U.S. patent application number 11/824534 was filed with the patent office on 2008-06-19 for adhesive n, o-carboxymethylchitosan coatings which inhibit attachment of substrate-dependent cells and proteins.
Invention is credited to Clive Elson, Timothy D.G. Lee.
Application Number | 20080146521 11/824534 |
Document ID | / |
Family ID | 23224634 |
Filed Date | 2008-06-19 |
United States Patent
Application |
20080146521 |
Kind Code |
A1 |
Elson; Clive ; et
al. |
June 19, 2008 |
Adhesive N, O-carboxymethylchitosan coatings which inhibit
attachment of substrate-dependent cells and proteins
Abstract
The present invention relates to a method of inhibiting cellular
and protein attachment to substrates by applying a composition
containing an effective amount of adherent
N,O-carboxymethylchitosan to a substrate with such that cellular
and protein attachment are prevented or greatly reduced.
Inventors: |
Elson; Clive; (Halifax,
CA) ; Lee; Timothy D.G.; (Halifax, CA) |
Correspondence
Address: |
Edwards Angell Palmer & Dodge LLP
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Family ID: |
23224634 |
Appl. No.: |
11/824534 |
Filed: |
June 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11103354 |
Apr 11, 2005 |
7238678 |
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11824534 |
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10672072 |
Sep 25, 2003 |
6894035 |
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11103354 |
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09315480 |
May 20, 1999 |
6645947 |
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10672072 |
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Current U.S.
Class: |
514/55 |
Current CPC
Class: |
C12N 5/0068 20130101;
C12N 2533/72 20130101; A61L 29/085 20130101; A61L 31/042 20130101;
A61L 27/34 20130101; A61L 29/085 20130101; C08L 5/08 20130101; C08L
5/08 20130101; C08L 5/08 20130101; C08L 5/08 20130101; C08L 5/08
20130101; A61L 31/10 20130101; A61L 31/10 20130101; A61K 31/722
20130101; A61L 31/042 20130101; A61L 27/34 20130101; A61L 24/08
20130101; A61L 24/08 20130101 |
Class at
Publication: |
514/55 |
International
Class: |
A61K 31/722 20060101
A61K031/722 |
Claims
1. A method of inhibiting attachment of substrate-dependent cells
to a substrate comprising applying an adherent
N,O-carboxymethylchitosan coating to said substrate such that
attachment of substrate-dependent cells is inhibited.
2-42. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/315,480, entitled "ADHESIVE
N,O-CARBOXYMETHYLCHITOSAN COATINGS WHICH INHIBIT ATTACHMENT OF
SUBSTRATE-DEPENDENT CELLS AND PROTEINS," filed May 20, 1999, the
disclosure of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The attachment of cells and proteins to substrates is a
well-known problem that has presented itself in a number of
contexts. For example, in cell cultures to produce antibodies,
fibroblasts attach to extracellular matrix proteins bound to the
tissue culture substrate. Similarly, in urinary catheters,
bacterial cells attach to the walls of the catheter; in arterial
catheters, platelets attach to the tip of the catheter; and in
contact lenses, proteins coat the surfaces of the lenses.
[0003] Various bioadhesives are known in the art. U.S. Pat. No.
4,615,697, issued to Robinson et al., defines a bioadhesive as a
material that requires a force of at least about 50 dynes/cm.sup.2
to separate two adhered, freshly excised pieces of rabbit stomach,
following the procedure disclosed therein. The bioadhesive
disclosed in Robinson et al. is a water-swellable, but water
insoluble, fibrous, cross-linked carboxy-functional polymer.
[0004] Various attempts to ameliorate the problem of attachment of
cells and proteins to substrates have been employed, but none have
been found to be satisfactory. It would be desirable to solve this
problem using a biocompatible substance that is adherent to
substrates and inhibits cellular and protein attachment.
[0005] Certain cells, such as macrophages and fibroblasts, are
referred to as "substrate-dependent cells" because they are active
and proliferate only when attached to a surface or substrate. The
attachment occurs via a family of proteins ("attachment molecules
or proteins"), such as vitronectin and fibrinectin, which are found
in the extracellular matrix. A surface that is coated with a
material that is strongly adhesive may inhibit the attachment of
substrate dependent cells by blocking attachment of extracellular
matrix proteins. Hence, adhesive materials, as described herein,
are useful in compositions or can form devices that inhibit the
attachment of certain proteins and certain types of cells.
SUMMARY OF THE INVENTION
[0006] The present invention features a method of inhibiting
cellular attachment to substrates. The invention is based, in part,
on the discovery of adherent coatings of N,O-carboxymethylchitosan
("NOCC"), and in particular that adherent coatings of NOCC may be
applied to various substrates, such as mammalian tissue, so as to
inhibit attachment of other cells, such as substrate dependent
cells. Further, it has been discovered that these adherent coatings
of NOCC may be used in other areas where inhibition of cell or
protein attachment is desirable, such as in the preparation of cell
populations, on medical devices, and with cell-based products. The
invention also has application to the inhibition of the attachment
of proteins to surfaces.
[0007] The present invention provides a composition that is
adherent to a variety of synthetic materials and mammalian tissues.
The present invention also provides a method of inhibiting cellular
and protein attachment to a substrate by applying adherent coatings
of NOCC to the substrate such that the attachment of cells and
proteins is inhibited. The amount of adherent NOCC in the
composition should be effective to inhibit the attachment of
substrate-dependent cells, preferably in a concentration of 0.05-5%
(w/v), most preferably in a concentration of 0.1-2.5% (w/v).
