U.S. patent application number 14/445566 was filed with the patent office on 2016-06-23 for electrospun dextran fibers and devices formed therefrom.
The applicant listed for this patent is The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Virginia Commonwealth University. Invention is credited to Gary Bowlin, James Bowman, Stephen Rothwell, David Simpson.
Application Number | 20160175477 14/445566 |
Document ID | / |
Family ID | 52826805 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160175477 |
Kind Code |
A9 |
Bowlin; Gary ; et
al. |
June 23, 2016 |
ELECTROSPUN DEXTRAN FIBERS AND DEVICES FORMED THEREFROM
Abstract
The invention generally relates to dextran fibers which are
preferably electrospun and devices formed from such fibers. In
particular, such devices may include substances of interest (such
as therapeutic substances) associated with the electrospun fibers.
Upon exposure to a liquid the electrospun fibers dissolve
immediately and the substances of interest are released into the
liquid. Exemplary devices include bandages formed from electrospun
dextran fibers and associated agents that promote hemostasis, such
as thrombin and fibrinogen.
Inventors: |
Bowlin; Gary;
(Mechanicsville, VA) ; Simpson; David;
(Mechanicsville, VA) ; Bowman; James; (Richmond,
VA) ; Rothwell; Stephen; (Richmond, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Virginia Commonwealth University
The Henry M. Jackson Foundation for the Advancement of Military
Medicine, Inc. |
Richmond
Rockville |
VA
MD |
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150112287 A1 |
April 23, 2015 |
|
|
Family ID: |
52826805 |
Appl. No.: |
14/445566 |
Filed: |
July 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12937322 |
Feb 9, 2011 |
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PCT/US09/40182 |
Apr 10, 2009 |
|
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14445566 |
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61044165 |
Apr 11, 2008 |
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Current U.S.
Class: |
604/304 ; 156/60;
424/447; 424/94.64; 514/13.6 |
Current CPC
Class: |
A61L 2300/254 20130101;
A61L 15/38 20130101; C08L 5/02 20130101; A61L 15/44 20130101; A61L
15/28 20130101; A61L 15/32 20130101; A61L 2300/418 20130101; A61L
15/28 20130101; A61L 2400/04 20130101; A61L 2300/252 20130101; Y10T
156/10 20150115 |
International
Class: |
A61L 15/28 20060101
A61L015/28; A61L 15/32 20060101 A61L015/32; A61L 15/44 20060101
A61L015/44; A61L 15/38 20060101 A61L015/38 |
Claims
1. A hemostatic product comprising a plurality of hemostatic layers
that each comprise: a dextran support; and at least one hemostatic
agent that is selected from the group consisting of thrombin and
fibrinogen, wherein the hemostatic layers are arranged in a stacked
configuration to form the hemostatic product.
2. The hemostatic product of claim 1, wherein the plurality of
hemostatic layers comprise a first hemostatic layer and a second
hemostatic layer, wherein the thrombin is associated with the first
hemostatic layer and wherein the fibrinogen is associated with the
second hemostatic layer.
3. The hemostatic product of claim 1, wherein the at least one
hemostatic agent is placed on a surface of the dextran support and
further comprising a top dextran support that covers the surface of
the dextran support.
4. The hemostatic product of claim 1, wherein the hemostatic
product comprises between about 5 and 10 of the hemostatic
layers.
5. The hemostatic product of claim 1, wherein the dextran support
comprises dextran fibers.
6. The hemostatic product of claim 5, wherein the dextran fibers
are electrospun.
7. The hemostatic product of claim 1, wherein the dextran support
has a moisture content of up to 5% by weight and wherein contact
between the at least one hemostatic agent and the moisture causes
the at least one hemostatic agent to be retained on the dextran
support.
8. The hemostatic product of claim 1, wherein the hemostatic
product further comprises a support material selected from the
group consisting of gauze, electrospun dextran, polyglycolytic acid
polymers, polylactic acid polymers, caprolactone polymers and
charged nylon.
9. The hemostatic product of claim 1, wherein the dextran support
is compressed.
10. A method of preparing a hemostatic product comprising:
fabricating a plurality of hemostatic layers that each comprise: a
dextran support; and at least one hemostatic agent associated with
the dextran support, wherein the at least one hemostatic agent is
selected from the group consisting of thrombin and fibrinogen; and
arranging the hemostatic layers in a stacked configuration.
11. The method of claim 10, wherein the plurality of hemostatic
layers comprise a first hemostatic layer and a second hemostatic
layer and wherein the method further comprises: associating the
thrombin with the first hemostatic layer; and associating the
fibrinogen with the second hemostatic layer.
12. The method of claim 10, and further comprising: placing the at
least one hemostatic agent is placed on a surface of the dextran
support; and covering the surface of the dextran support with a top
dextran support.
13. The method of claim 10, wherein the hemostatic product
comprises between about 5 and 10 of the hemostatic layers.
14. The method of claim 10, and further comprising placing one of
the hemostatic layers on a support material that is selected from
the group consisting of gauze, electrospun dextran, polyglycolytic
acid polymers, polylactic acid polymers, caprolactone polymers and
charged nylon.
15. The method of claim 10, and further comprising compressing the
hemostatic layer to a height that is less than an initial height of
the hemostatic layer.
16. The method of claim 10, wherein the dextran support comprises
dextran fibers.
17. The method of claim 16, wherein the dextran fibers are
electrospun.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 12/937,322, filed Feb. 9, 2011, which claims priority to
PCT/US09/40182, filed Apr. 10, 2009, which claims priority to U.S.
Applic. No. 61/044,165, filed Apr. 11, 2008, the contents of which
are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention generally relates to dextran fibers,
preferably electrospun dextran fibers, and devices formed
therefrom. In particular, such devices may be bandages which
include therapeutic substances associated with the electrospun
fibers, which, upon exposure to a liquid that dissolves the
electrospun fibers, are released into the liquid.
BACKGROUND OF THE INVENTION
[0003] The body's natural response to stem bleeding from a wound is
to initiate blood clotting via a complex process known as the
coagulation cascade. The cascade involves two pathways that
ultimately lead to the production of the enzyme thrombin, which
catalyzes the conversion of fibrinogen to fibrin. Fibrin is then
cross-linked to form a clot, resulting in hemostasis. For wounds
that are not severe, and in individuals that have no countervening
conditions, the body is usually able to carry out this process
efficiently in a manner that prevents excessive loss of blood from
the wound. However, in the case of severe wounds, or in individuals
in whom the clotting mechanism is compromised, this may not be the
case. For such individuals, it is however possible to administer
components of the coagulation cascade, especially thrombin and
fibrinogen, directly to the wound to bring about hemostasis.
Bandaging of bleeding wounds is also a usual practice, in part to
isolate and protect the wounded area, and also to provide a means
to exert pressure on the wound, which can also assist in
controlling bleeding.
[0004] While these methods may be carried out satisfactorily in
cases of mild trauma or under conditions of "controlled" wounding
(e.g. surgery), many situations in which such treatments are most
needed are also those in which it is the most difficult to provide
them. Examples of such wounds include, for example, those inflicted
during combat, or unanticipated wounds that occur as the result of
accidents. In such circumstances, survival of the wounded
individual may depend on stopping blood loss from the wound and
achieving hemostasis during the first few minutes after injury.
Unfortunately, given the circumstances of such injuries,
appropriate medical intervention may not be immediately
available.
[0005] In particular, the treatment of penetrating wounds such as
bullet wounds or some wounds from shrapnel is problematic. This is
due to the difficulty in placing a bandage and/or therapeutic
agents at the actual site of injury, which includes an area that is
well below the body surface and difficult or impossible to access
using conventional techniques.
[0006] Jiang et al. (Biomacromolecules 2004, 5, 326-333) teaches
electrospun dextran fibers. Agents associated with the fibers (e.g.
BSA, lysozyme) are directly electrospun into the fibers. The fibers
may also include other polymers electrospun with the dextran.
[0007] Jiang et al. (2006, Journal of Biomedical Materials Research
Part B: Applied Biomaterials, 50-57, Wiley Periodicals, Inc.)
discloses electrospun fibers which are a composite of
poly(c-caprolactone) as a shell and dextran as a core. These fibers
provide the slow release of agents (bovine serum albumin, BSA)
which are also electrospun into the fibers.
[0008] U.S. Pat. No. 6,753,454 to Smith et al. (Jun. 22, 2004)
discloses electrospun fibers comprising a substantially homogeneous
mixture of a hydrophilic polymer and a polymer which is at least
weakly hydrophobic, which may be used to form a bandage. The
bandage may comprise active agents (e.g. dextran). However, the
disclosed fibers are not readily soluble in liquid.
[0009] U.S. Pat. No. 6,762,336 to MacPhee et al. (Jul. 13, 2004)
teaches a hemostatic multilayer bandage that comprises a thrombin
layer between two fibrinogen layers. The bandage may contain other
resorbable materials such as glycolic acid or lactic acid based
polymers or copolymers. Neither electrospun fibers nor dextran
fibers are taught as components of the bandage.
[0010] U.S. Pat. No. 6,821,479 to Smith et al. (Nov. 23, 2004)
teaches a method of preserving a biological material in a dry
protective matrix, the matrix comprising fibers such as electrospun
fibers. One component of the fibers may be dextran, but homogeneous
dextran fibers are not described.
[0011] U.S. Pat. No. 7,101,862 to Cochrum et al. (Sep. 5, 2006),
teaches hemostatic compositions and methods for controlling
bleeding. The compositions comprise a cellulose containing article
(e.g gauze) to which a polysaccharide is covalently or ionically
crosslinked. The crosslinked polysaccharide may be dextran.
However, the compositions are not electrospun and exogenous
clotting agents are not included in the compositions.
[0012] United States patent application 2004/0018226 (Wnek et al.,
published Jan. 29, 2004) discloses fibers produced by an
electroprocessing technique such as electrospinning. The fibers
comprise enclosures within the fibers for containing substances
that are not miscible with the fibers. Dextran is not taught as a
fiber component.
[0013] United States patent application 2007/0160653 (Fisher et
al., published Jul. 12, 2007) teaches a hemostatic textile
comprising hemostatic factors (e.g. thrombin, fibrinogen) but the
fibers are formed from electrospun glass plus a secondary fiber
(e.g. silk, ceramic, bamboo, jute, rayon, etc.)
[0014] United States patent application 2008/0020015 (Carpenter et
al., published Jan. 24, 2008) teaches wound dressing comprised of
various biodegradable polymers and hydrogels having allogenic or
autologous precursor cells (e.g. stem cells) dispersed within the
polymers. The polymers may be prepared by electrospinning, and one
polymer component may be dextran. However, the polymers cannot be
immediately soluble upon contact with liquid, as they must provide
a scaffolding for delivery of the cells over time, even though the
polymers eventually biodegrade in situ.
[0015] United States patent application 2008/0265469 (Li et al.,
priority date: Nov. 10, 2006) describes electrospun nanofibers
which may comprise dextran. However, the nanofibers are not
described as readily soluble in liquids.
[0016] United States patent application 2009/0053288 (Eskridge et
al., published Feb. 26, 2009) teaches a woven hemostatic fabric
comprised of about 65% fiberglass yarn and about 35% bamboo yarn.
The fiberglass component may be electrospun, and hemostatic factors
such a thrombin may be associated with the fabric, e.g. by soaking
the material in a solution of thrombin. Dextran may be added as a
hygroscopic agent.
[0017] There is an ongoing need to provide improved methods and
means to initiate blood clotting in wounds in order to stop or at
least slow blood loss. In particular, there is an ongoing need to
improve the capability to readily promote hemostasis in severe
wounds in a facile manner, especially under circumstances where
immediate treatment by medical personnel is limited or
unavailable.
