Note: Descriptions are shown in the official language in which they were submitted.
CA 02219399 1997-10-24
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BULK FORMATION OF MQ~-Cr-TTHIC
POLY-C'~C~ARTnR-BAsED ~yl~O~~ c
P~CK~ROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to the bulk formation of
monolithic polysaccharide-based hydrogels. More par-
ticularly, polyacidic or polybasic polysaccharide mate-
rials are respectively dissolved in basic or acidic
media and are gelated through the in si tu uniform neu-
tralization and pH-mediated induction of a 3D continu-
ous networks of hydrogen bonds.
(b) DescriPtion of Prior Art
In recent developments, natural or artificial
biodegradable polymers as well as their derivatives
have been increasingly selected for satisfying the new
challenges that were proposed by recent medical
advances. Polypeptides and polysaccharides which repre-
sent a large range of natural macromolecules have gen-
erally received particular attentions, such as Colla-
gen, Gelatin, Fibronectin, Laminin, Tubulin, Fibrin,
Haemoglobin, Algar, Alginates, Carrageenan, Chitin,
Chitosan, Hyaluronic Acid, Xanthan Gum and the like.
Amino-containing and carboxyl-containing polysaccha-
rides, being among either natural or chemically-modi-
fied polysaccharides, have been commonly selected,
transformed and investigated as biomaterials in the dry
or hydrated state.
Among amino-containing polysaccharides, a spe-
cial interest has been given to explore the processingand use of D-glycosamine units which are commonly found
in Chitosan or Hyaluronic Acid. In Chitosan, D-gly-
cosamine units are generated through catalyzed N-deace-
tylation of Chitin which in turn is extracted from
marine living animals or organisms (shell-fish), par-
ticularly from shell crab, or biosynthesized by natural
CA 02219399 1997-10-24
organisms such as the zygomycete fungi. Like other ani-
mal polysaccharides, Chitosan is expected to have good
viscoelastic properties which combined to its tissue
compatibility and biodegradability, makes it ideal for
bioactive and bioresorbable implants. In other
respects, Chitosan has already been demonstrated a
great potential as structural materials for scaffolding
or supporting engineered artificial tissues. This poly-
D-glucosamine is also known to be able to attach a
large number of proteoglycan molecules and coexists
with fibrous Collagen to form aqueous gels. It is
believed that the role of proteoglycans within the gel
is to retain water and supply appropriate viscoelastic-
ity. The resulting extracellular matrix is in turn
expected to offer compatible environment for cells pro-
liferation and tissue formation, particularly for skin
or cartilage cells. This characteristic must be viewed
with particular attention and may constitute a poten-
tial application of Chitosan in the medical or surgical
field.
Among Carboxyl-Polysaccharides, the D-man-
nuronic acid, L-guluronic acid, D-galacturonic acid and
D-glucuronic acid are the well-known units of typical
carboxyl-containing Alginate (mannuronic and
guluronic), Pectins (galacturonic), Hyaluronic Acid
(glucuronic) polysaccharides. These carboxyl-polysac-
charides from marine (Alginate), animal (Hyaluronic
Acid) and vegetal (Pectins) origin are currently iso-
lated, processed and applied in industrial fields, such
as the biotechnological, biomedical or food industry.
Alginate and Pectin polysaccharides have been currently
gelled through a controlled chelation with bivalent
cations such the Barium (Ba2+), Calcium (Ca2+) and
Strontium (Sr2+) cations. Alginate and pectin have been
reported to form gel structures that can be studied by
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a ~egg-box~ or dimerisation of chains model. Pectin
gels were also processed at low pH by formation of
aggregates of chains, and were found to imply non-cova-
lent or non-electrostatic bonding forces and to appears
as being thermoreversible gels. Alginate gelation is
frequently applied to the encapsulation of living bio-
logical such as animal or human cells for cellular
biology experiments and engineering as well as duplica-
tions of organ functions. Pectin gelation found its
applications in the manufacture of jellies and jams.
Hyaluronic Acid and its derivatives products are now
well-recognized materials in medicine and surgery, as
viscoelastic materials, viscosupplementation and thera-
peutic products as well as dry or hydrated scaffolding
biomaterials of tissues and organs.
Whatever they were microbial or from marine
organisms, Chitin, Chitosan and partially-acetylated
Chitosan derivatives were extensively investigated as
therapeutic substances or biomaterials.
Production of gelled polysaccharide materials
has rapidly attracted much interest due to the variety
of applications for the expected resulting products,
e.g. drug delivery systems, encapsulating, thickening
or gelling agents, etc... Many techniques have been
proposed for gelling polysaccharides or cross-linking
polysaccharides into hydrated materials. Polysaccha-
rides can be gelled currently through ionic, physical
or chemical pathways. Neutral or ionic polysaccharides
were transformed or gelled for forming drug delivery
systems, encapsulation of living biological dressing
materials or surgical products: linear polysaccharides
such the agarose or gelose were gelled with triazine-
based products; D-glucose, L-fucose or D-glucuronate
containing polysaccharides were gelled by cations,
alginate-based gels were obtained by cationic cross-
CA 02219399 1997-10-24
linking in presence of a dialdehyde or diamine or by a
pH-controlled complexation with a protein such the Col-
lagen and a dehydration or chemical cross-linking.
