U.S. patent application number 11/509915 was filed with the patent office on 2007-03-01 for hydrophilized bactericidal polymers.
This patent application is currently assigned to Purdue Research Foundation. Invention is credited to Philippe Sellenet, Jeffrey P. Youngblood.
Application Number | 20070048249 11/509915 |
Document ID | / |
Family ID | 37804426 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048249 |
Kind Code |
A1 |
Youngblood; Jeffrey P. ; et
al. |
March 1, 2007 |
Hydrophilized bactericidal polymers
Abstract
A bactericidal polymeric composition includes a hydrophilic
first comonomer copolymerized to a second comonomer to produce a
polymeric composition that is more hydrophilic or more bactericidal
in an aqueous solution than either of the comonomers alone. Methods
for identifying bactericidal polymers, methods for rendering
materials bactericidal, and methods for using bactericidal
compositions to kill or reduce bacterial growth are also described.
Applications for the inventive compositions include their use in
catheters, stents, medical devices, contact lenses; root canal
fillers; and/or wound dressings.
Inventors: |
Youngblood; Jeffrey P.;
(Crawfordsville, IN) ; Sellenet; Philippe; (Nancy,
FR) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Assignee: |
Purdue Research Foundation
|
Family ID: |
37804426 |
Appl. No.: |
11/509915 |
Filed: |
August 24, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60711234 |
Aug 24, 2005 |
|
|
|
Current U.S.
Class: |
424/78.3 ;
435/32 |
Current CPC
Class: |
C08F 220/281 20200201;
C12Q 1/18 20130101; C08F 226/06 20130101; A01N 43/40 20130101; C08F
220/28 20130101; C08F 220/286 20200201 |
Class at
Publication: |
424/078.3 ;
435/032 |
International
Class: |
A61K 31/787 20070101
A61K031/787; C12Q 1/18 20060101 C12Q001/18 |
Claims
1. A polymeric composition comprising: a hydrophilic first
comonomer polymerized to a second comonomer to form a polymeric
composition, where the polymeric composition is more hydrophilic
than either of the first comonomer or the second comonomer alone
and/or where the polymeric composition is more bactericidal than
either of the first comonomer or the second comonomer alone.
2. The composition of claim 1, where the hydrophilic first
comonomer comprises hydroxyethylmethacrylate.
3. The composition of claim 1, where the hydrophilic first
copolymer comprises poly(ethyleneglycol) methacrylate.
4. The composition of claim 1, where the second comonomer comprises
polycationic species, polycationic derivatives or combinations
therefrom.
5. The composition of claim 1, where the second comonomer comprises
a plurality of quaternary ammonium groups.
6. The composition of claim 1, where the second comonomer comprises
quaternized poly(4-vinylpyridine).
7. A method for killing bacteria or rendering a medium bactericidal
comprising: providing a polymeric composition comprising a
hydrophilic first comonomer polymerized to a second comonomer,
where the polymeric composition is more hydrophilic than either of
the first comonomer or the second comonomer alone and/or where the
polymeric composition is more bactericidal than either of the first
comonomer or the second comonomer alone; applying the polymeric
composition to a medium to form a treated medium, where the
polymeric composition is applied in an amount sufficient to kill at
least one bacterium or significantly reduce bacterial growth in or
on the treated medium compared to an untreated medium.
8. The method of claim 7, where the polymeric composition is
applied as a coating to at least one surface of the medium.
9. The method of claim 7, where the treated medium is in contact
with an aqueous environment.
10. The method of claim 7, where the treated medium is in contact
with air.
11. The method of claim 7, where the treated medium is included in
or coated onto a catheter, stent, implantable medical device,
contact lens, root canal filler, or wound dressing.
12. The method of claim 7, further comprising the step of
contacting the treated medium with bacteria, where the treated
medium comprises the polymeric composition in an amount sufficient
to kill at least one bacterium or significantly reduce bacterial
growth in or on the treated medium compared to an untreated
medium.
13. The method of claim 12, where the treated medium is formulated
to kill or significantly reduce the growth of Gram-positive
bacteria.
14. The method of claim 12, where the treated medium is suitably
formulated to kill or significantly reduce the growth of
Gram-negative bacteria.
15. A method of identifying a polymeric composition suitable for
use in a bactericidal composition comprising: a) providing a first
comonomer, where the first comonomer is soluble in an aqueous
solution; b) providing a second comonomer, where the second
comonomer is bactericidal or capable of being rendered
bactericidal; c) polymerizing the first comonomer in step (a) to
the second comonomer in step (b) to form a polymeric composition;
d) treating or applying the polymeric composition in step (c) to a
medium to form a first treated medium; e) separately applying a
comonomer from step (a) or step (b) to the medium of step (d) to
form a second treated medium; f) separately contacting the first
treated medium and the second treated medium with a plurality of
bacteria; and g) determining whether the first treated medium is
more bactericidal than the second treated medium, where the
polymeric composition is suitable for use in a bactericidal
composition if the first treated medium is more bactericidal than
the second treated medium.