[0008] In one embodiment, the invention provides a composition and
method of inhibiting attachment of substrate-dependent cells to a
substrate by applying a composition containing adherent NOCC to a
substrate such that attachment of substrate-dependent cells is
inhibited. The method may be applied to inhibit substrate-dependent
cell attachment to mammalian tissue, medical devices, fermentation
units, bioreactors and solid supports. In preferred embodiments,
the substrate-dependent cells which are inhibited include
fibroblasts, macrophages, epithelial cells, and endothelial
cells.
[0009] In another embodiment, the invention provides a composition
and method of inhibiting attachment of proteins to a substrate by
applying a composition containing adherent NOCC to a substrate such
that attachment of proteinaceous material is inhibited. The method
may be applied to inhibit protein attachment to contact lenses,
medical devices, fermentation units, bioreactors and solid
supports.
[0010] In another embodiment, the invention may be used in a method
of obtaining a population of cells, e.g., mammalian cells, by
supplementing culture media with adherent NOCC, growing the
population of cells in the supplemented media, and allowing the
cells to grow or differentiate, such that substrate-dependent cells
do not proliferate within the cell population.
[0011] In another embodiment, the invention provides a method of
obtaining cells suitable for use in protein or antibody production
by supplementing culture media with adherent NOCC and growing the
cells in the supplemented media, such that intercellular attachment
(or clumping) within the cell population is inhibited and
production of proteins or antibodies is enhanced.
[0012] In yet another embodiment, the invention provides a method
of inhibiting attachment of inflammatory cells and platelets to a
medical device by coating said device with a composition containing
adherent NOCC, such that platelet or inflammatory cell attachment
to the medical device is inhibited. In preferred embodiments, the
internal medical device is either a stent or shunt. In other
preferred embodiments, the inflammatory cell includes fibroblasts,
macrophages, and monocytes.
[0013] In still another embodiment, the invention includes a method
of inhibiting fibroblast attachment in a cell-based product in
contact with a solid support by introducing adherent NOCC into the
cell based product such that fibroblast attachment is
inhibited.
[0014] In another embodiment, the invention provides a composition
and method of delivering drugs, proteins, and other therapeutic
agents from an adhesive device or composition that is adherent to
soft (mucosal or non-mucosal) tissue or hard tissue. In preferred
embodiments, the adherent delivery device can be used as a buccal,
oral, vaginal, inhalant, or the like delivery system. The device
can be in a variety of forms including solutions, creams, pellets,
particles, beads, gels, and pastes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a schematic of the apparatus used in Example
1.
[0016] FIG. 2 is a bar graph showing the results of Example 1.
[0017] FIG. 3 is a schematic of the procedure used in Example
2.
[0018] FIG. 4 is graph showing the total volume of .sup.125I-NOCC
adhered to rat femur, as calculated using Equation 1.
[0019] FIG. 5 is graph showing the total volume of .sup.125I-NOCC
adhered to rat femur, as calculated using Equation 3.
[0020] FIG. 6 shows the morphological difference between
fibroblasts grown in the absence of NOCC (6a) and fibroblasts grown
in the presence of NOCC (6b).
[0021] FIG. 7 is a bar graph showing a comparison of adherence by
fibroblasts grown in the presence and absence of NOCC, under
various pre-coating conditions.
[0022] FIG. 8 is a bar graph showing a comparison of adherence by
fibroblasts grown in the presence and absence of NOCC, under
various pre-coating conditions.
[0023] FIG. 9 is a bar graph showing a comparison of adherence by
fibroblasts grown in the presence and absence of NOCC, as
determined by .sup.51Cr release assay.
[0024] FIG. 10 is a bar graph showing a comparison of adherence by
fibroblasts grown in the presence and absence of NOCC, as
determined by .sup.51Cr release assay.
[0025] FIG. 11 is a bar graph showing a comparison of adherence by
fibroblasts grown in the presence and absence of NOCC under various
pre-coating conditions, as determined by .sup.51Cr release
assay.
[0026] FIG. 12 is a bar graph showing a comparison of adherence by
fibroblasts grown in plates that were pre-coated with and without
NOCC, as determined by using cells labeled with tritiated
thymidine.
[0027] FIG. 13 is a bar graph showing a comparison of adherence by
epithelial cells grown in plates that were pre-coated with and
without NOCC, as determined by using cells labeled with tritiated
thymidine.
[0028] FIG. 14 is a bar graph showing a comparison of adherence by
macrophages grown in plates that were pre-coated with and without
NOCC, as determined by using cells labeled with tritiated
thymidine.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention relates to the inhibition of cellular
and protein attachment to various substrates. The method of the
invention uses an adherent coating of N,O-carboxymethylchitosan
("NOCC") which provides unexpected benefits in inhibiting cellular
and protein attachment.
[0030] NOCC is a derivative of chitin, which is found in the shells
of crustaceans and many insects. Chitin and its derivatives are
normally biocompatible, naturally resorbed by the body, and have
previously been used for sustained drug release, bone induction and
hemostasis (Chandy and Sharma, Biomat. Art. Cells & Immob.
Biotech. 19:745-760 (1991); Klokkevold, P. et al., J. Oral
Maxillofac. Sur. 50:41-45 (1992)). Due to its prevalence, chitin
may be obtained relatively cheaply, largely from waste products. As
disclosed in U.S. Pat. No. 4,619,995, issued to Hayes, the entire
contents of which are hereby incorporated by reference, NOCC has
carboxymethyl substituents on some of both the amino and primary
hydroxyl sites of the glucosamine units of the chitosan stricture.