SUMMARY OF THE INVENTION
[0018] Electrospun dextran fibers (EDFs) are demonstrated herein to
be useful as a temporary "scaffolding" to sequester and transport
one or more associated substances of interest to a location of
interest. A liquid solvent is present at or will be present at the
location of interest, and the scaffolding is temporary because the
electrospun dextran fibers dissolve upon contact with the liquid,
releasing the associated substances of interest into the liquid. In
one embodiment of the invention, the EDFs are fabricated into a
bandage which also includes therapeutically beneficial active
agents, usually bioactive agents. For example, the bandage may
include substances that promote hemostasis such as thrombin and
fibrinogen. In this embodiment, the thrombin and fibrinogen are
present within the bandage in forms that are active upon contact
with liquid such as blood. Application of the bandage to a bleeding
wound results in dissolution of the dextran fibers in the liquid
blood, and release of active thrombin and fibrinogen directly into
the wound bed. The active thrombin rapidly catalyzes the conversion
of the fibrinogen to fibrin, and formation of a hemostatic plug or
clot from fibrin ensues, resulting in hemostasis.
[0019] The use of electrospun dextran for the delivery of
substances of interest to a liquid environment of interest has many
advantages over the prior art. For example, electrospun dextran
fibers are extremely light-weight and flexible (malleable), and
thus devices made from electrospun dextran fibers have very small
footprints. This is important in many situations. For example,
bandages made according to the invention are readily accommodated
in, e.g., the gear carried by a soldier, without adding excessive
weight or bulk. In addition, upon contact with liquid, electrospun
dextran dissolves instantaneously (or nearly instantaneously) and
completely. Therefore, when placed, for example, on or over a
bleeding wound, agents included in the bandage are rapidly and
directly delivered to the wound site, and a clot is formed. There
is no need to remove the dextran components of the bandage,
possibly disrupting the clot, because the dextran component
dissolves. Advantageously, implementation of this potentially
life-saving technique does not require the use of sophisticated
instrumentation or a high level of training or expertise; such
devices can be administered by a novice, and may even be
self-administered if need be. For example, a wounded soldier may be
able to self-administer a dextran bandage in order to staunch the
flow of blood from a wound, while waiting for medical personnel to
further assist him or her.
[0020] In some embodiments, the electrospun dextran fiber device
includes a non-soluble or partially or slowly soluble support
material to provide additional stability to the "cottony"
electrospun fibers. The support material may also be formed from
electrospun material, such as compressed electrospun dextran, or
other electrospun fibers, or from another type of material
altogether. If compressed dextran fibers are used, in general a
full size bandage contains 5-10 grams of dextran and is perhaps
from about 2.5 to about 4 inches in width and length, and bout 2
inches high (depth), and such a bandage can be compressed to about
0.5 inches in depth to form a support material. Advantages of
including a support material are, for example, that the device may
be somewhat more robust and able to withstand manipulations
(packaging, packing, handling, etc.). In the case where the device
is a bandage, the presence of the support material makes it
possible to apply pressure through the bandage and onto a wound to
which the bandage is applied, even though the dextran fibers and
active agents dissolve immediately upon exposure to liquid
blood.
[0021] It is an object of this invention to provide a method of
delivering one or more agents of interest to a location of
interest. The method comprises the step of applying or delivering
to said location of interest a device, the device comprising 1)
electrospun dextran fibers which dissolve upon contact with liquid;
and 2) one or more agents of interest associated with the
electrospun dextran fibers. The step of applying or delivering
results in dissolution of the electrospun dextran fibers in liquid
at the location of interest, thereby releasing the one or more
agents of interest into the liquid. In some embodiments, the
electrospun dextran fibers have a diameter ranging from 0.75
microns to 1.00 micron. In some embodiments, the location of
interest is a site on a body of a subject; and the liquid at the
site may be a bodily fluid. In addition, the one or more agents of
interest may be bioactive agents.
[0022] The invention further provides a bandage comprising
electrospun dextran fibers which have a diameter ranging from 0.75
microns to 1.25 microns, and which dissolve upon contact with
liquid; and at least one of thrombin and fibrinogen are associated
with the electrospun dextran fibers in the bandage. In one
embodiment of the invention, the thrombin and fibrinogen are salmon
thrombin and salmon fibrinogen, which may be in particulate form.
Alternatively, the thrombin and fibrinogen may be present within
electrospun droplets. In some embodiments of the invention, both
thrombin and fibrinogen are associated with the electrospun dextran
fibers. In other embodiments, the bandage further comprises one or
more additional bioactive agents. In yet other embodiments, the
bandage comprises a support material. In some embodiments, the
support material comprises a material selected from gauze,
compressed electrospun dextran, polyglycolytic acid polymers,
polylactic acid polymers, caprolactone polymers and charged
nylon.
[0023] The invention also provides a method of inducing hemostasis
in a wound. This method comprises the step of applying to the wound
a hemostatic bandage which comprises: 1) electrospun dextran fibers
which have a diameter ranging from 0.75 microns to 1.25 microns,
and which dissolve upon contact with liquid; and 2) at least one of
thrombin and fibrinogen associated with the electrospun dextran
fibers. The step of applying results in dissolution of the
electrospun dextran fibers in blood within the wound and release of
the thrombin and fibrinogen into the blood within said wound,
thereby inducing hemostasis in the wound. In some embodiments, both
thrombin and fibrinogen are associated with the electrospun dextran
fibers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1. Schematic of the electrospinning apparatus. The key
elements of the electrospinning system include a high voltage power
supply, a source reservoir for the polymer and a grounded mandrel.
This system utilizes a cylindrical target mandrel; however the
electrospinning process can be adapted to produce much more complex
shapes. Single and/or multiple polymers can be independently or
simultaneously delivered to the electric field from one or more
source reservoirs. Electrospinning distinct and unique polymers
from separate sources in a temporal sequence can be used to produce
a laminated structure.
[0025] FIGS. 2 A and B. A, schematic of air brush based dextran
processing; B, dextran fibers produced by electroaerosol
processing. The amount of material depicted is probably enough
material for about two bandages. Note the loft of the material. An
electric field was used to target the dextran to the mandrel.
[0026] FIG. 3. Scanning electron micrograph of electrospun dextran
fibers. The nominal average cross sectional diameter of the
individual fibers was 1 micron, providing a large surface area.
[0027] FIG. 4A-E. Schematic representations of exemplary bandages
formed form electrospun dextran fibers. A, bandage with
non-permeable support material as a backing; B, bandage with
net-like support material; C, bandage with non-permeable backing
and a net-like support material holding the electrospun fibers in
place on the backing; D, bandage (device) for delivery of
therapeutics to a deep wound; E, alternative embodiment of a device
for delivery of therapeutics to a deep wound.
[0028] FIGS. 5A and B. Changes in cytokine levels in animals
exposed to the salmon fibrinogen/thrombin bandage. (A) Levels of
IL-1.beta., IL-6, TNF-.alpha., IFN-.gamma., IL-4 and IL-10 are
shown as the log ratio of the cytokine level determined in blood
drawn at the initial surgery to implant the vascular port compared
to peak levels following exposure. Changes were seen in both
pro-inflammatory responses (IL-1.beta., IL-6, TNF-.alpha.,
IFN-.gamma.) and humoral responses (IL-4 and IL-10). (B) Changes in
the cytokines within an individual animal show that initial
exposure (first arrow) and the subsequent intravenous infusion of
proteins (second arrow) elicited a response that could be detected
in samples taken at the next blood draw.
[0029] FIG. 6A-F. Qualitative assessment of immunoglobulin
production by swine in response to salmon proteins by Western
blotting. (A) PAGE of salmon (Sal), human (Hu) and swine (Sw)
fibrinogen preparations and corresponding Western blots with serum
from two animals (B and C). Serum from pre-exposure and final
euthanasia blood draws are presented in these panels. IgG isotypes
present in the serum were visualized by specific HRP anti-swine IgG
second antibodies and are detected as binding to the proteins in
the gel samples. Arrows indicate the positions of the IgG heavy and
light chains components in the swine protein lanes which are also
recognized by the 2nd antibody. Molecular weights are show to the
left (kDal.times.10-3). (D) PAGE of salmon (Sal), human (Hu) and
swine (Sw) thrombin preparations and corresponding Western blots
with serum from the same animals shown in (C and D). In these
animals, thrombin was not recognized in E, but there is a faint
reaction in the salmon protein lane in F (arrow). The camera in the
detection system detected the heavy swine thrombin protein on the
membrane as a white band in F.
[0030] FIG. 7A-D. Time course of antibody development in animals
exposed to salmon thrombin/fibrinogen bandages through the dermal
patch protocol. ELISAs were performed using anti-IgG reagents. The
following antigens were used as the targets in the ELISAs: (A)
salmon fibrinogen, (B) salmon thrombin, (C) human fibrinogen and
(D) human thrombin. The increases in absorbance observed at the
later samples panels A, B, C occurred following intravenous
infusion of salmon proteins. Each curve represents data from a
different animal.
[0031] FIG. 8A-D. Time course of antibody development in animals
exposed to salmon thrombin/fibrinogen bandages through the
abdominal patch protocol. ELISAs were performed using anti-IgG
reagents. The following antigens were used as the targets in the
ELISAs: (A) salmon fibrinogen, (B) salmon thrombin, (C) human
fibrinogen and (D) human thrombin.
[0032] FIG. 9A-D. Progression of dermal healing following
full-thickness wound. Images from samples taken at 7 days from
control (A) and salmon bandage-treated (B) injuries show a
fibrinonecrotic coagulum filling the wound defect (*) and an
epithelial cell projection towards wound center in both cases as
wound healing progresses following initial clotting. (H&E
staining, bars=100 um). Samples taken at 28 days from control (C)
and salmon bandage-treated (D) injuries show complete
re-epithelialization by a hyperplastic and hyperkeratotic
epidermis. (H & E staining, bars=100 um). FIG. 10. Schematic of
the coagulation cascade.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The invention provides dextran fibers, especially
electrospun dextran fibers. The electrospun dextran fibers (EDFs)
may be formed into a variety of devices for a variety of purposes.
Generally, one or more substances of interest are associated with
the EDFs in the device, usually for the purpose of delivering the
one or more substances of interest to a liquid of interest. Upon
contact with the liquid, the EDFs dissolve almost immediately and
the associated substances are released into the liquid milieu.
[0034] In one embodiment of the invention, the EDFs are formed into
a bandage. The bandage generally includes active agents associated
with the EDFs, the active agents being delivered to a site of
action (e.g. a wound) via application of the bandage to the site.
The site of action contains or will contain a liquid, and when the
bandage is applied to the site of action, the EDFs of the bandage
dissolve in the liquid, and the active agents associated with or
sequestered in or around the mat of dextran fibers are released
into the liquid. In one embodiment, the site of action is a wound
bed, and the active agents that are delivered by the bandage are
factors or agents that participate in the coagulation cascade such
as thrombin and fibrinogen. Application of an EDF bandage to a
wound results in dissolution of the dextran fibers in blood within
the wound bed, which in turn results in release or delivery of the
active agents at or into the site. Thrombin and fibrinogen that are
associated with the bandage are in forms that are biologically
active when they come into contact with blood. Hence, upon
dissolution, the thrombin acts on the fibrinogen, converting it to
fibrin, which then forms a clot within the wound, staunching the
flow of blood. In some embodiments of the invention, only spun
dextran fibers are utilized and thus after clot formation, there is
no need to disturb the clot in order to remove bandage components,
since none remain at the site. In other embodiments, as described
below, the bandage may comprise other materials such as support or
backing material, which, after initial rapid application of the
bandage, may later be removed for further treatment of the wound by
conventional methods.
[0035] Electrospinning is a non-mechanical processing strategy and
can be scaled to accommodate the large volumes necessary to meet
the needs of commercial processing. A schematic representation of
one type of set-up for electrospinning is provided in FIG. 1. In
this process a polymer solution, or melt, is injected with current
to create a charge imbalance. The charged solution is then placed
in proximity to a grounded target (in FIG. 1, a grounded mandrel).
At a critical voltage the charge imbalance begins to overcome the
surface tension of the polymer source, forming an electrically
charged jet. Within the electric field, the jet is directed towards
the grounded target and the carrier solvent evaporates. Depending
upon reaction conditions, and the polymers used in the process,
electrospinning can be utilized to produce a fine aerosol of
material or a continuous non-woven mat of fibrillar material, as
shown in FIG. 1. For many polymers, the nature of the
electrospinning process intrinsically provides a high degree of
control over the diameter of the resulting fibers. Micron to
nanoscale diameters can be selectively achieved simply by
regulating the starting concentrations of the polymers present in
the electrospinning solutions. By controlling the motion of the
ground target with respect to the source solution, fibrils may be
deposited into a random matrix or into aligned arrays that are
oriented along a defined axis.
