "about"
for each value. Where present, all ranges are inclusive and
combinable. That is, reference to values stated in ranges includes
each and every value within that range.
[0077] It is to be appreciated that certain features of the
invention which are, for clarity, described herein in the context
of separate embodiments, may also be provided in combination in a
single embodiment. Conversely, various features of the invention
that are, for brevity, described in the context of a single
embodiment, may also be provided separately or in any
subcombination. Finally, while an embodiment may be described as
part of a series of steps or part of a more general composition or
structure, each said step may also be considered an independent
embodiment in itself.
[0078] Plasmas, referred to as the "fourth state of matter," are
ionized gases having at least one electron that is not bound to an
atom or molecule. In recent years, plasmas have become of
significant interest to researchers in fields such as organic and
polymer chemistry, fuel conversion, hydrogen production,
environmental chemistry, biology, and medicine, among others. This
is, in part, because plasmas offer several advantages over
traditional chemical processes. For example, plasmas can generate
much higher temperatures and energy densities than conventional
chemical technologies; plasmas are able to produce very high
concentrations of energetic and chemically active species; and
plasma systems can operate far from thermodynamic equilibrium,
providing extremely high concentrations of chemically active
species while having a bulk temperature as low as room
temperature.
[0079] Plasmas are generated by ionizing gases using any of a
variety of ionization sources. Depending upon the ionization source
and the extent of ionization, plasmas may be characterized as
either thermal or non-thermal. Thermal and non-thermal plasmas can
also be characterized by the temperature of their components.
Thermal plasmas are in a state of thermal equilibrium, that is, the
temperature of the free electrons, ions, and heavy neutral atoms
are approximately the same. Non-thermal plasmas, or cold plasmas,
are far from a state of thermal equilibrium; the temperature of the
free electrons is much greater than the temperature of the ions and
heavy neutral atoms within the plasma.
[0080] The initial generation of free electrons may vary depending
upon the ionization source. With respect to both thermal and
non-thermal ionization sources, electrons may be generated at the
surface of the cathode due to a potential applied across the
electrode. In addition, thermal plasma ionization sources may also
generate electrons at the surface of a cathode as a result of the
high temperature of the cathode (thermionic emissions) or high
electric fields near the surface of the cathode (field
emissions).
[0081] The energy from these free electrons may be transferred to
additional plasma components, providing energy for additional
ionization, excitation, dissociation, etc. With respect to
non-thermal plasmas, the ionization process typically occurs by
direct ionization through electron impact. Direct ionization occurs
when an electron of high energy interacts with a valence electron
of a neutral atom or molecule. If the energy of the electron is
greater than the ionization potential of the valence electron, the
valence electron escapes the electron cloud of the atom or molecule
and becomes a free electron according to:
e.sup.-+A.fwdarw.A.sup.++e.sup.-+e.sup.-.
As the charge of the ion increases, the energy required to remove
an additional electron also increases. Thus, the energy required to
remove an additional electron from A.sup.+ is greater than the
energy required to remove the first electron from A to form
A.sup.+. A benefit of non-thermal plasmas is that because complete
ionization does not occur, the power to the ionization source can
be adjusted to increase or decrease ionization. This ability to
adjust the ionization of the gas provides for a user to "tune" the
plasma to their specific needs.
[0082] An exemplary thermal plasma ionization source is an arc
discharge. Arc discharges have been otherwise used for applications
such as metallurgy, metal welding and metal cutting and are known
per se. Arc discharges are formed by the application of a potential
to a cathode, and arc discharges are characterized by high current
densities and low voltage drops. Factors relevant to these
characteristics are the usually short distance between the
electrodes (typically a few millimeters) and the mostly inert
materials of the electrodes (typically, carbon, tungsten,
zirconium, silver, etc). The majority of electrons generated in arc
discharges are formed by intensive thermionic and field emissions
at the surface of the cathode. That is, a much larger number of the
electrons are generated directly from the cathode as opposed to
secondary sources such as excited atoms or ions. Because of this
intense generation of electrons at the cathode, current at the
cathode is high, which leads to Joule heating and increased
temperatures of the cathodes. Such high temperatures can result in
evaporation and erosion of the cathode. The anode in arc discharges
may be either an electrode having a composition identical or
similar to the cathode or it may be another conductive material.
For example, the anode in arc discharges used in metal welding or
cutting is the actual metal to be welded or cut. Typical values for
parameters of thermal arc discharges can be seen in Table 1:
TABLE-US-00001 TABLE 1 Arc Discharge Parameters Parameters of a
Thermal Arc Discharge Typical Values Gas Pressure 0.1 to 100 atm
Arc Current 30 A to 30 kA Cathode Current Density 10.sup.4 to
10.sup.7 A/cm.sup.2 Voltage 10 to 100 V Power per unit length ~1
kW/cm Electron Density 10.sup.15 to 10.sup.19 cm.sup.-3 Gas
Temperature 1 to 10 eV Electron Temperature 1 to 10 eV
[0083] Although thermal plasmas are capable of delivering extremely
high powers, they have several drawbacks. In addition to the
electrode erosion problems discussed above, thermal plasmas do not
allow for adjusting the amount of ionization, they operate at
extremely high temperatures, and they lack efficiency.
[0084] Non-thermal plasma ionization sources have alleviated some
of the above-mentioned problems. Exemplary ionization sources for
non-thermal plasmas include glow discharges, dielectric barrier
discharges, and gliding arc discharges, among others. In contrast
to thermal plasmas, non-thermal plasmas provide for high
selectivity, high energy efficiencies, and low operating
temperatures. In many non-thermal plasma systems, electron
temperatures are about 10,000 K while the bulk gas temperature may
be as cool as room temperature.
[0085] A glow discharge is a plasma source that generates a
non-equilibrium plasma between two electrodes under a direct
current. There are several types of glow discharges; a common one
is the fluorescent light. This glow discharge is established in a
long tube with a potential difference applied between an anode at
one end of the tube and a cathode at the other end. The tube is
filled with an inert or reactive gas often under pressure. Due to
the potential difference between the electrodes, electrons are
emitted from the cathode and accelerate toward the anode. The
electrons collide with gas atoms in the tube and form excited
species. These excited species decay to lower energy levels through
the emission of light (i.e., glow). The ionized species generated
by the collision of electrons with gas atoms travel toward the
cathode and release secondary electrons, which are then accelerated
toward the anode. This generation of electrons, referred to as
secondary emission, is in contrast to the intensive formation of
electrons at the surface of the cathode in thermal plasma
generation. Typical parameters of a glow discharge as described
above are shown in Table 2:
TABLE-US-00002 TABLE 2 Parameters of Glow Discharge Parameters of a
Glow Discharge Typical Values Discharge Tube Radius 0.3 to 3 cm
Discharge Tube Length 10 to 100 cm Plasma Volume About 100 cm.sup.3
Gas Pressure 0.03 to 30 Torr Voltage Between Electrodes 100 to 1000
V Electrode Current 10.sup.-4 to 0.5 A Power Level ~100 W
[0086] Dielectric barrier discharge (DBD) may be generated using an
alternating current at a frequency of from about 0.5 kHz to about
500 kHz between a high voltage electrode and a ground electrode. In
addition, one or more dielectric barriers are placed between the
electrodes. DBDs have been employed for over a century and have
been used for the generation of ozone in the purification of water,
polymer treatment (to promote wettability, printability, adhesion),
and for pollution control. DBDs prevent spark formation by limiting
current between the electrodes.
[0087] Several materials can be utilized for the dielectric
barrier. These include glass, quartz, and ceramics, among others.
The clearance between the discharge gaps is typically between about
0.1 mm and several centimeters. The required voltage applied to the
high voltage electrode varies depending upon the pressure and the
clearance between the discharge gaps. For a DBD at atmospheric
pressure and a few millimeters between the gaps, the voltage
required to generate a plasma is typically about 10 kV. In certain
embodiments, the ground electrode of the DBD may be an external
conductive object, such as a human body. This is known as
floating-electrode DBD (FE-DBD). FE-DBD has recently been utilized
in medical applications.