NOCC may be used in an uncrosslinked form as a solution or may be
cross-linked or complexed into a stable gel. Because of its
advantageous physical properties, and its relative low cost, NOCC
presents advantageous properties for use in inhibiting cellular and
protein attachment.
Definitions
[0031] The term "inhibit," or any form thereof, is defined in its
broadest sense and includes minimize, prevent, repress, suppress,
curb, constrain, restrict and the like.
[0032] The terms "adherent NOCC" or "an adherent coating of NOCC"
mean a coating or composition of NOCC that exhibits an adhesion
between freshly excised tissue of at least about 100
dynes/cm.sup.2, using the procedure described in Example 1.
[0033] The term "substrate" refers to any object to which cells can
attach. Examples of substrates include, without limitation,
mammalian tissue (including both hard tissue, such as bone, and
soft tissue, such as mucosal and non-mucosal tissue), non-mammalian
tissue, mammalian and non-mammalian cells (including both
eukaryotic and prokaryotic organisms), medical devices,
fermentation units, bioreactors, and solid supports, such as cell
culture plates.
[0034] The term "substrate-dependent cells" means cells that are
only active when attached to a substrate. Examples of substrate
dependent cells include, without limitation, fibroblasts,
macrophages, epithelial cells, somatic cells, and endothelial
cells.
[0035] The term "medical device" means any device which is
implanted in the body for medical reasons or which has a portion of
the device extending into the body (like a catheter) as well as
devices which provide a medical benefit when attached to, or are in
contact with, the body. Examples of medical devices include,
without limitation, catheters, contact lenses, stents, shunts,
breast implants and pacemakers.
[0036] The term "inflammatory cell" means a cell involved in the
nonspecific immune response to any type of body injury. Examples of
inflammatory cells include, without limitation, fibroblasts,
macrophages, eosinophils, neutrophils, monocytes and
lymphocytes.
[0037] The term "cell-based product" means any product that
contains cell. Examples of cell-based products include, without
limitation, blood, plasma, aliquots of cell cultures, and the
like.
[0038] The invention provides a method of inhibiting attachment of
substrate-dependent cells or proteins to a substrate by applying a
composition containing adherent NOCC to a substrate such that
attachment of the substrate-dependent cells or protein is
inhibited. In preferred embodiments, the method is applied to
inhibit substrate-dependent cell attachment to mammalian tissue,
medical devices, fermentation units, bioreactors and solid
supports. In preferred embodiments, the substrate-dependent cells
which are inhibited include fibroblasts, macrophages, epithelial
cells, and endothelial cells.
[0039] The invention also may be used in a method of obtaining a
population of cells, e.g., mammalian cells, by supplementing
culture media with adherent NOCC, growing the population of cells
in the supplemented media, and allowing the cells to grow or
differentiate, such that substrate-dependent cells do not
proliferate within the cell population.
[0040] The invention further provides a method of increasing the
efficiency of protein or antibody production by supplementing
culture media with adherent NOCC and growing the cells in the
supplemented media, such that intercellular attachment within the
cell population is inhibited and production of protein or
antibodies is enhanced.
[0041] The invention also provides a method of inhibiting
attachment of inflammatory cells or proteins to a medical device by
coating said device with a composition containing adherent NOCC,
such that inflammatory cell or protein attachment to the medical
device is inhibited. In preferred embodiments, the medical device
is a catheter, a contact lens, a stent, pacemaker, breast implant,
or a shunt. The method is useful for preventing attachment of a
variety of inflammatory cells including fibroblasts, macrophages,
monocytes, as well as proteins such as albumin.
[0042] In still another embodiment, the invention includes a method
of inhibiting fibroblast attachment in a cell-based product in
contact with a solid support by introducing the cell based product
to an adherent coated solid support such that fibroblast attachment
is inhibited.
[0043] The adherent NOCC used in the present invention may take
many forms. For example, adherent NOCC may be used in a solution, a
hydrogel, a paste, a rehydratable film, cream, foam, or a sponge.
These forms are prepared by methods well known to those of ordinary
skill in the art.
[0044] The adherent NOCC used in the present invention may be the
parent compound or may be cross-linked. Cross-linked adherent NOCC
may be either covalently cross-linked or ionically cross-linked.
Various methods of cross-linking NOCC are known in the art and are
within the scope of this invention. In addition, the degree to
which the adherent NOCC is cross-linked may be optimized for
specific applications by one of ordinary skill without undue
experimentation. It has been found that the degree of cross-linking
is roughly inversely proportional to the adhesiveness of the
coating. That is, the greater the degree of cross-linking of the
adherent NOCC, the lesser degree of adherence. In preferred
embodiments, the degree of cross-linking is less than 1:5 (moles
cross-linking agent to moles, NOCC monomer), more preferably
between 1:100 and 1:1000 on a molar basis.
