U.S. patent application number 16/368038 was filed with the patent office on 2019-09-19 for ionic liquids that sterilize and prevent biofilm formation in skin wound healing devices.
The applicant listed for this patent is The Arizona Board of Regents on Behalf of Northern Arizona University, Dixie State University. Invention is credited to Rico Del Sesto, Robert Kellar, Andrew Koppisch, Nathan Christopher Nieto.
Application Number | 20190282728 16/368038 |
Document ID | / |
Family ID | 61757547 |
Filed Date | 2019-09-19 |
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United States Patent
Application |
20190282728 |
Kind Code |
A1 |
Kellar; Robert ; et
al. |
September 19, 2019 |
IONIC LIQUIDS THAT STERILIZE AND PREVENT BIOFILM FORMATION IN SKIN
WOUND HEALING DEVICES
Abstract
Compositions for enhancing wound healing are disclosed herein.
Also disclosed are methods of making the compositions and methods
of using the compositions for the prevention of biofilm formation
and for the inhibition of pathogen growth and proliferation.
Inventors: |
Kellar; Robert; (Flagstaff,
AZ) ; Nieto; Nathan Christopher; (Flagstaff, AZ)
; Koppisch; Andrew; (Flagstaff, AZ) ; Del Sesto;
Rico; (St. George, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Arizona Board of Regents on Behalf of Northern Arizona
University
Dixie State University |
Flagstaff
St. George |
AZ
UT |
US
US |
|
|
Family ID: |
61757547 |
Appl. No.: |
16/368038 |
Filed: |
March 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15725213 |
Oct 4, 2017 |
10293080 |
|
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16368038 |
|
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62404369 |
Oct 5, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
D01F 1/103 20130101;
A61L 26/0038 20130101; A61L 26/0033 20130101; A61K 31/14 20130101;
A61K 9/0014 20130101; D01D 5/0015 20130101; A61L 2300/404 20130101;
A61L 26/0042 20130101; A61K 38/39 20130101; A61L 15/44 20130101;
A61L 15/32 20130101; A61K 31/20 20130101; D01D 5/003 20130101 |
International
Class: |
A61L 26/00 20060101
A61L026/00; A61K 38/39 20060101 A61K038/39; A61K 31/20 20060101
A61K031/20; A61K 31/14 20060101 A61K031/14; A61L 15/32 20060101
A61L015/32; A61K 9/00 20060101 A61K009/00; A61L 15/44 20060101
A61L015/44; D01F 1/10 20060101 D01F001/10; D01D 5/00 20060101
D01D005/00 |
Claims
1. An electrospun wound care composition, comprising: an ionic
liquid (IL), wherein the ionic liquid is choline geranate (CAGE),
and wherein the ionic liquid is present in an amount of about 0.01%
w/w to about 60% w/w; and a protein solution comprising collagen,
agarose, albumin, alginate, casein, fibrin, fibroin, gelatin,
keratin, pectin, elastin, tropoelastin, cellulose, chitosan,
chitin, or combinations thereof, wherein the ionic liquid is
electrospun with the protein solution.
2. The wound care composition of claim 1, wherein the ionic liquid
is present in an amount ranging from about 0.01% w/w to about 40%
w/w.
3. The wound care composition of claim 2, wherein the ionic liquid
is present in an amount of ranging from about 0.01% w/w to about
20% w/w.
4. The wound care composition of claim 2, wherein the ionic liquid
is present in an amount of about 0.2% w/w.
5. The wound care composition of claim 2, wherein the protein
solution is present in an amount of about 1% w/v to about 20%
w/v.
6. The wound care composition of claim 2, wherein the protein
solution is present in an amount of about 10% w/v.
7. The wound care composition of claim 2, comprising about 0.2% w/w
choline geranate (CAGE) and about 10% w/v protein solution.
8. The wound care composition of claim 2, wherein the wound care
composition is incorporated with or impregnated into or coated onto
a wound dressing, a bandage, a gauze, a patch, a pad, tape, or a
wrap.
9. A wound dressing comprising: a wound care composition of claim
2; and a wound dressing material.
10. The wound dressing of claim 9, wherein the wound dressing
material is a bandage, a wipe, a sponge, a mesh, a dressing, a
gauze, a patch, a pad, tape, or a wrap.
11. The wound dressing of claim 9, wherein the wound care
composition is present in an amount of about 0.005% v/w to about 2%
v/w.
12. The wound dressing of claim 9, wherein the wound care
composition is present in an amount of about 0.625% v/w.
13. The wound dressing of claim 9, wherein the wound care
composition is incorporated into or coated onto or impregnated with
the wound dressing material.
14. A method of making the wound care composition according to
claim 2 comprising an ionic liquid (IL) and a protein solution, the
method comprising: providing a protein solution comprising
collagen, agarose, albumin, alginate, casein, fibrin, fibroin,
gelatin, keratin, pectin, elastin, tropoelastin, cellulose,
chitosan, chitin, or combinations thereof; providing an ionic
liquid, wherein the ionic liquid is choline geranate (CAGE); mixing
the ionic liquid with the protein solution; and electrospinning the
mixture of ionic liquid and protein solution.
15. The method of claim 14, wherein the solvent is the ionic liquid
or an organic solvent.
16. The method of claim 15, wherein the organic solvent is
hexafluoro-2-propanol (HFIP).
17. The method of claim 14, wherein the mixing is performed at a
temperature of about 5.degree. C. to about 100.degree. C.
18. The method of claim 14, wherein the electrospinning is
performed at a temperature of about 5.degree. C. to about
100.degree. C.
19. The method of claim 14, wherein the electrospinning is
performed at a voltage of about 10V to about 50V.
20. The method of claim 14, wherein the electrospinning is
performed at a flow rate of about 0.1 mL/hr to about 5 mL/hr.
21. The method of claim 14, wherein the electrospinning is
performed at a flow rate of about 1 mL/hr.
22. The method of claim 14, further comprising desiccating the
electrospun material to dryness.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/725,213, filed Oct. 4, 2017, which claims
the benefit of priority to U.S. Provisional Application No.
62/404,369, filed Oct. 5, 2016, the disclosure of each of which is
hereby expressly incorporated by reference in its entirety.
FIELD
[0002] The present disclosure relates generally to the field of
wound healing. In particular, the disclosure relates to
compositions for enhancing wound healing. Such compositions include
an ionic liquid and a protein scaffold. The compositions can be
impregnated into a wound healing device, such as a bandage, or
dressing. The disclosure also relates to methods of making the
compositions and using the compositions to treat wounds.
BACKGROUND
[0003] Wound healing is a complex cascade of events that attempts
to maintain homeostasis in the wounded tissue. Closing of the wound
is essential to the healing process as it protects the tissue
itself from infection with foreign agents, such as opportunistic
bacterial pathogens. Biofilms established by these pathogens are a
common cause of chronic infections that slow the process of wound
healing. The bacterial biofilms themselves are challenging to
eliminate with conventional antibiotics due to an extensive
exopolymeric layer covering the pathogen that limits diffusion of
these drugs into the biofilm. Ionic liquids, such including deep
eutectic solvents (DESs), is a family of molecules with diverse
chemical properties.
[0004] When considering dermal wound healing, there is a driving
need for the organism to close the wound from the environment as a
form of protection. If the wound does not close in a sufficient
amount of time, foreign agents can enter the body and have serious
pathological effects. Additionally, in a clinical environment,
various devices and/or materials are implanted into the integument
(skin) to treat the skin or other tissues and organs. In these
cases, altering the skin environment can present significant risks
to the organism (patient) for bacteria, viral, or fungal
contamination of the compromised skin.