[0088] Aspects of the present subject matter are directed to
contacting an organic or aqueous-based material with a non-thermal
plasma to form a disinfection composition. It should be noted that,
as discussed previously, the presently disclosed subject matter is
not limited to any one type of plasma. The following description is
based upon the use of non-thermal plasma for descriptive purposes
only and is not intended to limit the scope of the presently
disclosed subject matter to non-thermal plasma. The disinfection
composition may be a liquid and the surface may be contacted with
the disinfection solution to at least partially disinfect the
surface. Plasmas have been found to be useful for disinfection and
sterilization of materials such as water, air, medical devices and
tissues. Traditionally, a plasma is applied directly to the
material for disinfection/sterilization ("direct plasma
sterilization"). In some cases UV (Ultra-Violet radiation) produced
in the plasma is the active agent responsible for inactivation of
micro-organisms. In other cases, plasma produced ozone or radicals
such as OH or NO are the active disinfecting agents. Intense sound
waves produced by plasma in liquids are also suspected to kill
bacteria. FIGS. 1 and 2 show the percentage of surviving bacteria
cells after a traditional direct plasma sterilization. Depending
upon the bacteria, at a plasma energy of 0.78 J/cm.sup.2,
approximately 50% of the bacteria cells survive the plasma
treatment. In order to accomplish lower percentages of surviving
cells, high plasma energies must be applied. Use of higher plasma
energies in undesirable because of increases costs and the
potential to damage surrounding areas of the material.
[0089] In certain embodiments, the non-thermal plasma, is a
dielectric barrier discharge (DBD). A DBD may be generated using an
alternating current at a frequency of from about 0.5 kHz to about
500 kHz between a high voltage electrode and a ground electrode. It
should be noted that in certain configurations, a single pulse may
be used. Therefore, the present subject matter may be preferably
used in applications ranging from a single pulse to about 500 kHz.
In addition, one or more dielectric barriers are placed between the
electrodes. Exemplary surface power density outputs may be between
about 0.001 Watt/cm.sup.2 to about 100 Watt/cm.sup.2.
[0090] Various materials can be utilized for the dielectric
barrier. These include plastic, glass, quartz, and ceramics, among
others. The clearance between the discharge gaps is typically
between about 0.01 mm and several centimeters. The required voltage
applied to the high voltage electrode varies depending upon the
pressure and the clearance between the discharge gaps. For a DBD at
atmospheric pressure and a few millimeters between the gaps, the
voltage required to generate a plasma may vary, but in some
configurations, is about 10 kV.
[0091] In certain embodiments, the plasma may be generated having
an energy of at least about 0.1 J/cm.sup.2. In other embodiments,
the plasma may have an energy of at least about 0.5 J/cm.sup.2 or
at least about 1 J/cm.sup.2 or at least about 5 J/cm.sup.2. The
energy of the plasma may vary depending upon the surface being
treated, the extent of disinfection required, and the type and
amount of organic material in the disinfection composition.
[0092] Aspects of the present subject matter offer alternative
methodologies for plasma based disinfection and sterilization. As
used herein, the terms "disinfect," "disinfecting," or the like
refer to the ability or render pathogens less active, or to kill,
inactivate, inhibit the growth, or otherwise render pathogens
innocuous or less active, where pathogens include bacteria,
microbes, fungi, or yeasts. Exemplary, albeit non-limiting,
pathogens include Acinetobacter baumannii, Escherichia coli,
Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus
aureus (including drug resistant strains such as any variety of
MRSA), Staphylococcus epidermidis, Candida albicans, and Candida
glabrata. Disinfection can be quantified using standard tests,
including Safranin assay, LIVE/DEAD BacLight BacterialViability
Assay, or XTT assay.
[0093] A benefit of aspects of the present subject matter compared
to traditional direct plasma sterilization techniques is the
ability to apply the disinfection composition remote from the
plasma source. For example, the material to be plasma-treated may
be contacted with a plasma to form a disinfection composition and
the disinfection composition may be subsequently transported to
another location for contacting with a surface. For example, the
disinfection composition may be formed and transported to a
different location within a laboratory of surgical room, or it may
be transported to an entirely different building. Thus, in certain
embodiments, the surface may not be contacted with the disinfection
composition for a period of time after the disinfection composition
is formed. In certain embodiments, the period of time may be at
least about 20 minutes, at least about 30 minutes or at least about
60 minutes, or at least about 90 minutes, or at least about 120
minutes.
[0094] In other embodiments, a surface may be contacted with the
disinfection composition immediately after the disinfection
composition is formed. For example, the disinfection composition
may be formed at a "disinfection station" wherein surfaces, such as
medical devices and other components may be disinfected, for
example, near or in an operating room or hospital. In certain
embodiments, the surface is contacted with the disinfection
composition in less than about 1 minute after the disinfection
composition is formed, or for example, in less than about 10
seconds, or in less than about 1 second, or in less than about 0.1
seconds.
[0095] The material to be plasma-treated may be contacted by a
plasma for different periods of time. This period of time may vary
depending upon factors such as the material to be plasma-treated,
the type of plasma, and the plasma intensity among other. In
certain embodiments, the material to be plasma-treated may be
contacted with the plasma for at least about 10 seconds, or at
least about 60 seconds, or for at least about 90 seconds. Once a
surface is contacted with a disinfection material, the disinfection
material may remain in contact with the surface for a period of
time that may be referred to as a "hold time." In certain
embodiments, the hold time may be at least about 5 seconds, or at
least about 30 seconds, or at least about 60 seconds, or at least
about 600 seconds.
[0096] The extent of disinfection depends upon factors such as the
type and amount of plasma-treated material, plasma energy, and
exposure time, among others. In certain embodiments, the surface is
at least about 50% disinfected upon contact with the disinfection
composition, or, for example, at least about 75%, or, for example,
at least about 90%, or, for example, at least about 95%
disinfected.
[0097] In some embodiments, the disinfecting composition comprise
plasma treated aqueous or organic fluids (e.g., ethanol or
isopropanol) or other organic molecules (in solution, or as gels or
solids), which then may act as the active anti-microbial agents.
The organic molecules may be contacted by the plasma to create a
disinfection composition for the disinfection of a surface. In
certain embodiments, the organic molecule may be dispersed in a
liquid. Suitable liquids include saline, deionized water, and
phosphate buffered saline (PBS), among others. A plasma may be
created electrically in the gas phase near a gas/liquid interface.
The organic molecule dispersed in the liquid may be contacted by
the plasma to form a liquid disinfection composition. This liquid
can, for example, be used to wash living tissues as well as other
material surfaces for the purposes of disinfection or
sterilization.
[0098] A liquid disinfection composition provides benefits over a
traditional direct plasma sterilization or gaseous composition. For
example, a liquid disinfection composition may be particularly
suited for disinfection of devices having inner cavities difficult
to reach with a plasma or gas disinfection composition. For
example, a liquid disinfection composition may be used to disinfect
the inside of devices such as intravenous tubing, heplocks,
intravenous catheters, urinary catheters, wounds, or other
indwelling medical devices. Additionally, a liquid disinfection
composition may provide for visualization that an entire surface
has been contacted with the disinfection solution. In this latter
regard, the liquid disinfecting composition may also include a
colorant or other detectable additive, provided the additive does
not interfere with the disinfectant character of the plasma-treated
liquid in any appreciable way (i.e., the plasma-treated liquid
provides at least a portion the activity associated with a
comparable composition without the additive).
[0099] In certain embodiments, the disinfection composition may be
aerosolized. An aerosolized disinfection composition may provide
for a thin coating of the disinfection composition over an entire
surface. Exemplary uses may be for the disinfection/sterilization
of medical devices or other components used in a medical situation,
such as, an operating room. In yet other embodiments, the
disinfection composition may be used to charge a nebulizer for the
treatment/disinfection of airways of an individual with respiratory
diseases or infections such as cystic fibrosis or pneumonias.
[0100] Various organic molecules may be suitable for the present
subject matter. In a preferred embodiment, the organic molecule is
an antioxidant. Suitable antioxidants include, for example,
N-Acetyl Cysteine (NAC), ascorbic acid, glutathione, lipoic acid,
uric acid, carotenes, .alpha.-tocopherol, carotenes, ubiquinol, and
melatonin, among others. In a more preferred embodiment, the
organic molecule is NAC. NAC is known to have antioxidant activity
for humans providing protection against often harmful oxidative
stress. It is also known to have some anti-microbial activity
(FIGS. 3-5B).
[0101] Among plasma-treated fluids, N-acetyl-cysteine solution
stood out as a powerful antibacterial and antifungal agent. In
certain embodiments, such fluids can disinfect (as evidenced by
their ability to inactivate biofilms) Acinetobacter baumannii,
Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa,
Staphylococcus aureus (including drug resistant strains such as any
variety of MRSA), Staphylococcus epidermidis, Candida albicans, and
Candida glabrata. In various embodiments, this disinfection can
occur in less than 60, 45, 30, 15, 10, or 5 minutes of
holding/contact time. In other embodiments, plasma-treated
N-acetyl-cysteine solution are extremely stable, capable of
exhibiting the equivalent of more than 6 months, or 1 or 2 years of
shelf life at temperatures of 50.degree. C. See, for example, the
results shown below in the section, Experiments With Added Organic
Materials--Series 2.
