U.S. patent application number 17/070914 was filed with the patent office on 2021-02-11 for anti-infective antimicrobial-containing biomaterials.
This patent application is currently assigned to ATRIUM MEDICAL CORPORATION. The applicant listed for this patent is ATRIUM MEDICAL CORPORATION. Invention is credited to Jocelyn CHOROSZY, Keith M. FAUCHER, Anthony Richard HORTON, Paul MARTAKOS, Hilda N. RONO.
Application Number | 20210038772 17/070914 |
Document ID | / |
Family ID | 1000005170139 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210038772 |
Kind Code |
A1 |
FAUCHER; Keith M. ; et
al. |
February 11, 2021 |
ANTI-INFECTIVE ANTIMICROBIAL-CONTAINING BIOMATERIALS
Abstract
A biomaterial including a plurality of fatty acid chains
cross-linked together and elemental silver metal formed within the
material. Method(s) for forming the material are also included. The
silver-containing biomaterial can be utilized alone or in
combination with a medical device for the release and local
delivery of one or more anti-infective agents.
Inventors: |
FAUCHER; Keith M.; (Milford,
NH) ; RONO; Hilda N.; (Chelmsford, MA) ;
HORTON; Anthony Richard; (Bedford, NH) ; CHOROSZY;
Jocelyn; (Waltham, MA) ; MARTAKOS; Paul;
(Pelham, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ATRIUM MEDICAL CORPORATION |
Merrimack |
NH |
US |
|
|
Assignee: |
ATRIUM MEDICAL CORPORATION
Merrimack
NH
|
Family ID: |
1000005170139 |
Appl. No.: |
17/070914 |
Filed: |
October 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14281682 |
May 19, 2014 |
10864304 |
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17070914 |
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12539282 |
Aug 11, 2009 |
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14281682 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 29/14 20130101;
A61L 15/46 20130101; A61K 31/765 20130101; A61K 31/232 20130101;
A01N 61/00 20130101; A61L 2300/404 20130101; A61L 2300/45 20130101;
A61L 31/16 20130101; A61K 31/155 20130101; A61L 29/16 20130101;
A61K 31/09 20130101; A61L 2300/104 20130101; A61L 27/50 20130101;
A61L 2300/602 20130101; A61L 31/14 20130101; A61L 2420/06 20130101;
A61L 31/005 20130101; A61K 31/202 20130101; A61L 2300/624 20130101;
A61L 27/54 20130101; A61L 31/08 20130101; A61L 2300/22 20130101;
A61L 15/34 20130101; A61K 45/06 20130101; A61K 33/38 20130101; A61L
2300/406 20130101; A61K 31/35 20130101; A01N 37/36 20130101 |
International
Class: |
A61L 31/08 20060101
A61L031/08; A61L 31/16 20060101 A61L031/16; A61L 31/14 20060101
A61L031/14; A61K 31/202 20060101 A61K031/202; A61L 15/46 20060101
A61L015/46; A61K 31/765 20060101 A61K031/765; A61L 27/50 20060101
A61L027/50; A61L 29/14 20060101 A61L029/14; A01N 37/36 20060101
A01N037/36; A61K 33/38 20060101 A61K033/38; A61K 31/232 20060101
A61K031/232; A61K 31/35 20060101 A61K031/35; A61L 15/34 20060101
A61L015/34; A61L 27/54 20060101 A61L027/54; A01N 61/00 20060101
A01N061/00; A61L 29/16 20060101 A61L029/16; A61K 31/155 20060101
A61K031/155; A61K 45/06 20060101 A61K045/06; A61K 31/09 20060101
A61K031/09; A61L 31/00 20060101 A61L031/00 |
Claims
1-17. (canceled)
18. A fatty-acid derived biomaterial incorporated with reduced
silver, wherein the biomaterial is derived from a formulation
comprising: an oil comprising a plurality of fatty acid chains; a
pre-cure of the oil, wherein the plurality of the fatty acid chains
are at least partially cross-linked together; and a silver
solution, wherein the biomaterial results from oxidizing the
formulation so that additional fatty acid chains have cross-linked
and so that reduced silver metal forms within the biomaterial.
19. The biomaterial of claim 18, wherein the silver solution
comprises silver nitrate or silver acetate.
20. The biomaterial of claim 19, wherein the silver solution is an
aqueous solution.
21. The biomaterial of claim 18, wherein the reduced silver metal
formed within the biomaterial comprises nanoparticles of silver
metal.
22. The biomaterial of claim 18, wherein the fatty acid chains are
omega-3 fatty acids and the oil is a fish oil.
23. The biomaterial of claim 22, wherein the fish oil and pre-cured
fish oil of the formulation are in a ratio of approximately 50:50
with each other.
24. The biomaterial of claim 18, wherein the oil and the pre-cure
of the oil are in a ratio of approximately 50:50 with each
other.
25. The biomaterial of claim 18, wherein further comprising an
additional antimicrobial compound.
26. The biomaterial of claim 25, wherein the antimicrobial compound
is selected from the group consisting of triclosan, chlorhexidine
and gentamicin.
27. The biomaterial of claim 18, wherein FTIR analysis of the
biomaterial does not possess a peak corresponding to the presence
of fatty acid salts.
28. The biomaterial of claim 18, wherein the biomaterial has a
silver release profile in .mu.g/cm.sup.2 as shown in FIG. 10.
29. The biomaterial of claim 18, wherein the biomaterial is a solid
or a gel.
30. A coating or film comprising the biomaterial of claim 18
disposed on a medical device.
31. The coating or film of claim 30 having an additional
antimicrobial compound applied to an outer surface of the coating
or film.
32. The coating or film of claim 30, wherein the medical device is
a graft, a catheter balloon, or a stent.
33. A method for forming the biomaterial of claim 18, wherein the
method comprises the steps of: mixing the oil, the pre-cure of the
oil and the silver solution with intermittent heating so as to form
an emulsion; thermally curing the emulsion so that additional fatty
acid chain cross-linking occurs; and oxidizing silver of the silver
solution so that reduced silver metal forms within the
biomaterial.
34. The method of claim 33, wherein the silver solution mixed with
the oil and the pre-cure of the oil is an aqueous silver acetate
solution or an aqueous silver nitrate solution.
35. The method of claim 33, wherein the pre-cure of the oil is
formed by at least partially cross-linking fatty acids of the oil
so that the pre-cure of the oil has a higher viscosity than the
oil.
35. The method of claim 33, wherein the silver solution is an
aqueous silver solution, and thermally curing the emulsion includes
forming hydroperoxides in the emulsion, and silver ions react with
the hydroperoxides to form the reduced silver metal from the
aqueous silver solution.
36. The method of claim 33, further comprising the step of: casting
the emulsion onto a medical device before thermally curing the
emulsion on the medical device.
37. The method of claim 36, wherein the medical device is a graft,
a catheter balloon, or a stent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/281,682 filed on May 19, 2014 (now U.S.
Pat. No. 10,______ B2), which claims priority to and is a
continuation of co-pending U.S. patent application Ser. No.
12/539,282, filed Aug. 11, 2009, which patent applications and
patent are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Implantable medical devices are indispensable in the ability
to treat a variety of medical conditions in critically and
chronically ill patients. Catheters can be used to deliver drugs or
nutrients to a patient or to safely remove waste products. Stents
can be used to open blocked coronary arteries and restore blood
flow to the heart. Vascular grafts can also be used to restore
blood flow in addition to providing easier access and improved
treatment of a patient with kidney failure by dialysis. Hernia mesh
devices allow for improved patient outcomes in the treatment of
abdominal wounds by providing additional strength to the surgical
repair.
[0003] One complication in the use of implantable medical devices
is the risk of these devices becoming colonized with bacteria
during surgical implantation (see, e.g., A. M. Carbonell et al.
Surg Endosc. 2005; Vol. 19, pgs 430-435; T. Bechert et al. Nature
Medicine. 2000; Vol. 6, No. 8, pgs 1053-1056; R. Kuijer et al.
Biomaterials. 2007; Vol. 28, pgs 5148-5154; C. R. Arciola et al.
Biomaterials. 2008; Vol. 29, pgs 580-586). Once a patient shows
signs of device infection, the surgeon is often required to perform
several additional surgical procedures to treat the device
infection, such as drainage of the infection site and the local
administration of antibiotics. In cases where the infection is not
successfully resolved, the surgeon will be required to remove the
device from the implanted surgical site until the infection is
resolved with the treatment of oral and/or intravenous antibiotics
to the patient. Thus, an infected medical device not only results
in increasing medical costs, but also results in increased risk of
morbidity and mortality to the patient (A. M. Carbonell et al.,
2005; T. Bechert et al, 2000; R. Kuijer et al., 2007).
[0004] Another important aspect in the use of medical devices is
the biological response to a medical device after surgical repair
of an in-vivo injury (see, e.g., Y. C. Cheong et al. Human
Reproduction Update. 2001; Vol. 7, No. 6, pgs 556-566). A typical
biological response to the surgical site includes inflammation of
native tissue followed by migration and proliferation of cells to
mitigate the inflammatory response, including platelets and
macrophages, and a subsequent healing phase that includes fibrin
deposition and the formation of fibrin matrix followed by tissue
remodeling. In the case of hernia repair, abnormal peritoneal
healing can occur when there is the expression of inflammatory
cytokines from macrophages (e.g., .alpha.-TNF) that can result in
an inability of the fibrin matrix to be properly broken down and
can result in the formation of adhesions (Y. C. Cheong et al.,
2001). Abdominal adhesions formed after hernia repair can result in
pain, bowel strangulation, infertility and in some cases death (Y.
C. Cheong et al., 2001).
[0005] Accordingly, there remains a need for medical devices that
have a reduced susceptibility to colonization by bacteria and other
microorganisms, or other health complications, such as
adhesions.
SUMMARY OF THE INVENTION
[0006] What is desired is a material (e.g., a device coating, gel
or stand-alone film) that can be utilized to prevent or diminish
chronic inflammation due to the hydrolysis products of the coating,
as well as to reduce infection resulting from surgical implantation
of medical devices. Furthermore, it is desirable that the material
release and deliver therapeutic agents (e.g., an antimicrobial,
e.g., an antibiotic agent) in a sustained and controlled fashion.
When in the form of a stand alone film, such a device can be useful
in wound healing applications.
[0007] Thus, provided herein is a composition comprising an
antimicrobial agent, a fatty acid, and a glyceride, wherein the
fatty acid and glyceride components are cross-linked. Such a
composition can be referred to as an "antimicrobial
agent-containing biomaterial." When the antimicrobial agent is a
silver compound, the composition can be referred to as a
"silver-containing biomaterial." This composition has both
anti-inflammation and anti-infection properties, and can come in
many forms, e.g., a coating for a medical device, a stand-alone
film, a gel, a particle, or an emulsion. When the composition is a
coating, it can be placed on the surface of a medical device in
order to prevent bacterial colonization and infection. The
antimicrobial-containing biomaterial can also be used to prevent
tissue adhesion, as well as to facilitate general wound healing,
when, e.g., in the form of a film.
[0008] In one aspect, provided herein is a composition comprising
an antimicrobial compound, a fatty acid, and a glyceride, wherein
the fatty acid and glyceride components are cross-linked. In one
embodiment, the antimicrobial compound is a silver compound, and
the silver compound is ionic silver or elemental silver. The silver
can be elemental silver and is in the form of a silver
nanoparticle. In another embodiment, the silver is ionic silver and
is selected from the group consisting of silver nitrate, silver
chloride, silver fluoride, silver bromide, silver oxide, silver
sulfate, silver carbonate, silver cyanide, silver
tetrafluoroborate, silver sulfide, silver acetate, silver lactate,
silver benzoate, silver cyclohexanebutyrate, silver
diethyldithiocarbamate, silver trifluoromethanesulfonate and
mixtures thereof. In another embodiment, the silver is ionic silver
and is selected from the group consisting of silver nitrate, silver
acetate, silver oxide, and mixtures thereof.
[0009] In another embodiment, the antimicrobial compound is
selected from the group consisting of diamidines, iodine and
iodophors, peroxygens, phenols, bisphenols, halophenols, biguanides
and silver compounds. In still another embodiment, the
antimicrobial compound is selected from the group consisting of
triclosan, chlorhexidine, triclocarban, hexachlorophene,
dibromopropamidine, chloroxylenol, phenol and cresol.
[0010] In another embodiment, the antimicrobial compound is an
antibiotic compound. The antibiotic compound can be selected from
the group consisting of gentamicin sulfate, penicillin g,
ephalothin, ampicillin, amoxicillin, augmentin, aztreonam,
imipenem, streptomycin, gentamicin, vancomycin, clindamycin,
erythromycin, azithromycin, polymyxin, bacitracin, amphotericin,
nystatin, rifampicin, tetracycline, doxycycline, chloramphenicol,
nalidixic acid, ciprofloxacin, sulfanilamide, gantrisin,
trimethoprim, isoniazid, para-aminosalicylic acid, and
minocycline.
[0011] In another embodiment of the biomaterial provided herein,
the cross-linked fatty acid and glyceride are derived from oil
containing at least one omega-3 fatty acid, such as fish oil.
[0012] In another embodiment, the antimicrobial-containing
biomaterial contains an additional antimicrobial compound. The
additional antimicrobial compound can be selected from the group
consisting of diamidines, iodine and iodophors, peroxygens,
phenols, bisphenols, halophenols, biguanides and silver compounds.
In another embodiment, the additional antimicrobial compound is
selected from the group consisting of elemental silver, silver
nanoparticle, silver nitrate, silver chloride, silver fluoride,
silver bromide, silver oxide, silver sulfate, silver carbonate,
silver cyanide, silver tetrafluoroborate, silver sulfide, silver
acetate, silver lactate, silver benzoate, silver
cyclohexanebutyrate, silver diethyldithiocarbamate, silver
trifluoromethanesulfonate, triclosan, chlorhexidine, triclocarban,
hexachlorophene, dibromopropamidine, chloroxylenol, phenol and
cresol.
[0013] In still another embodiment, the antimicrobial-containing
biomaterial contains an additional antibiotic compound. In one
embodiment, the antibiotic compound is selected from the group
consisting of gentamicin sulfate, penicillin g, ephalothin,
ampicillin, amoxicillin, augmentin, aztreonam, imipenem,
streptomycin, gentamicin, vancomycin, clindamycin, erythromycin,
azithromycin, polymyxin, bacitracin, amphotericin, nystatin,
rifampicin, tetracycline, doxycycline, chloramphenicol, nalidixic
acid, ciprofloxacin, sulfanilamide, gantrisin, trimethoprim,
isoniazid, para-aminosalicylic acid, and minocycline.