[0005] A specific patient population at significant risk for open
wound contamination by biofilm formation includes unmanaged
diabetic patients who develop chronic, non-healing wounds. Diabetic
foot ulcers affect an estimated 1.4 million people in the U.S.,
resulting in enormous health care expenditures estimated at more
than $176 billion per year for 2012 (Margolis et al., Incidence of
diabetic foot ulcer and lower extremity amputation among Medicare
beneficiaries, 2006 to 2008, Data Points Publication Series, Feb.
17, 2011; Mathieu, D. (Ed.). Handbook on hyperbaric medicine (Vol.
27). New York: Springer. 2006; Menke, et al., Impaired wound
healing. Clinics in dermatology, 25(1), 19-25. 2007. Washington
health system: Washington hospital. (2014)). Diabetic patients who
have chronic disease may experience persistent wounds for months to
years. Furthermore, if healing occurs, the healed tissue has
substantial scarring and the scar may fail, resulting in recurrence
of the ulcer.
[0006] Chronic diabetic wounds pose different pathophysiological
abnormalities that contribute to a complex wound microenvironment
that varies from the "normal" wound healing cascade (Falanga, V.
Wound healing and its impairment in the diabetic foot. The Lancet,
366(9498), 1736-1743. 2005). This variation leads to a loss of
synchrony of events indicative of rapid healing. There is a
pathogenic triad of events that are predisposed in diabetic
patients consisting of neuropathy, ischemia and trauma that hinders
normal healing. Each of these factors affects each other in a way
that results in an impaired ability to fight infection and presents
difficulties in closing chronic skin wounds.
[0007] Currently, the clinical treatment of full thickness skin
wounds presents a significant challenge. Therapies for the
treatment of these skin wounds include autologous tissue grafts and
fat transplantation (replacing burnt or severely traumatized tissue
with a patient's own skin & fat tissue--taken from a distant
site), and alloplastic (synthetic) implants. However, these methods
present significant problems for the patient including donor site
morbidity, implant migration, rupture, volume reduction, and
foreign body reaction (Sterodimas, Aris, et al. Tissue engineering
with adipose-derived stem cells (ADSCs): current and future
applications. Journal of Plastic, Reconstructive & Aesthetic
Surgery 63.11: 1886-1892, 2010; Stosich, et al. Adipose tissue
engineering from human adult stem cells: clinical implications in
plastic and reconstructive surgery. Plastic and reconstructive
surgery 119.1: 71, 2007).
[0008] Furthermore, a lack of subcutaneous adipose tissue in full
thickness skin wounds contributes to the aesthetically unappealing
post-operative appearance (Shill K, et al. (2011) Ionic liquid
pretreatment of cellulosic biomass: enzymatic hydrolysis and ionic
liquid recycle. Biotechnology and bioengineering 108(3):511-520).
There remains a major clinical demand for better methods of skin
tissue healing of diabetic foot ulcers but also following severe
burns, tumor excision, and trauma (Sterodimas, Aris, et al. Tissue
engineering with adipose-derived stem cells (ADSCs): current and
future applications. Journal of Plastic, Reconstructive &
Aesthetic Surgery 63.11: 1886-1892, 2010).
[0009] In the clinical treatment of wounds, it is well established
that open skin wounds colonize with bacteria; therefore, optimized
wound care targets rapid wound closure in efforts to prevent
infection and possible sepsis in severe cases (Wysocki, Annette B.
Evaluating and managing open skin wounds: colonization versus
infection. AACN Advanced Critical Care 13.3: 382-397, 2002). Today,
wound care to prevent progression from colonization to infection
remains the paramount objective of health care providers. However,
this progression has become progressively difficult to combat due
to emergent antimicrobial resistance (Davis, S. C., et al.
Microscopic and physiologic evidence for biofilm-associated wound
colonization in vivo. Wound Repair and Regeneration, 2008, 16:
23-29).
SUMMARY
[0010] The present disclosure generally relates to wound care
compositions and methods of making and using the same.
[0011] Accordingly, in some embodiments is provided a wound care
composition including an ionic liquid and a protein scaffold. In
some embodiments, the wound care composition is formulated for
incorporation into a wound dressing, a bandage, gauze, a patch, a
pad, tape, or a wrap.
[0012] In some embodiments, the wound care composition includes an
ionic liquid and a protein scaffold. In some embodiments, the ionic
liquid is a deep eutectic solvent (DES). In some embodiments, the
ionic liquid is antimicrobial. In some embodiments, the ionic
liquid includes choline geranate (CAGE). In some embodiments, the
ionic liquid is present in an amount of about 0.01% w/w to about
99% w/w, such as 0.01, 0.05, 0.1, 0.5, 1.0, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% w/w or
greater, or a value within a range defined by any two of the
aforementioned values. In some embodiments, the ionic liquid is
present in an amount of about 40% w/v.
[0013] In some embodiments, the wound care composition includes a
scaffold. In some embodiments, the scaffold includes a protein or
polysaccharide scaffold including any protein or polysaccharide in
solution, for example, collagen, agarose, albumin, alginate,
casein, fibrin, fibroin, gelatin, keratin, pectin, elastin,
tropoelastin, cellulose, chitosan, chitin, or combinations thereof.
In some embodiments, the protein scaffold is electrospun. In some
embodiments, the protein scaffold is present in an amount of about
1% w/w to about 99% w/w, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, or 99% w/w, or greater, or a value
within a range defined by any two of the aforementioned values. In
some embodiments, the protein scaffold is present in an amount of
about 10% w/v.
[0014] In some embodiments, the wound care composition includes 40%
w/w choline geranate (CAGE) and 10% w/w gelatin.
[0015] Some embodiments provided herein relate to a wound dressing.
In some embodiments, the wound dressing includes a wound care
composition as described herein and a dressing material. In some
embodiments, the wound care composition includes an ionic liquid
and a protein scaffold. In some embodiments, the wound dressing
material is a bandage, a wipe, a sponge, a mesh, a dressing, a
gauze, a patch, a pad, tape, or a wrap. In some embodiments, the
wound care composition is present in an amount of about 0.005%
vol/w % to about 2% vol/w %, such as 0.005, 0.006, 0.007, 0.008,
0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, or 2% volume of wound care composition/weight
wound dressing material (vol/w %), or greater, such as 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 30, 40, or 50% vol/w. In some embodiments, the
wound care composition is present in an amount of about 0.625%
vol/w %.
[0016] In some embodiments is provided a method of making a wound
care composition in one of any embodiment described herein. In some
embodiments, the method of making a wound care composition includes
providing a scaffold solution. In some embodiments, the scaffold
solution includes a protein scaffold or polysaccharide scaffold in
a solution. In some embodiments, the scaffold is any protein or
polysaccharide solution, for example, collagen, agarose, albumin,
alginate, casein, fibrin, fibroin, gelatin, keratin, pectin,
elastin, tropoelastin, cellulose, chitosan, chitin, or combinations
thereof. In some embodiments, the solution is the ionic liquid or
an organic solvent. In some embodiments, the organic solvent is a
polyamide, a polyacrylonitrile, a polyacetal, a polyester, or a
polyketone, or a combination thereof. In some embodiments, the
organic solvent is ethanol, ethyl formate, hexafluoro-2-propanol
(HFIP), cyclic ethers, acetone, acetates of C2 to C5 alcohol, glyme
or dimethoxyethane, methylethyl ketone, dipropyleneglycol methyl
ether, lactones, 1,4-dioxane, 1,3-dioxolane, ethylene carbonate,
dimethylcarbonate, diethylcarbonate, benzene, toluene, benzyl
alcohol, p-xylene, N-methyl-2-pyrrolidone, dimethylformamide,
chloroform, 1,2-dichloromethane (DCM), morpholine,
dimethylsulfoxide (DMSO), hexafluoroacetone sesquihydrate (HFAS),
anisole and mixtures thereof. In some embodiments, the method of
making a wound care composition includes providing an ionic liquid.