[0102] In other embodiments, the material to be activated by the
non-thermal plasma is an alginate or other gelatinous material.
Alginates are gelatinous substances obtained from certain seaweeds
and used as stabilizers and water retainers in beverages, ice
cream, ices, frozen custard, emulsions, desserts, baked goods, and
confectionery ingredients. Alginic acid is a polysaccharide complex
built from mannuronic acid units. Salts such as iron, magnesium and
ammonium alginates form viscous solutions. Alginates hold large
amounts of water and are useful as thickeners, stabilizers, and
gelling, binding and emulsifying agents in ice-cream, synthetic
cream etc. Alginates such as pure sodium alginate and potassium
alginate, with low calcium content, create firm, fairly heat stable
gels. Sodium alginate is available from cold storage with top gel
strength, and white color (food grade) and produces varying
viscosities. Sodium alginate is also used as gum and edible films
by food industries. In presence of calcium salts it gives thermally
irreversible gels. Major Monosaccharides are D-mannuronic acid,
L-gluluronic acid and the anionic seaweed polysaccharides are often
linear polymer of D-mannuronic acid and L-guluronic acid. Since
1983 the Alginates have been used as food ingredients, and the Food
and Drug administration (FDA) has been pursuing tests for its
safety. Alginate is obtained commercially from three genera of the
marine brown algae, Phaeophyceae (Macrocystis pyrifera, Laminaria
digitata, and Laminaria saccharina). The alginate molecule is a
linear polysaccharide consisting of chains of repeating
(D)-mannuronic acid (M blocks), (L)-guluronic acid (G blocks), and
a combination of the two (MG blocks). Each type of chain is present
in a specific ratio (M:G ratio) depending upon the species of
algae. This ratio determines the characteristics of the gel, with G
blocks capable of forming stronger, more brittle gels with good
heat stability whereas high M content produces weaker more-elastic
gels. Sodium alginate is soluble in water. However, when the sodium
is replaced with calcium, the ionic bond with calcium cross-links
the polymer chains resulting in an insoluble gel. This gel is the
basis for a variety of wound dressings. Other dressings are created
using small fibers of calcium alginate that are applied as a paste
or are pressed or woven to form a stronger fabric, and others are
produced from freeze-dried alginate.
[0103] Using the presently disclosed subject matter, alginates may
also be used in wound dressings. Currently available dressing
material is hard and sticks to wounded dressed area and causes
injury to healing granulation tissues when removed. Furthermore it
is impregnated with chemicals or antibiotic-like antimicrobial
agents (having a short lived effect). In some applications, an
alginate activated according to the presently disclosed subject
matter may be softer and more flexible with good moisture retention
capabilities making it very easy to remove when dressings are
changed. The Alginate gels or gels possessing similar properties
when treated with non-thermal (DBD) plasma may simultaneously
inhibit microbial growth and improve healing.
[0104] For example, the results of experiments described below (see
Experiments with Alginate Gels--Series 1 and Series 2, below)
demonstrate that calcium alginate gels treated by exposure to
non-thermal plasma have the potential to be clinically useful wound
dressings based on their broad spectrum of antimicrobial activity.
They are a viable alternative to silver-containing dressings
because of the faster rate of killing. This is important because of
the potential for bacteria to develop resistance to slower-acting
agents. With regard to silver, the fastest rate of kill quoted in
the literature is 30 minutes, and as long as 24 hours. E coli
reproduces about every 20 minutes (depending on the strain)
correlating to .about.10.sup.21 colonies over 24 hours. Each new
generation produced in an environment with a constant level of
antibiotic has the potential to select for mutations that confer
resistance. The chlorhexidine-containing dressing used for
comparison in this study acts far more rapidly than silver, and
chlorhexidine in solution has been shown to have 3.5-log reduction
in E coli after only 30 seconds. While the plasma-treated gels do
not act this quickly, the 10-minute killing time demonstrated is
shorter than the average reproductive cycle of bacteria and is
therefore likely not to lead to the development of resistance.
[0105] In various embodiments, the present invention includes those
alginate gels which exhibit the disinfectant characteristics,
including those described herein. Such gels include gels such as
those prepared by the methods described herein. Further, use of
these gels to disinfect surfaces and the methods of using these
gels treated with plasma according to these conditions are also
embodiments of the present invention.
[0106] In certain embodiments, the alginate gels of the present
compositions or the methods derived from the gels described in
previous paragraphs. These gels may further comprise least one of
the organic materials described elsewhere within this specification
or may be free of such organic materials. Likewise, these gels may
comprise inorganic salts such as described herein, or may be free
of any added inorganic salts or buffers.
[0107] In certain embodiments, the disinfecting alginate gels are
prepared by exposing the gels to plasmas having energies of at
least about 0.2 J/cm.sup.2, at least about 0.4 J/cm.sup.2, at least
about 0.6 J/cm.sup.2, at least about 0.8 J/cm.sup.2, at least about
1 J/cm.sup.2, at least about 1.5 J/cm.sup.2, at least about 2
J/cm.sup.2, at least about 3 J/cm.sup.2, at least about 4
J/cm.sup.2, at least about 5 J/cm.sup.2, at least about 8
J/cm.sup.2, at least about 10 J/cm.sup.2, at least about 12
J/cm.sup.2, at least about 14 J/cm.sup.2, at least about 16
J/cm.sup.2, at least about 18 J/cm.sup.2, at least about 20
J/cm.sup.2, or at least about 24 J/cm.sup.2.
[0108] In certain embodiments, these gels and methods are capable
of killing the pathogens described above, and including Escherichia
coli, Staphylococcus aureus, Staphylococcus epidermidis,
Acetinobacter baumanii, Candida albicans, and Candida glabrata. In
other independent embodiments, these gels and methods are capable
of at least partially (including completely) sterilizing surfaces
(inactivating pathogens) described herein within 5 secs, 10 secs,
30 secs, 1 min, 3 min, 5 min, 10 min, and 30 minutes of contacting
the plasma treated gels and the pathogens.
[0109] In other independent embodiments, the gels and methods are
capable of maintaining at least a portion of their ability to
inactivate pathogens (antimicrobial activity) for periods of 0.5,
1, 2, 4, 6, 8, 10, 12, or 24 hours, 1, 2, 3, 4, 5, or 6 days, 1, 2,
3, 4, 5, or 6 weeks after exposure to plasma treatment.
[0110] FIG. 6 shows a comparison of cells treated with traditional
direct plasma sterilization, NAC alone, and an exemplary
disinfection composition comprising NAC contacted with a plasma.
Disinfection is improved when the NAC is contacted with a plasma as
compared to the treatments with plasma alone and NAC alone. This
significant improvement is also shown in FIGS. 8 and 9 in which
traditional direct plasma treatment is compared to treatment with
an exemplary disinfection composition of the current subject
matter. In additional embodiments, a surface may be first treated
with a plasma and may subsequently be treated with an NAC
composition as exemplified in FIG. 11.
[0111] The concentration of organic material may vary depending
upon the surface being treated, the extent of disinfection
required, and the type and amount of organic material in the
disinfection composition. In certain embodiments, the concentration
of organic material may be at least about 2.5 mM, at least about 5
mM, at least about 10 mM, at least about 30 mM, or at least about
50 mM.
[0112] In some embodiments, the disinfection composition may be
generated and subsequently dried to produce a powder for later use.
The powder may be distributed, reconstituted to sterilize or
disinfect when applied in a manner similar to a disinfection
composition recently created. See section titled Preparing and
Reconstituting Dry Powder Disinfectants below. Certain embodiments
provide for kits containing the powder to be reconstituted and
instructions as to how to accomplish this reconstitution.