[0045] The bioadhesive strength of several adherent NOCCs was
compared to that of polycarbophil, a cross-linked acrylic acid
polymer available from B.F. Goodrich. As more fully described in
Example 1, solutions of low and high viscosity NOCC were prepared,
as well as hydrogels of high viscosity NOCC. The bioadhesive was
applied to stomach and cecal tissue samples and the bioadhesive
strength was measured according to a modified version of the
procedure disclosed in U.S. Pat. No. 4,615,697, which is hereby
incorporated by reference. The transfer of polymer to both tissue
surfaces indicated that the adhesive force of the polymer exceeded
the cohesive force. A summary of results appears in Tables 1 and 2,
and FIG. 2. In preferred embodiments, the bioadhesive strength of
adhesive NOCC coatings of the invention is desirably greater than
at least about 1000 dynes/cm.sup.2, more preferably greater than at
least about 2000 dynes/cm.sup.2, and most preferably greater than
at least about 3000 dynes/cm.sup.2.
[0046] Both the low viscosity and high viscosity NOCC polymer
solutions in citrate buffer behaved similarly to polycarbophil when
applied as a coating to the mucosal surface of stomach tissue
(Table 1). This was also true for similar solutions of NOCC using
phosphate buffered saline instead of citrate buffer as well as
non-mucosal, cecal tissue (Table 2). It was observed that as NOCC
was cross-linked the cohesion of the materials increased and the
adhesion decreased. The loss of adhesion was dependent on the
extent of cross-lining. These findings are likely attributable to
the fact that cross-linking adherent NOCC introduced more structure
into the polymer, which consequently restricted interactions with
the tissue surface. The cross-linking also joined the polymer
chains together, resulting in increased cohesiveness.
[0047] The ability of NOCC to adhere to bone tissue was also
studied. The results indicate that NOCC adheres to bone tissue
(FIG. 5). After the third wash, 9.5.times.10.sup.-3.+-.0.002
.mu.L/mm.sup.2 (or about 0.1 .mu.g NOCC/mm.sup.2) of .sup.125I
labeled NOCC remained adhered to the rat femur.
[0048] Surprisingly, the adhesive NOCC coatings of the present
invention have been shown to inhibit cellular attachment of
substrate dependent cells. The adherent NOCC coatings of the
present invention thus have applicability in a multitude of areas.
In addition, adherent NOCC coatings may be applied to either hard
or soft mammalian tissue, such as bone or stomach tissue.
Alternatively, adherent NOCC coatings may be applied to
non-biological substrates, such as medical devices and solid
supports. Examples of such substrates include stents, shunts,
contact lenses, microtiter plates, and cell culture plates.
[0049] Typically, fibroblasts in a cell or tissue culture adhere to
extracellular matrix (ECM) proteins that are bound to the culture
substrate (usually plastic). The ECM proteins in culture typically
come from the culture medium, which is supplemented with serum to
provide these proteins as well as other factors necessary for cell
growth. Alternatively, if there are no ECM proteins in the culture
medium, fibroblasts will secrete their own ECM proteins and adhere
to them. A normal, adhered fibroblast has a very characteristic
morphology: it flattens and exhibits cellular appendages or
processes extending from the cell over the substrate surface, which
indicates fibroblast adherence to the substrate (FIG. 6a).
[0050] The present invention takes advantage of the observation
that substrate-dependent cells, e.g., fibroblasts, plated in tissue
culture media in the presence of adherent NOCC coating do not have
the characteristic morphology and do not exhibit processes
indicating attachment of the cell (FIG. 6b).
[0051] Initial observations of fibroblast morphology in the
presence or absence of adherent NOCC in the medium, revealed that
when fibroblasts were plated in serum-free media without adherent
NOCC they consistently displayed an "adherent" morphology, viz. the
cells were flattened with complex processes. As described more
fully in Example 3, fibroblasts were plated on tissue culture
plates, either in the presence or absence of adherent NOCC, under
four different coating conditions. Irrespective of the coating
treatment, approximately 80% of cells observed looked like normal
cultured fibroblasts in the absence of adherent NOCC. In contrast,
when fibroblasts were plated in the presence of adherent NOCC, the
number of cells displaying the adherent morphology was
significantly reduced. In fact, in the instance where no ECM
proteins were present, no cells adhered in the presence of adherent
NOCC. Where ECM proteins were present, some cell adherence was
observed, but the adherence was significantly less than that which
occurred in the absence of adherent NOCC (FIG. 7).
[0052] Hyaluronic acid (HA) was also tested in this system, to
determine whether it had similar effects to NOCC. When a similar
morphological examination was performed on cells plated in sfRPMI
(serum free or protein free RPMI medium) containing 0.1% HA, it was
observed that the HA did not have the same effect on fibroblast
morphology (FIG. 8).
[0053] Fibroblast adherence was also measured quantitatively, using
a .sup.51Cr adhesion assay (the .sup.51Cr release assay). The
results confirmed that adherent NOCC blocks adhesion of 3T3
fibroblasts to plastic, by more than 90% using this assay (FIG. 9).
This result, taken together with the previous work, suggests that
adherent NOCC adheres to the substrate and interferes with the
deposition of ECM proteins in a competitive manner.
[0054] A competitive assay was performed using media supplemented
with varying concentrations of fetal calf serum (FCS), which
contains the ECM proteins of interest, and in the presence or
absence of NOCC. As expected, it was found that the presence of FCS
in the plating medium reversed the inhibitory effect of adherent
NOCC on fibroblast adhesion in a dose dependent manner, where 10%
FCS fully restored binding of 3T3 to the plates in the presence of
NOCC (FIG. 10). There are two possible explanations for this
effect: 1) adherent NOCC prevents fibroblast adhesion to the ECM
proteins which bind to the plate, or 2) adherent NOCC prevents the
binding of ECM proteins to the plate in a competitive manner.