In some embodiments, the ionic liquid is a deep eutectic solvent
(DES). In some embodiments, the ionic liquid is choline geranate
(CAGE). In some embodiments, the method of making a wound care
composition includes mixing the ionic liquid with the protein
scaffold solution. In some embodiments, the method of making a
wound care composition include electrospinning the mixture of ionic
liquid and protein scaffold solution.
[0017] In some embodiments, mixing the ionic liquid with the
protein scaffold solution is performed at a temperature of about 0,
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100.degree. C. or greater, or a temperature within a
range defined by any two of the aforementioned values. In some
embodiments, mixing the ionic liquid with the protein scaffold
solution is performed at a temperature of about 40.degree. C.
[0018] In some embodiments, electrospinning is performed at a
temperature of about 0, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100.degree. C. or
greater or a temperature within a range defined by any two of the
aforementioned values. In some embodiments, electrospinning is
performed at a temperature of about 26.degree. C. In some
embodiments, electrospinning is performed at a humidity of about 0,
5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50,
55, 60, 65, or 70% or greater, or a humidity within a range defined
by any two of the aforementioned values. In some embodiments,
electrospinning is performed at a humidity of about 19%. In some
embodiments, electrospinning is performed at a voltage of about 5,
10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or
50V, or greater, or a value within a range defined by any two of
the aforementioned values. In some embodiments, electrospinning is
performed at a voltage of about 25V. In some embodiments,
electrospinning is performed at a flow rate of about 0.01, 0.05,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mL/hr, or
greater or a value within a range defined by any two of the
aforementioned values. In some embodiments, electrospinning is
performed at a flow rate of about 1 mL/hr.
[0019] In some embodiments, the method of making a wound care
composition includes desiccating the material to dryness following
the electrospinning.
[0020] In some embodiments is provided a method of preventing or
inhibiting biofilm formation in a subject. In some embodiments, the
method of preventing or inhibiting biofilm formation in a subject
includes contacting a wound with a wound care composition as
described herein. In some embodiments, treatment with a wound care
composition prevents biofilm formation. In some embodiments,
treatment with a wound care composition reduces bacterial growth
and bacterial populations. In some embodiments, treatment with a
wound care composition inhibits the growth of S. aureus or P.
aeruginosa.
[0021] In some embodiments is provided a method of inhibiting,
reducing, or preventing the growth of a pathogen. In some
embodiments, the method includes contacting a wound with a wound
care composition as described herein. In some embodiments, the
wound includes a burn, an abrasion, a laceration, a lesion, an
ulcer, or a sore. In some embodiments, the wound is infected with a
pathogen. In some embodiments, the wound is not infected with a
pathogen. In some embodiments, the pathogen is S. aureus or P.
aeruginosa.
[0022] In some embodiments is provided a method of enhancing wound
healing. In some embodiments, the method includes contacting a
wound with a wound care composition as described herein. In some
embodiments, the wound comprises a burn, an abrasion, a laceration,
a lesion, an ulcer, or a sore. In some embodiments, the wound
comprises a diabetic foot ulcer. In some embodiments, enhancing
wound healing includes improving, accelerating, or ameliorating
wound healing.
[0023] Some embodiments provided herein relate to a wound care
composition including a scaffold. In some embodiments, the scaffold
exhibits anti-proliferative activity towards bacterial or fungal
cells, but does not prevent human or mammalian cells from adhering,
associating with, or proliferating on the surface.
[0024] These features, together with other features herein further
explained, are described in greater detail in the following
description of the drawings and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The foregoing and other features of the present disclosure
will become more fully apparent from the following description,
taken in conjunction with the accompanying drawings. Understanding
that these drawings depict only some embodiments in accordance with
the disclosure and are, therefore, not to be considered limiting of
its scope, the disclosure will be described with additional
specificity and detail through use of the accompanying
drawings.
[0026] FIG. 1 illustrates the chemical components of one embodiment
of ionic liquids (ILs).
[0027] FIG. 2 depicts a graphical representation of the ability of
various ionic liquids to kill a biofilm of P. aeruginosa. BL=bleach
and NT=no treatment. Percent biofilm survival after treatment is
shown in the Inset on a log10 scale to show differences between the
most efficacious ILs. Choline geranate (CAGE) is shown as Ionic
Liquid 11.
[0028] FIG. 3 depicts a scanning electron micrograph of an
electrospun gelatin (5% w/v) scaffold, spun at a flow rate of 0.8
mL/hr, with an electric field of 2.91 kV/m.
[0029] FIG. 4 shows an image of a 0.2% w/v ionic liquid with
gelatin scaffold during electrospinning.
[0030] FIG. 5 shows an image of a 1.0% w/v ionic liquid with
gelatin scaffold during electrospinning.
[0031] FIG. 6A shows an image of an electrospun product having 10%
w/v gelatin scaffold in hexafluoro-2-propanol (HFIP), spun at 30V,
at a flow rate of 1 mL/hr, with a distance of the needle to target
of 12 cm, at 26.degree. C. in 18% humidity. FIG. 6B shows an image
of an electrospun product having 10% w/v gelatin scaffold in HFIP
plus 0.2% ionic liquid, spun at 25V, at a flow rate of 1 mL/hr,
with a distance of the needle to target of 9 cm, at 26.degree. C.
in 18% humidity. FIG. 6C shows an image of an electrospun product
having 10% w/v gelatin scaffold in HFIP plus 0.7% ionic liquid,
spun at 25V, at a flow rate of 1 mL/hr, with a distance of the
needle to target of 9 cm, at 26.degree. C. in 18% humidity. FIG. 6D
shows an image of an electrospun product having 10% w/v gelatin
scaffold in HFIP plus 1.0% ionic liquid, spun at 25V, at a flow
rate of 1 mL/hr, with a distance of the needle to target of 9 cm,
at 26.degree. C. in 18% humidity. Each of FIGS. 6A-6D show the
scaffold before being removed from the foil (left) and after
removal from foil (right).
[0032] FIG. 7 depicts structures of geranate (top left) and choline
(top right), with an NMR spectrum of choline geranate (CAGE).
[0033] FIG. 8 depicts NMR spectra of the ionic liquid-incorporated
scaffolds, indicating the presence of choline geranate incorporated
scaffold (top), compared to choline geranate (CAGE) alone
(bottom).
[0034] FIG. 9A depicts effects of a particular ionic liquid that
includes choline geranate (CAGE) against P. aeruginosa on gauze.
FIG. 9B depicts the percentage of P. aeruginosa colonies remaining
on the gauze at 30 minutes or two hours following treatment
(*=p<0.05, **=p<0.01, ***=p<0.001).
[0035] FIG. 10A depicts effects of CAGE against Enterococcus on
gauze. FIG. 10B depicts the percentage of Enterococcus colonies
remaining on the gauze at 30 minutes or two hours following
treatment (*=p<0.05, **=p<0.01, ***=p<0.001).
[0036] FIG. 11A depicts effects of CAGE against K. pneumoniae on
gauze. FIG. 11B depicts the percentage of K. pneumoniae colonies
remaining on the gauze at 30 minutes or two hours following
treatment (*=p<0.05, **=p<0.01, ***=p<0.001).
[0037] FIG. 12A depicts effects of CAGE against
methicillin-sensitive Staphylococcus aureus (MSSA) on gauze. FIG.
12B depicts the percentage of MSSA colonies remaining on the gauze
at 30 minutes or two hours following treatment (*=p<0.05,
**=p<0.01, ***=p<0.001).
[0038] FIGS. 13A and 13B graphically depict the effects of CAGE
scaffolds on P. aeruginosa at 30 minutes (FIG. 13A) and 2 hours
(FIG. 13B) after treatment.