[0113] In still further embodiments, the disinfection compound may
be or comprise water, alcohols (e.g., ethanol or isopropanol) or
other appropriate liquid. The water may include or be free of added
salts/buffering agents. Suitable aqueous liquids include saline,
deionized water, tap water, and phosphate buffered saline (PBS),
among others. Similarly, the alcohols may include any of the
organic or additive materials described above, or be free from
added additives or other materials. For example, disinfecting or
sterilizing a surface may be performed by contacting liquid in the
form of a mist with a non-thermal plasma formed in a gas to form a
disinfection composition and contacting a surface with the
disinfection composition, wherein the surface is at least partially
disinfected upon contact with the disinfection composition. The
liquid may be water or other appropriate liquid. The surface to be
disinfected or sterilized may include, but is not limited to,
steel, polyethylene, or polytetrafluoroethylene. The types of
organisms that may be killed include, but are not limited to,
Escherichia coli, Staphylococcus aureus (including
methicillin-resistant S. aureus, MRSA), Staphylococcus epidermidis,
Acetinobacter baumanii, Candida albicans, and Candida glabrata, P.
aeruginosa, and K pneumonia. In various embodiments, the
plasma-treated water is applied to the pathogen as a mist, spray,
or aerosol. Some sample mist flow rates range from 0.1 ml/min to
160 ml/min.
[0114] In certain embodiments, the disinfecting water or aqueous
mixtures are prepared by exposing the water or aqueous mixtures to
plasmas having energies of at least about 0.2 J/cm.sup.2, at least
about 0.4 J/cm.sup.2, at least about 0.6 J/cm.sup.2, at least about
0.8 J/cm.sup.2, at least about 1 J/cm.sup.2, at least about 1.5
J/cm.sup.2, at least about 2 J/cm.sup.2, at least about 3
J/cm.sup.2, at least about 4 J/cm.sup.2, at least about 5
J/cm.sup.2, at least about 8 J/cm.sup.2, at least about 10
J/cm.sup.2, at least about 12 J/cm.sup.2, at least about 14
J/cm.sup.2, at least about 16 J/cm.sup.2, at least about 18
J/cm.sup.2, at least about 20 J/cm.sup.2, or at least about 24
J/cm.sup.2.
[0115] In certain embodiments, these disinfecting water or aqueous
mixtures are capable of killing the pathogens described above, and
including Escherichia coli, Staphylococcus aureus, Staphylococcus
epidermidis, Acetinobacter baumanii, Candida albicans, and Candida
glabrata. In other independent embodiments, these disinfecting
water or aqueous mixtures are capable of at least partially
(including completely) sterilizing surfaces (inactivating
pathogens) described herein within 5 secs, 10 secs, 30 secs, 1 min,
3 min, 5 min, 10 min, and 30 minutes of contacting the plasma
treated gels and the pathogens.
[0116] For example, and as described below (see Experiments Without
Organic Materials Added--Series 3), free living planktonic forms of
E. coli, S. aureus and MRSA, exposed to DBD plasma-treated
phosphate buffered saline (PBS) were rapidly inactivated under
clinically useful scenarios.
[0117] In some embodiments, the methods and systems described
herein may be used to sterilize or disinfect biologics such as
hands, arms, or face, and including at least one finger, palm,
wrist, forearm, elbow, and/or upper arm. FIG. 27 is an exemplary
device that may be used to disinfect (or sterilize) the arm or hand
of a person. Shown is a disinfection or sterilization system having
power supply 204 and disinfection station 202. Station 200
generates a non-thermal plasma using power supply 204 in the
presence of one or more aerosols or mists of liquids, such as water
or NAC. The plasma activates the one or more aerosols or mists of
liquid, with the mist (or aerosol) being directed to disinfection
area 208. A person places their hand 202 in the area 208 while
station 200 is generating the plasma for disinfection or
sterilization purposes. To further the ability to sterilize or
disinfect, an additional solution, solution 206, may be added.
Solution 206 may be the liquid aerosol or may be an additional
liquid that provides for additional sterilization or disinfection
capabilities.
[0118] Further embodiments provide devices for at least partially
disinfecting a human body part, comprising a power supply for
generating a plasma; a plasma-treating chamber in which a fluid and
plasma can be introduced to form a disinfection composition; a
disinfection chamber for contacting a human body part or medical
device with the disinfection composition, wherein the human body
part is at least partially disinfected upon contact with the
disinfection composition.
[0119] The plasma may be generated by a dielectric barrier
discharge, a corona discharge, or a pulsed corona discharge, ark,
spark, gliding arc, radio frequency discharge, microwave discharge
or any combination thereof and, in some cases, the plasma may be of
different type applied at different times and in different
locations.
[0120] The power supplies of such devices may generate non-thermal
plasmas having an intensity of at least about 0.1 J/cm.sup.2, or at
least about 0.5 J/cm.sup.2, or at least about 1 J/cm.sup.2, or at
least about 5 J/cm.sup.2.
[0121] In certain embodiments, the devices may contain a means for
aerosolizing or misting a liquid feed stream, such that the fluid
takes the form of an aerosol or mist either before or while
contacting the plasma. As with other embodiments described herein,
the fluid may comprise water, saline, phosphate buffer composition,
or a combination thereof. The water may be deionized or distilled
or be obtained without treatment from clean commercial sources
("tap water"), and may contain or be free of added organic
materials or additives. Various alcohols, preferably ethanol or
isopropanol, may also be used in such devices.
[0122] The devices may use water or alcohol containing at least one
added organic material as described herein, preferably comprising
one or more amino acid, anti-oxidant, and/or an alginate gel, and
preferably N-Acetyl Cysteine. The organic material may be partially
or completely dissolved in the fluid. If present, separate
embodiments provide that the organic material in the disinfection
composition may be at a concentration of at least about 2.5 mM, at
least about 5 mM, or at least about 10 mM.
[0123] As shown below, the devices may be used to disinfect medical
devices (e.g., intravenous tubing, heplocks, intravenous catheters,
a nebulizer, and urinary catheters) or human body parts (e.g.,
hand, arm, or portion thereof, including at least one finger, palm,
wrist, forearm, or elbow).
[0124] Such devices may be configured such that the surface is
contacted with the disinfection composition remote from the plasma.
This can be accomplished, for example, by configuring the device
such that the plasma-treating chamber is spatially separate from
the disinfection chamber, and the two chambers are in fluid
communication with one another. Alternatively, the plasma-treating
chamber and the disinfection chamber may be the same chamber, and
the device is configured to form the disinfection composition
before allowing contact of the disinfection with the human body
part or medical device (i.e., temporally separated).
[0125] These concepts may be exemplified in FIGS. 27 and 28. That
is, tools or other devices may be sterilized using a station
similar to station 200 of FIG. 27. Referring to FIG. 28, station
218 is configured to sterilize or disinfect items placed into
portal 212, which is similar to the disinfection area 202 of FIG.
27. As described with regards to station 200 of FIG. 27, station
218 uses power from power supply 214 to generate a non-thermal
plasma in the presence of one or more aerosols or mists of liquids,
such as water, alcohol, or NAC. The plasma activates the one or
more aerosols or mists of liquid, with the mist (or aerosol) being
directed to disinfection area 212. A device (not shown) is placed
in area 212 while station 218 is generating the plasma for
disinfection or sterilization purposes. To further the ability to
sterilize or disinfect, an additional solution, solution 216, may
be added. Solution 216 may be the liquid aerosol or may be an
additional liquid that provides for additional sterilization or
disinfection capabilities.
[0126] The present subject matter is further defined in the
following Examples. It should be understood that these examples,
while indicating preferred embodiments of the subject matter, are
given by way of illustration only. From the above discussion and
these examples, one skilled in the art can ascertain the essential
characteristics of this subject matter, and without departing from
the spirit and scope thereof, can make various changes and
modifications of the subject matter to adapt it to various usages
and conditions. Such modifications are considered to be within the
scope of the present invention.
EXAMPLES
[0127] Experiments with Added Organic Materials--Series 1
[0128] FIG. 12 shows a schematic of experimental conditions for
exemplary disinfection compositions for disinfection and
sterilization of solutions comprising Escherichia coli cells. As
used herein, "plasma treatment time" refers to the amount of time
the organic material is contacted with the plasma to form a
disinfection composition; "delay time" refers to the duration of
time between formation of the disinfection composition and
contacting a surface with the disinfection composition; and "hold
time" refers to the amount of time the surface is contacted with
the disinfection composition. The nomenclature for the following
examples is as follows: plasma treatment time (seconds):delay time
(seconds):hold time (seconds):NAC Concentration (mM). Thus, 0:0:0:0
stands for a plasma treatment time of 0 seconds, a delay time of 0
seconds, a hold time of 0 seconds, and a NAC concentration of 0 mM.
Equal amounts of Escherichia coli cells were placed in Petri dishes
(approximately 1,400 cells per dish) and were treated in duplicate
per the parameters of FIG. 12. The plasma used for treatment of NAC
was a DBD. Surviving Escherichia coli cells were visually observed
and counted. If the number of surviving cells was too numerous to
count these trials are indicated as "TNTC."