[0055] To address these possibilities, tissue culture plates were
pre-coated with serum-free medium containing varying concentrations
of FCS. The presence of adherent NOCC did not interfere with the
adhesion of fibroblasts to the FCS coated plates, which confirmed
that adherent NOCC does not inhibit adhesion of fibroblasts to ECM
proteins already deposited on the plate (FIG. 11). This result was
confirmed by coating tissue culture plates with an adherent NOCC
coating. The results (FIG. 12) demonstrate that fibroblast
adherence to adherent NOCC coated plastic is eliminated in the
presence of FCS. This result supports the hypothesis that adherent
NOCC binds to the plastic plate surface and prevents the deposition
or attachment of ECM proteins. Thus, adherent NOCC may inhibit
cellular attachment by preventing the deposition of ECM proteins
rather than by inhibiting the adhesion of fibroblasts to the ECM
proteins. In the absence of the ECM protein network, fibroblasts
are unable to bind to a substrate.
[0056] The following, non-limiting examples will further elucidate
the invention.
EXAMPLE 1
[0057] In this example, the bioadhesive strength of several
adherent NOCC coating compositions is compared to that of
polycarbophil. Polycarbophil (B.F. Goodrich, Akron, Ohio) was
prepared as a 4% w/v solution in both 0.2M citrate buffer (pH 4.8)
and 0.9% saline (pH 6.8). Low viscosity ("LV") NOCC (240 cps,
Brookfield spindle 3, 50-100 rpm) was prepared as 4% w/v solution
in citrate buffer (pH 5.6). High viscosity ("HV") NOCC (P78NOCC1)
was prepared as 2.5% w/v solution in citrate buffer (pH 5.6). High
viscosity NOCC was prepared as 1% and 2.5% in citrate buffer (pH
5.6-5.7), autoclaved and cross-linked (1:500). HV NOCC was also
prepared as 2.5% solution in phosphate buffered saline (PBS). Gels
were formed from 1% HV NOCC by cross-linking (1:100) in PBS and by
cross-linking (1:250) in saline following autoclaving.
[0058] Both stomach and cecal tissues from Sprague-Dawley rats were
harvested immediately prior to testing and were kept moist in
saline solution, Tissue samples were mounted on circular plastic
disks with the inner surfaces of stomach tissues and the outer
surfaces of cecal tissues exposed. Tissue samples were held in
place with a suture around the end of the plastic disks. The
plastic disks were obtained from the plungers of 3 and 5 ml
syringes; the diameters of the disks were 7.0 (surface area of 38.5
mm.sup.2) and 9.5 mm (surface area of 70.9 mm.sup.2), respectively.
The tissue holders were attached to a cantilever load cell and to
the actuator of an MTS servohydraulic material testing machine (see
FIG. 1).
[0059] The temperature compensated load cell was wired into a
Daytronic 3720 Strain Gauge Conditioning Unit in a half bridge
configuration. Data collection was performed using a Macintosh
Centris 650 computer equipped with labVIEW software and a 12 bit
NB-MIO-16 data acquisition board. The cantilever load cell was
calibrated over the working range of 0-3 grams using a series of
proving masses (0.1, 0.23, 0.5, 1 to 3.0 g) verified on a Mettler
PJ 360 balance. A least squares calibration curve was determined to
convert the resulting output from volts to grams force.
[0060] The smaller diameter tissue of the pair of fresh tissue
samples received 30 .mu.l of test material. The software was
designed to take a zero reading after attaching the tissue samples
and applying a coating of the bioadhesive. The testing system
actuator was then manually advanced using the displacement
potentiometers to bring mating faces into compression while
visually monitoring the resulting load level on the computer
monitor. The mating faces were allowed to remain compressed at a
nominal load of 0.9 g for one minute. The computer then displaced
the actuator at a constant rate of 12.0 mm/min, monitoring the
distraction force with time. After failure the computer determined
the peak distraction load and saved the loading curves to a
spreadsheet file.
[0061] For repeated testing of the same samples, the tissues were
scraped with the side of a syringe needle, rinsed with citrate
buffer or water as appropriate and a new aliquot of the same
polymer was applied. Fresh tissues were used for each different
polymer sample; all samples in citrate buffer were tested on
stomach tissue and all samples at neutral pH were tested on cecal
tissue. All testing was performed in air.
[0062] All polymer samples were applied to the smaller surface area
tissue sample at a rate of approximately 1 .mu.l per sq.mm.
Following distraction of the actuator, the transfer of polymer to
both tissue surfaces indicated that the adhesive force of the
polymer exceeded the cohesive force. For example, polycarbophil was
adhesive to both cecal and stomach tissue and required a tensile
force of 2300-2800 dynes/cm.sup.2 to cause failure. The failure was
cohesive rather than adhesive since polymer was observed on both
tissue surfaces after separation. A summary of results appears in
Tables 1 and 2 and FIG. 2.
[0063] Both the low viscosity and high viscosity adherent NOCC
polymer solutions in citrate buffer behaved similarly to
polycarbophil when applied as a coating to the mucosal surface of
stomach tissue. Both adherent NOCC samples failed cohesively and
required larger forces to achieve tissue separation than for
polycarbophil. However, when high viscosity NOCC solutions were
cross-linked to form hydrogels, they became more cohesive and
failed by detaching from the larger diameter disk at forces of 85%
(1% gel) and 53% (2.5% gel) of that of polycarbophil.