[0039] FIGS. 14A and 14B graphically depict the effects of CAGE
scaffolds on Enterococcus at 30 minutes (FIG. 14A) and 2 hours
(FIG. 14B) after treatment.
[0040] FIGS. 15A and 15B depict micrographs showing human dermal
neonatal fibroblasts (P9) on control scaffolds (FIG. 15A) or
CAGE-incorporated scaffolds (FIG. 15B).
DETAILED DESCRIPTION
[0041] In the following detailed description, reference is made to
the accompanying drawings, which form a part hereof. In the
drawings, similar symbols typically identify similar components,
unless context dictates otherwise. The illustrative embodiments
described in the detailed description, drawings, and claims are not
meant to be limiting. Other embodiments may be utilized, and other
changes may be made, without departing from the spirit or scope of
the subject matter presented herein. It will be readily understood
that the aspects of the present disclosure, as generally described
herein, and illustrated in the drawings, can be arranged,
substituted, combined, separated, and designed in a wide variety of
different configurations, all of which are explicitly contemplated
herein.
[0042] As summarized above, aspects of the wound care compositions
and methods making and using the compositions are provided
herein.
[0043] It is to be understood that this disclosure is not limited
to particular embodiments described, as such may, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting.
[0044] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0045] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the present disclosure belongs.
For purposes of the present disclosure, the following terms are
defined below.
[0046] In some embodiments, provided herein is a method of treating
a skin wound. A method of treating a skin wound includes contacting
a wound with a wound care composition. In some embodiments, the
wound care composition includes an ionic liquid-incorporated
scaffold. As used herein, a wound refers to a burn, an abrasion, a
laceration, a lesion, an ulcer, or a sore. Wounds include diabetic
foot ulcers, severe burns, tumor excision, or trauma. A wound
includes damage to the skin, and can include, for example damage
caused by trauma, burn, surgery, or other type of damage. In some
embodiments, the method of treating a skin wound enhances wound
healing, prevents pathogen biofilm formation, inhibits pathogen
growth and proliferation, inhibits sepsis, and prophylactically
prevents the formation of biofilm or growth of pathogens. In some
embodiments, the wound is skin damage, a burn, an abrasion, a
laceration, an incision, a sore, a puncture wound, a penetration
wound, a gunshot wound, or a crushing injury.
[0047] In some embodiments, the wound healing composition is
applied to the wound at least once daily. In some embodiments, the
wound healing formulation is applied to the wound three times a
day. In some embodiments, applying the wound healing formulation
provides a wound healing rate that is accelerated in comparison to
a healing rate of a non-treated wound. In some embodiments, the
formulation is applied in an amount effective to accelerate wound
healing, promote wound closure, or cause wound regression. In some
embodiments, the applying the formulation reduces, attenuates, or
prevents bacterial growth or infection in the wound. In some
embodiments, the composition does not prevent human or mammalian
cells from proliferating.
[0048] In some embodiments is provided a wound care composition
that includes an ionic liquid and a protein scaffold. In some
embodiments, the composition may be used alone, for example, for
direct application to a wound. Thus, for example, the composition
may be formulated as an ointment, a cream, a spray, a spritz, a
mist, a liquid, a gel, a lotion, or a solution. In some
embodiments, the composition may be topically applied to a wound.
In some embodiments, the composition may be administered
subcutaneously.
[0049] In some embodiments, the composition may be configured for
incorporation into a wound dressing material, including a bandage,
a wipe, a sponge, a mesh, a dressing, a gauze, a patch, a pad,
tape, or a wrap, or other wound dressing material. The composition
may be used to saturate, impregnate, cover, coat, or otherwise be
incorporated into a wound dressing material.
[0050] As referred to herein, the term "ionic liquid" (IL) refers a
family of molecules commonly composed of an organic alkyl cation
paired with either an organic or inorganic anion. These materials
all have a melting point below 100.degree. C., and are frequently
described as "molten salts". The chemical nature of the cation and
anion components of an IL are readily modified, and as such, the
physiochemical properties of the salt as a whole can be "tuned" for
optimal use within a variety of applications (Hassan et al.,
Studies on the dissolution of glucose in ionic liquids and
extraction using the antisolvent method. Environmental science
& technology 47(6):2809-2816, 2013; Frederix M, et al.,
Development of a native Escherichia coli induction system for ionic
liquid tolerance. PloS one 9(7):e101115, 2014; Eisenberg, Ionic
interactions in biological and physical systems: a variational
treatment. Faraday discussions 160:279-296, 2013; Cao Y, et al.,
Separation of soybean isoflavone aglycone homologues by ionic
liquid-based extraction. Journal of agricultural and food chemistry
60(13):3432-3440, 2012; De Diego et al., A recyclable enzymatic
biodiesel production process in ionic liquids. Bioresource
technology 102(10):6336-6339, 2012); each of which is incorporated
by reference herein in its entirety). As used herein, "deep
Eutectic Solvents" (DESs) are broadly defined as a mixture of
charged and neutral species, either in equimolar or imbalanced
ratios, that, when combined, have a much lower melting point than
the individual component. DESs are mixtures of compounds and
neutral molecules and ILs are themselves ionic compounds. The ionic
liquid described herein, including DESs are intimately related on a
chemical level and both are considered part of a larger class of
molecules.
[0051] A number of ILs have shown a propensity to disrupt the
noncovalent bonds within the structure of recalcitrant biopolymers
such as cellulose or keratin (Lovejoy, et al. (2011)
Tetraalkylphosphonium-Based Ionic Liquids for a Single-Step Dye
Extraction/MALDI MS Analysis Platform. Anal Chem 83(8):2921-2930;
Lovejoy et al. (2012) Single-Pot Extraction-Analysis of Dyed Wool
Fibers with Ionic Liquids. Anal Chem 84(21):9169-9175; Shill K, et
al. (2011) Ionic liquid pretreatment of cellulosic biomass:
enzymatic hydrolysis and ionic liquid recycle. Biotechnology and
bioengineering 108(3):511-520; each of which is incorporated by
reference herein in its entirety).
[0052] This aspect of IL chemistry has played an important role in
the processing, disruption and dissolution of biopolymers for
applications in renewable energy and forensics (Zhang J, et al.
(2014) Understanding changes in cellulose crystalline structure of
lignocellulosic biomass during ionic liquid pretreatment by XRD.
Bioresource technology 151:402-405; Uju, et al. (2013) Peracetic
acid-ionic liquid pretreatment to enhance enzymatic
saccharification of lignocellulosic biomass. Bioresource technology
138:87-94; Varanasi P, et al. (2013) Survey of renewable chemicals
produced from lignocellulosic biomass during ionic liquid
pretreatment. Biotechnology for biofuels 6(1):14; Lovejoy, et al.
(2011) Tetraalkylphosphonium-Based Ionic Liquids for a Single-Step
Dye Extraction/MALDI MS Analysis Platform. Anal Chem
83(8):2921-2930; Lovejoy, et al. (2012) Single-Pot
Extraction-Analysis of Dyed Wool Fibers with Ionic Liquids. Anal
Chem 84(21):9169-9175; each of which is incorporated by reference
herein in its entirety).
[0053] The ability of ILs to disrupt biopolymers has also enabled
their use as novel antibiotic agents that specifically target
bacterial biofilms and have a bactericidal effect in general.