TABLE-US-00003 TABLE 3 Survival of Cells After Contact With
Disinfection Composition With 60 Second Plasma Treatment Time
Parameters Trial A Trial B 60:0:0:0 TNTC TNTC 60:0:0:10 TNTC TNTC
60:0:0:30 350 440 60:0:600:0 TNTC TNTC 60:0:600:10 500 300
60:0:600:30 6 7 60:60:0:0 TNTC TNTC 60:60:0:10 TNTC TNTC 60:60:0:30
TNTC TNTC 60:60:600:0 TNTC TNTC 60:60:600:10 502 0 60:60:600:30 0
0
TABLE-US-00004 TABLE 4 Survival of Cells After Contact With
Disinfection Composition With 10 Second Plasma Treatment Time
Parameters Trial A Trial B 10:0:0:0 TNTC TNTC 10:0:0:10 TNTC TNTC
10:0:0:30 TNTC TNTC 10:0:600:0 TNTC TNTC 10:0:600:10 TNTC TNTC
10:0:600:30 1 20 10:60:0:0 TNTC TNTC 10:60:0:10 TNTC TNTC
10:60:0:30 TNTC TNTC 10:60:600:0 TNTC TNTC 10:60:600:10 TNTC TNTC
10:60:600:30 0 0
TABLE-US-00005 TABLE 5 Survival of Cells After Contact With
Disinfection Composition With 0 Second Plasma Treatment Time
Parameters Trial A Trial B 0:0:0:0 TNTC TNTC 0:0:600:10 TNTC TNTC
0:0:600:30 TNTC TNTC
[0129] Sterilization increased with an increased plasma treatment
time and increased concentration of NAC. Additionally, increased
"hold time" had an impact on increased sterilization.
[0130] Experiments with Added Organic Materials--Series 2
[0131] Plasma Treatment of Liquids.
[0132] The non-thermal plasma DBD plasma generator used in this
work employed copper electrode (38 mm.times.64 mm) covered with a
1-mm glass slide (Fischer Scientific Inc., Pittsburgh, Pa.); a 2 mm
fixed discharge gap; and a customized liquid container was designed
to maintain a liquid column of 1 mm. De-ionized water (DIW) (MP
Biomedicals Inc., Solon, Ohio), PBS, or N-acetyl-cysteine (NAC)
(Sigma Chemical Co., St. Louis, Mo.) was treated separately at
different time points. A stock solution of NAC (100 mM) was
prepared in 1.times. sterile PBS and sterilized through a filter;
aliquots were stored at -20.degree. C. until used. A freshly
prepared working solution of 5 mM NAC in PBS was used for
subsequent experiments. All liquids were treated for 0, 1, 2, and 3
min under these conditions.
[0133] Culture and Isolates of Bacterial Pathogens.
[0134] Escherichia coli (ATCC25922), Staphylococcus aureus
(ATCC25923), Acinetobacter baumannii (ATCC19606), and
Staphylococcus epidermidis (ATCC12228) strains were purchased from
American Type Culture Collection (ATCC, Manassas, Va.). All strains
were maintained and used as overnight cultures in trypticase soy
broth (TSB) for primary inoculations according to the supplier's
guidelines. Reference strains of Candida albicans and Candida
glabrata (obtained from Dr. Thomas Edlind, Drexel University
College of Medicine) were grown in yeast extract-peptone-dextrose
(YPD) medium. Hydrogen peroxide (Sigma) or 70% ethyl alcohol was
used as the known biocide agent and either TSB alone or PBS alone
was used as the negative control, as appropriate.
[0135] Plasma Fluid-Mediated Bactericidal and Fungicidal
Activity.
[0136] A given pathogen was cultured overnight, inoculated (10
.mu.l) into TSB medium (10 ml), and incubated at 37.degree. C. for
4 hours on an orbital shaker incubator; the optical density at 600
nm (OD.sub.600) was adjusted to 0.2 before use. The culture
dilution thus prepared (1:100) was mixed with plasma-treated
liquids (50 .mu.l:50 .mu.l) and held together at room temperature
for 0- to 15-min intervals. After holding (holding time), the
culture was diluted appropriately with sterile 1.times.PBS and
spread on trypticase soy agar plates to incubate at 37.degree. C.
for 24 hours. After the culture was incubated, the colony forming
units (CFU) were counted to quantify surviving pathogen cells. Some
of the experiments were carried out using 10.sup.6 to 10.sup.9
CFU/ml (as initial cell numbers) to determine cell
density-dependent rates of inactivation. Plates that did not show
any growth were incubated further up to 72 h and observed every 24
h for possible growth. Similarly, the cultures of 0.2 OD.sub.600
were diluted (1:100) and exposed to plasma-treated fluid (50
.mu.l:50 .mu.l), mixed, and held for variable times. The sample was
centrifuged at 8000 rpm for 10 min; the supernatant was removed to
collect the cell pellet. The pellet was re-suspended in XTT reagent
to carry out the XTT assay. The untreated or treated biofilms were
processed for XTT assay to determine whether the bacteria embedded
in the biofilms had been inactivated. Ethanol (70%) was used as a
positive control for the biofilm experiments.
[0137] Delay Time, Holding Time, and Fluid-Aging Experiments.
[0138] Holding time (also known as contact time) was defined as the
time that plasma-treated liquid came in contact with the bacterial
suspension. To evaluate the effect of holding time, plasma-treated
fluid was exposed to bacteria for variable periods of time. For 0
min holding time, plasma-treated fluid was exposed to bacteria and
then immediately mixed thoroughly by micropipetting; a standard
colony-counting assay was performed. For longer holding times, the
plasma-treated fluid and bacteria were mixed and held together for
the desired time in the same tube at room temperature; after the
desired time, the standard colony-counting assay was performed.
[0139] Delay time is defined as the time that starts immediately
after treatment with plasma until the exposure of the
plasma-treated liquid to bacteria. To evaluate the effect of delay
time, different time points were selected (0 min-3 mo). For 0 min
delay time, plasma-treated liquid was exposed to bacteria
immediately after plasma treatment, and the standard
colony-counting assay was performed. For prolonged delay time
points, plasma-treated liquid was stored either at +4.degree. C.
(in the refrigerator) or at room temperature in microtubes sealed
with parafilm. Once the tube containing the sample was opened, to
avoid contamination of the fluid, the sample was used for the
experiments at hand and never reused.
[0140] For aging experiments, the plasma-treated NAC solution was
kept in a thermostatically controlled incubator set to an elevated
temperature (55.degree. C.) and incubated over time. The protocol
of the U.S. Food and Drug Administration (FDA) was used for aging
pharmaceutical compounds. In brief, 1 ml of plasma-treated NAC
solution was immediately transferred into 3-ml glass vials; the
screw caps were replaced; the vials were sealed with parafilm and
put upright in racks kept in the incubator. At the indicated time
point, one vial was removed, and the antimicrobial property of the
liquid was tested using the colony count assay described above.
[0141] Data Analysis.
[0142] All experiments had built-in negative and positive controls
as stated. The initial concentrations of bacteria (untreated
samples or 0 time treatment samples) were taken as 100% surviving
cells to calculate percent inactivation (unless specifically
stated). Wherever needed, Prism software v4.03 for Windows
(Graphpad, San Diego, Calif.) was used for analysis. AP value was
derived using pair comparisons between two bacterial groups with a
student t test and one-way analysis of variance for multiple
comparisons. A P value of <0.05 was considered statistically
significant.
[0143] Results: Plasma-Treated Liquids Inactivated Bacteria in a
Concentration-Dependent Manner.
[0144] FIG. 16B shows that all three plasma-treated liquids (NAC
solution, PBS, and de-ionized water) carry strong antimicrobial
properties; and less than 3 min of treatment with plasma generated
a sufficient amount of energy transfer into the liquids to effect
complete inactivation of planktonic E. coli. Even by the end of 2
min, there was significant inactivation by the NAC solution
(P<0.05), PBS (P<0.05), or de-ionized water (P<0.05)
compared with the respective controls (0 min or no treatment). The
plasma-treated NAC solution had the most powerful antimicrobial
effect. With the treatment for 1 min (14.5 J/cm.sup.2), it
inactivated a highly significant amount of E. coli (P<0.05)
compared to de-ionized water or PBS alone for comparable times.