[0064] The strengths of adhesion to the external surface of the
cecum (Table 2) again demonstrated that a solution of NOCC
(2.5%-high viscosity) was comparable to polycarbophil. It was also
observed that as adherent NOCC was cross-linked the cohesion of the
materials increased and the adhesion decreased. The loss of
adhesion was dependent on the extent of cross-linking.
[0065] It should be noted that polycarbophil measured under the
present conditions exhibited twice the adhesive force as reported
in U.S. Pat. No. 4,615,697. This is presumably due to testing in
air rather than in solution. For both stomach and cecal tissues,
adherent NOCC solutions were either comparable to or exceeded the
performance of polycarbophil: the force required to achieve failure
was equal to or larger than that of polycarbophil and failure was
due to cohesion not adhesion.
[0066] NOCC hydrogels on both types of tissue were adhesive;
however, they were significantly less adhesive than materials that
were not cross-linked. They demonstrated an adhesive failure rather
than cohesive; also it was observed that increasing the extent of
cross-linking decreased the adhesive force. These findings were not
surprising since cross-linking adherent NOCC introduced more
structure into the polymer which restricted interactions with the
tissue surface and also joined the polymer chains together
resulting in increased cohesiveness.
[0067] Another finding was that both the 2.5% high viscosity NOCC
solution and the 1% NOCC gel in citrate were more adhesive than its
counterparts in PBS. Without limitation to the present invention,
this difference may possibly be explained by the influence of the
citric acid environment. At neutral pH, NOCC exists as an anionic
species resulting from the presence of negatively-charged
carboxylate groups (--COO); the free amines on NOCC are primarily
uncharged. By contrast, in acidic citrate buffer (pH 5.6) the amine
groups are protonated to form positively-charged ammonium sites
(--NH.sub.3+) which ionically bind citrate ions. Such salts are
described in U.S. Pat. No. 5,412,084, the disclosure of which is
incorporated herein by reference. Since citrate has 3 carboxylate
groups, 2 of which are negatively-charged at pH 5.6, the net result
is that NOCC in acidic citrate has an increased number of
carboxylate groups associated with the polymer and, hence, displays
an increased bioadhesiveness.
TABLE-US-00001 TABLE 1 Bioadhesion of NOCC Formulations to Stomach
Tissue. Force to Separate Adhesive or Tensile Failure Tissue
Cohesive Polymer Sample Force (grams) (dynes/sq mm) Failure 4%
Polycarbophil 0.901 .+-. 0.035 2295 .+-. 170 Cohesive 4% LV NOCC
1.007 .+-. 0.107 2567 .+-. 270 Cohesive solution 2.5% NOCC(HV)
1.513 3857 Cohesive 1% NOCC gel 0.770 .+-. 0.280 1961 .+-. 410
Adhesive 2.5% NOCC gel 0.481 1226 Adhesive Notes: Error limits are
one average deviation based on 2-3 determination and values without
error limits result from a single measurement.
TABLE-US-00002 TABLE 2 Bioadhesion of NOCC Formulations to Cecal
Tissue. Force to Separate Adhesive or Tensile Failure Tissue
Cohesive Polymer Sample Force (grams) (dynes/sq mm) Failure 4%
Polycarbophil 1.113 2837 Cohesive 2.5% NOCC (HV) 0.992 .+-. 0.060
2567 .+-. 140 Cohesive solution 1% NOCC gel 0.302 .+-. 0.010 770
.+-. 30 Adhesive (1:100) 1% NOCC gel 0.410 1045 Adhesive (1:250)
Notes: Error limits are one average deviation based on 2-3
determination and values without error limits result from a single
measurement.
EXAMPLE 2
[0068] This example illustrates the adherent property of an
adherent NOCC coating of the present invention.
[0069] Six female rats were anaesthetized using sodium
pentobarbitol (60 mg/kg) and subsequently sacrificed by cervical
dislocation. Twelve femurs were harvested and stripped of
connective tissue by sharp dissection. Excess connective tissue was
removed from the rat femur by immersing the rat femurs in boiling
water for thirty minutes. The femurs were then rinsed and air
dried.
[0070] Each femur was immersed in 1 ml of .sup.125I labeled NOCC
such that half the surface area of the femur was in direct contact
with the .sup.125I NOCC solution (FIG. 3). The other half of the
femur was used to manipulate the femur. Subsequently, the femur was
either placed directly into a scintillation vial and then placed in
a .gamma.-counter rack, or the femur was subjected to a uniform
"wash" before being placed into a scintillation vial and the
.gamma.-counter rack.
[0071] Four groups of three .sup.125I NOCC treated femurs were
subjected to either one wash, two washes, three washes or no
washes. A wash consisted of the uniform agitation of the femur in
approximately 150 ml of PBS for five seconds. Two washes consisted
of a wash, removing the femur from PBS for one second, and then
repeating a wash. Hence, three washes consisted of a wash, removal
of the femur, a wash, removal of the femur, and one last wash. The
PBS solution was replaced for each group of femurs.
[0072] The activity of .sup.125I NOCC was evaluated by a Beckman
.gamma.-counter. The amount of .sup.125I NOCC adhered to a rat
femur was calculated using Equation 1, which uses the activity of 1
ml of .sup.125I NOCC (7.2.times.10.sup.7 CPM and the activity of
the .sup.125I NOCC on the femur, (detected by the .gamma.-counter).