Biofilms are communities of microbes that secrete a thick layer of
exopolymeric material (including polysaccharides, proteins and
nucleic acids) that effectively serve as a physical barrier to
treatment with antibiotics (Donlan et al. (2001) Biofilms and
device-associated infections. Emerg Infect Dis 7(2):277-281;
Flemming et al. (2010) The biofilm matrix. Nature Reviews
Microbiology 8(9):623-633; each of which is incorporated by
reference herein in its entirety). Typical applications of
prescribed antibiotics do not efficiently pass through this
physical barrier, and thus, biofilm bacteria are often 500-1000
times less susceptible to antibiotic treatment than their
planktonic counterparts. Bacterial biofilms are responsible for
most hospital-acquired infections, and the CDC has estimated the
cost of combatting these maladies alone exceeds $10 billion per
year (Bickers et al. (2006) The burden of skin diseases: 2004--A
joint project of the American Academy of Dermatology Association
and the Society for Investigative Dermatology. J Am Acad Dermatol
55(3):490-500). Antibacterial ILs disrupt the biofilm's protective
exopolymeric layer and re-enable efficient antibiotic delivery to
the cells within it, and sometimes have antibacterial properties
themselves (Zakrewsky et al. (2014) Ionic liquids as a class of
materials for transdermal delivery and pathogen neutralization. P
Natl Acad Sci USA 111(37):13313-13318; Lovejoy et al. (2012)
Utilization of Metal Halide Species Ambiguity to Develop Amorphous,
Stabilized Pharmaceutical Agents As Ionic Liquids. Cryst Growth Des
12(11):5357-5364; each of which is incorporated by reference herein
in its entirety).
[0054] As used herein, the term "choline geranate" refers to a
specific DES that combines choline with geranate anion and geranic
acid. This formulation of choline geranate is referred to herein as
CAGE. CAGE shows potent antibiofilm properties against pathogens
associated with common skin infections (including, for example,
Staphylococcus aureus, Pseudomonas aeruginosa), including
multi-drug resistant isolates (e.g. Methicillin resistant S.
aureus, or MRSA). CAGE exhibits low toxicity toward human
epithelial cells and C3H/HeJ mice systematically exposed to CAGE
(50 .mu.L/day, for seven days, n=4) do not show any local or
systemic inflammation following exposure. Further, CAGE not only
neutralizes biofilms in vitro, but is also capable of passing
through the dermis to address infections that lie below the outer
layers of the skin. This aspect of the compound has significant
ramifications for its use as an antibiofilm agent to treat
established skin infections (such as necrotizing infections,
chronic wounds, or diabetic ulcers) but also as a prophylactic to
insure that infections do not establish in surgical wounds during
the process of wound healing. CAGE is particularly effective both
at neutralizing bacterial biofilms in vitro and traversing dermal
layers to treat biofilms that exist within skin (Zakrewsky et al.
(2014) Ionic liquids as a class of materials for transdermal
delivery and pathogen neutralization. P Natl Acad Sci USA
111(37):13313-13318). The development of ionic liquid-modified
wound healing scaffolds will render the scaffolds themselves
resistant to contamination with biofilms and enhance healing in
difficult to treat patients. ILs are capable of preventing biofilm
formation on wound healing devices without changing the structure
and efficacy of the device.
[0055] In some embodiments, composition including an IL is added,
incorporated, coated on, applied to, saturated with, impregnated
with, covered with, or otherwise be incorporated or contacted with
a wound dressing material in an amount of 0.005% to 2% volume IL to
weight gauze (vol/w %), such as 0.005, 0.006, 0.007, 0.008, 0.009,
0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, or 2% volume of wound care composition/weight wound
dressing material (vol/w %), or greater, such as 3, 4, 5, 6, 7, 8,
9, 10, 15, 20, 30, 40, or 50% vol/w or an amount within a range
defined by any two of the aforementioned values. In some
embodiments, the wound care composition is present in an amount of
about 0.625% vol/w %. In some embodiments, the IL is contacted with
a wound dressing in an amount of 0.05 .mu.g per mg wound dressing
material to 20 .mu.g per mg wound dressing material, such as 0.05,
0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 .mu.g/mg, or an amount within a range defined by any two
of the aforementioned values. In some embodiments, the wound care
composition is present in an amount of about 6.25 .mu.g/mg. Thus,
in some embodiments, the composition including an IL is
incorporated into a wound dressing material, for example, to
saturate, impregnate, cover, coat, or otherwise be incorporated
into the wound dressing material. Coating can include, for example,
dip coating or surface modifying a material with an IL.
[0056] In some embodiments, the amount of IL incorporated into an
electrospun scaffold can be 0.01% w/w to about 99% w/w, such as
0.01, 0.05, 0.1, 0.5, 1.0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% w/w or greater, or a
value within a range defined by any two of the aforementioned
values. In some embodiments, the IL is present in an amount of
about 40% w/v.
[0057] As used herein, pathogens can include opportunistic
pathogens associated with skin infections, including, for example,
bacterial infections (including, for example, Staphylococcus
aureus, Pseudomonas aeruginosa, methicillin resistant S. aureus
(MRSA), Streptococcus pyogenes), viral infections, fungal
infections, or yeast infections. In some embodiments, the
compositions provided herein prevent pathogenic growth. In some
embodiments, the compositions do not prevent human or mammalian
cell proliferation. In some embodiments, the compositions do not
prevent human or mammalian cells from adhering, associating with,
or proliferating on a scaffold or surface.
[0058] As used herein a scaffold refers to a material that acts as
a foundation or structure for the formulation of the wound care
compositions as described herein. The scaffold may be a protein
scaffold or a polysaccharide scaffold and may include a molecule
for structural formation, including, for example any protein
solution, including, for example, one or more of collagen, agarose,
albumin, alginate, casein, elastin, fibrin, fibroin, fibronectin,
gelatin, keratin, laminin, pectin, elastin, tropoelastin,
cellulose, chitosan, chitin. In some embodiments, the wound care
composition is prepared by electrospinning a protein scaffold
incorporated with IL. In some embodiments, the amount of protein
scaffold, can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,
0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,
1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,
16%, 17%, 18%, 19% or 20% w/v or greater, or a value within a range
defined by any two of the aforementioned values.
[0059] The protein scaffold can be in solution, for example, in a
solvent. In some embodiments, the solvent is the IL. In some
embodiments, the solvent is an organic solvent. In some
embodiments, the organic solvent is a polyamide, a
polyacrylonitrile, a polyacetal, a polyester, or a polyketone, or a
combination thereof. In some embodiments, the organic solvent is
ethanol, ethyl formate, hexafluoro-2-propanol (HFIP), cyclic ethers
(tetrahydrofuran (THF), and 2,5-dimethylfuran (DMF)), acetone,
acetates of C2 to C5 alcohol (such as ethyl acetate and butyl
acetate), glyme or dimethoxyethane (monoglyme, ethyl glyme,
diglyme, ethyl diglyme, triglyme, butyl diglyme, tetraglyme,
dimethyl glycol, ethylene glycol dimethyl ether, dimethyl
cellosolve, and DME), methylethyl ketone (butanone),
dipropyleneglycol methyl ether, lactones (such as
.delta.-valerolactone, .gamma.-valerolactone, b-butyrolactone,
g-butyrolactone), 1,4-dioxane, 1,3-dioxolane, ethylene carbonate,
dimethylcarbonate, diethylcarbonate, benzene, toluene, benzyl
alcohol, p-xylene, N-methyl-2-pyrrolidone, dimethylformamide,
chloroform (trichloromethane, methyl trichloride),
1,2-dichloromethane (DCM), morpholine, dimethylsulfoxide (DMSO),
hexafluoroacetone sesquihydrate (HFAS), anisole (methoxybenzene)
and mixtures thereof.
[0060] Electrospun materials have been described since 1934 (Garg
et al. Electrospinning jets and nanofibrous structures."
Biomicrofluidics 5.1: 013403, 2011). Since then a plethora of
proteins and polymers have been electrospun into scaffolds for use
in clinical and research realms. The principle behind
electrospinning is to eject a solubilized protein through a charged
nozzle onto an oppositely charged target. When the surface tension
forces are balanced with the electric field, the protein droplet
elongates forming a funnel shape known as a Taylor cone (Taylor.