During exposure to plasma-treated liquid, free-floating planktonic
bacteria (10.sup.7 CFU/ml) were found to be inactivated. Therefore,
cell suspensions of various CFUs of E. coli were exposed to
plasma-treated liquids. FIG. 16C shows colony assays that indicate
rapid inactivation of bacterial cells. The CFUs of the initial cell
suspensions were considered to be 100%, and the rates of
inactivation were compared. The treated liquid has shown 100%
inactivation (sterilization) when exposed to
.about.1.times.10.sup.8 CFU/ml and .about.25% inactivation with
5.times.10.sup.8 to 1.times.10.sup.9 CFU; it exhibits strong
antimicrobial effects (P<0.05) compared with the respective
untreated samples.
[0145] Plasma-Treated NAC Solution is a Powerful Antimicrobial
Agent Against a Range of Pathogens.
[0146] A range of common pathogens, such as S. aureus, S.
epidermidis, A. baumannii, C. albicans, and C. glabrata, in
addition to E. coli, were tested at concentrations of NAC (1 mM-20
mM) with or without plasma treatment. It was found that 5 mM was
sufficient and showed a linear relationship during inactivation
studies in colony assays. Higher concentrations of NAC did not show
significantly different efficacy (data not shown). Studies in
planktonic forms showed that the NAC solution completely
inactivated all of the pathogens tested (FIG. 16D-E) after 3 min of
treatment with plasma. Only C. glabrata required 3.2 min (195 sec)
of treatment with the liquid to achieve 100% inactivation
(sterilization) of this fungal pathogen (data not shown). Most of
the pathogens in their biofilm form were equally or slightly more
sensitive to the biocidal effect of the treated NAC solution.
[0147] Determination of holding time, delay time, and accelerated
aging of solution.
[0148] The contact time (holding time) of the antimicrobial agent
with the pathogen is critical. Often, the biocidal effect is
proportional to the initial contact time. FIG. 16F shows that, on
exposure to the plasma-treated NAC solution, significant
inactivation of E. coli occurred from 2 min of holding time onward
(P values for 2, 3, and 5 min against 0 min<0.05); after about 5
min, the bacteria were .about.98% inactivated; after less than 15
min, the bacteria were completely inactivated (sterilized), upon.
Similarly, to determine how long plasma-treated fluids retain
antimicrobial effect at room temperature, delay time experiments
were done. That is, mixing the post-treatment fluid (NAC solution)
with the pathogen was delayed to determine whether the fluid lost
its antimicrobial properties. FIG. 16G shows the antimicrobial
efficacies of treated liquid delayed over time, post-treatment. It
is apparent that treated fluid retained significant antibacterial
effect (P<0.05; against control, C) for up to 90 days and showed
complete inactivation of E. coli.
[0149] Experiments were also conducted to assess the shelf life of
the plasma-generated antimicrobial properties of the NAC solution,
using accelerated aging scenarios for medically important solutions
and devices at 37.degree. C. and 50.degree. C. over time,
equivalent to 2 years of delay time at room temperature. FIG. 16H
illustrates days of accelerated aging time at the respective
temperatures and equivalent delay time at room temperature.
Complete inactivation (sterilization) was observed when E. coli at
107 CFU/ml were exposed to the solution. Because there were no
colonies, the data are not shown. This result indicates that the
plasma-treated NAC solution has the potential to act as an
antimicrobial solution for local application.
[0150] Experiments with Alginate Gels--Series 1
[0151] Freshly prepared calcium alginate gels were directly treated
with plasma (0.6 W/cm2; 1250 Hz) over time at room air, and
pathogen was inoculated onto the alginate dressing. After a contact
time of 15 minutes, the pathogen cells were harvested and tested
for viability. Standard colony and XTT assays were used to evaluate
biocidal efficacies. Scanning electron microscopy (SEM) was used to
identify any gel surface-associated changes. Staphylococcus aureus,
S. epidermidis, Acinetobacter baumannii, Escherichia coli, Candida
albicans, and C. glabrate were tested as representative pathogens
(10.sup.6-10.sup.9 CFU/mL). The thickness of the dressing was 1 mm
and the distance between plasma probe and the gel surface was kept
1 mm constant.
[0152] The results showed that plasma treatment of alginate gels
generated a strong biocidal property by completely inactivating
(sterilizing) all of the pathogens tested within 15 seconds of
plasma treatment. Optimization experiments revealed that 15 minute
contact time was sufficient for complete inactivation of the
pathogens even at 10.sup.8 CFU/mL. In 1 min of plasma treatment and
15 minutes of contact time, 10.sup.9 CFU/mL were completely
inactivated. The biocidal effect was retained in the dressing for
more than a month at room temperature. SEM imaging revealed no
damage of the gel surfaces even upon plasma treatment of 3
minutes.
[0153] Experiments with Alginate Gels--Series 2
[0154] Dressing Preparation--
[0155] Sodium alginate (M/G Ratio 1.6, Sigma-Aldrich, USA) was
suspended in deionized H.sub.2O, sterile filtered, poured into a
mold, and then cross-linked with calcium to form circular hydrogels
(26 mm diameter). Hydrogels were then treated with non-thermal,
dielectric barrier discharge, micropulse plasma (1.2 Watt/cm.sup.2,
31 kV, 1500 Hz) for three minutes. The plasma was generated using
an electrode constructed with a 38.times.64 mm copper plate
insulated in silicone and a 1 mm-thick glass sheet. The discharge
gap between the bottom of the quartz and the treated sample surface
was fixed 1 mm for treatment. Silver-(Acticoat.RTM. Smith &
Nephew, a dressing made from alginate fibers coated in silver
nanoparticles) and chlorhexidine-containing (Tegaderm CHG.RTM.3M, a
dressing made from sodium alginate with chlorhexidine in
suspension) dressings were used for comparison to the test
dressings. Sontara (DuPont) a sterile, nonwoven absorbent material
was used as a control. 26 mm discs were prepared in an aseptic
manner and kept in sterile vials for use in the log reduction
assays.
[0156] Log Reduction Assay--
[0157] Untreated alginate hydrogel and Sontara were used as
controls. For aerobic bacteria, colonies from an overnight culture
were inoculated in 5 mL of tryptic soy broth and incubated for 4 to
6 hours at 37.degree. C. to reach an ODU 600 of 0.2 (0.5 McFarland
turbidity). Volumes of 50 .mu.l of the test bacterial suspensions
were transferred onto the dressings in sterile vials. After 15
minutes of incubation at room temperature, samples were washed for
5 minutes with 10 mL sterile PBS with gentle agitation to
homogenize the solution, out of which triplicate samples were
taken. Bacterial survival numbers were obtained using a standard
plate count procedure, and the log reduction was calculated as the
difference between the log numbers of bacteria surviving on the
control dressings and the test dressings. Plates remained incubated
72 hours to assess for delayed reactivation.
[0158] SEM Observations--
[0159] The morphologies of alginate hydrogels before and after
exposure to non-thermal plasma and of bacteria on the surface of
those hydrogels were observed using the Zeiss Supra 50VP Scanning
Electron Microscope. Samples were fixed, dehydrated, and
sputter-coated with a platinum/palladium alloy prior to
imaging.
[0160] Results--
[0161] Gels exposed to as few as 15 seconds of non-thermal plasma
treatment resulted in complete inactivation of 10.sup.7 CFU/mL E.
coli (control material yielded only a 2.7-log reduction and
untreated alginate gels yielded a 2.3 log-reduction). The
bactericidal effect was also complete with longer durations of
plasma treatment (FIG. 17). Comparison of SEM images suggests that
exposure to non-thermal plasma does not alter the hydrogel surface
(image 1). SEM micrographs of bacteria on the surface of treated
hydrogels show extensive damage as compared to those on an
untreated hydrogel (not shown).
[0162] In order to determine the killing capacity of the
plasma-treated gel dressings, increasing concentrations of bacteria
were exposed to gels treated with either 15 seconds or 3 minutes of
plasma exposure. As illustrated in FIG. 18, gels treated with 3
minutes of gel exposure completely inactivated 10.sup.9 CFU/mL E.
coli, while gels treated for 15 seconds were able to completely
inactivate 10.sup.8 CFU/mL E. coli, but any greater concentration
exceeded the bactericidal capacity.
[0163] In order to test the spectrum of activity, a variety of
pathogens were exposed to the plasma-treated gel dressings.
Pathogens tested were: Escherichia coli ATCC 25922, Staphylococcus
aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228,
Acetinobacter baumanii ATCC 19606, Candida albicans, and Candida
glabrata (Candida specimens generously provided by Dr. Thomas
Edlind, PhD, Drexel University College of Medicine). As
demonstrated in FIG. 19, the gels treated with 15 seconds or longer
of plasma exposure resulted in complete inactivation of all
pathogens at 10.sup.7th concentration.