16. The method of claim 15, where each comonomer from step (a) and
step (b) is separately applied to the medium of step (d) to form a
second treated medium and a third treated medium, respectively,
where the polymeric composition is suitable for use in a
bactericidal composition if the first treated medium is more
bactericidal than each of the second and third treated mediums.
17. The method of claim 15, where the molecular weight(s) of one or
more more monomers in the first comonomer in the first polymeric
composition of the first treated medium is modified compared to the
corresponding molecular weight(s) of one or more monomers in a
second polymeric composition of a fourth medium, such that the
fourth treated medium is more bactericidal than the first treated
medium.
18. The method of claim 15, where a molar ratio(s) of one or one or
more monomers in the first comonomer in the first polymeric
composition in a first treated medium is modified compared to the
corresponding molar ratio(s) of one or more monomers in a second
polymeric composition of a fourth medium, such that the fourth
treated medium is more bactericidal than the first treated
medium.
19. The method of claim 15, where the second comonomer is
quaternized after the second comonomer is polymerized to the first
comonomer.
20. The method of claim 15, where step (g) comprises a luminescence
assay, optical density determination or microscopic determination.
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) to U.S. Provisional Application No.
60/711,234, filed Aug. 24, 2005, which is hereby incorporated by
reference in its entirety.
BACKGROUND
[0002] There is an ever-growing demand for materials suitable for
killing harmful microorganisms. Such materials could be used to
coat surfaces of common objects touched by people to render them
antiseptic so as to prevent transmission of bacterial infections or
to facilitate the killing of microorganisms in solution.
[0003] Various polycations are known to have bactericidal
properties. However, their bactericidal properties can be strongly
influenced by whether the polycation or a composition containing
the polycation is soluble. In some instances the bactericidal
property is most apparent in an insoluble form, which is not
particularly amenable to killing microorganisms. In other instances
the bactericidal activity is lost when the polycation is
cross-linked or otherwise rendered insoluble. Application of
bactericidal polymers may also be limited by their use in brushes,
their insolubility in solution, or by their unfavorable
biocompatibility characteristics. Accordingly, there is a need for
bactericidal formulations possessing having improved bactericidal,
hydrophilicity/wettability and biocompatibility characteristics
suitable for rendering materials or areas bactericidal and for
killing airborne and/or waterborne microorganisms.
BRIEF SUMMARY
[0004] The present invention is directed to polymeric compositions
providing improved bactericidal, hydrophilicity/wettability, and
biocompatibility characteristics. In particular, the present
invention provides a bactericidal composition, including a
hydrophilic first comonomer polymerized to a second comonomer to
form a polymeric composition, where the polymeric composition is
more soluble and/or more bactericidal in an aqueous solution than
either of the first comonomer or the second comonomer alone.
[0005] In a particular example, the present invention provides a
quaternized bactericidal composition, in which
poly(4-vinylpyridine) (PVP) is copolymerized with
hydroxyethylmethacrylate (HEMA) or poly(ethyleneglycol)
methacrylate (PEGMA).
[0006] In another example, the present invention provides a method
for rendering a material or area bactericidal in which a
bactericidal composition of the present invention is applied to a
medium or device in an amount suitable for killing or significantly
reducing the number of bacteria in or on the treated medium or
device compared to an untreated medium or device.
[0007] In another example, the present invention provides a method
for killing or significantly reducing the number of bacteria on a
material or area treated with a bactericidal composition of the
present invention.
[0008] In a further example, the present invention provides a
method for identifying a polymer having suitable bactericidal
activity in which a hydrophilic first comonomer is polymerized to a
second comonomer to form a bactericidal polymeric composition,
where the polymeric composition is determined to have suitable
bactericidal activity if the polymeric composition has a higher
bactericidal activity in an aqueous solution than either of the
hydrophilic first comonomer or second comonomer alone (or treated
similarly as the polymeric composition).
[0009] Applications for the inventive compositions include their
use in catheters, needles, sutures, stents and other implantable
medical devices, contact lenses, root canal fillers, wound
dressings, burn dressings, tissue culture plates, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic showing (A) the radical polymerization
of P(VP-co-HEMA) and (B) quaternization of P(VP-co-HEMA)-HB.
[0011] FIG. 2 is graph of bactericidal results for surface testing
of P(VP-co-HEMA)-HB.
[0012] FIG. 3 is a graph of advancing and receding contact angles
for P(VP-co-HEMA).
[0013] FIG. 4 is a graph of bactericidal results for testing of
P(VP-co-PEGMA1100).
DETAILED DESCRIPTION
[0014] In order to provide a more clear and consistent
understanding of the specification and claims, the following
definitions are provided. Unless defined otherwise, all technical
and scientific terms have the same meaning as is commonly
understood by one of skill in the art to which this invention
belongs.