Electrically driven jets. Proceedings of the Royal Society of
London A: Mathematical, Physical and Engineering Sciences. Vol.
313. No. 1515. The Royal Society, 1969). However, the stability of
the cone was a problem until 1987 when Hayati discovered that
semiconducting insulating liquids created more stability at higher
voltages (Hayati et al., Investigations into the mechanisms of
electrohydrodynamic spraying of liquids: I. Effect of electric
field and the environment on pendant drops and factors affecting
the formation of stable jets and atomization. Journal of Colloid
and Interface Science 117.1: 205-221, 1987). The stable cone and
ejection of the solubilized protein causes the solvent to evaporate
before the target is reached and the creation of nanofibers being
laid upon the target in a nonwoven pattern.
[0061] In 1971, Baumgarten determined that by varying the
concentration of protein and changing the applied voltage, the
diameter of the nanofibers could be manipulated, with a higher
concentration producing larger continuous fibers and lesser
concentrations producing shorter and finer fibers (Baumgarten,
Electrostatic spinning of acrylic microfibers. Journal of colloid
and interface science 36.1: 71-79, 1971).
[0062] Provided herein are electrospinning methods for the creation
of novel protein scaffolds that serve as wound healing agents.
These scaffolds are fabricated using native skin proteins and as a
result, they more closely match the composition and architecture of
skin, thereby enhancing wound healing.
[0063] The novelty of the embodiments and alternatives described
herein includes the combination of ILs and protein scaffolds to
enhance the wound healing process. The development of IL-modified
wound healing scaffolds mitigates biofilm formation on these
scaffolds and enhances healing in difficult to treat patients.
[0064] As used herein, the term "treatment" refers to an
intervention made in response to a wound, such as a burn or other
wound, including difficult to treat wounds, in a subject in need.
The aim of treatment may include, but is not limited to, one or
more of the alleviation or prevention of symptoms, slowing or
stopping the progression or worsening of a wound, enhancement of
the wound healing, and the remission of the wound. In some
embodiments, "treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already affected by a wound as well as those in which
an infection of the wound is to be prevented.
[0065] As used herein, the terms "treating," "treatment,"
"therapeutic," or "therapy" do not necessarily mean total cure or
abolition of the disease or condition. Any alleviation of any
undesired signs or symptoms of a disease or condition, to any
extent can be considered treatment and/or therapy.
[0066] Treatment may include administration of a wound care
composition alone or a wound dressing material that has a wound
care composition incorporated therein. When used alone, the wound
care composition may be administered topically, orally,
subcutaneously, or in other means in order to properly treat the
wound. When the wound care composition is incorporated into a wound
dressing material, the wound dressing material is applied to the
wound to treat the wound.
[0067] As used herein, a "subject" refers to an animal that is the
object of treatment, observation or experiment. "Animal" includes
cold- and warm-blooded vertebrates and invertebrates such as fish,
shellfish, reptiles and, in particular, mammals. "Mammal" includes,
without limitation, mice, rats, rabbits, guinea pigs, dogs, cats,
sheep, goats, cows, horses, primates, such as monkeys, chimpanzees,
and apes, and, in particular, humans. A subject in need includes a
subject that is suffering from a wound, including a burn (including
a severe, moderate, or minor burn), trauma, surgical wound, a
laceration, a lesion, an ulcer, or a sore. In some embodiments, the
subject suffers from a diabetic foot ulcer.
[0068] As used herein, the term "prevention" refers to any activity
that reduces the burden of the individual later expressing wound
symptoms. This can take place at primary, secondary, and/or
tertiary prevention levels, wherein: a) primary prevention avoids
the development of symptoms/condition; b) secondary prevention
activities are aimed at early stages of the condition/symptom
treatment, thereby increasing opportunities for interventions to
prevent progression of the condition/symptom and emergence of
symptoms; and c) tertiary prevention reduces the negative impact of
an already established condition/symptom by, for example, restoring
function and/or reducing any condition/symptom or related
complications.
[0069] The articles "a" and "an" are used herein to refer to one or
to more than one (to at least one) of the grammatical object of the
article. By way of example, "an element" means one element or more
than one element.
[0070] By "about" is meant a quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight or length
that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1% to a reference quantity, level, value, number, frequency,
percentage, dimension, size, amount, weight or length.
[0071] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises," and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0072] By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of." Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they materially
affect the activity or action of the listed elements.
[0073] In certain embodiments, the "purity" of any given agent in a
composition may be specifically defined. For instance, certain
compositions may include, for example, an agent that is at least
80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure,
including all decimals in between, as measured, for example and by
no means limiting, by high pressure liquid chromatography (HPLC), a
well-known form of column chromatography used frequently in
biochemistry and analytical chemistry to separate, identify, and
quantify compounds.
EXAMPLES
[0074] Some aspects of the embodiments discussed above are
disclosed in further detail in the following examples, which are
not in any way intended to limit the scope of the present
disclosure. Those in the art will appreciate that many other
embodiments also fall within the scope of the disclosure, as it is
described herein above and in the claims.
Example 1: Choline Geranate Synthesis and Preparation
[0075] The following example demonstrates one embodiment of a
method of synthesizing CAGE for use in an IL-incorporated wound
dressing.
[0076] CAGE was synthesized from commercially available reagents
using equipment standard to the organic chemistry laboratory.
Geranic acid typically requires purification via recrystallization
from acetone. The product was characterized with standard methods
(including, for example, NMR, UV, or IR) to verify purity. CAGE was
synthesized through salt metathesis of 1:2 molar ratio choline
bicarbonate and geranic acid, and the final product possessed both
fluidity and transparency at room temperature. Two equivalents of
neat geranic acid (50.0 g, 0.297 moles, Sigma-Aldrich, St. Louis,
Mo.), were recrystallized five times at -70.degree. C. in acetone,
in a 500-mL round bottom flask and added to one equivalent of
choline bicarbonate (80 wt% solution, 30.7 g, 0.149 moles,
Sigma-Aldrich, St. Louis, Mo.). The mixture was stirred at room
temperature until CO.sub.2 evolution ceased. Residual H.sub.2O was
removed by rotary evaporation at 60.degree. C. for 2 h and drying
in a vacuum oven for 24 h at 60.degree. C.
[0077] Physical characterization at 25.degree. C. was in good
agreement with published values and was as follows: density,
0.989.+-.0.001 g mL.sup.-1; and conductivity, 0.0427.+-.0.0005 mS
cm.sup.-1.
[0078] Physicochemical properties were identical to those
previously published confirming purity (Zakrewsky et al., PNAS,
2014, 111, 13313; incorporated by reference herein in its
entirety). CAGE has unique properties based upon the 1:1:1 ratio of
cation:anion:protonated acid. This is important since it gives rise
to the low conductivity determined for CAGE (0.043 mS cm.sup.-1)
and is a potential indicator of anti-biofilm efficacy but, also
provides insight to minimized skin irritation potential.
[0079] Composition was confirmed with nuclear magnetic resonance
spectroscopy (NMR). NMR assignments were also in good agreement
with published assignments and were as follows: 1 H NMR
(DMSO-d.sub.6), .delta. 5.57 (s, 2H), 5.07 (t, J=6.1, 2H), 3.85 (t,
J=6.6, 2H), 3.42 (t, J=6.6, 2H), 3.17 (s, 9H), 2.60 (m, 4H), 2.00
(m, 4H), 1.93 (s, 6H), 1.70 (s, 2H), 1.64 (s, 6H), and 1.57 (s,
6H); .sup.13C NMR (DMSO-d.sub.6), .delta. 170.3, 150.4, 131.5,
124.0, 121.7, 67.6, 55.6, 53.5, 40.4, 32.8, 25.8, and 17.8.