[0164] Speed of killing was examined by carrying out the
log-reduction test while varying the duration of contact between
the test dressing and bacteria (the holding time). The
plasma-treated gel dressings (3 minutes of plasma treatment pre
exposure) were compared to the commercially available chlorhexidine
and silver based dressings listed above. The chlorhexidine dressing
resulted in the most rapid killing with a 1.5-log reduction at 1
minute and complete inactivation at 5 minutes (FIG. 20). The
plasma-treated gels resulted in total inactivation at 10 minutes.
The performance of the silver containing dressing and the untreated
alginate were roughly equivalent over the 15-minute time period
examined.
[0165] The antimicrobial effect of the plasma-treated gel dressings
started to dissipate by four weeks of storage (FIG. 21). After
treatment, the gels were stored in either a non-airtight container
(where the treated surface did not come in contact with anything
but air) placed in a refrigerator and kept at constant temperature
and humidity or in a vacuum-sealed container (where the treated
surface came in contact with the plastic container). Regardless of
storage method, the gels maintained the antimicrobial effect for 21
days but it was diminished by 28 days.
[0166] Experiments without Organic Materials Added--Series 1
[0167] FIG. 22 shows changes in of E. coli concentration as a
function of plasma treatment time for two different initial
concentrations of 3.times.10.sup.5 and 2.times.10.sup.3 CFU/mL. For
this experiment, 25 .mu.l of E. coli was dried on a glass slide (to
prevent an interference of organics that might have been present in
E. coli medium) then mixed with plasma treated water, after 2 min
(holding time) was plated and counted following 24 hour incubation.
In both cases bacteria were fully inactivated by plasma treated
water for treatment time of 90 sec (11.7 J/cm.sup.2).
[0168] Experiments without Organic Materials Added--Series 2
[0169] Experimental Conditions--
[0170] E. coli aliquots of 9-, 8-, 7-, and 6-Log 10 CFU/ml were
prepared from fresh overnight cultures in trypticase soy broth
(TSB) to generate killing curves. For biofilms, the overnight
culture was diluted to 1:100 in TSB and allowed to established
biofilms for 24 h/37.degree. C. (detected by microplate/Safranin
assay). Phosphate-buffered saline (PBS) was exposed to plasma for
various time (amount of plasma energy) and the culture aliquot or
biofilm bearing surface was then treated with it, and processed
further for the demonstration of percent surviving cells. In
another set, the 1:100 diluted overnight-cultures was mixed with
plasma-treated PBS and incubated as above. Standard colony count
assay, Safranin assay, XTT assay and Live/Dead BacLight bacterial
viability kit were used for this purpose. The Plasma energy was
generated corresponding to energies (J/cm.sup.2) as a function of
time of 0 (0 sec), 1.95 (15 sec), 3.9 (30 sec), 7.8 (60 sec), and
11.7 (90 sec), 15.6 (120 sec), 23.4 (180 sec), 39 (300 sec).
[0171] Results--
[0172] A plasma (energy) dose-dependent and bacterial cell
density-dependent killing responses were observed in all three
conditions of E. coli. To 30 second's plasma exposure (3.9
J/cm.sup.2), planktonic forms resulted in about 60%, 89%, 97% and
100% killing of respectively 9, 8, 7, and 6 Log 10 cells.
Biofilm-embedded E. coli were 100% killed (treatment of biofilms)
in 120 seconds, and were comparable to 70% ethanol treated biofilms
(for 1 h). The exposure of plasma-treated PBS resulted in
significant reduction (p<0.05) in biofilm formation (prevention
of biofilms). The findings of colony assay, XTT and BacLight assay
were comparable.
[0173] Experiments without Organic Materials Added--Series 3
[0174] Culture and Isolates of Bacterial Pathogens:
[0175] E. coli, S. aureus (also referred as methicillin-sensitive
S. aureus, MSSA, occasionally), MRSA USA300 (BAA-1680) and MRSA
USA400 (BAA-1683) strains were purchased from American type culture
collection (ATCC, Manassas, Va.). Clinical isolates of MRSA
(referred as MRSA95) was isolated from Hahnemann University
Hospital's clinical laboratory from patients with skin and soft
tissue infection, and identified by API and VITEK 2 automatic
systems (BioMerieux, Inc., Durham, N.C.). All strains were
maintained and used as overnight cultures in trypticase soy broth
(TSB) for primary inoculations. Hydrogen peroxide or 70% Ethyl
alcohol were used as known biocide agents, and either TSB alone or
PBS alone as negative controls, as appropriate.
[0176] DBD-Plasma Generating Device, Parameters and Conditions:
[0177] Experiments were done using a Floating-electrode DBD
(FE-DBD) plasma generator, using a first electrode (which was a
dielectric protected powered electrode) and a second electrode (the
sample-carrying surface). The second electrode was not grounded and
remains floating having potential to ignite discharge plasma when
the powered (first) electrode approaches the surface to be treated.
The diameter of plasma generating probe was 25 mm (sufficient for
15 mm diameter area under treatment). A low-frequency alternating
current (120V) was generated and the desired output voltage and
frequency (Hz) was obtained through a step transformer across the
interface. A typical plasma power of 0.13 W/cm.sup.2 was used for
variable times of exposures in order to deposit desired amount of
plasma energy (J/cm.sup.2) to the sample to be treated. The
micro-pulse mode has fair uniformity, and therefore uniform
deposition of plasma energy is possible. In these experiments, 100
.mu.l of phosphate-buffered saline (PBS, 150 mM sodium chloride and
150 mM sodium phosphate, pH 7.2 at 25.degree. C.) was exposed to
plasma being discharged, and 80 .mu.l of treated PBS immediately
applied (no delay time) to the surface or suspension of interest.
The plasma energy (J/cm.sup.2) levels which were used in the
present research (otherwise stated) are as follows: 0 (0 seconds,
s), 0.39 (3 s), 0.78 (6 s), 1.56 (12 s), 1.95 (15 s), 3.12 (24 s),
3.9 (30 s), 7.8 (60 s), 11.7 (90 s), 15.6 (120 s), and 19.5 (150
s), etc.
[0178] Plasma Bactericidal Activity and Planktonic Forms:
[0179] The cultures of interest were inoculated as 10 .mu.l primary
cultures into 10 ml TSB medium and incubated at 37.degree. C. for
24 h at 180 rpm and then appropriately diluted to obtain the
desired number of cells (CFU/ml). Concurrently, the optical
densities at 580 nm and colony count assays were performed in
parallel to select the desired cell densities. In some of the early
experiments, overnight cultures were used to prepare bacterial
lawns on trypticase soy agar plates, and after plasma treatment or
non-treatment, plates were incubated at 37.degree. C. up to 48 h
and observed periodically at 18 h, 24 h, and 48 h for zone of
inhibition of growth. Samples of 25 .mu.l of PBS was exposed to
plasma and then immediately mixed with 25 .mu.l of 2.times. cell
suspension (to have final 1.times. cell suspension in PBS), held
for 5 min before serial dilution for colony count. The plates were
observed periodically as above, and colonies were counted to
calculate total CFU/mL.
[0180] Bacterial Pathogens as Biofilms:
[0181] Biofilms were developed for E. coli, S. aureus (MSSA),
MRSA-95, MRSA-USA300 and -USA400 in sterile 96 well plates (all
Costar, Corning Inc., Corning, N.Y.). An overnight culture was
diluted to 1:100 with sterile TSB, and 200 .mu.l of suspension was
applied to the 96-well plates, in triplicates to incubate for 24 h
at 37.degree. C. without shaking. The next day, fluid from the
biofilm containing 96-well plates was gently aspirated, and washed
three times with PBS, and either left untreated or treated with the
plasma-treated PBS, and then washed two times post-treatment with
PBS before treatment and then used for appropriate staining or
detection of metabolic activity.
[0182] Safranin Assay:
[0183] Biofilms developed in a 96-well plate were washed as above,
and dried briefly by holding at 37.degree. C. (20 min) to stain by
the known Safranin microtiter plate method. The biofilms were
stained with 200 .mu.l of 0.1% aqueous solution of Safranin for 15
min. The excess stain was removed by washing three times with PBS.