[0014] The antimicrobial-containing biomaterial composition
provided herein can be further combined with an oil containing an
omega-3 fatty acid. Examples of such oils include, but are not
limited to, fish oil, olive oil, grape oil, palm oil, or flaxseed
oil.
[0015] In another embodiment, when the biomaterial composition is
associated with a tissue of a subject, the silver compound is
released from the cross-linked fatty acid at a controlled release
rate.
[0016] In another aspect, provided herein is a film comprising an
antimicrobial compound, a fatty acid, and a glyceride, wherein the
fatty acid and glyceride components are cross-linked.
[0017] The antimicrobial-containing biomaterial provided herein can
be associated with a medical device, such as a bandage, a stent, a
graft, a shunt, a catheter, a surgical mesh, or a balloon.
[0018] In another aspect, provided herein is a medical device at
least partially coated with the antimicrobial-containing
biomaterial provided herein. In one embodiment, the medical device
is a bandage, a stent, a graft, a shunt, a catheter, or a balloon.
In another embodiment, the mesh is a surgical mesh.
[0019] In another aspect, provided herein is a method of forming a
composition comprising an antimicrobial compound and a cross-linked
fatty acid, comprising:
[0020] curing a starting material comprising a fatty acid and a
glyceride according to a first curing condition to form a second
material; and associating the second material with an antimicrobial
compound that is dissolved in a solution by immersing the second
material in the antimicrobial compound solution or aerosolizing the
antimicrobial compound solution onto the second material;
[0021] such that the composition is formed.
[0022] In one embodiment of this method, the fatty acid starting
material is fish oil. In another embodiment, the antimicrobial is a
silver compound, and the silver compound is selected from the group
consisting of a silver nanoparticle, silver nitrate, silver
chloride, silver fluoride, silver bromide, silver oxide, silver
sulfate, silver carbonate, silver cyanide, silver
tetrafluoroborate, silver sulfide, silver acetate, silver lactate,
silver benzoate, silver cyclohexanebutyrate, silver
diethyldithiocarbamate, silver trifluoromethanesulfonate and
mixtures thereof. In another embodiment of the method, before
curing the fatty acid starting material, the starting material is
associated with a medical device, such as a bandage, a stent, a
graft, a shunt, a catheter, or a balloon. In another embodiment,
the composition is associated with a surgical mesh.
[0023] In another aspect, provided herein is a method of forming a
composition comprising an antimicrobial compound and a cross-linked
fatty acid, comprising:
[0024] curing a starting material comprising a fatty acid and a
glyceride according to a first curing condition to form a second
material;
[0025] associating the second material with a fatty acid-containing
oil and an antimicrobial compound that is dissolved in a solution
to form a third material; and
[0026] curing the third material according to a second curing
condition;
[0027] such that the composition is formed.
[0028] In one embodiment of this method, a portion of the fatty
acids of the second material are cross-linked. In still another
embodiment, the fatty acid starting material and the fatty
acid-containing oil are fish oil. In still another embodiment, the
antimicrobial compound is a silver compound, and the silver
compound is selected from the group consisting of a silver
nanoparticle, silver nitrate, silver chloride, silver fluoride,
silver bromide, silver oxide, silver sulfate, silver carbonate,
silver cyanide, silver tetrafluoroborate, silver sulfide, silver
acetate, silver lactate, silver benzoate, silver
cyclohexanebutyrate, silver diethyldithiocarbamate, silver
trifluoromethanesulfonate and mixtures thereof. In another
embodiment of this method, before curing the third material, the
third material is associated with a medical device, such as a
bandage, a stent, a catheter, a surgical mesh, or a balloon.
[0029] In another aspect, provided herein is a method of forming a
composition comprising an antimicrobial compound and a cross-linked
fatty acid, comprising:
[0030] associating a fatty acid- and glyceride-containing oil with
an antimicrobial compound to form a second material; and
[0031] curing the second material according to a first curing
condition;
[0032] such that the antimicrobial-containing composition is
formed.
[0033] In one embodiment of this method, the fatty acid-containing
oil is a fish oil. In still another embodiment, the fatty acid- and
glyceride-containing oil is a mixture of fish oil, and pre-cured
fish oil. In another embodiment of this method, the antimicrobial
compound is a silver compound, and the silver compound is ionic
silver in aqueous solution. In still another embodiment, the
antimicrobial compound is a silver compound, and the silver
compound is in the form of a silver nanoparticle.
[0034] In yet another embodiment of this method, an additional
antimicrobial compound is dissolved in solvent to form a third
composition, and the third composition is coated onto the outer
surface of the antimicrobial-containing composition. In one
embodiment, the third composition is coated onto the outer surface
of the antimicrobial-containing composition by spraying the third
composition or submerging the antimicrobial-containing composition
in the third composition. The additional compound can be an
antimicrobial compound, and the antimicrobial compound is selected
from the group consisting of diamidines, iodine and iodophors,
peroxygens, phenols, bisphenols, halophenols, biguanides and silver
compounds. In another embodiment, the additional antimicrobial
compound, and the antimicrobial compound is selected from the group
consisting of elemental silver, silver nanoparticle, silver
nitrate, silver chloride, silver fluoride, silver bromide, silver
oxide, silver sulfate, silver carbonate, silver cyanide, silver
tetrafluoroborate, silver sulfide, silver acetate, silver lactate,
silver benzoate, silver cyclohexanebutyrate, silver
diethyldithiocarbamate, silver trifluoromethanesulfonate,
triclosan, chlorhexidine, triclocarban, hexachlorophene,
dibromopropamidine, chloroxylenol, phenol and cresol.In still
another embodiment of this method, the additional compound is an
antibiotic compound, and wherein the antibiotic compound is
selected from the group consisting of gentamicin sulfate,
penicillin g, ephalothin, ampicillin, amoxicillin, augmentin,
aztreonam, imipenem, streptomycin, gentamicin, vancomycin,
clindamycin, erythromycin, azithromycin, polymyxin, bacitracin,
amphotericin, nystatin, rifampicin, tetracycline, doxycycline,
chloramphenicol, nalidixic acid, ciprofloxacin, sulfanilamide,
gantrisin, trimethoprim, isoniazid, para-aminosalicylic acid, and
minocycline.
[0035] In another embodiment of this method, before curing the
second material, the second material is associated with a medical
device, such as a bandage, a stent, a catheter, a surgical mesh, or
a balloon.
[0036] In another aspect, provided herein is a method of treating
or preventing tissue adhesion in a subject in need thereof,
comprising administering the subject the antimicrobial-containing
biomaterial provided herein.
[0037] In another aspect, provided herein is a method of treating
or preventing a bacterial infection in a subject in need thereof,
comprising administering the subject the antimicrobial-containing
biomaterial provided herein.
[0038] In another aspect, provided herein is an emulsion comprising
an antimicrobial compound, a fatty acid, and a glyceride, wherein
the fatty acid and glyceride components are cross-linked.
[0039] In another aspect, provided herein is a composition
comprising a silver compound, a fatty acid, and a glyceride,
wherein the fatty acid and glyceride components are
cross-linked.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The foregoing and other aspects, embodiments, objects,
features and advantages of the invention can be more fully
understood from the following description in conjunction with the
accompanying drawings. In the drawings, like reference characters
generally refer to like features and structural elements throughout
the various figures. The drawings are not necessarily to scale,
emphasis instead being placed upon illustrating the principles of
the invention.
[0041] This includes various embodiments that are based on observed
antimicrobial properties of medical articles augmented with fatty
acid antimicrobial compositions containing various pharmaceutical
and anti-infective agents. The examples set forth in this
specification are not intended to be limiting and merely set forth
some of the many possible embodiments of the claimed invention.
[0042] FIG. 1 is a schematic of reactions that result in the
formation of ester bonds;
[0043] FIG. 2 shows bar graphs showing similarity of fatty acid
composition between fatty acid-derived biomaterial coating and
biological tissue;
[0044] FIG. 3 illustrates the silver release profile of the fatty
acid-derived biomaterial coating hydrated in silver acetate over 4
hours;
[0045] FIG. 4 illustrates the silver release profile of the fatty
acid-derived biomaterial coating hydrated in silver nitrate over 4
hours;
[0046] FIG. 5 depicts a bar graph of the total silver in
.mu.g/cm.sup.2, assayed from Fatty Acid Biomaterial samples
hydrated in aqueous silver nitrate and aqueous silver acetate over
a period of 6 hours;
[0047] FIGS. 6A-6B represent FTIR analysis of fatty acid-derived
biomaterial samples hydrated in silver nitrate and silver acetate
between the regions of 900-1850 cm.sup.-1 illustrating an increase
in silver fatty acid salts with increased hydration time as
indicated by the peaks at 1512 cm.sup.-1;
[0048] FIG. 7 is a bar graph of the recorded log reduction of
colony forming units per cm.sup.2 of silver nitrate hydrated fatty
acid-derived biomaterial samples against a control fatty
acid-derived biomaterial in a three day in-vitro S. aureus biofilm
study;
[0049] FIG. 8 illustrates the silver release profile of a fatty
acid-derived biomaterial sample spray coated with layer of
silver;
[0050] FIG. 9 depicts a bar graph of the log reduction of colony
forming units per cm.sup.2 of a fatty acid-derived biomaterial
sample coated with a layer of silver together with a control fatty
acid-derived biomaterial sample in a three day in-vitro S. aureus
biofilm study;
[0051] FIG. 10 illustrates the silver release profile in
.mu.g/cm.sup.2 of a fatty acid-derived biomaterial sample
incorporated with reduced silver;
[0052] FIG. 11 graphically depicts different zone sizes in mm in a
zone of inhibition in-vitro study achieved from fatty acid-derived
biomaterial samples prepared with different concentrations of
reduced silver;
[0053] FIG. 12 depicts a bar graph of the log reduction of colony
forming units per cm.sup.2 of a fatty acid-derived biomaterial
sample incorporated with reduced silver together with a control
fatty acid-derived biomaterial sample in a three day in-vitro S.
aureus biofilm study;
[0054] FIG. 13 represents FTIR analysis of fatty acid-derived
biomaterial with reduced silver and a fatty acid-derived
biomaterial control between the regions of 800-1800 cm.sup.-1
without the presence of the silver fatty acid peak at approximately
1512 cm.sup.-1 , showing a variation of the samples analyzed in
FIGS. 9A and 9B;
[0055] FIG. 14 depicts silver release data of the silver
nanoparticle coated fatty acid-derived biomaterial in aqueous media
as described in Example 3;
[0056] FIG. 15 illustrates the silver assay loading of different
samples with different concentrations of silver nanoparticles in
the fatty acid-derived biomaterial coating as discussed in Example
4;
[0057] FIG. 16 depicts silver release data of the silver
nanoparticle fatty acid film-derived biomaterial as described in
Example 3;
[0058] FIG. 17 depicts silver release data of the silver
nanoparticle coated fatty acid-derived biomaterial with gentamicin
added in-situ as described in Example 3;
[0059] FIG. 18 depicts gentamicin release data of the silver
nanoparticle coated fatty acid-derived biomaterial with gentamicin
added in-situ as described in Example 3;
[0060] FIG. 19 provides a bar graph of zones measured in mm of
Silver nanoparticle fatty acid-derived biomaterial surface coated
with gentamicin, a Silver nanoparticle fatty acid-derived
biomaterial and a fatty acid-derived biomaterial control in a three
day zone of inhibition study as described in Example 5;
[0061] FIG. 20 illustrates a bar graph of the colony forming units
per mL enumerated by extracting samples of a Silver nanoparticle
fatty acid-derived biomaterial surface coated with gentamicin, a
Silver nanoparticle fatty acid-derived biomaterial and a fatty
acid-derived biomaterial control in a bacterial adherence study as
described in Example 5;
[0062] FIG. 21 depicts the drug release data of gentamicin in an
aqueous media from gentamicin Silver nanoparticle fatty acid
film-derived biomaterial samples with different gentamicin
concentrations loaded onto the samples as described in Example
5;
[0063] FIG. 22 depicts the gentamicin assay loading data in
.mu.g/cm.sup.2 in gentamicin Silver nanoparticle fatty acid
film-derived biomaterial samples containing different gentamicin
concentrations as described in Example 5;
[0064] FIG. 23 shows a bar graph of zones measured in mm from a
three day bacterial zone of inhibition study of Silver nanoparticle
fatty acid-derived biomaterial surface coated with chlorhexidine
diacetate and a Silver nanoparticle fatty acid-derived biomaterial
control;
[0065] FIG. 24 is a bar graph of a bacterial adherence assay of
silver nanoparticle fatty acid biomaterial surface coated with
chlorhexidine diacetate , chlorhexidine-silver fatty acid
biomaterial surface coated with chlorhexidine diacetate together
with fatty acid biomaterial and silver nanoparticle fatty acid
biomaterial controls;
[0066] FIG. 25 shows triclosan release data from a Silver
nanoparticle fatty acid-derived biomaterial sample surface coated
with Triclosan;
[0067] FIG. 26 illustrates the zones measured from a Silver
nanoparticle fatty acid-derived biomaterial sample surface coated
with triclosan over approximately three weeks;
[0068] FIG. 27 is a S. aureus bacterial suspension assay plot of
Silver nanoparticle fatty acid-derived biomaterial sample surface
coated with Triclosan;
[0069] FIG. 28 portrays a S. aureus zone of inhibition plot showing
the zone sizes measured from a Triclosan-Silver nanoparticle fatty
acid-derived biomaterial triclosan surface coated sample over two
weeks;
[0070] FIG. 29 is a S. aureus bacterial adherence plot of Silver
nanoparticle fatty acid-derived biomaterial sample surface coated
with Triclosan;
[0071] FIG. 30 portrays a S. aureus zone of inhibition plot showing
the zone sizes measured from a Triclosan-Silver nanoparticle fatty
acid-derived biomaterial sample over two and a half weeks; and
[0072] FIG. 31 illustrates the efficacy of different antimicrobial
formulations of fatty acid-based biomaterials containing different
doses of triclosan with and without silver nanoparticles as
evaluated in an in-vivo infectability model with a clinical strain
of MRSA.