Hydrogels for surgical uses were equally prepared by
blending polysaccharide/protein with acrylic or meth-
acrylic. In a same way, carboxyl-containing polysac-
charide gels were formed in acidic aqueous media at pH
2 to 5 by incorporating multifunctional epoxides and
used in the prevention of tissue adhesion.
Ionic polysaccharides such as Chitosan or Algi-
nate were gelled at the physiological pH through the
use of specific polyoxyalkylene polymers, then applied
to the reduction of post-surgical adhesion or as con-
tact lens. Chitosan granular gels were cross-linked by
polyfunctional agents and reduced by reacted for reduc-
ing agents for immobilizing insolubilized active
enzymes. Chitosan membrane gels were also formed from
acid glycerol-water gels by neutralization and used for
medicament carriers. In a same way, Chitosan-based gels
were used for immobilized and encapsulated living bio-
materials such as cells, bacteria and fungi (U.S. Pat-
ent No. 4,647,536 and International Patent Application
published under No. WO 93/24,112). Chitosan derivatives
were also gelled with poly(N-vinyl lactam) such as
polyvinylpyrrolidone for wound dressings, drug delivery
dressings or cosmetic products as well as with divalent
metal oxides and inorganic additives for bone paste
substitutes. Chitin hydrogels were proposed by Drohan
et al. for growth factors, plasma proteins or drugs
encapsulation (International Patent Application pub-
lished under No. WO 94/41,818).
It would be highly desirable to be provided
with bulk formation of monolithic polysaccharide-based
hydrogels which can gelate in situ.
CA 02219399 1997-10-24
SUMMARY OF THE INVENTION
One aim of the present invention is to provide
bulk formation of monolithic polysaccharide-based
hydrogels which can gelate in situ.
The present invention provides a novel method
which enables the gelation of ionic polysaccharide
solutions into three-dimensionally-shaped monolithic
massive hydrogels. This method can be applied on poly-
base polysaccharides in acidic aqueous solutions as
well as on polyacid polysaccharides in alkaline solu-
tions. It is based on an in situ uniform neutralization
(or pH variation) carried out through a) the introduc-
tion of a hydrolyzable chemical substance into the
polysaccharide aqueous solution able to induce pH
changes upon hydrolysis, b) the hydrolysis of the said
substance within the polysaccharide aqueous solution
such as the pH of the said solution is uniformly modi-
fied, and c) the uniform pH modification to a pH level
which induces the gelation of the polysaccharide solu-
tion, and d) the in situ bulk-gelation of the polysac-
charide into a three-dimensionally-shaped monolithic
massive hydrogel.
In accordance with the present invention there
is provided a monolithic ionic carboxyl-containing or
amino-containing polysaccharide hydrogel that is bulk-
formed by the in situ uniform modification of the pH
within the solution through the introduction of an
acid-releasing or base-releasing hydrolyzable chemical
substance and the controlled hydrolysis in solution of
the said hydrolyzable chemical substance.
The polysaccharide hydrogel may be charac-
terized by a continuous uniform three-dimensional mas-
sive structure and obtained by the combination of an in
situ chemical hydrolysis of a hydrolyzable substance or
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a mixture thereof and a progressive uniform pH increase
throughout the solution or structure.
The polysaccharide hydrogel may be a
polycationic polymer with amino groups on its consti-
tutive monomers, such amino groups being free aminegroups (-NH2) or amino groups from acetyls (-NH-).
The polysaccharide hydrogel may contain D-gly-
cosamine, N-deacetylated-D-glycosamine, D-galactosamine
or N-deacetylated-D-galactosamine units.
The polysaccharide hydrogel may be a Chitin or
Chitosan polymer and their derivatives, being
essentially made of monomeric beta-(1-4)-D-glucosamine
linked units and of monomeric beta-(1-4)-N-acetyl-D-
glucosamine linked units, whatever the degree of N-de-
acetylation within the said Chitosan.
The polysaccharide hydrogel may be synthetic,
or produced biologically, either microbially or by
natural marine organisms.
The polysaccharide hydrogel may consists in a
pure low-molecular weight polysaccharide or a pure
medium-molecular weight polysaccharide or a pure high-
molecular weight polysaccharide or a mixture thereof.
The polysaccharide hydrogel may further include
a water-soluble chemical component or a mixture of
water-soluble components is introduced in the aqueous
polysaccharide solution prior to the gelation whatever
this said component or mixture of components is
rendered later water-nonsoluble within the
polysaccharide hydrogel.
Such water-soluble chemical components may
include, without limitation,
a) dimethyl sulfoxide, glycerin, glycerol,
cyclodextrin, sorbitan esters, mannitol or
sorbitol and their derivatives; and/or
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b) poly(vinyl alcohol), poly(vinyl phosphate),
poly(ethylene oxide), poly(ethylene glycol),
poly(propylene glycol), poly(N-vinyl lactam),
dextran, povidone, hydroxyethylcellulose,
methylcellulose, polysorbate polymers and their
derivatives; and/or
c) in inorganic materials or a mixture of inor-
ganic materials such as silica or titanium
based inorganics.