[0080] CAGE may be incorporated into a wound care composition, such
as a dressing or bandage. For example, CAGE may be deposited onto
the surface of medical-grade gauze using standard dip-coating
protocols. Application of the IL at various concentrations (.mu.mol
per cm.sup.2) is established by dissolving the IL into an
appropriate solvent (e.g. acetone) at various concentrations after
which the dressings are coated and allowed to dry. Exemplary IL
concentrations include 0.005, 0.006, 0.007, 0.008, 0.009, 0.01,
0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, or 2% volume of wound care composition/weight wound dressing
material (vol/w %), or greater, such as 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 30, 40, or 50% vol/w, or an amount within a range defined
by any two of the aforementioned values. In some embodiments, the
wound care composition is present in an amount of about 0.625%
vol/w %. In some embodiments, the amount of IL incorporated into an
electrospun scaffold can be 0.01% w/w to about 99% w/w, such as
0.01, 0.05, 0.1, 0.5, 1.0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, or 90, 95, or 99% w/w or greater, or a
value within a range defined by any two of the aforementioned
values. In some embodiments, the IL is present in an amount of
about 40% w/v.
[0081] Extraction of a defined weight of the gauze, followed by
gravimetric and/or HPLC analysis (with a geranic acid standard) may
be used to verify deposition of the appropriate amounts of IL
within this procedure. CAGE is unreactive toward the cotton fibers
in gauze, but effects (if any) of the deposition on various
material may be determined by analysis of treated and un-treated
samples with SEM.
Example 2: Electrospinning of IL-Incorporated Wound Dressings
[0082] The following example demonstrates one embodiment of a
method for electrospinning IL-incorporated wound dressings.
[0083] Ionic liquids are incorporated into a protein solution and
electrospun onto targets to create novel wound dressings that
include CAGE, thereby forming IL wound dressings. The resulting
scaffolds are evaluated using SEM for porosity and fiber diameter
and compared to known control scaffolds to understand how the
inclusion of CAGE affects the architecture of the scaffold.
Additionally, mechanical strength testing and characterization are
also performed on the IL wound dressing. Last, using HPLC
techniques, the inclusion of IL into the wound dressing is
confirmed.
[0084] Electrospinning was performed under various conditions, as
set forth in Table 1. All samples were performed at 26.degree. C.,
18% humidity, and at a flow rate of 1 mL/hr. The distance refers to
the distance from the electrospin needle to the target. The % IL
refers to the final percentage in the product.
TABLE-US-00001 TABLE 1 Electrospinning Conditions % Ionic Liquid
(w/w) % Gelatin (w/v) Voltage (V) Distance (cm) 1 0 10 30 12 2 0.2
10 25 9 3 0.7 10 25 9 4 1.0 10 25 9
[0085] FIGS. 6A-6B show the product that result from each of the
conditions shown in Table 1.
[0086] The purpose of this experiment was to manipulate the
concentration of IL, CAGE, within a 5 mL, 10% gelatin in
hexafluoro-2-propanol (HFIP) solution to effectively incorporate
the IL into a final electrospun gelatin scaffold, in the effort of
providing the scaffold with an antimicrobial aspect. HFIP is toxic
and corrosive.
[0087] The method of electrospinning was carried out with a 0.02%
w/w concentration of IL in liquid mixture (0.2% w/w concentration
of IL in product). 0.5001 g of solid gelatin was weighed out and
placed into a glass bottle along with a magnetic stir bar. Next, 5
mL of HFIP was added to the bottle with the gelatin. The bottle was
then covered completely with aluminum foil to inhibit the IL from
reacting with the light upon being added to the solution. 1 .mu.L
of IL was then added to the bottle using a 1 .mu.L-10 .mu.L
pipette; the tip was removed after the IL was dispensed, and a 5 mL
syringe was used to push air through the tip in an effort to
release any liquid IL remaining in the tip. The bottle was capped
and placed in a water bath on top of a stir/heat plate. The
temperature of the bath was monitored to ensure it did not exceed
40.degree. C. (human body temperature). The solution was stirred
for approximately 30 minutes, where the heat was set to "low" while
the stir speed was set to about 4. The entire bath was covered with
foil and the laboratory lights were shut off during this period (to
avoid light exposure of the IL). Once the solution was mixed, until
it was a homogeneous mixture, it was placed in the fridge
overnight. The gelatin solution was stirred again for about twenty
minutes prior to electrospinning.
[0088] The solution containing 0.02% w/w IL (liquid form) was
electrospun following the general protocol for electrospinning,
while the parameters were as follows: temperature, 26.degree. C.;
humidity, 19%; distance from needle to target, 9 cm; voltage, 25V;
flow rate, 1 mL/hr.
[0089] Once the process commenced, the aluminum target was not
immediately covered with visible white solid product (FIG. 6B) in
comparison to a control gelatin solution without the incorporated
IL (FIG. 6A). However, once the scaffold's texture appeared to be
uneven with drops, concentrated product spread throughout the
scaffold. Because ILs are extremely conductive, the "crackling"
sound of the electricity throughout the system was increased, in
comparison to electrospinning a control.
[0090] Once an hour had passed while electrospinning, the gelatin
scaffold with 0.2% IL was placed in a desiccator for twenty-four
hours to ensure that the scaffold completely dried.
Data/Calculations (0.2% Ionic Liquid in Final Product)
[0091] Mass of gelatin: 0.5001 g
[0092] Volume of HFIP: 5 mL
[0093] Volume of ionic liquid: 1 .mu.L (0.2% in product)
[0094] Percent weight/volume (for mass of gelatin and volume of
HFIP)
[0095] 10%=10 g/100 mL
[0096] 0.5 g gelatin/5 mL HFIP
[0097] For volume/concentration of ionic liquid: 5 mL (total
volume).times.0.2% (desired concentration of ionic liquid in
product)=1.0 .mu.L ionic liquid.
[0098] FIG. 6B shows the 0.2% ionic liquid-concentrated gelatin
scaffold being electrospun.
[0099] The same procedure used for the 10% gelatin scaffold in 5 mL
HFIP with 0.2% incorporated IL may be used to create additional
mixtures with IL, for example, with concentrations of 0.7% and 1.0%
IL in the final gelatin scaffolds. The parameters for
electrospinning were kept the same for these additional mixtures. A
control scaffold was also spun, with the same amount of gelatin in
HFIP. The distance from the needle to the target was returned to 12
cm for the control.
Data/Calculations (0.7% Ionic Liquid in Final Product)
[0100] Mass of gelatin: 0.5006 g
[0101] Volume of HFIP: 5 mL
[0102] Volume of ionic liquid: 3.5 .mu.L
[0103] Percent weight/volume
[0104] 10%=10 g/100 mL
[0105] 0.5 g gelatin/5 mL HFIP
[0106] For volume/concentration of ionic liquid: 5 mL (total
volume).times.0.7% (desired concentration of ionic liquid in
product)=3.5 .mu.L ionic liquid.
[0107] Similar to the first IL-concentrated solution that was spun,
the aluminum target turned white after a longer period than normal.
The sound of the electricity again was more prominent than with a
control, but not much different from the first IL-spun scaffold.
The texture of the final product was not as smooth as a control
scaffold, with more droplet-structures upon the surface, as shown
in FIG. 6C.
Data/Calculations (1.0% Ionic Liquid in Final Product)
[0108] Mass of gelatin: 0.5001 g
[0109] Volume of HFIP: 5 mL
[0110] Volume of ionic liquid: 5.0 .mu.L
[0111] Percent weight/volume
[0112] 10%=10 g/100 mL
[0113] 0.5 g gelatin/5 mL HFIP
[0114] For volume/concentration of ionic liquid: 5 mL (total
volume).times.1.0% (desired concentration of IL in product)=5.0
.mu.L ionic liquid.