DETAILED DESCRIPTION
[0073] Implantable medical devices are designed to improve the
quality of life of patients with varying medical conditions. These
devices, however, are highly susceptible to infection and can
affect patient mortality and morbidity leading to high medical
costs. While such infection may occur during surgery, nosocomial
infections are also associated with medical device infections which
necessitate the use of antimicrobial compositions within or on the
surface of these medical devices in order to prevent bacterial
colonization and infection.
[0074] Provided herein is a biomaterial containing an antimicrobial
compound (an "antimicrobial-containing biomaterial"). In one
embodiment, the antimicrobial compound is a silver compound or
triclosan. In another embodiment, the antimicrobial compound is an
antibacterial compound, such as gentamicin. Embodiments of the
present invention may provide a biomaterial containing a silver
compound. The biomaterial can be made from a source of fatty acids
and glycerides (e.g., an oil, e.g., a fish oil) that is cured to
cross-link the fatty acids and glycerides. The biomaterial can be
further combined with other antimicrobial agents such as, but not
limited to, triclosan, chlorhexidine, gentamicin, gentamicin
sulfate, and other antibiotics. One advantage of the present
antimicrobial-containing biomaterial is the anti-inflammatory
properties derived from the fatty acids, while providing a suitable
vehicle for sustained controlled release of the antimicrobial
(e.g., silver-based agents, triclosan, chlorhexidine, gentamicin,
gentamicin sulfate, etc.). The antimicrobial-containing biomaterial
furthermore provides a favorable surface for adding topical layers
of additional antimicrobial agents that provide different sustained
release and efficacy properties.
[0075] When associated with a medical device, the
antimicrobial-containing biomaterial provided herein can reduce the
incidence of inflammatory and foreign body responses after
implantation of that medical device. The antimicrobial-containing
biomaterial can also provide the release of one or more therapeutic
agents (silver-based agents, triclosan, chlorhexidine, gentamicin,
gentamicin sulfate, etc.) over a period of time in order to reduce
the ability of bacteria to colonize during implantation and to
prevent a device-related infection. The antimicrobial-containing
biomaterial can also be used to prevent tissue adhesion, as well as
to facilitate general wound healing. When in the form of a
stand-alone film, or when associated with a bandage, the
antimicrobial-containing biomaterial can be used in wound healing
applications. The antimicrobial-containing biomaterial can also be
metabolized via a bioabsorption mechanism.
[0076] The antimicrobial-containing biomaterial can be utilized
alone or optionally in combination with a medical device for the
release and local delivery of one or more antimicrobial agents to
prevent colonization of bacteria onto the device during surgical
implantation. Methods of forming and tailoring the properties of
said biomaterials are also provided. Additionally, due to the
unique properties of the underlying chemistry of the biomaterial,
the biomaterial (e.g., coating or stand-alone film) contains
specific chemical components that assist in reducing a foreign body
response and inflammation at the site of tissue injury during
implantation that improves its in-vivo performance
Antimicrobial-Containing Fatty Acid-Derived Biomaterials
[0077] In various aspects, the antimicrobial-containing biomaterial
can be, but is not limited to, a coating for a medical device, a
stand-alone film, a gel, a particle, or an emulsion. The
biomaterial can be a hydrophobic biomaterial comprising
cross-linked fatty acids and glycerides. In various embodiments,
the fatty acid/glyceride-derived biomaterial is non-polymeric. In
certain instances, as described herein, the source of the fatty
acid and glyceride is an oil, e.g., a fish oil. The oil can also be
olive oil, grape oil, palm oil, or flaxseed oil. In such an
instance, the antimicrobial-containing biomaterial can also be
referred to as an "oil-derived antimicrobial-containing
biomaterial" (e.g., an "oil-derived silver-containing
biomaterial").
[0078] Various methods can be used to prepare the
antimicrobial-containing biomaterial described herein. The
fatty-acid and glyceride (e.g., oil, e.g., fish oil) can be cured
(using heat, UV, etc.) to induce fatty-acid oxidation and
crosslinking of a portion, majority, or all of the fatty acids and
glycerides to form a gel. The antimicrobial component can be added
to the fatty acid before, during, or after the curing process. In
one embodiment, a pre-cure-derived biomaterial is combined with an
antimicrobial compound to create the antimicrobial-containing
biomaterial (e.g., coating or stand-alone film) described
herein.
[0079] A "pre-cure" component refers to fatty acids (e.g., from
fish oil) that are partially cured (using heat, UV, etc.) to induce
an initial amount of fatty-acid oxidation and crosslinking to form
a viscous fatty acid-derived gel. The pre-cured fatty acid
component (when the source of the fatty acid is an oil, such as
fish oil, the component can also be referred to as "pre-cured oil")
can be combined with an antimicrobial compound, wherein the
antimicrobial compound is optionally combined with an oil (e.g.,
fish oil). The resulting combination can be further cured, thereby
further cross-linking the fatty acids and glycerides of the oil, to
provide a fatty-acid based, pre-cure derived biomaterial that
contains an antimicrobial agent. Thus, in this instance, a portion
of the fatty acid-based biomaterial was pre-cured before
formulation, and then exposed to further curing in the presence of
an antimicrobial compound.
[0080] The process of creating a pre-cure (e.g., of a fish oil) has
the advantage of creating an initial platform of oxidized fatty
acid cross-links that will be hydrolyzed by human tissue. This
process results in a partially cross-linked composition, with
reduced oxidizable reactive sites (e.g., C.dbd.C bonds), that
contains no antimicrobial compound. After the pre-cure is formed,
the antimicrobial compound is then added.
[0081] Several methods are available to cure the oil starting
material containing one or more therapeutic agents to produce a
fatty acid-derived biomaterial for a drug release and delivery
coating or stand-alone film in accordance with the present
invention (for example, as described in US Patent Application
Publications 2008/0118550, 2007/0202149, 2007/0071798,
2006/0110457, 2006/0078586, 2006/0067983, 2006/0067976,
2006/0067975, all of which are incorporated herein by reference).
Preferred methods for curing the starting material to produce an
antimicrobial agent-containing, fatty acid-derived biomaterial of
the present invention include, but are not limited to, heating
(e.g., employing an oven, a broadband infrared (IR) light source, a
coherent IR light source (e.g., laser), and combinations thereof)
and ultraviolet (UV) irradiation. The starting material may be
cross-linked through auto-oxidation (i.e., oxidative
cross-linking).
[0082] In accordance with various embodiments described herein, the
drug release coatings and stand alone films of the present
invention are formed of a fatty acid-derived biomaterial, which can
be derived from saturated and unsaturated fatty acid compounds
(e.g., free fatty acids, fatty acid ester, monoglycerides,
diglycerides, triglycerides, metal salts, etc.). Preferably, the
source of fatty acids described herein is saturated and unsaturated
fatty acids such as those readily available in triglyceride form in
various oils (e.g., fish oils). One method of the formation of a
fatty acid-derived biomaterial is accomplished through autoxidation
of the oil. As a liquid oil containing unsaturated fatty acid is
heated, autoxidation occurs with the absorption of oxygen into the
oil to create hydroperoxides in an amount dependent upon the amount
of unsaturated (C.dbd.C) sites in the oil. However, the (C.dbd.C)
bonds are not consumed in this initial reaction. Concurrent with
the formation of hydroperoxides is the isomerization of (C.dbd.C)
double bonds from cis to trans in addition to double bond
conjugation. Continued heating of the oil results in the
solidifying of the coating through the formation of cross-linking
and by the further reaction of the hydroperoxides and the cleavage
of C.dbd.C double bonds, which convert them into secondary
oxidation byproducts including aldehydes, ketones, alcohols, fatty
acids, esters, lactones, ethers, and hydrocarbons which can either
remain within the coating and/or are volatilized during the
process.
[0083] The type and amount of cross-links formed during oil
oxidation can be tailored depending on the conditions selected
(e.g., coating thickness, temperature, metal composition, etc.).
For instance, heating of the oil allows for cross-linking between
the fish oil unsaturated chains using a combination of peroxide
(C--O--O--C), ether (C--O--C), and hydrocarbon (C--C) bridges (see,
e.g., F. D. Gunstone, "Fatty Acid and Lipid Chemistry." 1999.).
Heating at lower temperatures (below 150.degree. C.) results in the
formation of predominantly peroxide cross-links, where heating at
higher temperatures (i.e. above 150.degree. C.) results in the
thermal degradation of peroxides and C.dbd.C and ether cross-links
dominate (F. D. Gunstone, 1999).
[0084] In addition to thermal curing processes, oxidation of oils
can also be induced by light (e.g., photo-oxygenation).
Photo-oxygenation is limited to C.dbd.C carbon atoms and results in
a conversion from cis to trans C.dbd.C isomers during curing (as
occurs with heat initiated curing). However, photo-oxygenation
using UV is a relatively quicker reaction than autoxidation from
heat curing, in the realm of about 1000-1500 times faster. The
quicker reaction especially holds true for methylene interrupted
polyunsaturated fatty acids, such as EPA and DHA, which are found
in the fish oil based embodiments of the present invention.
[0085] An important aspect of UV curing when compared to heat
curing is that although the byproducts obtained by both curing
methods are similar, they are not necessarily identical in amount
or chemical structure. One reason for this is due to the ability of
photo-oxygenation to create hydroperoxides at more possible C.dbd.C
sites.
[0086] Photo-oxygenation, such as that which results from UV
curing, due to its enhanced ability to create inner hydroperoxides,
also results in the ability to form relatively greater amounts of
cyclic byproducts, which also relates to peroxide cross- linking
between fish oil hydrocarbon chains. For example, photo-oxygenation
of linolenate results in 6 different types of hydroperoxides to be
formed, whereas autoxidation results in only 4. The greater amount
of hydroperoxides created using photo-oxygenation results in a
similar, but slightly different, structure and amount of secondary
byproducts to be formed relative to autoxidation from heat curing.
Specifically, these byproducts are aldehydes, ketones, alcohols,
fatty acids, esters, lactones, ethers, and hydrocarbons.
[0087] Depending on the oil curing conditions and the fatty acid
composition of the starting oil, a fatty acid-derived biomaterial
can be produced by curing the oil so as to oxidize the double bonds
of the unsaturated fatty acid chains while predominantly preserving
triglyceride ester functional groups. The oxidation of the
unsaturated fatty acid chains results in the formation of
hydroperoxides, which, with continued curing, are converted into
and aldehydes, ketones, alcohols, fatty acids, esters, lactones,
ethers, and hydrocarbons. With continued heating of the oxidized
oil, the byproducts are volatilized, resulting in an increase in
the coating viscosity in addition to the formation of ester
cross-links. The formation of ester and lactone cross-links can
occur via different types of mechanisms (i.e., esterification,
alcoholysis, acidolysis, interesterification as described in F. D.
Gunstone, 1999, Chapter 8, incorporated herein by reference)
between the hydroxyl and carboxyl functional components in the
coating formed from the oxidation process (i.e., glyceride and
fatty acid). The cross-linking reaction can form different types of
ester linkages such as ester, anhydride, aliphatic peroxide, and
lactones. One possible mechanism for the formation of the fish-oil
derived biomaterial used herein can be provided as follows: fish
oil (triglycerides) absorbs oxygen; oxidation of the C.dbd.C bonds
in the oil occurs; fatty acid and glycerides (a mixture of mono,
di- and tri) are formed; continued curing results in dehydration of
neighboring carboxyl and hydroxyl functional groups; lactone/ester
cross-links are formed between fatty acids and glycerides; and the
oil solidifies into a bioabsorbable gel containing cross-linked
fatty acids and glycerides. The reaction chemistry of this
formation is as follows: isomerization and oxidation of C.dbd.C
bonds; volatilization of water, hydrocarbons and aldehydes,
resulting in an increase in coating viscosity; and ester and
lactone cross-links formed results in solidifying the coating into
a gel.
[0088] FIG. 1 provides a schematic of different methods to form
esters from oils for illustrative purposes, but is not meant to be
limiting in its scope to the invention.
[0089] In various aspects, provided herein is a means to load
different antimicrobial compounds (e.g., silver compounds) into
fatty acid-derived biomaterials. In one embodiment, these
antimicrobial-containing biomaterials can be combined with a
medical device in order to enhance the device's short term
antimicrobial activity. For example, the release of silver ions
from the oil silver-containing biomaterials (e.g., coating or
stand-alone film) can be altered depending on the silver compound
selected (e.g., whether it is an insoluble or soluble form of
silver) in addition to the method of loading silver into the
coating and/or formulation (e.g., silver compound encapsulated and
cured into coating, sprayed onto cured coating, or soaked into
cured coating). Depending on the method of incorporation of the
antimicrobial compounds into the biomaterial, the antimicrobial
release in water and chemical form of the antimicrobial can be
altered, which can alter the efficacy of the
antimicrobial-containing biomaterial.
[0090] In one embodiment, the antimicrobial behavior of the
compounds incorporated into the antimicrobial-containing
biomaterials (e.g., coatings or stand-alone films) can be altered
by not only the form of antimicrobial agent, but also by the
interaction between the antimicrobial compound and the biomaterial
itself. The antimicrobial-containing biomaterials described herein
are created by thermal oxidation of the unsaturated double bonds in
an oil, initially resulting in the formation of hydroperoxides,
which are then subsequently further oxidized into other byproducts
including fatty acids. Carboxyl and hydroxyl groups formed during
the oxidation of the oil (i.e., fatty acids and glycerides) can
react together upon further heating by esterification, which
results in the formation of lactone and ester cross-links to gel
the coating. Thus, depending on the type of antimicrobial compound
and the stage of incorporation of the compound into the coating,
different antimicrobial coatings (e.g., silver-containing coatings)
can be produced by not only the type of antimicrobial compound
selected, but also due to the final resultant chemistry produced by
the interaction of the antimicrobial compound with the different
molecular species present in the oil-derived coating. Additionally,
unlike traditional hydrophobic medical device materials such as
PTFE, the antimicrobial-containing biomaterial becomes readily
hydrated in an aqueous environment, which results in the release of
antimicrobial compounds (e.g., silver compounds) to be
observed.
[0091] In another embodiment, the method of incorporating an
antimicrobial compound into the biomaterial is to utilize an
antimicrobial compound (e.g., a silver compound) to create a
suspension into the oil starting material by either cryo-grinding
and/or sonication with vortexing. In one embodiment, of this
method, the antimicrobial compound is a silver compound, and in
another embodiment, the silver compound is in a nanoparticle size.