[0015] The term "monomer" refers to a relatively simple compound,
usually containing carbon and of low molecular weight, which can
react to form a polymer by combining with itself or with other
monomers.
[0016] The terms "polymer" and "polymeric composition" are used
interchangeably to denote a product of a polymerization reaction,
and are inclusive of homopolymers, copolymers, terpolymers,
etc.
[0017] The terms "polymerization" and "polymerization reaction" are
inclusive of homopolymerizations, copolymerizations,
terpolymerizations, and the like, and include all types of
copolymerizations such as random, graft, block, and the like. In
general, the polymers in the bactericidal composition on may be
prepared in accordance with any suitable polymerization process,
including slurry polymerization, solution polymerization, emulsion
polymerization, gas phase polymerization, and high pressure
polymerization and the like.
[0018] The term "comonomer" refers to a monomer, copolymer, or
polymer which can copolymerize with itself or with at least one
different monomer, copolymer, or polymer in a copolymerization
reaction, the result of which can be a polymer, copolymer or
polymeric composition.
[0019] The term "copolymer" refers to a polymer which can
copolymerize with itself or with at least one different comonomer,
polymer, or copolymer in a polymerization reaction or it can refer
to a product resulting from a polymerization reaction of two
comonomers. The copolymer may be identified or named in terms of
the monomer(s) from which the copolymer is produced.
[0020] The terms "corresponding comonomer," "corresponding
copolymer," and "corresponding polymer" are used to relate
comonomers, copolymers, or polymers, respectively, sharing a common
set of monomeric units between e.g. distinct polymeric
compositions. The common comonomers, copolymers, or polymer need
not be identical in terms of the molecular weight(s) or molar
ratio(s) of commonly shared monomeric units.
[0021] The phrase "corresponding molecular weight" is used to
relate molecular weight(s) of corresponding comonomers, copolymers,
or polymers, respectively, in distinct polymeric compositions in
which the common comonomers, copolymers, or polymers differ from
one another by molecular weight(s) or commonly shared monomeric
units within the corresponding comonomer, copolymer or polymer.
[0022] The phrase "corresponding molar ratio" is used to relate
molar ratio(s) of corresponding comonomers, copolymers, or
polymers, respectively, in distinct polymeric compositions in which
the common comonomers, copolymers, or polymers differ from one
another by molar ratio(s) or commonly shared monomeric units within
the corresponding comonomer, copolymer or polymer.
[0023] The term "bactericidal" is used to interchangeably denote
any one of the following: (i) a comonomer, polymer, copolymer,
polymeric composition suitably formulated to kill, reduce the
growth, number, viability and/or metabolic activity of one or more
bacteria; (ii) a material, substance, medium, device, or area
treated with a bactericidal comonomer, polymer, copolymer,
polymeric composition so as to kill, reduce the growth, number,
viability and/or metabolic activity of one or more bacteria.
[0024] The term "aqueous solution" refers to a solution in which
water is the solvent.
[0025] The term "medium" refers to a treatable material, treatable
substance, treatable device, or treatable area in which "treatable"
refers to a capacity to be rendered bactericidal by a bactericidal
comonomer, polymer, or copolymer. A treatable medium may have a
defined physical form, but may include liquid (e.g., water, aqueous
solution) or gaseous materials (e.g., air) also.
[0026] The phrases "significantly reducing the growth of bacteria"
and "significantly reducing bacterial growth" are used
interchangeably to denote one or more of the following conditions,
including (i) a condition in which the metabolic activity of at
least 50% of the microorganisms of a particular type exposed to a
treated medium is terminated or reduced compared to bacteria of
that particular type exposed to an untreated medium over a fixed
period of time; (ii) a condition where there is 50% or less of one
or more bacterial types present in and/or on a treated medium
compared to the number of bacteria exposed to an untreated medium;
and/or (iii) a condition resulting when one or more types of
bacteria adhere 50% less to a treated medium compared to an
untreated medium. The degree of bacterial growth reduction with
respective to conditions (i)-(iii) may range from 50% to greater
99.9%.
[0027] The phrase "significantly bactericidal" denotes a comonomer,
polymer, copolymer, composition, polymeric composition, material,
substance or treated area in which the bactericidal comonomer,
polymer, copolymer, composition, polymeric composition, material,
substance or treated area is suitably formulated to significantly
reduce the growth, number, viability and/or metabolic activity of
bacteria by at least 50%.
[0028] The term "biocompatible" refers to a material that is
substantially non-toxic in the in vivo environment of its intended
use, and that is not substantially rejected by the patient's
physiological system (i.e., is non-antigenic). This can be gauged
by the ability of a material to pass the biocompatibility tests set
forth in International Standards Organization (ISO) Standard No.