After addition of 70% ethanol the biofilm-containing wells were
held at room temperature for 15 min. The plate was read in
microtiter plate reader (Synergy, BioTek, Winooski, Vt.) after
brief shaking (3 sec) at 550 nm. The well containing TSB alone plus
Safranin was used as negative control to normalized readings. This
Safranin assay is used for semi-quantification of biofilms, and is
often the test used for detection of biofilms in clinical
isolates.
[0184] Other Assays--
[0185] The LIVE/DEAD BacLight Bacterial Viability assay kit is
routinely used for detection of efficacy of bactericidal agents,
and quantification of biofilms, and was used as recommended by the
supplier (Molecular Probes, Invitrogen, CA).
[0186] For each XTT assay, fresh XTT reagent solutions were
prepared as described earlier. From the aliquots, 0.5 mg XTT
(Molecular Probe) and 1 uM Menadione (Sigma Chemical Co.) working
solution was made up in 1.times.PBS. After appropriate treatment or
no treatment, the microtubes containing samples of planktonic form
of pathogen were spun at 8000 rpm/6 min, and supernatant discarded.
The cells were resuspended in 200 .mu.l of XTT reagent, mixed
thoroughly, and tubes incubated at 37.degree. C./2 h. After
centrifugation, the supernatant (100 .mu.l) containing orange
colored XTT metabolic product was measured by reading absorption at
492 nm using a microtiter plate reader (Synergy Mx, BioTek). The
readings were normalized and percent surviving cells were
calculated against untreated samples. Similarly, the XTT assay for
biofilms was carried out in the 96 well plates (200 .mu.l for each
well) and no spinning was involved.
[0187] Statistical Analysis of Data--
[0188] The data from the experiments were analyzed using Prism
software 4.03 for Windows (GraphPad, San Diego, Calif.), and
standard deviations calculated from minimum three sets of
experiments. All the experiments were repeated in triplicate. The
p-value was derived using pair comparisons between two bacterial
groups using Student t-test and one way ANOVA for multiple
comparisons wherever applicable (* indicates p value as a
statistically significant (p<0.05)).
[0189] Results--
[0190] Free living planktonic forms of E. coli, S. aureus and MRSA,
exposed to DBD plasma-treated phosphate buffered saline (PBS) were
rapidly inactivated under clinically useful scenarios. About
10.sup.7 bacterial cells were completely (100%) killed whereas
10.sup.8 and 10.sup.9 were reduced by about 90 to 95% and 40 to 45%
respectively, in less than 60 seconds (7.8 J/cm.sup.2) and
completely disinfected in <120 seconds. In established biofilms,
the susceptibility of MRSA USA400 was comparable to USA300, but
less susceptible than MRSA95 (clinical isolate), S. aureus, and E.
coli (p<0.05) to FE-DBD-plasma, and plasma was able to kill MRSA
more than 60% within 15 seconds (1.95 J/cm.sup.2). The killing
responses were plasma-exposure time- and cell density-dependent.
The plasma was able disinfect surfaces in a less than 120
seconds.
[0191] Dose-Dependent Responses of Planktonic Cultures and Surface
Contaminants to Non-Thermal Plasma--
[0192] The efficacy of plasma applications on representative
bacteria (one each from gram-positive and gram-negative groups)
were quantified using colony count assay methods described above.
The findings of the colony count assay were highly reproducible,
when carried in three to five sets, each in triplicate. As shown in
FIG. 23, E. coli was significantly more susceptible as compared to
S. aureus (which took little more plasma energy for comparable
inactivation (p<0.05). Sterilization of both the organisms (at
.about.10.sup.7 CFU/ml) was observed with PBS treated with 7.8
J/cm.sup.2 plasma.
[0193] Time to Plasma-Mediated Sterilization is Proportional to
Bacterial Load: The Cell-Density-Dependent Responses--
[0194] A series of experiments were conducted using 10.sup.9,
10.sup.8, 10.sup.7, and 10.sup.6 CFU/ml to evaluate plasma's
antibacterial efficacy. The inactivation process took a longer time
for all the organisms, viz. E. coli, S. aureus, and MRSA95 at
higher loads (10.sup.9 CFU/ml) versus sterilization at 10.sup.6
CFU/ml when exposed to PBS treated with for 30 sec to either direct
or indirect plasma. FIG. 24 indicates that plasma can be safely
applied to achieve 100% disinfection of these organisms (10.sup.6
CFU/ml), including multidrug resistant MRSA isolates in less than
30 sec (3.9 J/cm.sup.2).
[0195] Plasma Potential of Sterilization of MRSA-USA300 and
-USA400, in Both Planktonic & Embedded Biofilms Forms:
[0196] Tests were also conducted using a multidrug resistant
clinical MRSA isolate (MRSA95), and the most widely reported
virulent biofilm-producing strains, the USA300 and USA400 for their
comparative responses in the midst of biofilms. FIG. 25
demonstrates the percent survival for cells of all three MRSA
strains tested in this study during plasma exposure. For comparison
biofilms embedded E. coli and S. aureus (MSSA) strains were
included in these tests. Given that the XTT assay is a gold
standard to demonstrate viable cells, the XTT assay findings from
FIG. 25 show that E. coli in biofilm form offers more resistance to
plasma inactivation than S. aureus and USA 400 and USA 300 at
various plasma energy levels. MRSA USA400 was slightly more
resistant than MRSA USA300 in biofilm forms, when plasma was
applied directly, but nevertheless the methodology sterilized all
biofilm form of all the pathogens (all p<0.05) in less than 120
sec (15.6 J/cm.sup.2). These tests also showed that the biofilms
which are resistant to many of the powerful biocides were totally
disinfected in a short time (<120 sec/<15.6 J/cm.sup.2).
[0197] Experiments without Organic Materials Added--Series 4
[0198] An agar place of bacterial (containing colonies of 10.sup.5
E. coli) was placed on top of a grounded metal plate. A needled
syringe containing 98% ethanol and having a drip rate of
approximately 0.5 mL/min) was configured so that the needle was
positioned 5'' above the surface of the agar plate. A 10 kV
positive polarity DC corona charge was attached to the needle,
which generated an electric field between the tip of the needle and
the grounded plate, the resulting corona discharge causing the
dropping ethanol to aerosolize into a mist and be attracted to the
surface of the agar plate. In separate duplicate experiments,
treatment times of 5 sec, 15 sec, and 30 sec resulted in the
complete disinfection of the E. Coli. In control experiments (i.e.,
no treatment), the bacteria showed no evidence of disinfection. In
experiments wherein the alcohol was dispersed for 15 sec, but where
no corona discharge was applied, some spotty effects were observed,
but the sterilization was quantitatively less and less uniform than
when the plasma was applied.
[0199] Preparing and Reconstituting Dry Powder Disinfectants
[0200] A series of experiments were conducted to determine the
efficacy of powders obtained from plasma-treatment (either as
solutions or powders) and compositions prepared from reconstituting
the same. In these experiments, NAC was used as the disinfecting
agent. FIG. 26A shows the results of a series of experiments in
which NAC was partially dissolved in water to provide a dispersion
of solid NAC in 5 mM NAC solution. The mixture was treated with
plasma, the solid and liquid phases were separated, and the water
was removed at room temperature from the liquid phase to provide a
powder. The two solids were then separately rehydrated with PBS and
deionized water. Equal portions (50 .mu.L) of E. coli bacteria
suspension and the rehydrated NAC were mixed together. FIG. 26B
shows the results of testing when NAC powder was exposed to plasma
treatment, rehydrated and tested. The test conditions used to
generate FIGS. 26C &D were similar to those used to generate
FIG. 26A, except that the solutions were heated after plasma
treatment.
[0201] While the embodiments have been described in connection with
the preferred embodiments of the various figures, it is to be
understood that other similar embodiments may be used or
modifications and additions may be made to the described embodiment
for performing the same function without deviating therefrom.
Therefore, the disclosed embodiments should not be limited to any
single embodiment but rather should be construed in breadth and
scope in accordance with the appended claims.
[0202] As those skilled in the art will appreciate, numerous
modifications and variations of the present invention are possible
in light of these teachings, and all such are contemplated hereby.
For the sake of brevity, each and every combination is not provided
here, but the skilled artisan would appreciate that, in addition to
the embodiments described herein, the present invention
contemplates and claims those inventions resulting from the
combination of each and every feature of the invention cited herein
and those of the cited prior art references which complement the
features of the present invention. Similarly, it will be
appreciated that any described material, feature, or article may be
used in combination with any other material, feature, or article,
and such combinations are considered within the scope of this
invention.
[0203] The disclosures of each patent, patent application, and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
* * * * *