Thus, a silver-containing biomaterial (e.g., coating or stand-alone
film) can be produced by the incorporation of silver nanoparticles
into the oil starting material (e.g., fish oil) after sonicating
and vortexing to uniformly suspend the nanoparticles into the oil.
The oil-nanoparticle suspension is then thermally cured either as a
stand-alone product or as a coating onto a medical device. This
process results in encapsulating the silver nanoparticles in the
coating existing predominantly in their native silver metal form or
as silver oxide. The silver nanoparticles used in this invention
can be of different sizes and either be pure silver metal
nanoparticles or nanoparticles coated with a surface coating to
prevent agglomeration. This process can also be applied to other
antimicrobial agents (e.g., triclosan, chlorhexidine, gentamicin,
gentamicin sulfate).
[0092] As discussed above, the fatty-acid and glyceride (e.g., oil,
e.g., fish oil) can be cured to induce fatty-acid oxidation and
crosslinking of a portion, majority, or all of the fatty acids and
glycerides to form a gel. The antimicrobial agent can be added to
the fatty acid before, during, or after the curing process. In one
embodiment, the method to incorporate a silver antimicrobial
compound into a fatty-acid derived biomaterial is to combine a
water soluble form of silver with a formulation containing a
mixture of native oil with partially oxidized oil (i.e., a
pre-cured oil). This method for incorporating a silver compound
involves combining a formulation of an aqueous form of silver
(Ag.sup.+) in a formulation of fish oil and pre-cured fish oil. In
one embodiment, the formulation of fish oil and pre-cured fish oil
is approximately a 50:50 ratio of fish oil: pre-cured fish oil. The
silver ions in the silver nitrate solution then react with the
lipid hydroperoxides formed during the oxidation of the oil-derived
cross-linked gel and are reduced to silver metal.
[0093] In another embodiment, a method to load antimicrobial
compounds into a silver-containing biomaterial is to first create
the coating using a thermal cure process and then soaking the
coating in a water soluble form of silver (e.g., silver nitrate or
silver acetate). This process creates silver fatty acid salts
within the silver-containing biomaterial. Additionally, the amount
of silver fatty acid salts formed within the biomaterial can be
altered as a function of pH, silver compound selected and soak
time. Using this process, the amount of silver ions produced within
the biomaterial are released more rapidly, typically in less than a
week as measured using in-vitro dissolution testing methodology.
Accordingly, this process can be used with any other water soluble
antimicrobial agent.
[0094] It will be appreciated that the present invention can be
implemented as a coating, as well as a stand-alone material, or
other forms as described herein (e.g., a gel, a particle, or an
emulsion). As would be understood by those of ordinary skill in the
art these biomaterials will have an outer surface that interacts
with its environment upon implantation or application.
[0095] Antimicrobial-containing biomaterial coatings and
stand-alone films of the present invention are formed from an oil
component. The term "oil component" is also referred to herein as
the "oil acid-containing starting material" or "fatty
acid-containing starting material." The "fatty acid-containing
starting material" can be natural or derived from synthetic
sources. Preferably, the "oil containing starting material"
comprises unsaturated fatty acids. The oil component can be either
an oil, or an oil composition. The oil component can be a naturally
occurring oil, such as fish oil, flax seed oil, grape seed oil, or
palm oil, or other oils having desired characteristics. The oil can
also be a synthetic oil. One embodiment of the present invention
makes use of a fish oil in part because of the high content of
omega-3 fatty acids, which can provide healing support for damaged
tissue, as discussed herein. The fish oil can also serve as an
anti-adhesion agent. In addition, the fish oil maintains
anti-inflammatory or non-inflammatory properties as well. The
present invention is not limited to formation of the fatty
acid-derived biomaterial with fish oil as the oil. However, the
present application makes reference to the use of fish oil as one
example embodiment. Other naturally occurring oils or synthetic
oils can be utilized in accordance with the present invention as
described herein.
[0096] It should be noted that as utilized herein, the term "fish
oil" includes but is not limited to omega-3 fatty acid, unsaturated
fatty acid, polyunsaturated fatty acid, fish oil fatty acid, free
fatty acid, monoglycerides, di-glycerides, or triglycerides, esters
of fatty acids, or a combination thereof. The fish oil may include
one or more of arachidic acid, gadoleic acid, arachidonic acid,
eicosapentaenoic acid, docosahexaenoic acid or derivatives, analogs
and pharmaceutically acceptable salts thereof.
[0097] Furthermore, as utilized herein, the term free fatty acid
includes, but is not limited to, one or more of butyric acid,
caproic acid, caprylic acid, capric acid, lauric acid, myristic
acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid,
vaccenic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic
acid, behenic acid, erucic acid, lignoceric acid, analogs and
pharmaceutically acceptable salts thereof. The naturally occurring
oils, including fish oil, are cured as described herein to form a
hydrophobic cross-linked fatty acid-derived biomaterial, creating
the coating.
[0098] With regard to the aforementioned oils, it is generally
known that the greater the degree of unsaturation in the fatty
acids the lower the melting point of a fat, and the longer the
hydrocarbon chain the higher the melting point of the fat. An
unsaturated fat, thus, has a lower melting point, and a saturated
fat has a higher melting point. Those fats having a lower melting
point are more often oils at room temperature. Those fats having a
higher melting point are more often waxes or solids at room
temperature. Therefore, a fat having the physical state of a liquid
at room temperature is an oil. In general, unsaturated fats are
liquid oils at room temperature, and saturated fats are waxes or
solids at room temperature.
[0099] Polyunsaturated fats are one of four basic types of fat
derived by the body from food. The other fats include saturated
fat, as well as monounsaturated fat and cholesterol.
Polyunsaturated fats can be further composed of omega-3 fatty acids
and omega-6 fatty acids. Under the convention of naming, the
unsaturated fatty acid according to the position of its first
double bond of carbons, those fatty acids having their first double
bond at the third carbon atom from the methyl end of the molecule
are referred to as omega-3 fatty acids. Likewise, a first double
bond at the sixth carbon atom is called an omega-6 fatty acid.
There can be both monounsaturated and polyunsaturated omega fatty
acids.
[0100] Omega-3 and omega-6 fatty acids are also known as essential
fatty acids because they are important for maintaining good health,
despite the fact that the human body cannot make them on its own.
As such, omega-3 and omega-6 fatty acids must be obtained from
external sources, such as food. Omega-3 fatty acids can be further
characterized as containing eicosapentaenoic acid (EPA),
docosahexanoic acid (DHA), and alpha-linolenic acid (ALA). Both EPA
and DHA are known to have anti-inflammatory effects and wound
healing effects within the human body.
[0101] The antimicrobial-containing biomaterial described herein
may also be in the form of an emulsion. The term "emulsion," as
used herein, includes classic oil-in-water or water in oil
dispersions or droplets, as well as other lipid structures that can
form as a result of hydrophobic forces that drive polar residues
(i.e., long hydrocarbon chains, such as those found in fatty acids)
away from water and drive polar head groups toward water, when a
water immiscible oily phase is mixed with an aqueous phase. These
other lipid structures include, but are not limited to,
unilamellar, paucilamellar, and multilamellar lipid vesicles,
micelles, and lamellar phases. Such systems possess a finite
stability, generally defined by the application or relevant
reference system, which may be enhanced by the addition of
amphiphilic molecules or viscosity enhancers. Accordingly, the
fatty acid-based particles provided herein (with or without a
therapeutic agent) also can be loaded into fatty acid based liquids
(e.g., fish oil) or gels (e.g., partially cured fish oil) to create
an emulsion.
Antimcrobial Agents
[0102] As discussed above, provided herein are compositions
comprising an antimicrobial agent, a fatty acid, and a glyceride,
wherein the fatty acid and glyceride components are cross-linked.
As used herein the term "antimicrobial," "antimicrobial agent" or
"antimicrobial composition" refers to a composition that has the
effect of inhibiting the growth of bacteria, fungi, yeast, algae,
etc., or killing these micro-organisms. Specific non-limiting
examples of antimicrobials include elemental silver, silver
nanoparticle, silver nitrate, silver chloride, silver fluoride,
silver bromide, silver oxide, silver sulfate, silver carbonate,
silver cyanide, silver tetrafluoroborate, silver sulfide, silver
acetate, silver lactate, silver benzoate, silver
cyclohexanebutyrate, silver diethyldithiocarbamate, silver
trifluoromethanesulfonate, triclosan, chlorhexidine, triclocarban,
hexachlorophene, dibromopropamidine, chloroxylenol, phenol and
cresol. Examples of antimicrobial compounds include, but are not
limited to, diamidines, iodine and iodophors, peroxygens, phenols,
bisphenols, halophenols, biguanides and silver compounds.
[0103] An antibiotic is another example of an antimicrobial agent.
The term "antibiotic" as used herein refers to any compound known
to one of ordinary skill in the art that will inhibit the growth
of, or kill, bacteria. Non-limiting examples of antimicrobial
agents that can be used with the biomaterials provided herein
include gentamicin sulfate, penicillin g, ephalothin, ampicillin,
amoxicillin, augmentin, aztreonam, imipenem, streptomycin,
gentamicin, vancomycin, clindamycin, erythromycin, azithromycin,
polymyxin, bacitracin, amphotericin, nystatin, rifampicin,
tetracycline, doxycycline, chloramphenicol, nalidixic acid,
ciprofloxacin, sulfanilamide, gantrisin, trimethoprim, isoniazid,
para-aminosalicylic acid, and minocycline.
[0104] These anti-infective agents can be used either alone or in
combination with one another to create an infection-resistant fatty
acid based biomaterial .
[0105] When the antimicrobial agent is a silver compound, the
composition can be referred to as a "silver-containing
biomaterial." Silver exists in two forms, ionic (Ag.sup.+) and
elemental (Ag.sup.0), but only the ionic form of silver has
antimicrobial properties (S. Pal et al. 2007, "Applied and
Environmental Microbiology" Vol. 73 (6), 1712-1720; A. B. G.
Lansdown. 2006, "Curr. Probl. Dermatol." Vol 33, 17-34; R. O.
Darouiche, 1999, "Clinical Infectious Diseases" Vol. 29, 1371-1377;
V. Sambhy et al., 2006, "J. American Chem. Society. Vol. 128,
9798-9808; Matsumura, K et al., 2003, "Applied & Environmental
Microbiology" Vol. 69, No. 7, 4278-4281). The antimicrobial
activity of silver depends on the interaction of silver ions
(Ag.sup.+) with the bacteria's cellular membrane, which results in
preventing DNA replication and subsequent proliferation of
bacteria. All forms of silver produce silver ions, but the rate of
silver ionization and/or release in a hydrated environment is
dependent on the silver compound selected (A. B. G. Lansdown. 2006,
"Curr. Probl. Dermatol." Vol 33, 17-34; R. O. Darouiche, 1999,
"Clinical Infectious Diseases" Vol. 29, 1371-1377; V. Sambhy et
al., 2006, "J. American Chem. Society. Vol. 128, 9798-9808;
Matsumura, K et al., 2003, "Applied & Environmental
Microbiology" Vol. 69, No. 7, 4278-4281). For instance, silver
nitrate rapidly forms silver ions in an aqueous environment whereas
silver metal slowly forms silver ions. The amount of silver needed
to produce an antimicrobial effect has been previously reported to
be in the range of only 50 ppb (K. C. Chaw et al. 2005). Thus, only
small amounts of silver need to be loaded on the device to be
effective. The incorporation of the silver antimicrobial into the
coating is designed to protect the device from bacterial
colonization.
[0106] Antimicrobial silver compounds that can be used with the
biomaterials of the invention include, but are not limited to,
silver nitrate, silver chloride, silver fluoride, silver bromide,
silver oxide, silver sulfate, silver carbonate, silver cyanide,
silver tetrafluoroborate, silver sulfide, silver acetate, silver
lactate, silver benzoate, silver cyclohexanebutyrate, silver
diethyldithiocarbamate, silver trifluoromethanesulfonate and
mixtures thereof. In a particular embodiment, the silver is ionic
silver and is selected from the group consisting of silver nitrate,
silver acetate, silver oxide, and mixtures thereof.
[0107] The release of silver ions from the silver-containing
biomaterial can be altered depending on the silver compound
selected (e.g., whether it is an insoluble or soluble form of
silver) in addition to the method of loading silver into the
coating and/or formulation (e.g., silver compound encapsulated and
cured into coating, sprayed onto cured coating, or soaked into
cured coating). Depending on the chemical form of the silver
compound and the method of incorporation of the silver compounds
into the biomaterial, the silver ion's release in water and can be
altered, which can alter the biomaterial's efficacy.
[0108] As discussed above, the antimicrobial behavior of the silver
compounds incorporated into the silver-containing biomaterials can
be altered by not only the form of silver compounds that are
incorporated into a biomaterial, but also by the chemical
interaction between the silver compound and the biomaterial itself.
Depending on the type of silver compound and the stage of
incorporation of the silver compound into the coating, different
silver-containing biomaterials can be produced by not only the type
of silver compound selected, but also due to the final resultant
chemistry produced by the interaction of the silver compound with
the different molecular species present in the oil-derived
coating.
[0109] In another embodiment, the method of incorporating a silver
antimicrobial compound into the biomaterial is to create a
suspension of a silver compound in the oil starting material (e.g.,
fish oil). In one embodiment, the suspension is created by either
cryo-grinding and/or sonication with vortexing. The silver compound
utilized in this invention may or may not be in a nanoparticle
size. Specifically, an antimicrobial silver enhanced oil-derived
gel coating can be produced by the incorporation of commercially
available silver nanoparticles into the oil starting material after
sonicating and vortexing to uniformly suspend the nanoparticles
into the oil. The oil-nanoparticle suspension is then thermally
cured either as a stand-alone product or as a coating onto a
medical device. This process results in encapsulating the silver
nanoparticles in the coating existing predominantly in their native
silver metal form or as silver oxide. The silver nanoparticles used
in this invention can be of different sizes and either be pure
silver metal nanoparticles or nanoparticles coated with a surface
coating to prevent agglomeration. A coating containing silver metal
nanoparticles <150 nm was shown to release silver ions from the
coating out to a minimum of 65 days in a water dissolution testing
using an ICP silver assay and was effective at preventing biofilm
formation by a clinical isolate of MRSA in in-vitro studies for a
minimum of 3 days using this technique.