10993 and/or the U.S. Pharmacopeia (USP) 23 and/or the U.S. Food
and Drug Administration (FDA) blue book memorandum No. G95-1,
entitled "Use of International Standard ISO-10993, Biological
Evaluation of Medical Devices Part-1: Evaluation and Testing."
Typically, these tests measure a material's toxicity, infectivity,
pyrogenicity, irritation potential, reactivity, hemolytic activity,
carcinogenicity and/or immunogenicity. A biocompatible structure or
material, when introduced into a majority of patients, will not
cause a significantly adverse, long-lived or escalating biological
reaction or response, and is distinguished from a mild, transient
inflammation which typically accompanies surgery or implantation of
foreign objects into a living organism.
[0029] A bactericidal polymeric composition of the present
invention includes a hydrophilic first comonomer polymerized to a
second comonomer, where the polymeric composition is more soluble
and/or more bactericidal in an aqueous solution than either of the
first comonomer or the second comonomer alone. The polymeric
composition of the present invention were found to have unexpected
hydophilizing and/or wettabiliy properties providing enhanced
bactericidal activity compared to either comonomer alone.
[0030] The second comonomer may be inherently bactericidal or it
may be rendered bactericidal after a subsequent step (e.g.,
polymerization) and/or chemical modification (e.g., quaternization)
of alkyl groups. Where the polymeric composition is further
modified by chemical modification, such as quaternization,
preferably, the polymeric composition is more hydrophilic and/or
bactericidal than a similarly modified (by e.g., quaternization)
second comonomer alone.
[0031] Bactericidal comonomers or those capable of being rendered
bactericidal are copolymerized to a hydrophilizing comonomer.
Exemplary second comonomers for polymerization to a hydrophilizing
comonomer may include a variety of vinyl monomers capable of free
radical polymerization and/or quaternization. Accordingly, these
comonomers may include, but are not limited to, vinyl amines, such
as N,N-dimethylvinylamine; allyl amines; vinyl esters, such as
vinyl acetate; alkyl acrylates; and vinyl chloride. In a preferred
embodiment, a pyridinium-type comonomer, such as vinyl pyridine or
4-vinylpyridine, is quaternized after polymerization to a
hydrophilizing comonomer.
[0032] The second comonomer composition may include or be
chemically linked to a suitable bactericidal moiety, including, but
not limited to polycationic species, polycationic derivatives or
combinations therefrom. Polycationic species may contain two or
more quaternary ammonium groups with a molecular weight ranging
from several hundred Daltons to a few hundred thousand Daltons. The
quaternary ammonium groups may be part of a ring or they may be
acyclic. Examples include but are not limited to: polyionenes,
poly(diallyldimethylammonium chloride),
dimethylamine-epichlorohydrin copolymers and
imidazole-epichlorohydrin copolymers. Suitable bactericidal
comonomers for use in the present invention may include the
quaternary ammonium group-containing polymers disclosed in U.S.
Pat. No. 4,482,680, which are incorporated by reference herein.
[0033] Polycationic species may contain two or more amine groups.
The amine groups can be primary, secondary, tertiary, or mixtures
thereof. The amine groups may be part of a ring or they may be
acyclic. Examples include but are not limited to:
polyethyleneimines, polypropyleneimines, polyvinylamines,
polyallylamines, polydiallylamines, polyamidoamines,
polyaminoalkylmethacrylates, polylysines, and mixtures thereof.
[0034] The polycationic species may also be a modified polyamine
with at least one amine group substituted with at least one other
functional group. Examples include ethoxylated and alkoxylated
polyamines and alkylated polyamines. Other suitable bactericidal
comonomers or those that may be rendered bactericidal may be
identified and/or used in accordance with the applications and
objectives set forth in the specification and claims.
[0035] Quaternization may be carried out using alkylating agents,
including but not limited to alkyl halides (such as hexyl bromide),
alkyl sulfonates, alkyl mesylates, alkyl tosylates, or other
alkylating agents possessing a suitable leaving group.
Quaternization reduces self-polymerization of the bactericidal
comonomer upon polymerization with the hydrophilizing comonomer.
Quaternization may confer increased bactericidal activity and is
typically carried out after polymerization, since quaternized
polymers are unpolymerizable.
[0036] Quaternized alkyl groups and/or other cationic chains may be
attracted to and/or promote interaction and penetration negatively
charged bacterial cell walls on account of their lipophilic nature.
Alkyl chain lengths of quaternizing agents and overall
hydrophilic/lipophilic balance may affect bactericidal activity of
the polymeric compositions of the present invention. Accordingly,
these variables may be modified to optimize or improve bactericidal
activity of the polymeric compositions.
[0037] Hydrophilizing comonomers of the present invention confer
increased wettability or hydrophilicity to one or more surfaces of
the polymeric composition in aqueous solutions, including water.