[0036] A second schematic of an electrospinning apparatus is shown
in FIG. 2A. The key elements of the electrospinning system include
a high voltage power supply, a source reservoir for the polymer and
a grounded target mandrel. The system that is depicted utilizes a
cylindrical target mandrel; however the electrospinning process can
be adapted to produce much more complex shapes. Single and/or
multiple polymers can be independently or simultaneously delivered
to the electric field from one or more source reservoirs. In
addition, electrospinning distinct and unique polymers from
separate sources in a temporal sequence can be used to produce a
laminated structure. FIG. 2B shows the result of electrospinning
about 10 g of dextran dissolved in deionized water onto a round
mandrel target, as described in detail in the Example 1 below. FIG.
3 shows a scanning electron micrograph of electrospun dextran
fibers in which the average cross sectional diameter of the
individual fibers is about 1 micron.
[0037] Those of skill in the art will recognize that
electrospinning is not the only way to make dextran fibers. Such
fibers may be produced by other methods of aerosolization. However,
the electric field helps in the efficient collection of the fibers,
and electrospinning may yield more uniform fibers. Other
technologies which might also be employed for spinning dextran
fibers, including those described in U.S. Pat. No. 7,067,444 to Luo
et al. (Jun. 27, 2006); U.S. Pat. No. 6,116,880 to Bogue et al.
(Sep. 12, 2000); and U.S. Pat. No. 5,447,423 to Fuisz et al. (Sep.
5, 1995), the complete contents of each of which are hereby
incorporated by reference. In particular, so-called "cotton-candy
machines" (with or without applied electrostatic force) may be
suitable for use in fabricating the dextran fibers of the
invention. More detailed descriptions of methods of preparing the
dextran fibers of the invention are provided in Example 2 below.
Other methods include compressing a dextran solution between two
plates or other flat surfaces and drawing the plates or surfaces
away from each other, usually repeatedly. Dextran fibers form
between the two surfaces.
[0038] In some embodiments, substances other than dextran are used
to form fibers for use in the devices of the invention, especially
(but not exclusively) when a cotton-candy machine is employed.
Examples of such substances include but are not limited to sugars
such as dextrose, sucrose, etc.
[0039] The commercially available dextran that is used to produce
the electrospun fibers of the invention is synthesized from sucrose
by enzymes on the cell surface of certain lactic acid bacteria, the
best-known being Leuconostoc mesenteroides and Streptococcus
mutans. Dextran is a complex, branched glucan (a polysaccharide
made of many d-glucose molecules) composed of chains of varying
lengths (e.g. from 10 to 200 kilodaltons). The straight chain
consists of .alpha.-1,6 glycosidic linkages between glucose
molecules, while branches begin from .alpha.-1,4 linkages (and in
some cases, .alpha.-1,2 and .alpha.-1,3 linkages as well). Dextrans
are commercially available in a wide range of molecular weights
e.g. from about 10 kilodaltons (kDa) to about 200 kDa. Commercial
preparations are mixtures of dextrans of varying molecular weights,
usually in narrower weight ranges and may be provided, for example,
as "low" or "high" molecular weight dextrans. For example, "Dextran
40" has an average molecular weight of 40 kDa, "Dextrans 75" has an
average molecular weight of 75 kDa, etc. In the practice of the
present invention, the dextrans used for electrospinning are
typically in a molecular weight range of from about 10 to about 200
kDa, or from about 25 to about 200 kDa, or from about 50 to about
200 kDa, or from about 75 to 200 kDa, and usually from about 60 to
90 kDa, or from about 100 to about 200 kDa. Further, as would be
understood by those of skill in the art, the median size of the
dextran molecules in a dextran preparation also has an effect in
that if the median weight is high in a particular lot, less dextran
may be used to form the desired amount of fibers.
[0040] In general, the conditions for electrospinning dextran are
as follows: an ambient temperature of from about 60 to about
75.degree. F.; a relative humidity of from about 30% to about 40%,
and typically at least about 20%. The resulting fibers are
typically in the nanometer or mm range of cross-sectional diameter,
usually from about 0.75 microns to about 1.25 microns. The
electrospun fibers are "dry" and should be protected from exposure
to moisture to prevent premature dissolution. However, some water
is associated with the fibers and fiber compositions can contain
from about 7 to about 8% water, but must be less than about 5% when
the fibers are sterilized by x-ray irradiation.
[0041] The devices of the invention are usually formed of
substantially homogeneous spun dextran. The amount of dextran per
device can vary widely, depending on the size of device that is
being manufactured, with typical device formulations using from
about 5-10 g of dextran (usually 100,000-200,000 Mr) per device.
However, the range can be extended widely, e.g. from as low as
about 0.5 g or less (for small devices) to as high as 100 or more g
per device, for large devices. In some embodiments of the
invention, is may be helpful to use lesser amounts of dextran (e.g.
about 0.1 to about 0.5 g of dextran per device) in order to
concentrate the active agents that are delivered by the device into
a smaller volume. Of more consequence is the concentration of
dextran in the solution from which the fibers are spun. Generally,
a solution of dextran for electrospinning will be of a
concentration in the range of from about 0.1 to about 10 grams per
ml of solvent, or from about 0.5 to about 5 grams per ml, and
usually such a solution is at a concentration of about 1 gram per
ml, .+-.about 0.15 mg. A preferred range would be from about 0.9 to
about 1.1 grams of dextran per ml of solution that is to be spun.
Those of skill in the art will recognize that, due to the
variability of molecular weight ranges in dextran preparations, and
due to inherent variability from batch to batch of commercially
available preparations purporting to be of a particular molecular
weight range, it is typically necessary to test each batch of
dextran with respect to electrospinning properties. Such tests are
well within the purview of one of skill in the art, and usually
involve trials of electrospinning a range of concentrations of
dextran dissolved in a suitable solvent, in order to ascertain
which concentration(s) result(s) in the most desirable fiber
characteristics, e.g. stability (e.g. to heat, humidity, etc.),
uniformity, cross-sectional diameter, etc. Those of skill in the
art will recognize that critical indicators of success are very
obvious when trying a new batch of dextran. Too little dextran in
the spinning solution results in "spitting" from the needle,
whereas too much dextran results in the production of dried
droplets, or failure to spin at all. Likewise, when the humidity is
too low, similar results can occur, i.e. fibers fail to form and in
some cases fail to target efficiently to the ground. These
characteristics can be assessed according to methods that are well
known to those of skill in the art, including but not limited to
visual observation, testing of fiber strength and flexibility,
observation via electron microscopy, solubility testing, resistance
to heat and/or irradiation, color and tendency to discoloration,
etc. As would be understood by those of skill in the art, all such
testing may be carried out under varying conditions of heat,
humidity, etc. Formulations may also be assessed using animal
testing.
[0042] The area (length and width) of a device of the invention can
vary widely and can be adjusted by adjusting spinning parameters.
In addition, the mats of dextran fibers can be cut to a desired
size after spinning Generally, a device will be from about 0.5 cms
or less to about 30 cms or more in length and/or width, but larger
or smaller sizes are also contemplated. The height or thickness of
a device can likewise vary considerably, e.g. from 0.5 cm or less
(e.g. about 0.1, 0.2, 0.3, or 0.4 cm) up to any desired thickness,
e.g. from about 1 to about 30 cm, or usually less, e.g. from about
1 to about 20 cm, or from about 1 to about 10 cm, or even from
about 1 to about 5 cm, e.g. devices with a thickness of about 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 cm are usual. The thickness of the
device (which is related to the volume) may impact the rate of
dissolution of the dextran upon contact with liquid. For example, a
thin device (e.g. about 2 cm or less, or about 1 cm or less, or
even about 0.5 cm or less, e.g. about 0.4, 0.3, 0.2, or 0.1 cm),
e.g. a thin sheet, will dissolve more rapidly than a device that is
thicker, providing the loft of the fibers is comparable. In most
embodiments, dissolution of the dextran fibers is extremely rapid,
e.g. about 5 minutes or less after exposure to liquid, or about 4
minutes or less, or about 3 minutes or less, or about 2 minutes or
less, or about 1 minute or less, e.g. the device typically takes
only a few seconds to dissolve (e.g. from about 1 to about 60
seconds, or from about 1 to about 45 seconds, or from about 1 to
about 30 seconds, or from about 1 to about 20, 15, 10, or 5 seconds
or less to dissolve. This rapid dissolution may be referred to
herein as "instantaneous" or "immediate" dissolution. Compression
of an electrospun dextran mat may be used to modulate the rate of
dissolution, with greater levels of compression inversely impacting
the rate, i.e. generally, the greater the degree of compression,
the slower the rate of dissolution. The rapid rate of dissolution
is advantageous, particularly when delivering biologically active
agents (e.g. hemostatic agents) to a site of action such as a
wound. Rapid dissolution of the carrier dextran fibers provides
extremely rapid delivery of the hemostatic agents to the wound upon
deployment of the device.
[0043] Those of skill in the art will recognize that a plethora of
liquid solvents exist in which it is possible to dissolve dextran.
However, superior results for electrospinning dextran are generally
achieved when the solvent is water, especially deionized or
distilled or deionized, distilled (ddH2O) or other forms of
relatively pure water. In addition, there is far less environmental
impact associated with the use of water. It has been found that,
generally, high concentrations of salt in the solvent should be
avoided. Whereas salt is often used to facilitate the spinning of
some electrospun polymers, this is not the case for dextran. The
concentration of salts in the spinning solution should be kept at a
minimum to successfully form dextran fibers.
[0044] The one or more active agents that are associated with the
dextran fibers of the bandage may be any active agent that it is
desirable or advantageous to deliver to the site where the EDF
device is to be used or applied. In one embodiment of the
invention, the EDF device is a bandage and is used to deliver
beneficial agents, for example, to a wound. Such wounds include
wounds and breaches of body or tissue integrity that occur as a
result of trauma (e.g. accidental trauma, trauma resulting from
conflicts such as gunshot wounds, knives, etc.), as well as wounds
which are purposefully incurred, such as surgical incisions, body
piercings, etc. Usually the agents are bioactive agents that have a
beneficial or therapeutic effect at the wound site. In one
embodiment, the site is a bleeding wound at which it is desired to
form a blood clot in order to stop or slow the bleeding. In this
embodiment, the therapeutic substances of interest may include, for
example, thrombin and fibrinogen, although other agents active in
promoting hemostasis, including but not limited to capscian, may
also be included. In addition, electrospun or non-electrospun
collagen, agents that absorb water, various dry salts that would
tend to absorb fluids when placed in contact with e.g. blood;
engineered thrombin or thrombin mimics; engineered fibrinogen;
agents that cause vasospasm (e.g. ADP, 5-hydroxytryptamine, 5-HT
and thromboxane, (TXA-2) to help contract and seal a bleeding
vessel, etc. may also be included. In addition, other components of
the clotting cascade may be added to the bandage, for example:
tissue factors that are normally only expressed on the surface of
damaged cells and which start the normal clotting cascade;
serotonin which enhances platelet clumping and promotes vessel
constriction; and other agents that are used to replace missing
components of the clotting cascade in hemophilia, for example,
factor 7 (which activates the so called external extrinsic
coagulation cascade) and crude extracts of platelets. These agents
essentially work to "jump start" clotting by initiating the cascade
further down the reaction network, as illustrated in FIG. 10. In
FIG. 10, the various factors (and their alternative nomenclature
and/or characteristics and/or activities) are as follows:
[0045] Factor XII (Hageman factor): serine protease, plasma protein
binds collagen;
[0046] Factor XI (Plasma thromboplastin antecedent): serine
protease, plasma protein;
[0047] Factor IX (Christmas-Eve Factor): serine protease;
[0048] Factor VIII: Glycoprotein binds vWF, produced by endothelium
and liver;
[0049] Factor VII (Proconvertin): serine protease, Vitamin K
dependent synthesis in the liver;
[0050] Factor X (Stuart-Prowler Factor, Clotting Factor X): serine
endopeptidase, converts prothrombin to thrombin; and
[0051] Factor XIII (Fibrin stabilizing Enzyme): stabilizes fibrin
polymer. plasma protein, also present in platelets and monocyte
linage.