[0110] In various aspects, another method to incorporate a silver
antimicrobial compound into an oil-derived cross-linked gel is to
use a water soluble form of silver and combine that with a
formulation containing a mixture of native oil with partially
oxidized oil (a pre-cured oil). In one embodiment, the method for
incorporating a silver compound can involve combining a formulation
of aqueous silver nitrate (or silver acetate) in a mixture of fish
oil and pre-cured oil (e.g., a formulation of approximately 50:50
fish oil: pre-cured fish oil). The pre-cured fish oil can be
prepared by bubbling oxygen through fish oil while heating, e.g.,
approximately 18 hrs at 93.degree. C. The silver ions in the silver
nitrate solution then react with the lipid hydroperoxides formed
during the oxidation of the oil-derived cross-linked gel and are
reduced to silver metal (A. Kumar et al., "Nature Materials". 2008
Vol 7, pgs 236-241). Depending on the conditions employed, the size
of the reduced silver metal may or may not be in a nanoparticle
size. In addition to reducing the silver into metal form, the
presence of the silver nitrate solution with fatty acids in the
coating can also result in producing silver fatty acid salts, which
are antimicrobial (C. L. Fox et al., 1991; F. M. Mattei, 1980; M.
Roby, 2004). Oil-derived materials (e.g., coating or stand-alone
film) formed by this process were shown to release silver ions from
the coating out to a minimum of 60 days in water dissolution
testing using an ICP silver assay and were effective at preventing
biofilm formation by clinical isolates of MRSA and Staphylococcus
aureus in in-vitro studies for a minimum of 3 days using this
technique.
[0111] Another method to load silver antimicrobial compounds into
silver-containing biomaterial is to first create a coating or stand
alone film using a thermal cure process and then soak or spray a
water soluble form of silver (e.g., silver nitrate or silver
acetate) onto the coating. This is performed by soaking the
oil-derived gel into a solution of either silver nitrate or silver
acetate for a fixed period of time to load silver into the coating.
This process predominantly creates silver fatty acid salts within
the coating, which was confirmed using FTIR spectroscopy.
Additionally, the amount of silver fatty acid salts formed within
the coating or stand alone film can be altered as a function of pH,
silver compound selected (silver acetate or silver nitrate) and
soak time. Using this process, silver ions are released from the
coating more rapidly, typically in less than a week in a water
dissolution test using an ICP silver assay.
[0112] Another antimicrobial used with the biomaterials provided
herein can be triclosan. Triclosan
(2,4,4'-trichloro-2'-hydroxydiphenyl ether) is a broad spectrum
antimicrobial agent that is classified as a chlorinated bisphenol
that has long been used as a biocide in various products including
soap products, oral care products and cosmetics. Triclosan activity
covers a broad range of gram positive bacteria such as Bacillus
subtilis, Mycobacterium smegmatis, Staphylococcal aureus, gram
negative bacteria including E. coli, Salmonella typhimurium,
Shigella flexneri, yeasts and some fungi (M. J. Stewart et. al, "J.
Mol. Biol., 1999, Vol 290, pgs 859-865). At low concentrations,
triclosan is bacteriostatic and bactericidal at higher
concentrations and has been found to have a low toxicity profile
making it ideal for use in personal hygiene products and various
medical devices (A. D. Russell, Journal of Antimicrobial
Chemotherapy, 2004 Vol 53, pgs693-695). Triclosan's mechanism of
action targets a specific enzyme, enoyl-acyl carrier protein
reductase (ENR), involved in the lipid biosynthesis pathway by
acting as competitive inhibitor. ENR catalyzes the terminal
reaction in the fatty acid elongation cycle during fatty acid
biosynthesis. Enzyme inhibition studies using E. coli have
demonstrated that the enzyme-inhibitor complex formed between
triclosan and ENR are strong, slow to dissociate and relatively
irreversible (M. Kapoor, "Biochem. J." 2004, Vol 381, 719-724; J.
Stewart et. al, "J. Mol. Biol., 1999, Vol 290, 859-865). The
lipophilic nature of triclosan makes it an ideal antimicrobial
agent to incorporate into the fatty acid biomaterial coating as
described in the present invention.
[0113] The antimicrobial used with the biomaterials provided herein
can be chlorhexidine. Chlorhexidine is a biocide belonging to the
biguanides group. It is widely used as a preservative and as an
antiseptic agent in hand washing products, oral products and coated
in combination with silver-sulfadiazine on some medical devices
such as urinary catheters to prevent biofilm formation. Uptake of
chlorhexidine is pH dependent and its antibacterial action occurs
by diffusing into and attacking the bacterial cytoplasm after
damaging bacterial outer cell membranes (G. McDonnell et. al.,
"Clinical Biological Reviews." 1997, Vol 12(1), 147-179; S.
Bassetti, 2001, Vol 45(4), 1535-1538).
[0114] The antimicrobial used with the biomaterials provided herein
can be gentamicin. Gentamicin is a water soluble aminoglycoside
antibiotic that is active against a range of gram positive and gram
negative bacteria. The mechanism of transport of gentamicin across
cell membranes is not well known but it is hypothesized that
gentamicin interacts with some components of the cell membrane by
the interaction of gentamicin amino groups and free fatty aldehydes
via a Schiff-base reaction (D. E. Auslander et. al., "J Pharm Sci."
1975, 64(3), 516-519).
[0115] In one embodiment, the antimicrobial-containing biomaterial
contains more than one antimicrobial compound. For example, the
antimicrobial agent can be combined with the fatty acid/glyceride
starting material to form a composition, such that the composition
is cross-linked so the antimicrobial-containing biomaterial is
formed, and then an additional antimicrobial agent is added to the
surface of the biomaterial (by spraying, submersing, etc.). The
additional agent can be the same as the antimicrobial agent
incorporated in the cross-linked matrix, or different. In another
embodiment, two or more different antimicrobial agents are combined
with the fatty acid/glyceride starting material to form a
composition, such that the composition is cross-linked so an
antimicrobial-containing biomaterial is formed that that contains
two or more antimicrobial agents.
[0116] In another embodiment, provided herein is a
silver-containing biomaterial comprising fish oil, wherein the
fatty acids and glycerides of the fish oil are cross-linked, and
wherein the silver-containing biomaterial further comprises an
additional therapeutic agent, such as an antimicrobial agent. In a
particular embodiment, the silver-containing biomaterial further
comprises triclosan, chlorhexidine or gentamicin.
[0117] In another embodiment, the additional therapeutic agent that
is loaded into the antimicrobial-containing biomaterial (e.g.,
silver-containing biomaterial) is an anti-adhesive agent. As used
herein, the term "anti-adhesion agent" refers to any compound that
prevents adhesions or accretions of body tissues formed in response
to injury of various kinds, e.g., surgery, infection, chemotherapy,
radiation. Anti-adhesion agents include, but are not limited to,
hyaluronic acid, human plasma derived surgical sealants, and agents
comprised of hyaluronate and carboxymethylcellulose that are
combined with dimethylaminopropyl, ethylcarbodimide, hydrochloride,
PLA, and/or PLGA.
[0118] In various aspects, the additional antimicrobial agent is
sprayed onto the outside surface of the antimicrobial-containing
biomaterial (e.g., coating or stand-alone film). In one embodiment,
the antimicrobial agent is triclosan. Alternatively, the triclosan
can be sprayed in combination with a soluble silver compound onto
the outside surface of the silver-containing biomaterial (e.g.,
coating or stand-alone film).
[0119] In another embodiment, the fatty acid-derived biomaterial
can be cured with a silver nanoparticle or silver compound in the
base coating, and then an antimicrobial and/or antibiotic agent can
be sprayed onto the outer surface of the cured coating. In one
embodiment, the antimicrobial/antibiotic agent selected is
triclosan.
[0120] In another embodiment, the antimicrobial agent can be loaded
by incorporating the agent into the biomaterial during the thermal
cure process onto a finished device. The fatty acid-derived
biomaterial can contain either triclosan by itself and/or in
combination with a silver nanoparticle and/or silver compound.
Coating Hydrolysis and Bioabsorption Chemistry of Fatty
Acid-Derived Biomaterials
[0121] Biodegradable and bioabsorbable implantable materials with
ester, lactone, and anhydride functional groups are typically
broken down by either chemical and/or enzymatic hydrolysis
mechanisms (K. Park et al., "Biodegradable Hydrogels for Drug
Delivery." 1993; J. M. Andersen, "Perspectives on the In-Vivo
Responses of Biodegradable Polymers." in Biomedical Applications of
Synthetic Biodegradable Polymers, edited by Jeffrey O Hollinger.
1995, pgs 223-233). Chemical hydrolysis of an antimicrobial
-containing biomaterial occurs when the functional group present in
the material is cleaved by water. Enzymatic hydrolysis is the
cleavage of functional groups in an antimicrobial-containing
biomaterial caused by the reaction with a specific enzyme (e.g.,
triglycerides are broken down by lipases (enzymes) that result in
free fatty acids that can then be transported across cell
membranes). The length of time a biodegradable
antimicrobial-containing biomaterial takes to be hydrolyzed is
dependent on several factors such as the cross-linking density of
the material, the thickness, the hydration ability of the coating,
the crystallinity of the fatty acid-derived biomaterial, and the
ability for the hydrolysis products to be metabolized by the body
(K. Park et al., 1993 and J. M. Andersen, 1995).
[0122] It should be noted that a bioabsorbable substance is
different from a biodegradable substance. Biodegradable is
generally defined as capable of being decomposed by biological
agents, or capable of being broken down by microorganisms or
biological processes. Biodegradable substances can cause an
inflammatory response due to either the parent substance or those
formed during hydrolysis, and they may or may not be absorbed by
tissues. Some biodegradable substances are limited to a bulk
erosion mechanism for hydrolysis. For example, a commonly used
biodegradable polymer, PLGA (poly (lactic-co-glycolic acid))
undergoes chemical hydrolysis in-vivo to form two alpha-hydroxy
acids, specifically glycolic and lactic acids. Thus, in one
embodiment, the antimicrobial-containing biomaterial provided
herein is non-polymeric. Although glycolic and lactic acids are
byproducts of various metabolic pathways in the body, it has been
demonstrated in previous medical implant and local drug delivery
applications that a local concentration of these products results
in an acidic environment to be produced, which can lead to
inflammation and damage to local tissue (S. Dumitriu, "Polymeric
Biomaterials." 2002). Clinically, this can lead to impaired
clinical outcomes such as restenosis (D. E. Drachman and D. I.
Simon. Current Atherosclerosis Reports. 2005, Vol 7, pgs 44-49;
S.E. Goldblum et al. Infection and Immunity. 1989, Vol. 57, No. 4,
pgs 1218-1226) and impaired healing in a coronary stent application
which can lead to late-stent thrombosis or adhesion formation in an
abdominal hernia repair (Y. C. Cheong et al. Human Reproduction
Update. 2001; Vol. 7, No. 6: pgs 556-566). Thus, an ideal
antimicrobial -containing biomaterial should not only demonstrate
excellent biocompatibility upon implantation, but should also
maintain that biocompatibility during the life of its implantation
with its hydrolysis byproducts being absorbable by local
tissue.
[0123] The bio-absorbable nature of the antimicrobial-containing
biomaterial (derived, e.g., from fish oil containing triglycerides
and fatty acids) used as a stand-alone film, a coating for a
medical device, or in drug delivery applications results in the
biomaterial being absorbed over time by the cells of the body
tissue. In various embodiments, there are substantially no
substances in the biomaterial, or in vivo conversion by-products of
the biomaterial, which induce an inflammatory response, e.g., the
antimicrobial-containing biomaterial converts in vivo into
non-inflammatory components. For example, in various embodiments,
the antimicrobial-containing biomaterial of the present invention
upon absorption and hydrolysis do not produce lactic acid and
glycolic acid break-down products in measurable amounts. The
chemistry of the antimicrobial-containing biomaterial described
herein consists of predominantly fatty acid and glyceride
components that can either be hydrolyzed in-vivo by chemical and/or
enzymatic means which results in the release of fatty acid and
glyceride components that can be transported across cell membranes.
Subsequently, the fatty acid and glyceride components eluted from
the antimicrobial-containing biomaterials are directly metabolized
by cells (i.e., they are bio-absorbable). The bio-absorbable nature
of the coating and stand-alone film of the present invention
results in the biomaterial being absorbed over time, leaving only
an underlying delivery or other medical device structure that is
biocompatible. There is substantially no foreign body inflammatory
response to the biomaterial or its hydrolysis products in the
preferred embodiments of the present invention.
[0124] Because the materials of the invention (derived, e.g., from
fish oil containing triglycerides and fatty acids) are
biocompatible, and they hydrolyze into non-inflammatory components,
and are subsequently bio-absorbed by surrounding tissue, they are
referred to as "biomaterials."
Fatty Acid-Derived Biomaterial Biocompatibility and In-Vivo
Performance
[0125] The process of making the antimicrobial-containing
biomaterials (e.g., coating or stand-alone film) as described
herein led to some unexpected chemical processes and
characteristics in view of traditional scientific reports in the
literature about the oxidation of oils. Oil oxidation has
traditionally been of concern for oil curing procedures due to the
formation of reactive byproducts such as hydroperoxides and
alpha-beta unsaturated aldehydes that are not considered to be
biocompatible (H. C. Yeo et al. Methods in Enzymology. 1999, Vol.
300, pgs 70-78.; S-S. Kim et al. Lipids. 1999, Vol. 34, No. 5, pgs
489-496.). However, the oxidation of fatty acids from oils and fats
are normal and important in the control of biochemical processes
in-vivo. For example, the regulation of certain biochemical
pathways, such as to promote or reduce inflammation, is controlled
by different lipid oxidation products (V. N. Bochkov and N.
Leitinger. J. Mol. Med. 2003; Vol. 81, pgs 613-626). Additionally,
omega-3 fatty acids are known to be important for human health and
specifically EPA and DHA are known to have anti-inflammatory
properties in-vivo. However, EPA and DHA are not anti-inflammatory
themselves, but it is the oxidative byproducts they are
biochemically converted into that produce anti-inflammatory effects
in-vivo (V. N. Bochkov and N. Leitinger, 2003; L. J. Roberts II et
al. The Journal of Biological Chemistry. 1998; Vol. 273, No. 22,
pgs 13605-13612.). Therefore, by selecting the appropriate process
conditions, an antimicrobial-containing biomaterial (derived from,
e.g., fish oil) can be created and controlled using oil oxidation
chemistry with a final chemical profile that will have a favorable
biological performance in-vivo.