Preferably, the polymeric composition is more wettable than a
bactericidal comonomer or a comonomer rendered bactericidal by
quaternization, such as poly(4-vinylpyridine). Suitable
hydrophilizing monomers or copolymers, may include, but are not
limited to, ethylene glycol (ethylyene oxide); polyethylene glycol
derivatives, including poly(ethyleneglycol) methacrylate (PEGMA),
poly(ethyleneglycol) acrylate, and vinyl polyethylene glycol; vinyl
acetate; poly(vinyl alcohol); vinyl pyrrolidone and poly(vinyl
pyrrolidone); vinyl pyrrolidinone and poly(vinyl pyrrolininone);
vinyl oxazoline and poly(vinyl oxazoline); vinyl foramide and
poly(vinyl foramide); hydroxyalkyl acrylates and hydroxyalkyl
methacrylates, such as hydroxyethyl methacrylate (HEMA) and
hydroxyethyl acrylate; methacrylamide; acrylamide and
methacrylamide based monomers, such as acrylamide, N,N-dimethyl
acrylamide, N-ethyl acrylamide, N-isopropyl acrylamide, and
hydroxymethyl acrylamide; monomers containing one or more of the
following functional groups: hydroxy, amino, ammonium, ether,
carboxylate, amide, and sulfoamide groups; and combinations or
copolymers thereof. polyvinyloxazolines
[0038] Hydrophilic polymeric compositions and methods for
hydrophilizing polymeric materials, including the use of high
energy treatments, are disclosed in U.S. Pat. Appl. No.
20050008839, the contents of which are expressly incorporated by
reference in their entirety, also may be used.
[0039] Preferably, the hydrophilizing comonomer is biocompatible.
Standard assays may be utilized to evaluate biocompatibility,
including but not limited to viability/cytotoxicity mammalian cell
assays and the like. Representative hydrophilizing comonomers or
copolymers include hydroxyethylmethacrylate (HEMA) and
poly(ethyleneglycol) methacrylate (PEGMA).
[0040] HEMA is widely used in biomedical applications and devices,
most prominently soft contact lenses. HEMA, with 37.8% water per
weight, is typical of hydrogels. Preferably, the molar ratio of
HEMA comonomer in the polymeric composition is equal to or greater
than about 90 to 1.
[0041] PEGMA is a biocompatible polymer which possesses several
important properties, such as good solubility in both organic and
aqueous media, low toxicity, immunogenicity and
nonbiodegradability.
[0042] Preferably, the molar molecular weight of PEGMA comonomer in
the bactericidal composition is equal to or greater than 300, more
preferably between about 300 and about 2000, including but not
limited to 1100. Preferably, the molar ratio of PEGMA comonomer in
the polymeric composition is equal to or less than about 10 to 1;
equal to or less than about 25 to 1; equal to or greater than about
75 to 1; equal to or greater than about 95 to 1; equal to or
greater than about 99 to 1.
[0043] Hydrophilicity or wettability can be evaluated by any
suitable methodology known in the art, including contact angle
testing and tensionometry testing. Contact angle testing of
polymeric compositions may be carried out by dip coating microscope
slides in solutions with copolymer dissolved in chloroform and
methanol and obtaining contact angle measurements using e.g., a
Rame-Hart Advanced Goniometer. Contact angles may be characterized
as advancing or receding, the difference being whether or not the
angle is taken when moving onto a dry surface or moving off a wet
surface. Advancing angles may be used for surface energy
determinations, receding angles for characterizing other surface
characteristics.
[0044] Polymeric bactericidal compositions may be rendered
hydrophilic by engineering them to have advancing contact angles
with water of less than or equal to about 90 degrees, preferably
less than or equal to about 45 degrees, more preferably less than
or equal to about 30 degrees, less than or equal to 15 degrees
after 30 seconds of spreading.
[0045] The disclosed bactericidal compositions are suitably
formulated to significantly reduce the growth, number, viability
and/or metabolic activity of bacteria. A bactericidal composition
may be formulated to significantly reduce bacterial growth from a
treated medium by a factor of at least 50%. Further, a bactericidal
composition may be formulated to significantly reduce bacterial
growth from a treated medium by at least 60%, by at least 70%, by
at least 80%, by at least 90%, by at least 95%, by at least 99%, or
by at least 99.9%.
[0046] The bactericidal composition may be applied as a coating to
at least one portion or surface of a medium or medical device,
including but not limited to catheters, needles, stents, and other
implantable medical devices. Various methods may be used to apply
the comonomers or bactericidal polymers as a coating to the surface
of the medical device. Suitable methods for applying coatings may
include, but are not limited to the methods disclosed in U.S. Pat.
No. 5,509,899 and U.S. Pat. No. 6,221,425, the contents of which
are expressly incorporated by reference in their entirety.
[0047] Comonomers may be applied to a surface and subsequently
polymerized. Alternatively, the bactericidal polymer composition
may be applied directly to the surface of the medical device. In
particular, one or more comonomers or bactericidal polymers may be
combined with water and sprayed onto the medical device.