[0115] The observation of the loud sound of electricity noted from
the two previous scaffolds was again present. However, the target
began to turn white quicker, and again, the texture of the final
product consisted of droplets instead of being smooth, as shown in
FIG. 6D.
[0116] Upon removing the scaffolds from the desiccator eight days
later, the scaffolds were difficult to remove from the aluminum
targets, and therefore came off in broken-up pieces.
NMR Data
[0117] FIG. 7 shows the molecular structure of geranate (top left)
and choline (top right). The bottom photo is the NMR spectrum
showing the presence of the two ions in the gelatin scaffold.
[0118] To verify inclusion of CAGE within the IL-incorporated
scaffolds, a small portion (.about.1 mg) of the dried scaffold
material was placed into a test tube along with 0.7 mL of
deuterated acetone (acetone-d6, 99.9% ,Sigma-Aldrich) and the
mixture was incubated overnight at room temperature with periodic
mixing. A similar extraction was performed on a control scaffold
that was not incorporated with IL. The extracted acetone was
removed from the extraction vessel and placed into an NMR tube, and
analyzed via .sup.1H NMR (400 MHz, Bruker). The biological activity
of the extracted IL is also assessed using the broth microdilution
assay against Staphylococcus aureus and Pseudomonas aeruginosa.
[0119] FIG. 8 shows the .sup.1H NMR spectrum of the extracted
scaffold (top) and authentic CAGE (below) in acetone-d6.
[0120] As can be seen in FIG. 8, the spectrum for the extracted
scaffold shows characteristic resonances associated with the
geranate anion/geranic acid (singlets at .about.1.6 and 1.67 ppm,
multiplet at 2.0 ppm, singlets at 5.07 and 5.57 ppm) that are
similarly observed in the control spectrum. Characteristic
resonances of the choline cation are also observed (triplet at
3.85, triplet at 3.42 and singlet at 3.17) in the extracted
spectrum, but to a lower molar equivalence than that of the neat
CAGE. This is likely due to a relatively inefficient extraction of
the choline cation from the scaffold using this particular organic
solvent.
Example 3: Efficacy of Ionic Liquid-Incorporated Wound Dressings to
Resist Biofouling/Reduce Biofilms
[0121] The following example demonstrates the efficacy of the
IL-incorporated wound care compositions for the reduction of
biofilm formation and the prevention and inhibition of pathogen
proliferation and growth in wounds.
[0122] The effect of the IL-incorporated dressings to either resist
or to treat biofilms was quantified using in vitro assays with
pathogens associated with skin wounds (e.g. S. aureus) or diabetic
ulcers (e.g. P. aeruginosa). To examine the effect of IL-treatment
on the resistance of the dressings to biofouling, small (1 cm.sup.2
or less) samples of dressings were placed on a solid nutrient
medium and inoculated with actively growing bacteria (10.sup.5
cells). This method parallels a commonly employed assay known as
the colony biofilm test (Merritt et al. (2005) Growing and
analyzing static biofilms. Current protocols in microbiology
Chapter 1:Unit 1B 1; incorporated by reference herein in its
entirety). Biofilms were cultured on dressings with various amounts
of deposited CAGE and the number of viable cells existing on these
surfaces was determined by initial disruption of the biofilm with
sonication, followed by dilution and enumeration of the viable
bacteria. Biofilm formation was assayed using culture and qPCR of
the bacterial populations as a function of IL concentration and
time (24 to 72 hrs). Similarly, the ability of the IL-treated
dressings to reduce viability in an established biofilm was
assessed by overlaying small pieces of dressing upon biofilms that
had been previously cultured on a surface of equivalent size;
effectiveness was quantified as a function of treated material and
length of exposure.
Example 4: CAGE-Incorporated Wound Dressings
[0123] The following example demonstrates the efficacy of
CAGE-incorporated gauze for the reduction of biofilm formation and
the prevention and inhibition of pathogen proliferation and
growth.
[0124] Gauze was coated with an IL, CAGE, in various
concentrations. The CAGE-incorporated gauze was treated with
approximately 100 .mu.L of actively growing bacteria, including P.
aeruginosa, Enterococcus, K. pneumoniae, or methicillin sensitive
S. aureus (MSSA), at 10.sup.8-10.sup.9 cells per mL. Each of these
bacteria are clinically isolated pathogens commonly associated with
wounds and 10.sup.8-10.sup.9 cells/mL is a high concentration of
live bacteria. In terms of clinical practice, bacterial
concentrations in this amount are unlikely to be found, even in
cases of septicemia.
[0125] As shown in FIGS. 9 through 12, CAGE-incorporated gauze
effectively reduced and/or inhibited the growth of bacteria in the
gauze. FIGS. 9A and 9B show that the growth of P. aeruginosa was
prohibited at both 30 minutes and 2 hours following contact with
the gauze when CAGE was present in an amount ranging from 1%-10%
(1% is 1.25 .mu.g, 5% is 6.25 .mu.g, and 10% is 12.5 .mu.g CAGE).
In contrast, control gauze with no IL incorporation showed elevated
bacteria amounts. In addition, the percentage of surviving colonies
after contact with CAGE-incorporated gauze was reduced to values of
less than about 20% in all treatment groups, as compared to
percentage of surviving colonies that were untreated.
[0126] Similar results are achieved when the gauze is exposed to
Enterococcus (FIGS. 10A and 10B), K. pneumoniae (FIGS. 11A and
11B), and MSSA (FIGS. 12A and 12B).
Example 5: CAGE-Incorporated Scaffolds
[0127] The following example demonstrates the efficacy of
CAGE-incorporated scaffolds for the reduction of biofilm formation
and the prevention and inhibition of pathogen proliferation and
growth.
[0128] Scaffolds were prepared as described in Example 2, wherein
protein was mixed with CAGE to generate a CAGE-incorporated
scaffold. The CAGE scaffolds included various concentrations of
CAGE from 1% to 10% during the production process (1% is 1.25
.mu.g, 5% is 6.25 and 10% is 12.5 .mu.g CAGE). The CAGE scaffolds
were prepared by electrospinning. As shown in FIGS. 13 through 14,
CAGE incorporated into protein scaffolds reduced bacteria growth.
Specifically, FIGS. 13A and 13B show reduced colony forming units
(CFU) of P. aeruginosa in CAGE-incorporated scaffolds (at 10% and
5%) compared to control scaffold with no IL incorporation at both
30 minutes and 2 hours after exposure to the bacteria.
[0129] Similarly, FIGS. 14A and 14B show reduced CFU of
Enterococcus in CAGE-incorporated scaffolds (at 10% and 5%)
compared to control scaffold with no IL incorporation at both 30
minutes and 2 hours after exposure to the bacteria.
[0130] FIGS. 15A and 15B show the ability of human dermal neonatal
fibroblasts to attach and proliferate in general growth medium to
both the surface of control scaffolds (FIG. 15A) and
CAGE-incorporated scaffolds (40%) (FIG. 15B). Human dermal neonatal
fibroblasts (P9) were added to a well plate containing control
scaffolds or scaffolds containing 40% (v/w) CAGE. The control
scaffold was UV sterilized prior to seeding cells. These images
show that bacteria may be killed without harming or otherwise
preventing human cells from proliferating on the surface.
[0131] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
[0132] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0133] While various aspects and embodiments have been disclosed
herein, other aspects and embodiments will be apparent to those
skilled in the art. The various aspects and embodiments disclosed
herein are for purposes of illustration and are not intended to be
limiting, with the true scope and spirit being indicated by the
claims.
* * * * *