[0126] The process of making an antimicrobial-containing
biomaterial as described herein leads to a final chemical profile
that is biocompatible, minimizes adhesion formation, acts as a
tissue separating barrier, and is non-inflammatory with respect to
the material chemistry and the products produced upon hydrolysis
and absorption by the body in-vivo. The reason for these properties
is due to several unique characteristics of the
antimicrobial-containing biomaterials (e.g., coatings or
stand-alone films).
[0127] In some embodiments, no toxic, short-chained cross-linking
agents (such as glutaraldehyde) are used to form the
antimicrobial-containing biomaterials (e.g., coatings or
stand-alone films) of the invention. Short chain cross-linking
agents can elute during hydrolysis of biodegradable polymers and
cause local tissue inflammation. The process of creating
antimicrobial-containing biomaterials does not involve adding
external cross-linking agents because the oil is solely cured into
a coating using oil autoxidation or photo-oxidation chemistry. The
oxidation process results in the formation of carboxyl and hydroxyl
functional groups that allow for the fatty acid-derived biomaterial
to become hydrated and become slippery, which allows for frictional
injury during and after implantation to be significantly reduced
and/or eliminated. The methods of making the
antimicrobial-containing biomaterials described herein allow the
alkyl chains of the fatty acid, glyceride and other lipid
byproducts present in the antimicrobial-containing biomaterial to
be disordered, which creates a biomaterial that is flexible and
aids in handling of the material while being implanted. Thus, in
one embodiment, the antimicrobial-containing biomaterials (derived,
e.g., from fish oil) do not contain any cross-linking agents.
[0128] There are several individual chemical components of the
antimicrobial-containing biomaterial that aid in its
biocompatibility and its low to non-inflammatory response observed
in-vivo. The processes of creating a fatty acid-derived biomaterial
described herein result in low to non-detectable amounts of
oxidized lipid byproducts of biocompatibility concern, such as
aldehydes. These products are either almost completely reacted or
volatilized during the curing process as described in this
invention. The process of creating an antimicrobial-containing
biomaterial largely preserves the esters of the native oil
triglycerides and forms ester and/or lactone cross-links, which are
biocompatible.
[0129] In addition to general chemical properties of an
antimicrobial-containing biomaterial that assists in its
biocompatibility, there are also specific chemical components that
have positive biological properties. Another aspect is that the
fatty acid chemistry produced upon creation of a fatty acid-derived
biomaterial is similar to the fatty acid chemistry of tissue, as
presented in FIG. 2. Thus, as fatty acids are eluting from the
biomaterial they are not viewed as being "foreign" by the body and
cause an inflammatory response. In fact, C14 (myristic) and C16
(palmitic) fatty acids present in the coating have been shown in
the literature to reduce production of .alpha.-TNF, an inflammatory
cytokine. The expression of .alpha.-TNF has been identified as one
of the key cytokines responsible for "turning on" inflammation in
the peritoneal after hernia repair, which can then lead to abnormal
healing and adhesion formation. .alpha.-TNF is also an important
cytokine in vascular injury and inflammation, such as vascular
injury caused during a stent deployment. In addition to the fatty
acids just specified, there have also been additional oxidized
fatty acids identified that have anti-inflammatory properties.
Other components identified from the fatty acid-derived coatings as
described in this invention are delta-lactones (i.e., 6-membered
ring cyclic esters). Delta-lactones have been identified as having
anti-tumor properties.
[0130] These components identified are not meant to be limiting in
scope to this invention as changes in starting oil composition
and/or process conditions can invariably alter the fatty acid
and/or oxidative byproduct profiles and can be tailored as needed
depending on the intended purpose and site of application of the
fatty acid-derived biomaterial.
[0131] In summary, the biocompatibility and observed in in-vivo
performance of antimicrobial-containing biomaterials described
herein is not only beneficial as to prevent a foreign body response
in-vivo due to the similarity of the fatty acid composition of the
material to native tissue (i.e., a biological "stealth" coating),
but the specific fatty acids and/or other lipid oxidation
components eluting from the coating aid in preventing foreign body
reactions and reducing or eliminating inflammation, which leads to
improved patient outcomes. Additionally, the fatty acid and
glyceride components eluted from the antimicrobial-containing
biomaterials are able to be absorbed by local tissue and
metabolized by cells. Hence, the antimicrobial-containing
biomaterial (e.g., coating or stand-alone film) described in this
invention is also bioabsorbable.
Antimicrobial Properties of Antimicrobial-Containing
Biomaterials
[0132] In the present invention, the antimicrobial-containing
biomaterial is composed of various lipid components, free fatty
acids and glycerides that are derived from, e.g., fish oil. The
source of fatty acids in this invention may consist of but are not
limited to other oils including flaxseed oil, grapeseed oil, olive
oil, corn oil, peanut oil, safflower oil and soybean oil.
[0133] Generally, gram positive bacteria have been shown to be more
sensitive to the bactericidal effects of fatty acids with gram
negative bacteria being less sensitive. Longer chain unsaturated
fatty acids have greater bactericidal activity than shorter chain
saturated fatty acids, particularly against gram positive bacteria.
This difference in bacterial sensitivity towards long chain
unsaturated fatty acids and short chain saturated fatty acids are
likely a result of the differences in bacterial surface structure
and the mechanism of antimicrobial effects that occur between the
fatty acid and bacteria. Longer chain fatty acids may be able to
better insert the hydrocarbon chain into the bacterial phospholipid
bilayer thereby conferring membrane destabilization and subsequent
bactericidal effects. Monoglycerides have also been identified to
act as nonionic surfactants that alter membrane permeability by
penetrating the bacterial cell membrane causing plasma membrane
disintegration. Likewise, short chain and medium chain fatty acids
can enter bacterial cells by diffusion into bacterial cells in an
undissociated form and cause intracellular acidification by
dissociating within the bacterial protoplasm. The decreased
intracellular pH can in turn result in enzymatic inactivation and
interrupt amino acid transport which is important in protein
synthesis. An advantage of the antibacterial properties exhibited
by fatty acids and their respective monoglycerides is the decreased
potential to develop bacterial resistance due to the mechanisms by
which bacteria are killed.
Uses
[0134] The antimicrobial-containing biomaterials provided herein
can be used, for example, for wound healing in a subject, e.g., a
human or non-human animal. The process of wound healing involves
tissue repair in response to injury and it encompasses many
different biologic processes, including epithelial growth and
differentiation, fibrous tissue production and function,
angiogenesis, and inflammation. Accordingly, the
antimicrobial-containing material provides an excellent material
suitable for wound healing applications.
[0135] The antimicrobial-containing biomaterials provided herein
can be used, for example, to prevent tissue adhesion. The tissue
adhesion can be a result of blunt dissection. Blunt dissection can
be generally described as dissection accomplished by separating
tissues along natural cleavage lines without cutting. Blunt
dissection is executed using a number of different blunt surgical
tools, as is understood by those of ordinary skill in the art.
Blunt dissection is often performed in cardiovascular, colo-rectal,
urology, gynecology, upper GI, and plastic surgery applications,
among others.
[0136] After the blunt dissection separates the desired tissues
into separate areas, there is often a need to maintain the
separation of those tissues. In fact, post surgical adhesions can
occur following almost any type of surgery, resulting in serious
postoperative complications. The formation of surgical adhesions
involves complex inflammatory processes in which tissues that
normally remain separated in the body come into physical contact
with one another and attach to each other as a result of surgical
trauma.
[0137] It is believed that abdominal adhesions are formed when
bleeding and leakage of plasma proteins from damaged tissue deposit
in the abdominal cavity and form what is called a fibrinous
exudate. Fibrin, which restores injured tissues, is sticky, so the
fibrinous exudate may attach to adjacent anatomical structures in
the abdomen. Post-traumatic or continuous inflammation exaggerates
this process, as fibrin deposition is a uniform host response to
local inflammation. This attachment seems to be reversible during
the first few days after injury because the fibrinous exudates go
through enzymatic degradation caused by the release of fibrinolytic
factors, most notably tissue-type plasminogen activator (t-PA).
There is constant play between t-PA and plasminogen-activator
inhibitors. Surgical trauma usually decreases t-PA activity and
increases plasminogen-activator inhibitors. When this happens, the
fibrin in the fibrinous exudate is replaced by collagen. Blood
vessels begin to form, which leads to the development of an
adhesion. Once this has occurred, the adhesion is believed to be
irreversible. Therefore, the balance between fibrin deposition and
degradation during the first few days post-trauma is critical to
the development of adhesions (Holmdahl L. Lancet 1999; 353:
1456-57). If normal fibrinolytic activity can be maintained or
quickly restored, fibrous deposits are lysed and permanent
adhesions can be avoided. Adhesions can appear as thin sheets of
tissue or as thick fibrous bands.
[0138] Often, the inflammatory response is also triggered by a
foreign substance in vivo, such as an implanted medical device. The
body sees this implant as a foreign substance, and the inflammatory
response is a cellular reaction to wall off the foreign material.
This inflammation can lead to adhesion formation to the implanted
device; therefore a material that causes little to no inflammatory
response is desired.
[0139] Thus, the antimicrobial-containing biomaterial (e.g.,
coating or stand-alone film) can be used as a barrier to keep
tissues separated to avoid the formation of adhesions, e.g.,
surgical adhesions. Application examples for adhesion prevention
include abdominal surgeries, spinal repair, orthopedic surgeries,
tendon and ligament repairs, gynecological and pelvic surgeries,
and nerve repair applications. The antimicrobial-containing
biomaterial may be applied over the trauma site or wrapped around
the tissue or organ to limit adhesion formation. Other surgical
applications of the antimicrobial-containing biomaterial-based film
may include using the film as a dura patch, buttressing material,
internal wound care (such as a graft anastomotic site), and
internal drug delivery system. The antimicrobial-containing
biomaterial (e.g., coating or stand-alone film) may also be used in
applications in transdermal, wound healing, and non-surgical
fields. The antimicrobial-containing biomaterial may be used in
external wound care, such as a treatment for burns or skin ulcers.
The antimicrobial-containing biomaterial may be used without any
therapeutic agent as a clean, non-permeable, non-adhesive,
non-inflammatory, anti-inflammatory dressing, or the
antimicrobial-containing biomaterial may be used with one or more
therapeutic agents for additional beneficial effects. In another
embodiment, the antimicrobial-containing biomaterial is associated
with bandage or dressing for a wound. When used in any of the
aforementioned methods, the fatty acid-based material (in particle
form or particles pressed into a film) may or may not be associated
with a therapeutic agent.
[0140] In another embodiment, the antimicrobial-containing
biomaterials provided herein can be used as a coating on a medical
device to minimize the risk of these devices becoming colonized
with bacteria during surgical implantation. The formulations
described herein can also be spread onto a medical device surface
and allowed to cure for a period of time, thereby providing a
medical device coating comprising an antimicrobial-containing
biomaterial. Examples of such devices include, but are not limited
to, a graft, a catheter balloon, a stent or surgical mesh.
[0141] In still another embodiment, the antimicrobial-containing
biomaterials provided herein (e.g., in the form of a stand-alone
film) can be used for wound healing applications. The
antimicrobial-containing biomaterials provided herein can also be
used in combination with a bandage or dressing for wound healing
applications. The term "wound" refers broadly to injuries to the
skin and subcutaneous tissue initiated in any one of a variety of
ways (e.g., pressure sores from extended bed rest, wounds induced
by trauma, cuts, ulcers, burns and the like) and with varying
characteristics. Wounds are typically classified into one of four
grades depending on the depth of the wound: (i) grade I: wounds
limited to the epithelium; (ii) grade II: wounds extending into the
dermis; (iii) grade III: wounds extending into the subcutaneous
tissue; and (iv) grade IV (or full-thickness wounds): wounds
wherein bones are exposed (e.g., a bony pressure point such as the
greater trochanter or the sacrum). The antimicrobial-containing
biomaterials described herein can be used for treatment of any of
the aforementioned wounds.
[0142] The term "treat," "treated," "treating" or "treatment"
includes the diminishment or alleviation of at least one symptom
associated or caused by the state, disorder or disease being
treated. For example, treatment can be diminishment of one or
several symptoms of a disorder or complete eradication of a
disorder (e.g., bacterial infection, tissue adhesion, and/or
external wounds).
[0143] Various aspects and embodiments are further described by way
of the following Examples. The Examples are offered by way of
illustration and not by way of limitation.
EXAMPLES The following examples are for demonstration purposes and
are not meant to be limiting.
Example 1
Silver Fatty Acid Salts Within the Fatty Acid-Derived
Biomaterial
[0144] In accordance with the present invention, silver in its
aqueous form was used to hydrate a fatty acid-derived biomaterial
sample. One possible mechanism for the formation of the
antimicrobial-containing biomaterial hydrated in aqueous silver is
as follows: fish oil (triglycerides) absorbs oxygen into the oil;
oxidation of the C.dbd.C bonds in the oil occurs; fatty acid and
glycerides (a mixture of mono, di- and tri) are formed; continued
curing results in dehydration of neighboring carboxyl and hydroxyl
functional groups; lactone/ester cross-links are formed between
fatty acids and glycerides; the oil solidifies into a bioabsorbable
gel containing cross-linked fatty acids and glycerides; the
cross-linked fatty acid biomaterial is immersed in an aqueous Ag
solution; and the hydrated silver coated fatty acid biomaterial is
dried to evaporate the solvent.
[0145] The silver hydrated fatty acid-derived biomaterial was
prepared as follows.
[0146] Aqueous silver acetate was massed and dissolved to a final
concentration of 0.06M. In addition, a fatty acid biomaterial was
prepared by curing native fish oil, at a density of approximately
100 mg/inch.sup.2, onto polypropylene mesh at a temperature of
200.degree. F. for 24 hours to form a solid coating. To incorporate
silver by hydration, a 1 inch.sup.2 sample of the fatty acid
biomaterial was then immersed in the 0.06M aqueous silver acetate
solution for four hours. By immersing the sample in aqueous silver,
the fatty acid biomaterial becomes hydrated in an aqueous
environment over an extended period of time allowing for the
formation of silver fatty acid salts. Determination of free silver
content released in .mu.g/cm.sup.2 from the silver hydrated fatty
acid biomaterial sample was carried out in water and analyzed by
Inductively Coupled Plasma (ICP). The silver release profile
obtained is shown in FIG. 3. The results indicate that an immediate
burst of silver is released from the hydrated fatty acid-derived
coating. Similarly, FIG. 4 shows the silver release profile of
silver from a 1 inch.sup.2 fatty acid-derived biomaterial sample
hydrated in 0.07M silver nitrate. The free silver released from the
silver nitrate hydrated sample also occurs as a burst indicating
that high levels of silver would be released rapidly in an aqueous
environment.