The results appear in FIG. 4.
volume of 125 I N O C C adhered to femur = activity ( C P M ) of
sample 7.2 x 10 7 C P M X 1 mL Equation 1 ##EQU00001##
[0073] Next, the amount of .sup.125I NOCC per unit area of the
femur was calculated. The surface area that was in direct contact
with the .sup.125I NOCC solution was calculated for one
representative rat femur.
surface area in direct contact with 125 I N O C C = 2 .pi. r h 2 +
.pi. r 2 Equation 2 ##EQU00002## [0074] where h=the total height of
the femur; r=the radius of the femur
[0075] The amount of .sup.125I NOCC per unit area of then
calculated, using Equation 3, by dividing the surface area of the
rat femur in direct contact with .sup.125I NOCC into the amount of
.sup.125I NOCC adhered to the rat femur. The results appear in FIG.
5.
125 I N O C C per unit area of femur = .mu. L of 125 I N O C C
adhered to femur surface area in direct contact with 125 I N O C C
Equation 3 ##EQU00003##
[0076] The surface area of the rat femur was calculated to be 228
mm.sup.2, (radius=2.25 mm and total femur height 30 mm).
[0077] Table 3 outlines the number of washes each femur was
subjected to, the activity of .sup.125I NOCC, amount of .sup.125I
NOCC adhered to femur, and the amount of .sup.125I NOCC per unit
area of femur.
TABLE-US-00003 TABLE 3 volume of .sup.125I Number of activity
.sup.125I volume of .sup.125I NOCC (.mu.L)/ femur washes/
NOCC/femur NOCC adhered unit area of number femur (CPM) to femur
(.mu.L) femur (mm.sup.2) 1 0 2.3 .times. 10.sup.6 31.9 1.4 .times.
10.sup.-1 2 0 2.7 .times. 10.sup.6 37.5 1.6 .times. 10.sup.-1 3 0
2.9 .times. 10.sup.6 40.3 1.8 .times. 10.sup.-1 4 1 6.9 .times.
10.sup.5 9.6 4.2 .times. 10.sup.-2 5 1 5.1 .times. 10.sup.5 7.1 3.1
.times. 10.sup.-2 6 1 3.9 .times. 10.sup.5 5.4 2.4 .times.
10.sup.-2 7 2 1.4 .times. 10.sup.5 1.9 8.3 .times. 10.sup.-3 8 2
1.4 .times. 10.sup.5 1.9 8.3 .times. 10.sup.-3 9 2 2.9 .times.
10.sup.5 4.0 1.8 .times. 10.sup.-2 10 3 1.6 .times. 10.sup.5 2.2
9.6 .times. 10.sup.-3 11 3 1.3 .times. 10.sup.5 1.8 7.9 .times.
10.sup.-3 12 3 1.8 .times. 10.sup.5 2.5 11.0 .times. 10.sup.-3
[0078] The results indicate that .sup.125I NOCC adheres to rat
femur. After a third wash, it was found that 9.5.times.10-3+/-0.002
.mu.L/mm.sup.2 (or about 0.1 .mu.g NOCC/mm.sup.2) of .sup.125I NOCC
remained adhered to the rat femur.
EXAMPLE 3
[0079] This example illustrates the effect of adherent NOCC on
cellular attachment. In the first assay, 3T3 fibroblasts were
maintained in culture in RPMI culture medium supplemented with 10%
fetal calf serum (FCS), 20 mM HEPES, 100 U/ml
penicillin/streptomycin, 2 mM 1-glutamine, and 50 .mu.M
2-mercaptoethanol; referred to as complete RPMI (cRPMI). 3T3
fibroblasts were removed from the stock culture flask by treatment
with trypsin, washed and re-suspended to a concentration of
3.0.times.10.sup.5 cells/ml in serum free RPMI (sfRPMI; RPMI as
before but without the 10% FCS) either alone or with 0.1% NOCC.
[0080] These cells were then plated on 96 well Nunclon tissue
culture plates which had been pre-coated (overnight, room
temperature) with one of four different coating treatments. These
were: 1) phosphate buffered saline (PBS) as a control, 2)
vitronectin at a concentration of 15 .mu.g/ml in PBS (vitronectin
is an ECM protein that fibroblasts adhere to), 3) sfRPMI as a
control, and 4) cRPMI, which contains many ECM proteins. After
pre-coating, plates were washed three times in PBS to remove the
coating media. Cells were plated at a concentration of
3.0.times.10.sup.4 cells/well and incubated at 37.degree. C. for 90
minutes. Cells were then observed microscopically, and classed as
either adherent or non-adherent based on morphology. Two hundred
cells were counted in each well (each coating treatment was done in
triplicate), and a mean % adherence .+-.standard deviation (SD) was
calculated.
[0081] This second assay allows quantification of the effect of
adherent NOCC on fibroblast adhesion. In this assay, 3T3 cells were
labeled with radioactive chromium (.sup.51Cr, in the form of
Na.sub.2.sup.51CrO.sub.4) suspended in sfRPMI, added to the wells
of 96-well Nunclon delta plastic plates at a concentration of
2.times.10.sup.4 cells/well and allowed to adhere. After a 90
minute incubation at 37.degree. C., a large proportion of
fibroblasts will adhere to plastic. Washing of the plate with PBS
removed non- or loosely adherent cells. The number of remaining
adherent cells was assessed by lysis with 10% sodium dodecyl
sulfate (SDS; a detergent) and harvesting the well contents. The
lysate was then counted in a gamma counter and disintegrations per
minute were recorded. The level of radioactivity from the lysate
was compared to that present in 2.times.10.sup.4 labeled
fibroblasts and is indicative of the number of cells adhering to
each well. The adhesion assay was performed in the presence or
absence of 0.1% NOCC.