Alternatively, the medical device may be dipped into a solution
containing the bactericidal polymer. The comonomer or bactericidal
polymer may be present in the solution in an amount from about 50%
to about 98% by weight, particularly from about 70% to about 90% by
weight, and applied to the surface of the medical device.
[0048] The viscosity of the monomeric or polymeric solution can be
adjusted depending upon the particular application and
circumstances. In general, when dipping the medical device into the
solution, higher viscosities will cause more of the bactericidal
polymer to remain on the surface of the device. Thus, if thicker
coatings are desired, the viscosity can be increased. The viscosity
of the solution can be increased by minimizing the amount of water
in the solution. Additionally, thickeners, such as a
polyacrylamide, can be added to the solution. The viscosity of the
solution may also be increased by partially polymerizing the
monomer.
[0049] In another example, the present invention provides methods
for rendering a material or area bactericidal. In a further
example, the present invention provides a method for killing or
significantly reducing the number of bacteria on a material or area
treated with a bactericidal composition of the present
invention.
[0050] Accordingly, in one example, a bactericidal composition of
the present invention is applied to a medium or medical device in
an amount sufficient to kill or significantly reducing the number
of bacteria in or on the treated medium compared to an untreated
medium. In a further example, a bactericidal composition according
to the present invention is applied to a medium or medical device
in an amount sufficient to kill at least one bacterium or
significantly reduce bacterial growth compared to an untreated
medium.
[0051] The bacteria may be Gram-positive or Gram-negative. The
bactericidal composition may be is included in or coated onto a
catheter, stent, implantable medical device, contact lens, root
canal filler, or wound dressing. The treated medium may include
natural or synthetic materials, implantable devices, or bodily
surfaces. The treated medium may be contact with an aqueous
environment, such as water or the inside of a patient or other
vertebrate organism. Alternatively, the treated medium may be
contact with air or air and/or air borne bacteria in an external
environment or an enclosed bodily organ, such as lung.
[0052] Biocompatibility may be evaluated by any suitable
methodology known in the art, including biocompatibility tests set
forth in International Standards Organization (ISO) Standard No.
10993 and/or the U.S. Pharmacopeia (USP) 23 and/or the U.S. Food
and Drug Administration (FDA) blue book memorandum No. G95-1,
entitled "Use of International Standard ISO-10993, Biological
Evaluation of Medical Devices Part-1: Evaluation and Testing." In
addition, any of the viability/cytotoxicity assays known to those
of ordinary skill in the art may be used to evaluate lack of
toxicity for normal human cells.
[0053] In a further example, the present invention provides a
method for identifying a polymer having suitable bactericidal
activity. In this method, a hydrophilizing first comonomer may be
polymerized to a second comonomer and a bactericidal polymeric
composition is formed. The bactericidal polymeric composition may
be applied to a medium to form a first treated medium and the
medium may be separately treated with the second comonomer used in
the first treated medium. The first treated medium and the second
treated medium may be separately contacted with a plurality of
bacteria. Whether the first treated medium is more bactericidal
than the second treated medium may be determined.
[0054] In a further example, a first polymeric composition and a
second polymeric composition differing by molecular weight with
regard to one or more corresponding comonomers may be separately
applied to a medium and tested to identify a polymeric composition
having improved bactericidal activity.
[0055] Alternatively, a first polymeric composition and a second
polymeric composition differing by molar ratio of their
corresponding comonomers may be varied and may be separately
applied to a medium and tested to identify a polymeric composition
having improved bactericidal activity.
[0056] In the above disclosed methods, a given polymeric
composition may be rendered bactericidal by quaternization after
polymerizing the hydrophilizing first comonomer to the second
comonomer. Accordingly, the quaternized polymeric composition would
be deemed suitable for use in a bactericidal composition if a
medium containing or treated with the quaternized polymeric
composition is more hydrophilic and/or bactericidal than the same
medium containing or treated with the quaternized second comonomer
alone.
[0057] Bactericidal activity may be evaluated using any suitable
testing methodology used in the art, including, but not limited to,
luminescence, optical density, or microscopic evaluation of
bacterial growth or viability of coated and/or stained microscopic
slides, plates or cultures.
[0058] The following examples illustrate features in accordance
with the present invention, and are provided solely by way of
illustration. They are not intended to limit the scope of the
appended claims or their equivalents.
EXAMPLES
[0059] 1. Radical Polymerization and Quaternization. Copolymers
possessing suitable bactericidal properties and a suitable
hydrophilicity/biocompatibility profile were obtained using a
quaternized polymeric composition synthesized from 4-vinylpyridine
and a biocompatible, hydrophilic comonomer, such as
hydroxyethylmethacrylate (HEMA) or poly(ethyleneglycol)
methacrylate.