[0052] In FIG. 10, italic pathways denote inhibition and the
central role of thrombin in the activation of coagulation and
inactivation of coagulation processes is shown, where:
[0053] VI=Cofactor for Xa in the conversion of prothrombin to
thrombin;
[0054] APC=Activated Protein C, an extracellular signal molecule,
inhibits FVI (equivalent to FVa, a cofactor of XA in the conversion
of prothrombin to thrombin) and FVIIIa through a proteolytic event;
and
[0055] TAFI=Thrombin Activatable Fibrinolysis Inhibitor, an
inhibitor of clot lysis.
[0056] In addition, agents which function to promote late stages of
wound healing may also be included to, for example, facilitate cell
migration and remodeling. The incorporation of collagen is an
example of such an agent.
[0057] One or more of any of these agents may be used in the
practice of the present invention. The therapeutic agents must be
amenable to drying and are associated with the electrospun dextran
fibers in the dry state, since liquid would dissolve the fibers.
For example, the agents may be desiccated or lyophilized, or water
may be removed by some other means. Generally, the amount of water
that is present in the substances when they are associated with the
EDFs is less than about 5%, and preferably less that about 2%.
These substances retain full or partial activity when rehydrated,
e.g. in blood. Generally therapeutic substances associated with the
devices of the invention retain, upon contact with liquid, at least
about 25%, or about 50%, or even about 75 to 100% of their activity
before drying or desiccation, as compared to standard preparations
of the substance using standard assays that are known to those of
skill in the art.
[0058] In some embodiments, thrombin or fibrinogen, or both, are
associated with the bandage. In some embodiments, the thrombin and
fibrinogen are salmon thrombin and fibrinogen. Advantages of using
salmon as a source of these materials include but are not limited
to the lack of concern about transmission of etiologic agents (e.g.
viruses) that may occur when human and other mammalian sources of
thrombin or fibrinogen (e.g. bovine) are used. As demonstrated in
the Examples section below, salmon thrombin and fibrinogen are
highly efficacious and have no deleterious side effects, when used
in the pig model, which is a recognized animal model that is
considered to be indicative of results in humans. The quantity of
particulate fibrinogen added to the bandage is generally in the
range of from about 1 to about 3 grams per bandage, and usually
from about 1.5 to about 2 grams per bandage. For thrombin, the
quantity may be from about 100-10,000 units per bandage, and is
typically from about 4000-6000 units per bandage.
[0059] In some embodiments, the therapeutic agents may themselves
be electrospun, either with the dextran (i.e. they are dissolved in
and spun from the same solution as the dextran) or separately (they
are dissolved in and spun from a separate solution that does not
include dextran). In some embodiments, the agents may be
electrospun into fibers, as is the case for dextran. In other
embodiments, the active agents may be electrospun into other forms
such as droplets, beads, etc. In some applications, active agents
such as thrombin may be electrosprayed with sucrose to form sugar
droplets, which tends to stabilize thrombin and can also "trap"
other substances of interest for delivery to the bandage.
[0060] In particular, for thrombin and fibrinogen, in most
embodiments, these (or other) agents are associated with or added
to electrospun dextran fibers in a finely dispersed dry,
particulate or granular form e.g. as a fine powder or dust, as
electrospinning may tend to decrease their activity. In other
words, the agents are not electrospun either by themselves or with
the dextran. The provision of the substances in the form of a fine
powder provides a large surface area of contact for dissolution
when the materials come into contact with fluid. Generally, such
particles will have average diameters in the range of from about 1
to about 10,000 microns, and usually from about 10 to about 1000
microns. Such dry solid particles may be formed by any of several
means, including but not limited to grinding, pulverizing,
crushing, etc. However, those of skill in the art will recognize
that other forms of these agents may also be included in the
bandage, e.g. flakes, films, sheets, strings, etc. Further, in some
embodiments, thrombin and fibrinogen are in the form of electrospun
droplets when associated with the EDFs.
[0061] Association of substances of interest with the EDFs may be
accomplished by any of many suitable techniques that are known to
those of skill in the art, and will depend in part on the precise
form of the substance and the means at hand. For example, for
powdered, particulate thrombin and fibrinogen, association may be
carried out by sprinkling, shaking, blowing, etc. the agents onto a
layer of EDFs. Depending on the density of the fiber mat, the
substances of interest may become relatively evenly dispersed
throughout the woven mat of fibers or may be largely confined to
the topmost section of the fiber mat. If no backing is present, the
latter embodiment is preferable, to prevent the particulate
substance of interest from falling through and out of the mat. The
density of the fibrous mat can be adjusted (e.g. increased), for
example, by adjusting its thickness and/or by compressing the mat
under pressure so that the fibers are closer together. Other
techniques for association also exist, e.g. the placement of dry
but liquid soluble sheets or strips of material onto or between
layers of electrospun dextran, electrospinning the added materials
as a discrete layer or in discrete layers, etc., and any such
technique may be employed. The techniques for assembling the
devices of the invention may be carried out manually or may be
mechanized, or a combination of manual manipulation and
mechanization may be used. For thrombin in particular, 5000 units
of thrombin is a very small volume of powder. Therefore, inert
carriers or bulking agents such as dextrose may be added to insure
more complete dispersal of active agents in the bandage.
[0062] The association of substances of interest with the EDFs may
be carried out according to many different arrangements. For
example, a first layer of EDFs may be formed, and one or more of
the substances may be associated with the first layer. Then another
second layer of EDFs may be formed on top of the substance(s) of
interest, and the same or other substances of interest may be
associated with the second layer, and so on. A final or outermost
layer of EDFs may be added to prevent the dislodgement of
substances of interest from the layer(s) below. The number of
layers of EDFs that are used in a device of the invention may vary
widely, from as few as 1-2 to as many as several dozen, or even
several hundred, depending on the desired characteristics of the
device. Typically, a device will contain from about 1-2 and up to
about 5-10 layers. The very slight amount of moisture that is
present in a prepared bandage may help to trap and retain particles
of material on the surface of the bandage.
[0063] In some embodiments of the invention, the EDF devices also
include one or more support structures or support materials
incorporated therein. For example, a backing may be incorporated
into the device. The support material may be formed from various
electrospun materials such as polyglycolic acid (PGA), polylactic
acid (PLA), and their copolymers (PLGAs); charged nylon, etc. In
one embodiment, the support material is compressed electrospun
dextran fibers. By "compressed EDFs" we mean that EDFs are
compressed together under pressure. Compression of EDFs is carried
out, for example, under pressure between two plates (e.g. a vice),
and can compress a mat of fibers with a height (thickness) of about
3 inches to a sheet with a height of about 0.5 inches or even less
(e.g. about 0.1 to about 0.4 inches). In some embodiments, the EDFs
are electrospun directly onto a previously electrospun support
material, while in other embodiments, the support material and the
EDFs are associated after electrospinning of each, e.g. by joining
of one or more layers of each.
[0064] In other embodiments, the support material is not an
electrospun material but is some other (usually lightweight)
material on which EDFs can be formed, or associated with after
electrospinning Examples of such materials include but are not
limited to gauze; various plastics; hydrogels and other absorbent
materials that can facilitate absorption of blood and therefore
clot formation; etc.
[0065] The support material may or may not be soluble in liquid, or
may be slowly soluble in liquid, and may or may not be permeable to
liquid. Slowly soluble materials include those from which
absorbable or dissolving (biodegradable) stitches or sutures are
formed, included PGA, polylactic and caprolactone polymers. Such
support materials typically dissolve within from about 10 days to 8
weeks, depending on the material that is used, and provide the
advantage of, in some cases, not having to remove the bandage and
risk disrupting the clot. However, in any case, the support
material should not interfere with the immediate dissolution of the
EDFs and delivery of the active agents associated therewith into
the liquid that dissolves the EDFs. Thus, the support material
might be only on one side of the EDF device, so that when the
device is, for example, a bandage, and is applied to a wound, the
bandage is oriented so that the EDFs come into direct contact with
the blood in the wound bed and the support material does not, i.e.
the support material is the "top" or outermost surface of the
bandage when placed on the wound. This embodiment is illustrated,
for example, in FIG. 4A, in which EDFs 10 are shown as deposited
onto non-porous, liquid impermeable support material 20. When
applied to a wound, EDFs 10 would face downward into the wound, and
non-porous support material 20 would face away from the wound. This
arrangement could provide an advantage in that pressure could be
applied to the wound through the support material, to facilitate
the stoppage of bleeding. Alternatively, the support material may
contain pores, openings or spaces that allow liquid to access the
EDFs of the device even when the support material is present. For
example, the support material may be a net or web of material that
is insoluble (or slowly soluble) but that permits liquid to freely
access the EDFs and associated substances of interest. This
embodiment is illustrated schematically in FIG. 4B, which shows
EDFs 10 deposited on (or possibly under, or on and under, or woven
throughout) netting 40, which is shown partially in phantom where
covered by EDFs 10. In yet other embodiments, both a "backing" or
"top" support material and a second web-like support material may
be present in the devise. This embodiment is illustrated
schematically in FIG. 4C, which shows EDFs 10 deposited on
non-porous support material 50 and overlaid with net-like material
60, i.e. EDFs 10 are "sandwiched" between non-porous support
material 50 and net-like material 60.
[0066] One of skill in the art will be able to envision many other
combinations and shapes of EDF layers and support materials that
would provide advantages in particular scenarios. For example, EDFs
might be wrapped or wound around an elongated support such as a
filament or string, or wrapped around a particular form with the
shape of a cavity in which the device is likely to be placed, such
as a bullet hole, etc. The crux of the problem at the site of a
penetrating injury is that the wounded tissue is relatively
inaccessible. For example, for a bullet wound (e.g. in the leg or
thigh) bleeding does not occur as much at the surface but deeper
within the tissue, within a cavity formed by the bullet, where it
can not be easily treated by a bandage that is simply spread over
the external site of the injury (e.g. the point of entry of the
bullet, knife, shrapnel, sword, bayonet, etc., or other cause of
injury). This aspect of the invention solves the problems
associated with penetrating injuries, which can cause extensive
bleeding in the deep tissues, and takes advantage of the highly
soluble nature of the dextran bandage. A complicating factor in
this type of injury concerns the ability to deliver hemostatic
materials that are highly soluble to such a site. There may be
bleeding and other fluids evident at the entry site of the wound
and the application of a bandage to this superficial site may
result in the complete dissolution of the bandage at the
surface-without the delivery of the active materials to the
underlying source of the bleeding within the wound cavity. The
invention circumvents this occurrence by providing delivery of
active agents deep into the wound. Prior art bandages have failed
to adequately address this problem.
[0067] The present invention solves this problem by providing a
device, the shape and application of which can be adapted to use
with such wounds. For example, an elongated cylindrical
"cigar-shaped" device comprised of spun dextran as described
herein, and which also contains particulate thrombin or fibrinogen
or both, and which may contain support material, is provided. The
device is stored within a protective covering or packaging or tube.
This tube protects the bandage (device) from the ambient
environment. Both the bandage and the tube are preferably sterile,
and may be, for example, optionally further enclosed in an outer
wrapper of e.g. paper, polymer, blister pack, similar to that used
for disposable syringes, to prevent loss of sterility. When used,
the outer wrapping is torn open and the sterile tube containing the
bandage is accessed. In some embodiments, one end of the tube is
removed and placed over the outermost accessible portion of the
injury. The tube may also comprise a "plunger" or similar means
which enables the user to expel the bandage from the tube and into
the wound, in effect "injecting" the bandage into the wound. Means
such as those that are used for the vaginal delivery of, for
example, tampons, (i.e. a "cylinder within a cylinder") may be
employed, or a syringe-like means of delivery may be used. The
device can thus be introduced deep into the tissue along the wound
track and the therapeutic agents in the device are delivered to
where they are most needed, i.e. to the interior of the wound. In
other embodiments, a plunger per se is not included, but the tube
is fashioned so that both ends can be opened, and the spun dextran
device can be pushed into the wound from one open end by exerting
pressure on the opposite open end of the tube using any object that
fits at least partially into the tube, sufficiently to push the
device out of the tube and into the wound, e.g. a finger, stick,
etc. Such an object may be included with the device of the
invention. Those of skill in the art will recognize that, due to
the relatively high malleability of spun dextran, this embodiment
of the device may include support material around or within the
spun dextran (e.g. biologically compatible netting, rod, etc. that
will disintegrate via biodegradation) in order to render the device
more robust and less flexible as it is shunted down into the wound.