[0147] Silver assay of fatty acid-derived biomaterial samples
hydrated in silver acetate and silver nitrate over a six hour
period was performed by ICP. FIG. 5 illustrates that the silver
content in a fatty acid-derived biomaterial sample increases as a
function of hydration time using aqueous silver nitrate or aqueous
silver acetate as the hydration media. A greater amount of silver
was able to be loaded using silver acetate than silver nitrate
within the same hydration time as result of the additional
penetration of the silver acetate into the coating due to the
higher pH and the similarity of the acetate group to the fatty acid
components within the coating.
[0148] Analysis of the silver hydrated fatty acid-biomaterial
samples by FTIR, shows evidence of the formation of fatty acid
represented by the peak at 1512 cm.sup.-1, which increases with
increasing hydration time as shown in FIGS. 6A-6B for silver
nitrate and silver acetate loading solutions, respectively.
[0149] The efficacy of the silver nitrate hydrated fatty
acid-derived biomaterial was tested in a three day in-vitro S.
aureus biofilm study. A fatty acid-derived biomaterial sample,
without silver present, was used as a control and the results
obtained are shown in FIG. 7. These results show the silver
hydrated sample had an approximate 5 log reduction in colony
forming units (CFU's) per cm2 after one day and no CFU's were
present on days 2 and 3. The fatty acid-derived biomaterial control
sample had an approximate 3 log reduction in CFU's after day one as
a result of the antibacterial properties of short-chain fatty acids
(T. Kitahara, "Biol Pharm. Bull.: Second Edition." 2004, Vol 27(9),
pgs 1321-1326) but the CFU's gradually increased on day 2 and 3.
FIG. 7 demonstrates that adding silver to the fatty acid-derived
biomaterial coating through hydration in aqueous silver media
enhances the coating's ability to form a biofilm within an in-vitro
S. aureus infected environment.
Example 2
Fatty Acid-Derived Biomaterial Surface Coated with Silver
[0150] In a different embodiment of the present invention, silver
was coated onto the surface of a fatty acid-derived biomaterial.
Sample preparation was done as follows.
[0151] Silver nitrate was massed and dissolved in methanol to a
final concentration of 0.02M. In addition, a fatty acid biomaterial
was prepared by encapsulating polypropylene mesh with approximately
100 mg of fish oil per inch.sup.2 then thermally curing the fish
oil cast onto the polypropylene mesh at a temperature of
200.degree. F. for 24 hours to form a solid fatty acid-derived
coating. To incorporate silver on the surface, a 1 inch.sup.2
sample of the fatty acid biomaterial was then sprayed onto the
surface with the 0.02M silver nitrate solution and allowed to dry
to evaporate off the methanol solvent. One possible mechanism for
the formation of the antimicrobial-containing biomaterial coated
with aqueous silver is as follows: fish oil (triglycerides) absorbs
oxygen into the oil; oxidation of the C.dbd.C bonds in the oil
occurs; fatty acid and glycerides (a mixture of mono, di- and tri)
are formed; continued curing results in dehydration of neighboring
carboxyl and hydroxyl functional groups; lactone/ester cross-links
are formed between fatty acids and glycerides; the biomaterial
solidifies into a bioabsorbable gel containing cross-linked fatty
acids and glycerides; the cross-linked fatty acid biomaterial is
sprayed with an aqueous Ag solution; and the sprayed silver coated
fatty acid biomaterial is dried to evaporate the solvent.
[0152] The silver release properties of a fatty acid-derived
monofilament coated biomaterial sprayed with silver nitrate was
evaluated in water. The silver content in the dissolution media was
analyzed over 30 days by ICP. FIG. 8 shows the silver amounts
released into aqueous media depicting a dose dump of silver in the
dissolution media. The silver release profile of the sprayed fatty
acid-derived biomaterial is similar to the silver hydrated fatty
acid-derived biomaterial in FIGS. 3-4.
[0153] Results illustrating the efficacy of the fatty acid-derived
biomaterial sprayed with a layer of silver in a three day in-vitro
S. aureus biofilm study are shown in FIG. 9. A 5 log reduction in
colony forming units (CFU's) per cm.sup.2 was noted after one day
and no CFU's were present on days 2 and 3. The fatty acid-derived
biomaterial control sample had an approximate 3 log reduction in
CFU's after day one but the CFU's per cm.sup.2 gradually increased
on day 2 and 3 indicating that the addition of the surface silver
layer on the fatty acid-derived biomaterial helped reduce the
formation of a biofilm in this 3 day in-vitro study.
Example 3
Formation of Reduced Silver Within the Fatty Acid-Derived
Biomaterial
[0154] In this example, a different method of forming a silver
coated fatty acid-derived material is described. The method of
preparation is as follows:
[0155] Three components are used to make up a silver-containing
fatty acid emulsion formulation which is then thermally cured onto
polypropylene mesh to form a solid reduced silver fatty
acid-derived biomaterial coating. The three components that make up
the emulsion include an aqueous silver solution, native fish oil
and partially cured fish oil that has a viscous consistency. An
aqueous silver solution was prepared by massing silver nitrate or
silver acetate and dissolving it in water to give final
concentrations of 0.07M and 0.06M respectively. Separately,
partially cured fish oil was prepared by exposing native fish oil
to temperatures of approximately 90.degree. C. in the presence of
oxygen for 16 hours which results in a partially cured fish oil
component with a viscous consistency. The silver containing
emulsion is then prepared by massing out (w/w) 45% of the native
fish oil, 45% of the partially cured fish oil and 10% of the
aqueous silver solution in one vial and thoroughly mixing these
components with intermittent heating. The emulsified silver
formulation is then cast onto a medical device and thermally cured
at 200.degree. F. for approximately 24 hours to form a solid
reduced silver fatty acid-derived biomaterial coating. Thermal
curing is hypothesized to convert the aqueous silver present in the
coating to an insoluble form of silver within the fatty
acid-derived coating. This is accomplished by the interaction of
the silver ions in the silver nitrate solution with the lipid
hydroperoxides formed during the oxidation of the oil-derived
cross-linked gel, which are then reduced to silver metal (A. Kumar
et al., "Nature Materials". 2008 Vol 7, pgs 236-241). One possible
mechanism for the formation of the biomaterial coating containing
reduced silver is as follows: emulsified fish oil , pre-cured fish
oil and silver (Ag) absorb oxygen; oxidation of the C.dbd.C bonds
in the oil occurs; fatty acid and glycerides (mixture of mono, di-
and tri) and reduced silver are formed; continued curing results in
dehydration of neighboring carboxyl and hydroxyl functional groups;
lactone/ester cross-links are formed between fatty acids and
glycerides; and the material solidifies into a bioabsorbable gel
containing cross-linked fatty acids, glycerides and reduced Ag.
[0156] The silver release profile of a reduced silver fatty
acid-derived coating was evaluated in water as the dissolution
media. ICP was used to analyze the free silver content in the
dissolution media for a period of approximately two months. Silver
released in .mu.g/cm.sup.2 over time is depicted in FIG. 10. Silver
is released in steady gradual manner over an extended amount of
time in water indicating that the aqueous silver that was cured
with the fatty acid-derived biomaterial is converted to an
insoluble form. In an aqueous environment, a reduced silver fatty
acid-derived biomaterial sample would release low levels of silver
unlike Examples 1 and 2 where a rapid burst of silver is released
in an aqueous environment.
[0157] The reduced silver fatty acid-derived biomaterial was tested
for its bactericidal properties in a 2 day S. aureus zone of
inhibition assay using samples prepared using increasing
concentrations of aqueous silver nitrate. FIG. 11 shows the
graphical results of the zone of inhibition assay. Zones measured
on day 1 were found to be larger in samples prepared from a higher
concentration of silver. This demonstrates that the silver added to
the fatty acid-derived biomaterial is responsible for the growth
inhibiting effects observed. Day 2 of the zone assay had a
comparable size in all three samples tested. Similarly, a biofilm
assay was performed on a reduced silver fatty acid-derived
biomaterial sample and found to have no bacteria on the surface of
the device after three days as shown in FIG. 12. The overall
reduction in CPU's was approximately 5 log.
[0158] FTIR analysis of the reduced silver coating is shown in FIG.
13. The spectra of the fatty acid biomaterial control and the
reduced silver fatty acid sample have similar spectral profiles and
do not show the presence of fatty acid salts as seen in FIGS. 6A-6B
indicating that the aqueous silver in these samples interacts
differently after the thermal curing process.
Example 4
Formation of Silver Nanoparticle Fatty Acid-Derived Biomaterial
[0159] In a different embodiment of this invention, elemental
silver is used in the form of silver nanoparticles and added to a
fatty acid biomaterial to in order to enhance antibacterial
properties of the fatty acid biomaterial coating. The sample
preparation was done as follows:
[0160] Silver nanopowder surface coated with oleic acid was
purchased from American Elements, 99.9% silver metal. A fish oil
and silver nanopowder formulation was then prepared by mixing 1%
silver nano powder in 99% fish oil (w/w). The fish oil and silver
nanopowder was thoroughly mixed in a sonication bath for an hour
then cast onto a medical device and thermally cured at 200.degree.
F. for 24 hours to form a solid coating. One possible mechanism for
the formation of the nano-silver coated biomaterial coating is as
follows: fish oil and silver nanoparticles absorb oxygen; oxidation
of the C.dbd.C bonds in the oil occurs; fatty acid and glycerides
(mixture of mono, di- and tri) are formed; continued curing results
in dehydration of neighboring carboxyl and hydroxyl functional
groups; lactone/ester cross-links are formed between fatty acids
and glycerides; and the material solidifies into a bioabsorbable
gel containing cross-linked fatty acids, glycerides and Ag nano
particles
[0161] The silver release properties of the silver nanoparticle
fatty acid biomaterial samples in water are shown in FIG. 14. The
release profiles indicate that more silver is released as a
function of the concentration of silver nanoparticles added to the
fatty acid biomaterial coating. The silver nanoparticles in the
fatty acid biomaterial coating also produces a steady controlled
release of silver in aqueous media over a period of approximately
75 days. The release profiles in FIG. 20 are an improvement to the
quick burst of silver released when silver is sprayed onto the
fatty acid-derived biomaterial or hydration method is used to
incorporate silver fatty acid salts into the coating as described
in Example 1 and 2.
[0162] The amount of silver loaded into the fatty acid-derived
biomaterial was determined by ICP. The amounts of silver loaded
within the fatty acid biomaterial can be controlled by altering the
concentration of the silver nanoparticles in the coating as shown
in FIG. 15.
[0163] In a related method of this embodiment, a silver
nanoparticle fatty acid-derived film can be prepared separately and
cast onto a medical device to produce a variation of the mechanism
described in FIG. 13. One possible mechanism for the formation of
the silver nanoparticle-containing biomaterial coating is as
follows: fatty acid film particles and silver nanoparticles are
thoroughly mixed together; the blended particles and Ag
nanoparticles are pressed into a thin film under heat and pressure;
and the fatty acid silver nanoparticle film is cast onto a
polymeric device.
[0164] The silver release profile of this embodiment is shown in
FIG. 16, which also shows a sustained release profile in an aqueous
environment.
Example 5
Formation of Silver Nano-Particle Fatty Acid-Derived Biomaterial in
Combination with Gentamicin Sulfate
[0165] In this embodiment, the silver nanoparticle fatty
acid-derived biomaterial coating is combined with a pharmaceutical
agent such as gentamicin to enhance microbial stasis.
[0166] Silver nanopowder surface coated with oleic acid was
purchased from American Elements (Product Number AG-M-03M-NPC),
99.9% silver metal. gentamicin sulfate was purchased from Spectrum
Chemicals and Laboratory Products, part number G1174. The fish oil
and silver nanopowder formulation was then prepared by mixing 0.16%
silver nano powder and 2.57% gentamicin sulfate powder in 97% fish
oil (w/w). The gentamicin sulfate, silver nanopowder and fish oil
was then thoroughly mixed using a cryo-grinding apparatus under
cryogenic temperatures forming a cryoground formulation containing
fish oil gentamicin and silver. Cryogenic grinding (cryogrinding)
is a process carried out at extremely low temperatures enabled by
using liquid nitrogen (-196.degree. C.), to finely pulverize and
mix sample components by means of a magnetic grinding mechanism,
making the sample brittle under these low temperatures, thereby
yielding a thoroughly mixed homogenous formulation. A Cryogenic
impact grinder (6750 Freezer Mill) was used for homogenizing a fish
oil, gentamicin and silver formulation. The cryogenic grinding
apparatus' tub was filled with 4-5L of liquid nitrogen and set to
grind for eight cycles. Each cycle consists of 2 minutes of
grinding with a 2 minute cooling period at a rate of 15 impacts per
second during the grinding cycle.
[0167] Fish oil, gentamicin powder and silver nanoparticles were
appropriately massed into a cryogenic grinding polycarbonate center
cylinder vial capped on one end with a stainless steel end plug. A
stainless steel magnetic bar was added into the polycarbonate vial
containing the formulation components and the open end was capped
with a stainless steel end plug. The vial was then placed into the
cryogenic grinding apparatus programmed to produce an alternating
magnetic field that causes the stainless steel bar to impact the
stainless steel end plugs of the polycarbonate vial containing the
formulation components, resulting in a finely ground and
homogenized formulation. The cryoground formulation was allowed to
thaw, cast onto a medical device and thermally cured at 200.degree.
F. for 24 hours to form a solid coating. One possible mechanism for
the formation of the gentamicin and nano-silver oil-derived
biomaterial coating is as follows: fish oil, gentamicin and silver
nanoparticles absorb oxygen; oxidation of the C.dbd.C bonds in the
oil occurs; fatty acid and glycerides (mixture of mono, di- and
tri) are formed; continued curing results in dehydration of
neighboring carboxyl and hydroxyl functional groups; lactone/ester
cross-links are formed between fatty acids and glycerides; and the
coating solidifies into a bioabsorbable gel containing cross-linked
fatty acids, glycerides, gentamicin and Ag nano particles
[0168] The release of gentamicin and silver ions in .mu.g/cm.sup.2
was tested in aqueous conditions. FIG. 17 illustrates a plot of the
silver ions released over approximately three weeks whereas FIG. 18
shows the release profile of gentamicin in water. The silver
release profile was gradual but the gentamicin produced a burst of
the drug dose in less than 48 hours. This indicates that in this
particular embodiment, silver would be slow releasing over extended
time periods but the gentamicin would be fast releasing within a
relatively short amount of time in an aqueous environment.