[0060] Copolymers were synthesized by radical copolymerization with
AIBN as initiator. The reactants were stirred at 70.degree. C. for
48 hours under flowing N.sub.2 to prevent oxidation. As the monomer
contents were varied, the AIBN proportion was held constant to a
massic ratio VP+PEGMA:AIBN equal to 22:1. To investigate the
effects of hydrophilization, seven different compositions of VP
with PEGMA300, PEGMA 1100 and HEMA were synthesized, containing a
molar percentage of VP of 10, 25, 50, 75, 90, 95 and 99.
[0061] Copolymers were quaternized with a 3-fold excess of hexyl
bromide (HB) in a mixture of chloroform and methanol by reflux for
48 hr. They were precipitated in hexane, recovered and dried under
vacuum. A schematic of the radical polymerization and
quaternization process can be seen in FIG. 1.
[0062] Synthesis of P(VP-co-HEMA), P(VP-co-PEGMA300) and
P(VP-co-PEGMA 1100) was followed with FTIR and NMR. Spectroscopy
showed that the synthesis was successful and that the
quaternization went to near completion and that the resultant
products were relatively pure after work-up.
[0063] VP, HEMA and PEGMA were purchased from Sigma Aldrich Co.
(Milwaukee, USA). To avoid polymerization through heat or light,
these monomers were inhibited with hydroquinone (HQ),
4-Methoxyphenol (MEHQ), and 2,6-di-tert-butyl-4-methylphenol (BHT)
respectively. The HQ and MEHQ inhibitors were removed by means of
trap to trap while BHT was purified from PEGMA by column
chromatography on silica gel (70-270 mesh) stationary phase.
[0064] 2. Contact Angle and Bactericidal Testing. To evaluate
wettability or hydrophilicity, contact angle tests were conducted
by dip coating microscope slides in solutions with copolymer
dissolved in chloroform and methanol. Contact angle measurements
were obtained on a Rame-Hart Advanced Goniometer.
[0065] Bactericidal tests were performed with a small quantity of
the bacteria Escherichia coli O157:H7 in which the lux gene was
added for luminescence, which provides a measure of metabolic
growth or activity. A sample was taken from a culture and placed in
contact with the coated slides, by means of a pipette. The
intensity of the bioluminescence was recorded as a function of time
for two hours with a photomultiplier tube. Reduced bioluminescence
correlates with enhanced bactericidal activity.
[0066] 3. Bactericidal activity of P(VP-co-HEMA). The results of
the bactericidal tests on quaternized copolymers of VP and HEMA are
shown in FIG. 2. An initial increase of intensity is observed in
the control, due to the fast growth of the bacteria, called
blooming. After approximately 19 minutes, the intensity starts
decreasing as the bacteria start to die. PVP-HB, known to kill
bacteria, prevents blooming, as reflected by the fact that the
intensity never increases by more than 1 percent. The intensity
starts decreasing after only 7 minutes. Since this is much earlier
than the control, the death of the bacteria can be attributed to
the properties of the polymer. An uninterrupted blooming is
observed for a slide coated with PHEMA, and the number of bacteria
has quadrupled after two hours, following a lag-log behavior. This
indicates that PHEMA by itself is not bactericidal.
[0067] P(VP-co-HEMA)-HB 95/5 and P(VP-co-HEMA)-HB 90/10 exhibited
enhanced bactericidal activity compared to PVP-HB alone. The
luminescence recorded for P(VP-co-HEMA)-HB 99/1, is similar to, but
slightly less than that observed for PVP-HB alone. Accordingly,
this copolymer, having one molar percent HEMA, displays properties
similar to PVP-HB alone. However, a slide coated with
P(VP-co-HEMA)-HB 99/1 kills bacteria faster than one coated with
PVP-HB.
[0068] The wettability of dry, vitreous HEMA-based materials was
studied by contact angle measurements. The results for both
advancing and receding angles are given in FIG. 3. Contact angle
measurements showed an increase in hydrophilicity provoked by the
copolymerization. The surface energy was found to be minimal for
P(VP-co-HEMA) at 90/10 and slightly higher for P(VP-co-HEMA)-HB
99/1. This corresponds to the bactericidal behavior of the polymers
and suggests that the wettability plays a significant role in the
polymer's effectiveness. Being a hydrogel monomer, HEMA
hydrophilizes the copolymer.
[0069] Although not wishing to be bound by theory, it is believed
that coupling hydrophilization to bactericidal activity in the
polymer facilitates enhances bacterial killing, in part because of
the water-loving nature of bacteria: a hydrophilic growth medium is
better able to support uptake and killing by a hydrophilized
bactericidal polymer compared to an unhydrophilized bactericidal
polymer. Moreover, it is believed that the bactericidal polymers
are electrostatically attracted to the bacterial cell wall whereby
lipophilic side chains insert into the bacterial cell membrane,
disrupting it so that holes form therein.