Further, the outermost end of the device, that end on which
pressure is exerted (e.g. with a plunger) in order to expel the
device from the tube into the wound, may be reinforced with support
material so that the plunger or other object used to push on the
device can deliver sufficient force to remove the device from the
tube.
[0068] An exemplary schematic depiction of this embodiment of the
invention is provided in FIG. 4D, where device 100, comprised of
spun dextran fibers 110 and (optional) support material 120, and
having a first end 130 and second end 140 is illustrated as
enclosed within tube 200. Device 100 is enclosed within tube 200
but is not shown in phantom for the sake of clarity. Tube 200 has
openings 210 and 220, both of which may be capped prior to use
(caps not shown) or may be left open, especially if the entire
apparatus is packaged in sterile packaging 400. Sterile packaging
400 is removed or breached to provide access the apparatus prior to
use. In order to use the apparatus, openings 210 and 220 of the
tube must be open. To deliver device 10 to a penetrating wound, an
object such as plunger 300 in inserted into end 210 of the tube.
Pressure is exerted on device 100 as plunger 300 contacts device
end 130, and device 100 is consequently pushed out of tube 200 via
opening 220 (in the direction indicated by the arrows) and into the
penetrating wound (not shown). A second schematic representation of
such a device is provided in FIG. 4E. In this depiction, support
material is not included and the dry, sterile bandage material
(e.g. dextran fibers) with associated therapeutic agents is located
or positioned within a small, sealed cylinder with a cap at one end
and a plunger at the other. Upon deployment, the cap is discarded,
the open end of the cylinder is placed over the mouth of the wound
and may be inserted into the wound, and the plunger is depressed,
displacing or injecting the bandage material deeply into the wound.
Similar designs may be used to deliver the device of the invention
to orifices or channels such as the nasal passages, the ear canal,
the vagina, the anus, into blood vessels, etc. The dextran fibers
that are used in such an application will be formed into a device
that is on the order of about 1 to about 6 inches in length, and
from about 1/4inch to 1 inch in diameter, i.e. the dimensions will
be suitable for insertion through the external opening and deep
into an orifice or a wound cavity.
[0069] All such arrangements, shapes, and embodiments of EDF layers
and support materials as described herein are intended to be
encompassed by the invention.
[0070] The devices of the invention may be sterilized prior to use,
generally by using electromagnetic radiation, for example, X-rays,
gamma rays, ultraviolet light, etc. If thrombin is included in the
device, the moisture content of the device (e.g. a bandage) should
be reduced to 5% or less, in order to preserve thrombin activity
during sterilization. This can be achieved by drying the fabricated
bandage, e.g., under a vacuum, or by using a fabrication method
that reduces moisture content from the beginning Typically, the EDF
devices of the invention are sterilized using X-rays in a dose of
about 5 kilograys (kGray). Any method that does not destroy the
dextran fibers or the activity of substances associated with the
fibers, may be used to sterilize the devices of the invention.
[0071] When the device of the invention is a bandage, the
substances of interest that are associated with the fibers of the
bandage may include thrombin and fibrinogen, and the bandage may be
used to staunch bleeding. However, the range of active ingredients
may vary with the specific application of the bandage. For example,
bandages comprised of only dextran or only thrombin might be used
for small injuries or in combination with other interventions. In
addition, other therapeutically beneficial substances may also be
associated with the bandage, including but not limited to:
antibiotics, medicaments that alleviate pain, growth factors,
vasoactive materials (e.g. substances that cause vasospasms),
steroids to reduce inflammation, etc. In other embodiments, the
devices of the invention need not comprise agents that promote
clotting at all. Those of skill in the art will recognize that the
devices of the invention are highly suitable for delivering many
substances of interest to a desired liquid environment or location.
For example, the devices may be designed for delivery of
therapeutic or beneficial substances to any moist environment of
the body, where there is sufficient liquid to dissolve the EDFs and
release the active substance, and where dissolved dextran is not
problematic. Examples include but are not limited to oral, nasal,
tracheal, anal, lung, and vaginal delivery of substances such as
anti-microbial agents, analgesic agents, nutritional agents, etc.
Oral applications include the delivery of substances useful for
dental treatments, e.g. antibiotics, pain medications, whitening
agents, etc. Further, the devices of the invention may be ingested
to provide a quick release into the gastrointestinal tract of
substances such as nutritional supplements (vitamins, amino acids,
sugars, etc.). At the site of delivery, usually a bodily fluid is
or will be present, and the dextran fibers dissolve in the fluid,
thereby releasing or delivering the associated active agents or
interest to the site. Such bodily fluids include fluids that are
excreted or secreted from the body as well as fluids that normally
are not, examples of which include but are not limited to blood,
sweat, tears, mucus (including nasal drainage and phlegm), pleural
fluid, pus or other wound exudates, saliva, vaginal secretions, and
the like. However, in some embodiments, no bodily fluid is present
(or if insufficient body fluid is present) and the applied device
can be "activated" by wetting, e.g. by spraying, or by otherwise
applying a source of moisture (e.g. by exposing the device to a
moist material such as a sponge), or dropping devices into a liquid
(e.g. a body of water), in order to cause release of the agents of
interest associated with the dextran fibers. This embodiment of the
invention may be especially useful, for example, for the release of
materials at a site of interest, when activation is desired to
occur only upon exposure to liquid. Examples include but are not
limited to: delivering nutrients, fertilizers, insecticides, etc.
to plants or grass using sheets of dextran fibers; the application
of cosmetics; etc. The agents of interest may be relatively
innocuous (benign) materials for which maintenance or storage in a
dry state until use is desired; or they may be dangerous or toxic
materials which must be kept sequestered and in a dry state until
use. The devices of the invention are thus not limited to
therapeutic treatments. Any substance of interest that can be dried
sufficiently so as not to dissolve the EDFs prematurely (before
placement at a location of interest) and which retain activity upon
rehydration when delivered to a location of interest, may be
delivered by the methods and devices of the present invention.
[0072] Due to the small footprint and light-weight characteristics
of the devices of the invention, they are ideal for situations
where space and weight of supplies are at a premium. Examples of
such situations include but are not limited to: military operations
where the weight and size of the components of a soldier's gear are
an issue; in first aid kits; for emergency care during travel (e.g.
during space flight, camping, etc.); etc. However, this need not
always be the case. The devices may be used in a variety of
situations and for a variety of purposes in which space and weight
are not considerations. For example, the bandages of the invention
provide a convenient means to administer thrombin and fibrinogen to
surgical wounds in a conventional operating theater. The devices of
the invention may also be advantageously utilized whenever it is
desired to package and eventually release one or more dried
substances, but where it is unfeasible or undesirable to handle the
dried substances directly, e.g. where the quantity is extremely
small, or the substance is toxic. In such cases, the EDF devices of
the invention may serve as a "scaffolding" or carrier for
containing, storing and/or transporting the substance(s) until use,
i.e. until contacted with liquid that dissolves the EDFs,
concomitantly releasing the substances into the liquid. Such
substances may include, for example, enzymes or their precursors
(e.g. pro-enzymes or zymogens) and their substrates, substances
that activate a protein or enzyme (e.g. proteases, cofactors,
etc.), and the like.
EXAMPLES
Example 1
Bandage Fabrication
[0073] Approximately 5 g of dextran (100,000-200,000 Mr) was used
to produce a bandage that has a total weight of about 7 g after the
addition of 2 gms of particulate fibrinogen. Approximately 5 g of
dextran was suspended in distilled water at a concentration of 1.0
gm/ml. This solution was incubated overnight on a clinical rotator
set to rotate 1-5 rotations per minute. These conditions were used
to insure that the dextran is completely in solution. The dextran
solution was loaded into a syringe capped with a blunt tipped
needle, and approximately 5 mls per bandage was used in the
fabrication process. For electrospinning, the syringe was mounted
into a syringe driver calibrated to deliver 3-5 mls/hour to the
electric field. The syringe driver is used to meter the delivery of
material to the electric field and this setting does not
necessarily accurately reflect the rate of fabrication as the
electric field draws the dextran from the syringe.
[0074] Salmon fibrinogen was supplied by Sea Run Holdings (Evelyn
Sawyer, Freeport Me.) and 1.5-2.0 g of fibrinogen was used per
bandage. Salmon thrombin was supplied by Sea Run Holdings (Evelyn
Sawyer, Freeport Me.) and 4500 units of thrombin activity was used
per bandage. Salmon fibrinogen is supplied by Sea Run Holdings as a
dry powder. The raw material was removed and processed into a very
fine powder with a mortar and pestle. Approximately 1.5-2.0 gm of
fibrinogen is used per bandage. Salmon thrombin is provided by Sea
Run Holdings in a similar physical state (a dry powder). The
thrombin was added to the fibrinogen and processed into a fine
powder. The well blended, dry fibrinogen and dry thrombin mixture
were placed into a salt shaker or other sifting device.
[0075] For electrospinning, two separate, high voltage power
supplies were used to process the dextran into a fibrous matrix.
The positive pole of one power source was attached to the syringe
reservoir containing the dextran using an alligator clip. The
negative pole of the second power source was attached to the ground
target. A flat sheet of steel about 5.times.5 inches is typically
used as a target. A piece of paper was placed over the target, and
1 micron diameter fibers of dextran accumulated on the paper during
electroprocessing. The final product was easily removed from the
paper. One of skill in the art of electrospinning will recognize
that it is possible to switch the polarity of the power
supplies.
[0076] For electrospinning, the power supplies were set to
approximately +20-25 kV (kilovolts) on the syringe containing the
dextran solution and approximately -20-25 kV on the ground target.
Electrospinning was conducted across an air gap that can vary from
12 in to 24 inches, and the syringe driver was set to deliver the
dextran to the electric field at a rate of 3-5 mls/hr. Spinning was
initiated and a suitable volume of material was collected on the
ground target, perhaps 1 ml (or about 1 gm) of dextran was
delivered to the target where it collected as a small flattened
disk of material. The electric field was terminated at the ground
target and an aliquot of fibrinogen and thrombin was shaken onto
the forming dextran fibers.
[0077] Electrospinning was re-initiated until the
fibrinogen/thrombin mixture was covered with fresh dextran fibers.
The process was repeated until the bandage was completed. The
delivery of small volumes/amounts of fibrinogen in frequent small
aliquots of material was used to distribute the active ingredients
throughout the bandage. It is also possible to layer the fibrinogen
and thrombin into the forming bandage separately so as to separate
the active components by a layer of dextran. The final product
looks and feels very much like a cotton ball.
Example 2
Scaling of Electrospinning for Manufacturing
[0078] Electrospinning is a method of producing a fibrous matrix
composed of small diameter, nano-to-micron scale fibers. This
process can be scaled for manufacturing by at least 3 different
methods:
[0079] Multiple Jets. The use of multiple jets represents a simple
solution to scaling the manufacturing process. As depicted in FIG.
1, it is possible to use multiple jets to increase the throughput
of electrospinning. This can be done with dextran, however, the
humidity level of the processing environment must be controlled;
excess moisture limits the volume of material that can be collected
and small drops of overspray can cause the dextran to dissolve. The
level of humidity should be controlled to be from about 30% to
about 40%, and typically at least about 20%. In one embodiment,
multiple jets are arrayed above a conveyor belt. Each jet delivers
a set amount of dextran, the process stops, the belt may move
forward to allow a dose of e.g. fibrinogen and/or thrombin to be
added to each pile of dextran, the belt moves again, and
electrospinning is re-initiated.