[0169] Another method of incorporating gentamicin into the silver
nanoparticle fatty acid biomaterial coating is accomplished by
spraying a solution of gentamicin sulfate on a cured silver
nanoparticle fatty acid-derived biomaterial sample. The antibiotic
solution is prepared by dissolving gentamicin in water; the
concentration of the gentamicin can range between 1 and 50 mg/ml
depending on how much is drug to be loaded onto the surface of the
coating and how many sprayed layers are to be applied. The material
can be prepared as follows: fish oil and silver nanoparticles
absorb oxygen; oxidation of the C.dbd.C bonds in the oil occurs;
fatty acid and glycerides (mixture of mono, di- and tri) are
formed; continued curing results in dehydration of neighboring
carboxyl and hydroxyl functional groups; lactone/ester cross-links
are formed between fatty acids and glycerides; the material
solidifies into a bioabsorbable gel containing cross-linked fatty
acids, glycerides, and Ag nano particles; and gentamicin is sprayed
onto the surface of the cross-linked silver nanoparticle
biomaterial and allowed to dry.
[0170] The efficacy of the gentamicin sprayed silver nanoparticle
fatty acid-derived biomaterial coating was evaluated by zone of
inhibition (ZOI) and bacterial adherence tests shown in FIGS. 19
and 20. In the ZOI test, silver nanoparticle fatty acid biomaterial
coating and gentamicin sprayed silver nanoparticle fatty acid
biomaterial coating sample discs with a 0.5 inch diameter were
placed on a bed of S. aureus bacteria in an agar plate and
incubated for 24 hours before transferring the disc to a fresh
bacterial agar plate. In this particular test, the ZOI was done for
3 days comparing the silver nanoparticle fatty acid biomaterial
coating with and without the gentamicin sprayed layer. The plot in
FIG. 19 shows that both silver nanoparticle fatty acid biomaterial
coating with and without the gentamicin sprayed layer were
efficacious in reducing the number of bacteria growing around the
surface of the device but the sample with the added gentamicin
layer was more efficacious in this regard when compared to the
sample without a gentamicin layer. The bacterial adherence results
in FIG. 20 similarly show that significantly fewer bacteria adhered
to the surface of the gentamicin sprayed silver nanoparticle fatty
acid-derived biomaterial. Addition of gentamicin to the silver
nanoparticle fatty acid-derived biomaterial therefore improved the
antibacterial properties of the biomaterial coating.
[0171] In another embodiment of this invention, a fatty
acid-derived film containing silver nanoparticles and gentamicin is
prepared and cast onto a polymeric medical device. The mechanism
used to form the gentamicin and silver nanoparticle fatty
acid-derived biomaterial coating is as follows: fatty acid film
particles, genatmicin and silver nanoparticles thoroughly mixed
together; the blended particles, gentamicin and Ag nanoparticles
are pressed into a thin film under heat and pressure; and the
gentamicin fatty acid silver nanoparticle film is cast onto a
polymeric device.
[0172] gentamicin release in aqueous media occurred as a burst over
250 hours but the total amount released was able to be modified by
varying the total amount of gentamicin loaded onto the sample as
shown in FIG. 21. The total amount of gentamicin loaded per sample
can also be controlled by altering the amount of gentamicin added
to the initial formulation. Assay values of gentamicin in this
example were determined to be between 180 and 900 .mu.g/cm.sup.2
for concentrations of gentamicin ranging between 2 and 6% (FIG.
22).
Example 6
Formation of Silver Nano-Particle Fatty Acid-Derived Biomaterial in
Combination with Chlorhexidine Diacetate
[0173] In this example, chlorhexidine diacetate is used as an
additional antimicrobial agent to augment the silver nanoparticle
fatty acid-derived biomaterial. One possible mechanism for the
formation of the silver nanoparticle fatty acid biomaterial surface
coated with chlorhexidine diacetate is as follows: fish oil and
silver nanoparticles absorb oxygen: oxidation of the C.dbd.C bonds
in the oil occurs; fatty acid and glycerides (mixture of mono, di-
and tri) are formed; continued curing results in dehydration of
neighboring carboxyl and hydroxyl functional groups; lactone/ester
cross-links are formed between fatty acids and glycerides; the
coating solidifies into a bioabsorbable gel containing cross-linked
fatty acids, glycerides, and Ag nano particles; and chlorhexidine
is sprayed onto the surface of the cross-linked silver nanoparticle
biomaterial and allowed to dry.
[0174] The antimicrobial solution was prepared by dissolving
chlorhexidine diacetate in water to a final concentration of 10
mg/ml and was evenly sprayed onto the surface of the silver
nanoparticle fatty acid-derived biomaterial created using the
methodology described in Example 4.
[0175] Bacterial adherence and ZOI tests were performed on the
chlorhexidine sprayed silver nanoparticle fatty acid-derived
biomaterial samples. The ZOI test samples had measurable zones for
three days with the chlorhexidine sprayed silver nanoparticle fatty
acid-derived biomaterial samples having larger zones compared to
the control silver nanoparticle fatty acid biomaterial samples
without chlorhexidine as show in FIG. 23. Bacterial adherence test
results of the chlorhexidine sprayed silver nanoparticle fatty
acid-derived biomaterial samples showed that adding a chlorhexidine
layer significantly reduced the number of bacteria adhered to the
surface of the when compared to a silver nanoparticle fatty
acid-derived biomaterial control sample without chlorhexidine as
shown in FIG. 24.
Example 7
Formation of Silver Nano-Particle Fatty Acid-Derived Biomaterial in
Combination with Triclosan
[0176] This example describes the use of triclosan to augment the
antimicrobial properties of the silver nanoparticle fatty
acid-derived biomaterial coating. One possible mechanism for the
formation of the silver nanoparticle fatty acid-derived biomaterial
surface coated with triclosan is as follows: fish oil and silver
nanoparticles absorb oxygen; oxidation of the C.dbd.C bonds in the
oil occurs; fatty acid and glycerides (mixture of mono, di- and
tri) are formed; continued curing results in dehydration of
neighboring carboxyl and hydroxyl functional groups; lactone/ester
cross-links are formed between fatty acids and glycerides; the
coating solidifies into a bioabsorbable gel containing cross-linked
fatty acids, glycerides, and Ag nano particles; and triclosan is
applied to the surface of the solidified coating containing
cross-linked fatty acids, glycerides, and Ag nano particles
[0177] A triclosan solution was prepared by dissolving triclosan in
ethanol to a concentration ranging between 1 and 20 mg/ml and the
solution was then sprayed onto the silver fatty acid coating
surface created using the process described in Example 4. Multiple
layers were sprayed on the surface if lower triclosan solutions
were used and fewer triclosan layers were sprayed on the coating
surface if higher triclosan solutions were used. An illustration of
triclosan dissolution in a buffer solution is shown in FIG. 25
showing low level amounts of triclosan are gradually released in
this medium. The efficacy of the triclosan sprayed silver
nanoparticle fatty acid-derived biomaterial coating was evaluated
by zone of inhibition (ZOI) and bacterial adherence tests shown in
FIGS. 26 and 27. The plot in FIG. 26 showed improved zones measured
that were relatively consistent over a period of approximately 3
weeks. Previous ZOI tests of the silver nanoparticle fatty acid
biomaterial coating without surface sprayed Triclosan, exhibited
zones for approximately 3 days. Addition of triclosan to the
coating surface therefore significantly improved the bacteriostatic
capabilities of the silver nanoparticle fatty acid biomaterial
coating. FIG. 27 further depicts the number of colony forming units
enumerated per ml of media after performing a S. aureus bacterial
suspension assay using various fatty acid biomaterial samples
without silver, with silver and with both silver and triclosan
present. This assay monitors the ability of the antimicrobial to
elute from the device to the surrounding media or environment. FIG.
27a showed that the lowest number of colony forming units within
the suspension media was obtained from the fatty acid biomaterial
coated with both triclosan and silver. The results of the performed
tests also showed reduced bacterial colony forming units extracted
from the surface of the silver nanoparticle fatty acid biomaterial
silver compared to the control but the most significant reduction
was noted in the silver nanoparticle fatty acid biomaterial with
triclosan which had no bacteria adhered to the surface.
[0178] In a related embodiment, triclosan can be incorporated into
the silver nanoparticle fatty acid-derived coating and additional
sprayed with a layer of triclosan to further improve upon
antibacterial properties. The mechanism used to form this triclosan
silver nanoparticle fatty acid-derived biomaterial coating surface
coated with triclosan is as follows: fish oil, triclosan and silver
nanoparticles absorb oxygen; oxidation of the C.dbd.C bonds in the
oil occurs; fatty acid and glycerides (mixture of mono, di- and
tri) are formed; continued curing results in dehydration of
neighboring carboxyl and hydroxyl functional groups; lactone/ester
cross-links are formed between fatty acids and glycerides; the
coating solidifies into a bioabsorbable gel containing cross-linked
fatty acids, glycerides, gentamicin and Ag nano particles;
solidified coating containing fatty acids, triclosan, and Ag
nanoparticles is surface coated with an additional layer of
triclosan.
[0179] Sample preparation involves combining 1% triclosan powder,
1% silver nanoparticles and fish oil, thoroughly mixing the
formulation which is then cast onto a medical device and thermally
cured to solidify the coating. The surface of the solid coating
containing triclosan, silver nanoparticles and fatty acids is then
sprayed with a 1-20 mg/ml triclosan solution. FIG. 28 depicts a
plot of ZOI results obtained from triclosan silver nanoparticle
fatty acid-derived samples surface coated with triclosan.
Significant zones were measured through day 13 demonstrating a high
antimicrobial efficacy of these samples. The results of the
bacterial adherence test illustrated in FIG. 29 were acquired using
bacterial adherence counts extracted from the surface of the device
after 24 hours. The extraction bacterial counts obtained after 24
hours showed that the silver fatty acid biomaterial with triclosan
had the lowest number of colony forming units extracted from the
sample's surface.
[0180] In another related embodiment, triclosan is incorporated
into the silver nanoparticle fatty acid-derived biomaterial before
the coating formulation is thermally cured (i.e., triclosan not
sprayed on the surface of the finished device). One possible
mechanism for the formation of the triclosan and nano-silver
hydrophobic oil-derived cross-linked biomaterial coating is as
follows: fish oil, triclosan and silver nanoparticles absorb
oxygen; oxidation of the C.dbd.C bonds in the oil occurs; fatty
acid and glycerides (mixture of mono, di- and tri) are formed;
continued curing results in dehydration of neighboring carboxyl and
hydroxyl functional groups; lactone/ester cross-links are formed
between fatty acids and glycerides; the coating solidifies into a
bioabsorbable gel containing cross-linked fatty acids, glycerides,
triclosan and Ag nano particles.
[0181] ZOI test results showing antimicrobial properties of this
coating are displayed in FIG. 30 which illustrates zones measured
through day 19. This demonstrates that the coating has sufficient
antimicrobial efficacy to prevent bacterial growth on the device in
a S. aureus bacterial environment.
Example 8
Formation of Fatty Acid-Derived Biomaterial and Silver
Nano-Particle Fatty Acid-Derived Biomaterial in Combination with
Triclosan
[0182] The efficacy of triclosan fatty acid-derived biomaterials
were evaluated in an in-vivo infectability model using a clinical
strain of MRSA. An additional embodiment of this invention uses
triclosan coated onto the surface of a fatty acid-derived
biomaterial sample without silver present within the coating
matrix. Further yet, silver nanoparticles may be encapsulated
within the lipid coating matrix as described in Example 4 and then
surface coated with triclosan. The combination of triclosan and
silver in the fatty acid-derived biomaterial coating may be serve
to enhance the antimicrobial properties of the fatty acid-derived
biomaterial coating as seen in FIG. 31. The plot displays bacterial
counts scrapped from the samples' surface that represent 7 day
explant samples obtained from an in vivo infectability study. The
sample sets evaluated include fatty acid-derived biomaterial
samples surface coated with 20 ug/cm.sup.2 of triclosan and 60
ug/cm.sup.2 silver nanoparticles in the fatty acid-derived
biomaterial samples surface coated with 20 ug/cm.sup.2 and 40
ug/cm.sup.2 triclosan. While some efficacy was evident in all
sample sets tested in this study, FIG. 31 demonstrates that the
lowest bacterial counts were recorded in the sample sets which had
both silver nanoparticles and triclosan. Adding triclosan to the
surface of a fatty acid-derived biomaterial confers a benefit in
reducing bacterial adherence onto the surface of the device but
having both silver nanoparticles and triclosan present in the
coating results in a more significant reduction of bacterial
adherence as shown in FIG. 31. A possible synergistic effect
between the silver encapsulated in the lipid coating and the
surface coated triclosan may be attributed to the lower bacterial
counts in samples with both triclosan and silver nanoparticles.
[0183] Numerous modifications and alternative embodiments of the
present invention will be apparent to those skilled in the art in
view of the foregoing description. Accordingly, this description is
to be construed as illustrative only and is for the purpose of
teaching those skilled in the art the best mode for carrying out
the present invention. Details of the structure may vary
substantially without departing from the spirit of the invention,
and exclusive use of all modifications that come within the scope
of the appended claims is reserved. It is intended that the present
invention be limited only to the extent required by the appended
claims and the applicable rules of law.
[0184] All literature and similar material cited in this
application, including, patents, patent applications, articles,
books, treatises, dissertations and web pages, regardless of the
format of such literature and similar materials, are expressly
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials
differs from or contradicts this application, including defined
terms, term usage, described techniques, or the like, this
application controls.
[0185] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described in any way.
[0186] While the present invention has been described in
conjunction with various embodiments and examples, it is not
intended that the present teachings be limited to such embodiments
or examples. On the contrary, the present invention encompasses
various alternatives, modifications, and equivalents, as will be
appreciated by those of skill in the art.
[0187] The claims should not be read as limited to the described
order or elements unless stated to that effect. It should be
understood that various changes in form and detail may be made
without departing from the scope of the appended claims. Therefore,
all embodiments that come within the scope and spirit of the
following claims and equivalents thereto are claimed.
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