[0070] In P(VP-co-HEMA)-HB 90/10, the wettability effect is
particularly evident. This polymer exhibits a more optimal
bactericidal activity, reflected in the fact that all bacteria were
killed in 30 minutes. This further illustrates that that a slide
coated with P(VP-co-HEMA)-HB 90/10 copolymer is significantly more
bactericidal than pure PVP-HB.
[0071] 4. Bactericidal activity of P(VP-co-PEGMA). The bacterial
growth behavior for copolymers with PEGMA1100 can be seen in FIG.
4. Comonomer ratios of 90/10, 25/75, and 10/90 exhibited enhanced
bactericidal activity compared to PVP-HB alone. Extremely high
bactericidal activity was seen with ratios of 99/1, presumably due
to the large fraction of VP and improved wettability from
PEGMA1100. Copolymers with ratios ranging from 95/5 to 50/50
displayed bacterial results similar to PVP-HB.
[0072] P(VP-co-PEGMA1100)-HB 25/75 and 10/90 displayed a
surprisingly high antibacterial activity. Although
counterintuitive, this fact can have several explanations. The
molecular weight of P(VP-co-PEGMA1100)-HB 10/90 is much higher than
other copolymer formulations of this system. This could increase
bactericidal activity, because the copolymer possesses more alkyl
tails to traverse the bacterial membranes. The enhanced water
wettability of the polymer may enable the polymer to better
dissolve in and/or surround the bacteria in an aqueous medium, so
as to facilitate more efficient bacterial killing.
[0073] PPEGMA300 (graph not shown) alone does not kill bacteria and
actually improves growth due to its biocompatibility and
hydrophilicity. The improved biocompatibility and hydrophilicity is
carried over into the P(VP-co-PEGMA300) copolymers with ratios from
0/100 to 50/50 thereby improving bacterial growth. However, for
ratios greater 50/50, bactericidal activity was observed. The
optimum balance between spreading and VP content was found to be
75/25, in which half the bacteria were killed in the first 15
minutes. Overall, the bactericidal behavior of the PEGMA300 based
polymers were reduced compared to PEGMA1100 based polymers.
[0074] PEGMA1100 has a significantly larger PEG size than PEGMA300.
A smaller fraction of PEGMA 1100 is thus necessary to hydrophilize
P(VP-co-PEGMA 1100). However, even for some similarly hydrophilized
polymers, the PEGMA 1100 materials exhibit superior bactericidal
activity, possibly due to the enhanced protein resistance imparted
by longer PEG chains in the polymers.
[0075] The enhanced bactericidal activity exhibited by the HEMA and
PEGMA copolymers appears to result from enhanced wettability in
aqueous solutions, allowing the polymer to better surround and/or
gain access to the bacteria, so as to enhance bacterial
killing.
[0076] 5. Cytotoxity of P(VP-co-PEGMA). A viability/cytotoxicity
assay may be used to evaluate biocompatibility of the bactericidal
polymers for mammalian cells. In particular, FIG. 5 shows that an
exemplary bactericidal PEGMA 1100 copolymer is non-toxic to
mammalian cells. Corneal epithelial cells were seeded onto
polystyrene culture plates in phosphobuffered saline solution (PBS;
pH 7.2) at a density of 3,500 cells/cm.sup.2 for 24 hrs at
37.degree. C. The cells were co-incubated for 4 hrs. with
quaternized P(VP-co-PEGMA 1100) copolymer or PPEGMA control polymer
in PBS at a concentration of 2.5 mg/ml, along with a PBS negative
control media.
[0077] Live cells were distinguished from dead cells using a
fluorescence-based LIVE/DEAD viability/cytotoxicity assay system
(Molecular Probes, Invitrogen Detection Technologies). The assay
system includes two probes, calcein AM, a fluorogenic esterase
substrate producing a green fluorescent product in live cells
having intracellular esterase activity, and ethidium homodimer-1, a
high-affinity, red fluorescent dye only able to pass through and
stain the compromised membranes of dead cells. FIG. 5 plots the
fraction of dead epithelial cells as a function of added
bactericidal polymer or polymer control. As shown in FIG. 5,
treatment of epithelial cells with the bactericidal P(VP-co-PEGMA)
polymer did not exhibit a statistically significant level of
epithelial cell killing over that of the PEGMA polymer or PBS
negative controls.
[0078] It is to be understood that the above-described polymers and
methods for their use are merely representative embodiments
illustrating the principles of this invention and that other
variations in the polymers or methods, may be devised by those
skilled in the art without departing from the spirit and scope of
this invention. The foregoing detailed description and accompanying
drawings have been provided solely by way of explanation and
illustration, and are not intended to limit the scope of the
appended claims. Many variations in the presently preferred
embodiments illustrated herein will be apparent to one of ordinary
skill in the art, and remain within the scope of the appended
claims and their equivalents.
* * * * *