[0080] Electroaerosol. Dextran can be electro-aerosoled under
pressure from a paint sprayer and or an air brush. Dextran and gas
are delivered under pressure to the air brush. At the terminal
portion of the air brush, the dextran solution is pushed into the
air stream across a needle, forming a jet of material that is blown
into the electric field. The process is driven largely by the
pressure applied to the gas used to drive the reaction. This
technique is very rapid, requiring approximately 10 minutes to
produce a bandage-like volume of material. This is thus a very
effective method of producing large amounts of material very
quickly. If a room temperature air stream is used in the process,
the tip of the electrospinning jet may dry and clog the spraying
device. This can be managed by controlling the moisture content in
the gas used to drive the aerosol. An electric field is not
absolutely needed to form fibers when this method is used to
process dextran. Fibers will form in the aerosol jet, and the jet
appears to be dry enough to allow large amounts of material to
collect quickly. The electric field does, however, aid in targeting
the jet to a specific target. For example, a conveyor belt might be
used to collect sheets of fibrous dextran. By charging the solution
and the belt, the dextran can be effectively targeted to the
desired collecting site. The ground target can be placed, for
example, behind or under a conveyer belt. The ground does not have
to be in direct contact with the dextran. Multiple jets could also
be employed in this approach. By running multiple jets of aerosol
onto a moving conveyer belt that is interspersed with "sifters" (to
deliver the fibrinogen and thrombin), the powdered materials could
be added sequentially in discreet layers to the forming bandage.
Electrostatic Processing. Fabrication of dextran into fibers may be
carried out using a "cotton candy machine". A cotton candy machine
represents a cross between a true electrospinning device and an
aerosol-like device. Moisture content in the forming fibers can be
controlled by controlling the input temperature (and volume) of the
air delivered to the fabrication chamber where the fibers are
formed. An added advantage is that simple sugars can be processed
in to fibers using this type of instrument.
Example 3
Electrospun Dextran for Use as a Backing
[0081] PGA and PLA (2 synthetic, biocompatible polymers) are used
to provide strength to the backing material. Fibrinogen is added to
the PGA/PLA mixture and clearly represents a protein element of the
composition. A 90:10 mixture of hexafluoroisopropanol (HFP) is
added to aqueous (water, culture media, PBS, etc.) solvent
saturated with calcium chloride (the calcium helps to promote the
clotting cascade, or other salt) solution is prepared. The PGA/PLA
polymers and fibrinogen are added to this solution and allowed to
go into solution. Typical conditions are 100 mgs of PGA/PLA mixture
(ranging from 99:1.0 PGA/PLA to 1.0:99 PGA/PLA ratio), 10-50 mgs
fibrinogen per ml of electrospinning solution. Once in solution,
the composition is electrospun to form a fibrous mat of material.
The synthetic polymers provide strength; the fibrinogen component
provides cross-linkable sites for active ingredients to interact
with during clot formation. Ideally the clot will form at the
injury site as the active ingredients are released from the
bandage, the clot will adhere to the wound bed, surrounding tissue
and the backing of the bandage. The backing provides the structural
support necessary to stabilize the wound bed. The backing and
remnants of the bandage and clot may be surgically removed once the
patient is stable and or transported.
Example 4
Determination of Immune and Inflammatory Response to Salmon
Thrombin and Fibrinogen
[0082] The goal of experiments carried out in this Example was to
determine if salmon thrombin and fibrinogen would cause an adverse
immune and inflammatory response and to examine the cellular basis
for that response. We assessed the production of antibodies to the
salmon components and determined if the coagulation activity of the
swine was altered. We examined the histopathology to characterize
the tissue response to salmon dressings in swine after excisional
cutaneous surgery that created wounds with separated edges and
found a lymphocyte response that included cellular proliferation
and cytokine secretion. However, healing occurred normally and
there were no signs of adverse immunological reactions to the
dressings at the wound site.
Methods
Purification of Salmon Fibrinogen and Thrombin
[0083] Salmon proteins were purified from salmon blood as
previously described. [13] Briefly, salmon blood was drawn from 2-5
kg salmon and centrifuged to obtain plasma. The plasma was made 10
mM with benzamidine, 2 g/dL CaPO4 and 3 g/dL epsilon-aminocaproic
acid. The plasma was passed over a gelatin-sepharose column to
remove fibronectin. The fibrinogen was salt precipitated twice with
ammonium sulfate in a method modified from Mosher and Blout (Mosher
D F, Blout E R. J Biol Chem. 1973 Oct. 10; 248(19):6896-903).
Salmon thrombin was purified from the CaPO4 pellet by the method of
Michaud et al. (Michaud S E, Wang L Z, Korde N, Bucki R, Randhawa P
K, Pastore J J, et al. Thromb Res. 2002 Sep. 1; 107(5):245-54). The
pellet was dialyzed for 5 h against 20 mM Tris/HCl, pH 7.5, 0.15M
NaCl, 1 mM EGTA and 0.1M EDTA and then overnight against the same
buffer without EDTA. The resulting protein solution was then
subjected to two rounds of ammonium sulfate precipitation, first
with 35% and the second time with 70% ammonium sulfate. The
resulting pellet containing prothrombin was dialyzed against 20 mM
Tris/HCl 1 mM EGTA for 5 h and then against 20 mM Tris/HCl 1 mM
EGTA and 0.1 M NaCl. Finally, the solution was centrifuged at
12,000.times.g to remove contaminating particles. Fibrinogen was
used at a concentration of 19.4 mg/cm.sup.2 (2000 mg total) and
thrombin was used at a concentration of 50 U/cm.sup.2 (5200 U
total).
Electrophoresis, Western Blotting and ELISA
[0084] Immunological reactivity was determined by Western blotting
and ELISA. For electrophoresis, proteins were dissolved in 4.times.
sample buffer (Invitrogen Corp., Carlsbad, Calif.), heated to
80.degree. C. and separated for 45 minutes at 200V on Invitrogen
NuPAGE 4-12% Tris-Bis gels. Proteins were transferred from the gels
to PVDF membranes and the membranes were blocked in Tris-saline, 5%
dry milk for 1 hour. Following blocking, the membranes were
incubated with porcine serum diluted 1/10 in Tris-saline buffer,
with 4% bovine serum albumin. Antibody reaction was visualized
after incubation with secondary anti-swine horse radish
peroxide-conjugated antibody (HRP-swAB) and treatment with
Millipore Chemoluminescence reagent kit.
[0085] ELISAs were performed with thrombin or fibrinogen as the
substrate. Immunolon B1 plates were coated with 1 .mu.g
protein/well, the wells were blocked and then incubated with
porcine serum at 1/10 dilution. Titration curves were performed at
dilutions up to 1/5000. Antibody binding to salmon proteins was
quantified by incubation with horse radish peroxidase (HRP)-swAB
and Millipore substrate and read at OD450 with a Molecular Devices
plate reader.
[0086] Cytokine levels for IL1, IL2, IL4, IL6, IL8, IL10, IL12p40,
IFN.gamma. and TNF.alpha. were assayed by a commercial service,
Searchlight Cytokine Custom Multiplex Arrays, (Pierce
Biotechnology, Inc., Rockford, Ill.). The Protoarray Human Protein
Microarray, a 5000 protein array from Invitrogen Corp., was
analyzed to screen the serum from five animals for the presence of
anti-human antibodies generated following exposure of the swine to
the salmon proteins This assay would detect antibodies recognizing
proteins that are not included in the normal coagulation pathway
and, therefore, may not be detected by our standard assays. Serum
from blood taken at the time of surgery to implant the vascular
access ports (VAP) was compared to serum taken at euthanization of
the animals after the exposure to salmon proteins.
Surgical Preparation of Animals
[0087] Female Yorkshire swine (Sus scrofa Domestica) (25-28 kg)
were prepared for surgery and monitored during the procedure as
described previously. A vascular access port (VAP) catheter line
(Access Technologies, Skokie, Ill.) was inserted into the jugular
vein using a modified Seldinger technique (Knebel P, Frohlich B,
Knaebel H P, Kienle P, Luntz S, Buehler M W, et al. Trials. 2006;
7:20) to permit blood sampling. A 16-18-gauge, 2.5-3'' introducer
needle was inserted into the vein percutaneously, followed by a
j-wire. An expander catheter was fed onto j-wire through the skin
and into the jugular. The expander catheter was removed with the
j-wire remaining in the jugular vein and a central venous catheter
was fed onto the j-wire into jugular vein. The catheter was secured
to subcutaneous tissues in a simple interrupted pattern with 3-0
PDS and the catheter was flushed with a citrate anticoagulant
solution to verify placement into vein and to create a citrate
lock. The catheter was then attached to the port, which was buried
in a subcutaneous pocket on the shoulder.
[0088] Exposure to the salmon proteins was accomplished in several
ways. In the first approach, thrombin and fibrinogen were injected
intravenously through the vascular access ports. In the second
method, paired identical full thickness dermal wounds were
surgically created on the right and left dorsal skin surface,
paramedial to the spinal column in four pigs and monitored for 7
days. A second group of four pigs were subjected to a similar pair
of skin lesions and monitored for 28 days. The total number of
wounds to evaluate in each time point was eight. For animals in the
7 day group, the right dorsal lesion was bandaged with a dressing
composed of lyophilized fibrinogen produced by electrospinning the
protein onto a rotating mandrel (Nanomatrix, Inc, Baton Rouge,
La.). The dressings were applied to a full thickness dermal lesion
approximately 2.times.2 cm. The left dorsal lesion was dressed with
a commercially available, non-hemostatic bandage. For the 28-day
animals, this was reversed and an electrospun dressing containing
fibrinogen (500 mg) and thrombin (400 IU) was applied to the left
side. Animals in the 28-day group were injected with thrombin (60
IU) and fibrinogen (200 .mu.g) on day seven to simulate a
re-exposure to the dressing. At the end of the time period, the
animals in each group were euthanized and the carcass presented for
necropsy.
[0089] For the third exposure method, a midline abdominal incision
was performed and the fibrinogen/thrombin bandage was inserted into
the peritoneal cavity. The incision was sutured and the animal was
recovered. Animals were maintained for two weeks and blood was
drawn for analysis of antibody generation.
Tissue Preparation for Histological Examination
[0090] At necropsy, each pig was placed in lateral recumbency and
the skin defects measured, gross lesions noted and recorded, and
photographs were taken. The tissue of the salmon
fibrinogen/thrombin treated and non-hemostatic bandage treated
lesions, the pre-femoral lymph nodes, mesenteric lymph nodes and
spleen were harvested for histopathology. The tissue samples were
fixed in 10% neutral buffered formalin and then routinely trimmed,
processed, and embedded in paraffin wax for sectioning and staining
Histopathology assessment was performed in a non-blinded fashion
using a light microscope. Evaluation parameters on the skin
sections included comparison of the wound edge with the wound
center of the salmon fibrinogen/thrombin bandage treatment versus
the non-hemostatic bandage treatment for signs of inflammation,
re-epithelialization, granulation tissue, fibrosis, crust formation
and necrosis. Semi-quantitative scoring of the skin samples for
superficial and deep inflammation was performed, based on the
amount of inflammatory response evident on standard H&E stained
tissue sections. The scale was (1) minimal, (2) mild, (3) moderate,
and (4) marked (data not shown). The mesenteric lymph node,
pre-femoral lymph nodes and spleen from each animal were also
evaluated for histopathology.
Statistical Analysis Differences between groups were analyzed using
a two tailed T-test assuming equal variances. Values are expressed
as means.+-.the standard error. N values and p values are included
with each measurement.
Results
Inflammation and Re-Epithelialization of the Skin Lesion in the
7-Day Group
[0091] Paired dermal injuries were produced on the animals and the
injury sites were dressed on one side with the salmon fibrinogen
dressing and on the other with a non-fibrinogen standard dressing.
After 7 days, the animals were euthanized and taken for necropsy
("7-day group"). In the center of the wound, all four pigs on both
the left and right sides, exhibited a leading edge of epithelial
cells and superficial wound filling by a coagulum composed of
necrotic cellular debris, neutrophils, fibrin, hemorrhage, and
edema.