[0082] Visual inspection of fibroblast morphology in the presence
or absence of NOCC, demonstrated that when fibroblasts were plated
in sfRPMI, they consistently displayed an "adherent" morphology;
that is, the cells were flattened with complex processes.
Regardless of the coating treatment, approximately 800% of cells
observed looked like normal cultured fibroblasts. In contrast, when
fibroblasts were plated in sfRPMI with 0.1% NOCC, the number of
cells displaying the adherent morphology was greatly reduced. In
fact, wells pre-coated with PBS or sfRPMI (no ECM proteins
present), no cells adhered in the presence of NOCC. When wells were
pre-coated with vitronectin or cRPMI, some cells adhered but the
adherence was significantly less than that which occurred in the
absence of NOCC (FIG. 7).
[0083] Hyaluronic acid (HA) was also tested in this system, to
determine whether it had similar effects to NOCC. When a similar
morphological examination was performed on cells plated in sfRPMI
containing 0.1% HA, it was observed that the HA did not have the
same effect on fibroblast morphology (FIG. 8).
[0084] The .sup.51Cr adhesion assay was developed to achieve a more
quantitative method of measuring fibroblast adherence. The first
experiment using the .sup.51Cr adhesion assay confirmed the results
obtained by visual inspection examining the effect of adherent NOCC
on adhesion of 3T3 fibroblasts to uncoated Nunclon delta plates.
The results confirmed that adherent NOCC blocks adhesion of 3T3
fibroblasts to plastic, by more than 90% using this assay (FIG. 9).
This result, taken together with the previous work, suggests that
NOCC adheres to the plastic and interferes with the deposition of
ECM proteins in a competitive manner.
[0085] A competitive assay was performed to test these results with
varying concentrations of FCS, which contains the ECM proteins of
interest. The .sup.51Cr adhesion assay was performed using RPMI
supplemented with 2%, 5% or 10% FCS as a plating medium (in the
presence or absence of 0.1% NOCC). It was found that presence of
FCS in the plating medium reversed the inhibitory effect of
adherent NOCC on fibroblast adhesion in a dose dependent manner,
where 10% FCS fully restored binding of 3T3 to the plates in the
presence of adherent NOCC (FIG. 10). There were however, two
possible explanations for this effect: 1) adherent NOCC prevents
fibroblast adhesion to the ECM proteins which bind to the plate, or
2) adherent NOCC prevents the binding of ECM proteins to the plate
in a competitive manner.
[0086] To address these possibilities, plates were pre-coated with
ECM in the form of RPMI containing varying concentrations of FCS
ranging from 2% to 10% overnight at 4.degree. C. The unbound ECM
proteins were then washed off. The adhesion assay was performed
using these pre-coated plates and cells suspended in sfRPMI in the
presence or absence of 0.1% NOCC. The presence of adherent NOCC did
not interfere with the adhesion of fibroblasts to the coated
plates, thus confirming that adherent NOCC does not inhibit
adhesion of fibroblasts to ECM proteins already deposited on the
plate (FIG. 11).
[0087] To confirm that adherent NOCC competitively interferes with
deposition of ECM proteins on plastic surface, plates were
pre-coated with NOCC (in sfRPMI) overnight at 4.degree. C., and
washed. Fibroblasts suspended in RPMI supplemented with 2%, 5% or
10% FCS were allowed to adhere to such NOCC coated plates or
control uncoated plates. In this experiment, the adherence of
fibroblasts was determined after 1 hour by measuring the activity
of the tritiated thymidine-labelled cells that were attached to the
plates. The results (-FIG. 12) showed that fibroblast adherence to
NOCC coated plastic is eliminated in the presence of 2, 5 or 10%
FCS, supporting the hypothesis that adherent NOCC binds to the
plastic plate surface and prevents the deposition of ECM proteins.
In the absence of ECM network, fibroblasts are unable to bind to
the substrate.
EXAMPLE 4
[0088] In this Example, epithethial cells labeled with tritiated
thymidine were used to test whether NOCC could eliminate their
attachment to culture plates. Culture plates were pre-coated with
NOCC (in sfRPMI) overnight at 4.degree. C., and Washed. Labeled
epithelial cells were suspended in RPMI which was supplemented with
FCS and were allowed to adhere to the NOCC coated plates or control
uncoated plates. The adherence of the epithelial cells was
determined after 1 hour by measuring the activity of the tritiated
thymidine-labelled cells following lysis from the plates. The
results (shown in FIG. 13) show that the NOCC pre-coating prevents
the attachment of epithelial cells
EXAMPLE 5
[0089] In this Example, an experiment similar to that described in
Example 4 was carried out except instead of labeled epithelial
cells, tritiated thymidine labeled macrophages (1774M0) were used.
Again, the plates were precoated with 0.1% NOCC, and the
macrophages were suspended in RPMI and were allowed to adhere to
the NOCC coated plates or control uncoated plates. FIG. 14
illustrates that the NOCC precoating inhibits the attachment of the
macrophages to the plates.
[0090] The foregoing examples are merely exemplary and those
skilled in the art will be able to determine other modifications to
the described procedures which fall within the scope of the
invention. Accordingly, the invention is defined by the following
claims and equivalents thereof.
* * * * *