[0092] Inflammatory cells, granulation tissue and edema expanded
the superficial dermis on both the left and right sides subjacent
to this fibrinonecrotic coagulum. Histologically, this granulation
tissue was composed of many small caliber blood vessels lined by
hypertrophied endothelium and oriented perpendicular to the skin
surface. The edema separated the dermal collagen bundles and
fibroblasts. Evidence of fibroplasia, characterized by numerous
plump, activated fibroblasts with deposition of abundant collagen,
extended from the junction of the dermis deep to the panniculus
adiposus. This fibroplasia was moderate in severity with a
multifocal to diffuse distribution in all four pigs on both the
right and left sides.
[0093] Superficial inflammation of all eight wounds was moderate to
marked in severity and composed of primarily neutrophils, fewer
macrophages and rare multinucleate inflammatory giant cells
subjacent to the fibrinonecrotic scab. Deep inflammation was marked
in one salmon fibrinogen/thrombin treated lesion, and mild to
moderate in the remaining right side lesions. Deep inflammation in
the non-hemostatic treated lesions was marked in two cases and mild
to moderate in two cases.
[0094] An attempt at re-epithelialization on the wound edges was
evident in all eight wounds at the 7-day time point. Typical
findings at these margins included epidermal hyperplasia,
acanthosis, spongiosis, deep rete ridges and dermal pegs,
parakeratotic hyperkeratosis and projections of regenerative
epithelial cells toward the wound center.
[0095] To summarize the 7-day group, all wounds were filled with a
fibrinonecrotic coagulum. Each wound exhibited superficial
granulation tissue in the dermis on both the treated and untreated
sides. There were numerous neutrophils, fewer macrophages and
multifocal hemorrhage. Inflammation variably extended deep into the
subcutis and was composed of lymphocytes, plasma cells and
macrophages. Re-epithelialization at the margins and moderate
fibroplasia was evident in all eight wounds. It is noted that the
addition of other active materials such as growth factors or
collagen, as described herein, may be of help in reducing
inflammation and promotion healing.
Inflammation and Re-Epithelialization of the Skin Lesion in the
28-Day Group
[0096] To fully investigate the healing process, the dermal
injuries were repeated in a second set of animals and the course of
healing was followed for at least 28 days ("28-day group"). In this
28-day group, seven of eight wounds exhibited complete
re-epithelialization that was characterized by epidermal
hyperplasia and hyperkeratosis and multifocally there was a
superficial clot similar in cellular composition to the 7-day
group. In these seven wounds, the superficial inflammation was
minimal to mild. One non-hemostatic bandage treated wound in the
28-day group displayed incomplete re-epithelialization and the
wound defect was filled by a fibrinonecrotic coagulum along with
marked superficial inflammation. The wound edge in this case
exhibited similar epithelial cell hyperplastic changes as the 7-day
group. All eight wounds exhibited mild amounts of dermal
granulation tissue and deep inflammation that was composed of
perivascular lymphocytes and macrophages. Fibroplasia and collagen
deposition was brisk in comparison to the 7-day group. This change
extended from the junction of the dermis to the subcutaneous fat in
all eight wounds.
[0097] Superficial inflammation was minimal to mild in the four
non-hemostatic bandage treated lesions and generally composed of
few lymphocytes, plasma cells and macrophages. Superficial
inflammation in the salmon fibrinogen/thrombin bandage treated side
varied from minimal in one case, mild in two cases and marked in
one case. The minimal to mild cellular infiltrate was similar to
the non-hemostatic bandage treated wounds. However, the one marked
case of inflammation in one wound on the salmon fibrinogen/thrombin
bandage side was composed of numerous neutrophils with fewer dermal
macrophages, lymphocytes, plasma cells and eosinophils. Neutrophils
rarely formed intra-epidermal pustules. Additionally, there was
hemorrhage, fibrin and edema with necrosis in this wound.
[0098] Deep inflammation in the non-hemostatic bandage treatment
varied from minimal to mild in three cases and moderate in one
case. This subacute inflammation was predominantly clustered around
vessels. In the salmon fibrinogen/thrombin treatment, deep
inflammation was minimal in two cases and moderate in two cases;
subacute and primarily perivascular.
[0099] In summary, the 28-day group exhibited complete
re-epithelialization in seven wounds, which was covered by a
fibrinonecrotic scab. Granulation tissue was evident in the
superficial dermis. The inflammation was primarily composed of
mononuclear cells. Fibroplasia was abundant. One wound treated with
the salmon fibrinogen/thrombin dressing exhibited similar
histopathology lesions as the 7-day group, including incomplete
re-epithelialization and marked inflammation.
Immune Organ Involvement
[0100] The lymph nodes and the spleen were histologically examined
for signs of activation, including lymphoid follicle formation and
lymphocytolysis. The mesenteric lymph nodes and spleen were found
to be similar histologically among the four pigs in each time
point. The amount of white pulp (lymphoid tissue containing T and B
lymphocytes) contained in the spleen increased slightly in the
28-day samples compared to the 7-day samples.
[0101] Although mesenteric lymph nodes showed little difference
between the 7-day and 28-day groups, the pre-femoral lymph nodes
that drain the ipsilateral area of the skin wound did display
differences when examined at the 7-day time point. The node
draining the non-hemostatic bandage wound generally exhibited fewer
and smaller lymphoid follicles and decreased turnover of lymphoid
cells than the pre-femoral nodes that drained the salmon
fibrinogen/thrombin wound. By the 28-day time point, lymph nodes
from both sides showed equivalent size of germinal centers and
amount of lymphocytolysis.
Systemic Changes in the Immune Status as Determined by Cytokine
Levels
[0102] To determine if the morphological changes observed in the
lymph nodes and the spleen were reflected in the systemic
circulation of immunomodulatory or inflammatory signaling
molecules, levels of a panel of cytokines were measured in the
groups of animals exposed to the dermal wound and then
intravenously infused two weeks later with soluble salmon proteins.
Levels of IL-1.beta., IL-6, TNF-.alpha., IFN-.gamma., IL-4 and
IL-10 are shown in FIG. 5A as the log ratio of the cytokine level
determined in blood drawn at the initial surgery to implant the
vascular port compared to levels following exposure. Responses
varied between individual animals from almost no response to
20-30-fold increases. Changes were seen in both pro-inflammatory
responses (IL-1.beta., IL-6, TNF-.alpha., IFN-.gamma.) and humoral
responses (IL-4 and IL-10). Changes in the cytokines within an
individual animal are shown in FIG. 5B where it can be seen that
initial exposure and the subsequent infusion of proteins elicited a
response that could be detected in samples taken at the next blood
drawn.
Characterization of Antibody Production in Treated Animals Against
Thrombin and Fibrinogen
[0103] Blood drawn from animals (n=24) that had been exposed to
salmon thrombin and fibrinogen was analyzed for the generation of
antibodies using Western blotting and ELISA. Sera of animals
exposed to salmon protein by the dermal protocol and the abdominal
were assessed for the production of antibodies as described in
FIGS. 6A-F. Serum with detectable antibodies (+), undetectable
antibodies (-) and serum not assayed (NA) are listed in Table 1,
where "Abd" is abdomen. If antibodies were detectable as binding to
any fibrinogen subunit or thrombin or prothrombin, that serum was
counted as positive. The majority of animals (94%, see Table 1)
that were exposed to salmon proteins generated antibodies that
recognized salmon fibrinogen (FIG. 6) and 66% of the animals
developed antibodies that reacted with human fibrinogen. In
contrast, thrombin antibodies were low or undetectable after one
exposure but were detectable at low titers (1/10) after a second
exposure (FIG. 6) in some animals (4/24). These antibodies varied
in that some only recognized the prothrombin form while others
recognized the cleaved thrombin molecule. Isotypes of IgM and IgG
were detected for both antigens, but IgA antibodies were not
detected by either Western Blotting or ELISA.
TABLE-US-00001 TABLE 1 Antibody response in swine exposed to salmon
fibrinogen/thrombin dressing as accessed by Western blotting. Salm-
Hu- Salm- Hu- Animal on man Swine on man Swine number Procedure FIB
FIB FIB THR THR THR 12171 skin patch 12172 skin patch + - +* - - -
12173 skin patch + - +* - - - 12174 skin patch + + +* - - - 13085
Abd patch + + +* - + + 13086 Abd patch + + +* - - - 13087 Abd patch
** 13088 Abd patch + + +* + + - 14029 Abd patch .dagger. + + 14031
Abd patch + + - - - 14032 Abd patch 14033 Abd patch 14871 Abd patch
+ - + - - - 14872 Abd patch + - + - - - 14873 Abd patch 14874 Abd
patch - - + - - - 16954 Abd patch + - + - - - 16955 Abd patch + + +
- - - 16956 Abd patch + + + - - - 16957 Abd patch + + + - - - 17218
Abd patch + + + - 17219 Abd patch + + + + + 17220 Abd patch + + + +
+ 17222 Abd patch + + + + + +
[0104] The time course of antibody development was determined by
ELISA on sequential blood samples taken from two series of pigs,
one set subjected to the skin lesion animals and one set that
received the abdominal patch placement. The results were plotted as
optical density vs. blood collection time, starting with blood
collected at the implantation of the VAP until the termination of
the experiment. Animals that were exposed to the bandage via the
skin lesion modality (FIG. 7A-C) displayed slightly different
responses compared to animals that were exposed via the abdominal
placement of the patch (FIG. 8A-D). Animals that were in the
abdominal patch group had very low responses to human fibrinogen
and low responses to both salmon thrombin and human thrombin. The
blots in general proved to be more sensitive in detecting very low
levels of responses to the protein, but the ELISAs enabled us to
easily track the progression of the immune response and antibody
production in each animal.
Identification of Human Cross-Reactive Antibodies in the Serum of
Salmon Protein-Exposed Swine
[0105] The Protoarray Human Protein Microarray service from
Invitrogen Corp. was utilized to screen the serum from five animals
that had been exposed to the salmon proteins for the presence of
antibodies reactive against human proteins generated following
exposure of the swine to the salmon proteins by either the skin and
abdomen placement. These proteins could fall into two categories,
1) proteins that were part of the coagulation cascade and whose
function could possibly be inhibited by interfering antibodies or
2) proteins that have no perceived relationship to the coagulation
process, but may still react with antibodies induced during
exposure. Sera from animals was assayed by Invitrogen for
reactivity to a microarray displaying 5000 proteins to determine if
antibodies were being generated in the treated swine that may
recognize human protein that were not assayed by Western blotting.
As shown in Table 2, the array had 4 proteins that were part of the
coagulation cascade and 5 that were related to transglutaminase
(Factor XIII) activity or were coagulated related. All 5 of the
animals tested had strong initial reactions against the human
transglutaminase 2, but none showed an increase following exposure
and none of the animals had impaired coagulation responses (see
below). Most antibodies to coagulation proteins did not show
significant changes pre- to post exposure in this assay system. A
second group of proteins is also presented in Table 2. These are
proteins that showed significant increases in antibody responses
following exposure of the animals to the salmon proteins, but are
not related to the coagulation process. There were 15
non-coagulation proteins showing increased reactivity to antibodies
following exposure to the salmon proteins that fell into this class
with a p-value threshold (initial vs final value) of <0.05.
TABLE-US-00002 TABLE 2 Screening of human protein-swine antibody
interactions with the Invitrogen ProtoArray 5000 protein
microarray. Signal level (arbitrary units*) Factor Common name
Related ProtoArray Content Pre Post Coagulation or
coagulation-related proteins contained on the ProtoArray 5000 I
Fibrinogen fibrinogen-like 1, transcript variant 1 0.5 0.5 II
Prothrombin serine (or cysteine) proteinase 0.5 0.5 inhibitor,
clade C (antithrombin), member 1 III Tissue factor, tissue
coagulation factor III 0.5 0.5 thromboplastin (thromboplastin,
tissue factor) (F3) VIII Antihemophilic factor A coagulation factor
VIII, 0.5 0.5 (globulin) (AHG) procoagulant component (hemophilia
A) (F8) XIII Protransglutaminase, see below fibrin stabilizing
factor, fibrinoligase Other coagulation related multiple
coagulation factor 0.5 0.5 proteins deficiency 2 (MCFD2) Other
coagulation related tissue factor pathway inhibitor 0.5 0.5
proteins (lipoprotein-associated coagulation inhibitor) Other
coagulation related transglutaminase 2 (C polypeptide, 3-5 3-5
proteins protein-glutamine-gamma- glutamyltransferase), transcript
variant 2 Other coagulation related transglutaminase 4 (prostate)
0.1 0.1 proteins transglutaminase 1 (K polypeptide Other
coagulation related epidermal type I, protein- 0.2 0.1 proteins
glutamine-gamma- glutamyltransferase) (TGM1) Other coagulation
related CTL2114 TRANSGLUTAMINASE- 0.2 0.2 proteins known
Autoantigen ProtoArray Content Pre Post Unrelated proteins
contained on the ProtoArray 5000 showing reactivity SUMO/sentrin
specific protease, 8 0 4 WW domain binding protein 2 1 5 (WBP2)
cellular retinoic acid binding protein 2 1 5 RAB3A interacting
protein-like 1 0 4 nuclear factor I/A 1 5 secretogranin III 1 5
glycine-N-acyltransferase-like 2 0 5 B-cell CLL/lymphoma 7C 1 5
phosphatidylinositol-4-phosphate 5- 0 4 kinase mahogunin, ring
finger 1 1 5 serpin peptidase inhibitor, 6 0 4 ataxin 3 0 4 Casein
kinase 1, gamma 2 1 5 Ribosomal protein S6 kinase 0 4 NIMA-related
kinase 11 1 5
Discussion
[0106] Wound Healing in Animals Treated with Salmon
Fibrinogen/Thrombin Dressings.
[0107] Because of the introduction of foreign proteins derived from
the salmon blood into a wound site, we were concerned that the
wound healing process may be impeded and that coagulopathy may be
induced by initiation of an adverse immune response. To investigate
these possibilities, we treated full thickness skin wounds with
fibrinogen/thrombin dressings, control dressings, and compared the
progress of wound healing and the state of activation of the lymph
nodes and the spleen.
[0108] Excisional cutaneous wounds were surgically created in these
eight pigs, bandaged with two different types of dressings, and
monitored for two time points of seven or twenty-eight days
following surgery. The 7-day and 28-day time points generally
followed the well-established models of cutaneous wound healing by
second intention where there are separated edges and no surgical
opposition. Cutaneous wounds healed by second intention follow a
complex process in closing the defect. These types of wounds
display a robust, localized inflammatory response, form abundant
granulation tissue and have a thin epidermis overlying scar tissue.
As expected, the 28-day group exhibited complete
re-epithelialization in seven out of eight wounds. Over time, these
wounds would likely show some signs of scarring with contracture if
allowed to progress for additional weeks and months.
[0109] A notable histopathology difference at the 7-day time point
was increased activation of pre-femoral lymph nodes on the salmon
fibrinogen/thrombin bandage treated side in contrast to nodes of
the non-hemostatic treated side. This was not unexpected as it may
reflect a greater degree of immune stimulation on the side exposed
to the salmon protein bandages or it may be due to an increased
coagulative response at the time of the initial wound, resulting
from the fibrin/thrombin bandages.
The Immune Response of Swine Following Exposure to Salmon
Fibrinogen/Thrombin Dressings
[0110] Animals exposed to salmon fibrinogen/thrombin were monitored
for health complications and blood was drawn to determine the
immune response to the salmon proteins. The levels of the cytokines
that were measured gave a good representation of the status of the
response. The inflammatory cytokines (IL-1, IL-6, TNF-.alpha. and
INF-.gamma.) often mirrored the surgical manipulations while the
humoral signals (IL-4 and IL-10) increased consistently with
changes in the antibody titers. Our results show that the animals
routinely made immunoglobulins to fibrinogen and, as expected,
these were of the IgM and IgG classes. Furthermore, in most animals
these antibodies recognized all three of the fibrinogen antigens
that were assayed, salmon, swine and human fibrinogen. Thrombin, on
the other hand, did not induce as universal response as fibrinogen
did, with only 6 of the animals generating a thrombin response and
only 4 of them producing antibodies that recognized swine thrombin.
Titers of all of the antibodies were low and the Western blots were
conducted with a high concentration of the serum (1/10 dilution) to
permit visualization of the protein bands. There was some diversity
in the antigens recognized by the antibodies with some of the
antibodies only recognizing prothrombin and not thrombin, even
though the prothrombin was a relatively small percentage of the
sample.
[0111] Measurements of the coagulation parameters were conducted to
determine if the antibodies inhibited the coagulation process, but
all the measured values remained in the normal range. With the
rationale that there may be hidden effects on human coagulation
that was not detected in measurements of swine plasma clotting
times, we mixed swine and human plasma together. In this experiment
as well, we measured normal levels of coagulation. From a gross
assessment of the animals' health as well as the biochemical assays
described above, the antibodies did not seem to cause an adverse
response.
Generation of Inhibitory Coagulation Factors in the Clinical
Settings
[0112] Acquired coagulopathy is a rare but serious disorder that
can arise when a patient generates antibodies that recognize and
interfere with the normal function of components of the coagulation
pathway. Antibodies have been reported to recognize and inhibit a
range of coagulation proteins. Von Willebrand's disease is the most
widely inherited blood disorder (Rodeghiero F, Castaman G, Dini E.
Blood. 1987 February; 69 (2):454-9) and is most commonly due to low
expression levels of functional VWF. However, it can also be caused
by the inappropriate production of antibodies that can interfere
with the von Willebrand polypeptide itself or the protease
ADAMTS-13 (Shelat S G, Smith P, Ai J, Zheng X L. J Thromb Haemost.
2006 August; 4(8):1707-17) While the underlying cause for these
autoantibodies may be due to immune dysfunction in the patient or a
triggering health crisis that precipitates the autoimmune response,
the use of coagulation agents in surgery as hemostatic agents has
also been implicated as the cause of inhibitory antibodies that
processes the polypeptide. Autoantibodies to Factors VIII or IX,
although very rare, can lead to acquired hemophilia syndrome
(Franchini M. Rituximab, Critical reviews in oncology/hematology.
2007 July; 63 (1):47-52). The use of bovine thrombin as a topical
agent in all types of surgical procedures has been widespread with
estimates of over a million uses in 2006. Reports have now
documented adverse effects resulting from the antibodies developed
against thrombin, prothrombin, factor V and cardiolipin following
the use of these hemostatic agents. Many other cases of acquired
coagulopathy are associated with surgery without specific reference
to whether or not bovine thrombin had been used in those
procedures. One approach to combat the autoimmune response has been
to attempt to suppress the immune system and the drug rituximab, a
monoclonal antibody directed at CD20, has proven to be effective in
treating many case. A disadvantage of this treatment is the
increased risk of leukemia and other cancers that occur when the
host defense system is impaired. Clearly, any protein-based
pro-coagulative therapeutic agent will need to prove that the risk
of autoantibody induction and subsequent acquired coagulopathy is
low. Our study has demonstrated that the immune system of pigs,
while able to recognize the salmon proteins and generate
antibodies, has not mounted a response that leads to coagulopathy
and, in the short term, the animals remain healthy.
Example 5
Deployment of an Electrospun Dextran Bandage
[0113] Animals were generally prepped (anesthetized, instrumented
for surgery) as described in Example 4. Briefly, a midline incision
was performed to expose the abdominal aorta. This involves moving
the large and small intestines to one side and cutting through the
peritoneum to isolate the aorta. Aortic injury (4 mm punch) was
performed and bleeding was permitted for 3-4 seconds. Thereafter, a
bandage was applied with pressure for 4 min. Pressure was released
and the injury was checked for hemostasis, and blood loss and
physiological signs were measured. The experiment was terminated at
60 min or if mean arterial pressure fell below 20 mm Hg.
Results
[0114] Prior formulations of lyophilized bandages did not work
well. For example, when thrombin and fibrinogen were lyophilized
into the bandage, the bandage exhibited poor texture and 0/4
animals survived. Lyophilized bandages with added lipids resulted
in 5/7 survival for 60 minutes. When a reduced amount of material
was used to fabricate the bandages, 4/6 animals survived for 60
minutes.
[0115] Dextran bandages were first formulated with organic solvents
and the bandages were very hard in texture, and did not dissolve
well, and most animals (8/10 tested) died. The survival criteria
was increased to 3 hours because we noted that some the animals
listed as "surviving" at 60 min in previous experiments would not
have lasted much longer. Importantly, in vitro tests of thrombin
showed that the enzyme was inactive.
[0116] After bandage formulations were switched to use aqueous
buffers, the bandages and results were much improved. Bandage
texture was soft and pliable and the bandage could be folded and
shaped to fit into the wound site. In vitro measurements of
thrombin and clot strength showed great improvement. Thrombin was
active and caused fibrinogen polymerization. Of the animals tested,
7/9 animals survived. During experiments, blood pressures would
drop to mean arterial pressures of 30-50 mmHg and then recover to
50-70 mmHg. Heart rates typically increase to 160-180 bpm and
eventually drop back to 120 bpm or lower after the wound
stabilizes. Coagulation parameters remained normal over the three
hours.
[0117] The effect of the bandages on the coagulation and healing
process over time have also been examined for three different time
periods: 1 week, 4 weeks and 6 months. These animals were not
"injured" as described above but rather surgically exposed to the
bandages. During each time period, all coagulation values were
normal, all healing was normal (as tested by dermal wounds) and
abdominally implanted bandages were absorbed without adverse
effects. Necropsy showed minimal adhesion at the site of insertion.
All animals made antibodies against salmon fibrinogen. These
antibodies recognize human fibrinogen but not swine fibrinogen
(self antigens). Antibodies were produced at low titers against
salmon thrombin, but did not cross react with either human or swine
thrombin.
[0118] FIGS. 9A-D show that wound healing that proceeds normally
when treated with the bandage. Hematological parameters were
measured to determine if antibodies developed following exposure to
salmon proteins alter normal blood cellular composition or normal
coagulation values. Mean, standard deviation and p values are
presented in tabular form below, in Table 3A and B and Table 4. For
Table 4, human plasma and swine plasma from 2 exposed animals were
mixed and assayed for coagulation parameters to determine if there
may be cryptic factors that could interfere with clotting. As can
be seen, none of the parameters was significantly altered following
exposure to the bandages of the invention.
TABLE-US-00003 TABLE 3A AND B Changes in hematology and coagulation
parameters before and after exposure to the salmon
fibrinogen/thrombin dressing A. White Red Blood Red Blood Cells PLT
(.times.10.sup.9/L) (.times.10.sup.12/L) HCT (%)
(.times.10.sup.9/L) initial final initial final initial final
initial final Mean 19.88 17.30 5.79 5.98 29 30 444 426 n = 30
Standard 3.96 3.10 0.43 0.50 3.23 2.74 89 126 Deviation T-test 0.03
0.24 0.49 0.62 p value B. Activated partial thromboplastin Thrombin
Fibrinogen PT (sec) time (APTT) (sec) time (sec) (mg/dL) initial
final initial final initial final initial final Mean 13.75 14.66
38.46 40.57 23 23 164 155 n = 30 Standard 1.38 3.63 7.82 10.27 4.59
2.81 40.30 40.63 Deviation T-test 0.32 0.47 0.83 0.58 p value
TABLE-US-00004 TABLE 4 Effects of immunized swine plasma on
coagulation of human plasma Activated partial Fibrinogen
thromboplastin Thrombin concen- Prothrombin time (APTT) time
tration Sample time (sec) (sec) (sec) (mg/dL) Swine A alone 13.3
32.2 21.2 137 Human alone 13.0 29.5 16.7 313 Swine A/Human 11.9
26.5 22.2 215 1:1 ratio Swine B/Human 11.8 26.7 21.2 206 1:1 ratio
Swine A/Human 12.2 34.0 23.3 179 7:3 ratio
[0119] While the invention has been described in terms of its
preferred embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims. Accordingly, the present
invention should not be limited to the embodiments as described
above, but should further include all modifications and equivalents
thereof within the spirit and scope of the description provided
herein.
* * * * *