In accordance with the present invention there
is provided a method of preparing an aqueous amino-
containing polysaccharide solution capable upon heating
up to 80~C and then cooling up to 15~C of bulk-forming
a monolithic hydrogel of the present invention, which
method comprises:
a) providing the amino-containing polysaccharide
normally insoluble in water at pH superior to 6 but
soluble in acidic aqueous solution;
b) dissolving the polysaccharide in an acidic
aqueous solution at temperatures around the ambient
temperature and up to 80~C but lower than the decompo-
sition temperature of the polysaccharide to provide a
solution thereof; and
c) dissolving the desired amount of a hydrolyzable
chemical substance in the aqueous polysaccharide solu-
tion at temperatures around of the ambient temperature
and up to 80~C, and thereafter maintaining the aqueous
polysaccharide solution at a high temperature around
50-80~C so as to initiate the hydrolysis of the said
hydrolyzable chemical substance; and
d) while degasing the aqueous polysaccharide solu-
tion, maintaining the said solution at a temperature
ranging from 15~C to 80~C so as to hydrolyze completely
the hydrolyzable chemical substance and to increase
uniformly the pH to 6.4 and higher.
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The hydrolyzable chemical substance may be
introduced and hydrolyzed through a temperature-
controlled and/or acid-controlled process such as the
pH of the aqueous polysaccharide solution is increased
progressively and uniformly.
The hydrolysis of the chemical substance within
the aqueous polysaccharide solution generates enough
ammonium by-products to overall basify uniformly the
aqueous polysaccharide solution.
The hydrolysis of the chemical substance within
the aqueous polysaccharide solution generates ammonium
and degasable products.
The degasing of the aqueous polysaccharide
solution during the said hydrolysis of the chemical
substance controls in part the uniform pH increase
within the polysaccharide solution and the bulk-
formation of the polysaccharide hydrogel.
The method of Claim 16 wherein the said chemi-
cal substance consists in an amide, and specially a
carbamide.
The chemical substance consists in urea,
thiourea, guanadine, selenourea, ureids, carbamic acid,
cyanuric acid and their derivatives or in any low-
molecular weight ureathanized substances which are
hydrolyzable in an aqueous acidic solution at the
selected temperatures.
In accordance with the present invention, there
is also provided a polysaccharide hydrogel charac-
terized by a continuous uniform three-dimensional mas-
sive structure and obtained by the combination of an insitu chemical hydrolysis of a hydrolyzable substance or
a mixture thereof and a progressive uniform pH decrease
throughout the solution or structure.
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The polysaccharide may be a polyanionic polymer
with carboxyl (-COOH) groups on its constitutive
monomers.
The polysaccharide may contain D-mannuronic
acid, L-guluronic acid, D-galacturonic acid or D-
glucuronic acid units.
The polysaccharide may be an Alginate or Pectin
and their derivatives.
The polysaccharide may alkso be synthetic, or
produced biologically.
The polysaccharide consists in a pure low-
molecular weight polysaccharide or a pure medium-
molecular weight polysaccharide or a pure high-molecu-
lar weight polysaccharide or a mixture thereof.
A water-soluble or chemical component or a
mixture of water-soluble components may be introduced
in the aqueous polysaccharide solution prior to the
gelation whatever this said component or mixture of
components is rendered later water-nonsoluble within
the polysaccharide hydrogel.
Such water-soluble chemical components may
include, without limitation,
a) dimethyl sulfoxide, glycerin, glycerol,
cyclodextrin, sorbitan esters, mannitol or
sorbitol and their derivatives; and/or
b) poly(vinyl alcohol), poly(vinyl phosphate),
poly(ethylene oxide), poly(ethylene glycol),
poly(propylene glycol), poly(N-vinyl lactam),
dextran, povidone, hydroxyethylcellulose,
methylcellulose, polysorbate polymers and their
derivatives; and/or
c) inorganic materials or a mixture of inorganic
materials such as silica or titanium based
inorganics.
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In accordance with the present invention, there
is also provided a method of preparing an aqueous
carboxyl-containing polysaccharide solution capable of
bulk-forming a monolithic hydrogel within a temperature
range from 0~C to 80~C, which method comprises:
a) providing the carboxyl-containing polysaccha-
ride soluble in an alkaline aqueous solutions;
b) dissolving the polysaccharide in an alkaline
aqueous solution at temperatures around the ambient
temperature and up to 80~C but lower than the decompo-
sition temperature of the polysaccharide to provide a
solution thereofi and
c) dissolving the desired amount of a hydrolyzable
chemical substance in the aqueous polysaccharide solu-
tion at temperatures around 0~C and up to 80~C; and
d) maintaining the said solution at a temperature
ranging from 0~C to 80~C so as to hydrolyze completely
the hydrolyzable chemical substance and to decrease
uniformly the pH to 7 and lower.
The hydrolyzable chemical substance may be
introduced and hydrolyzed through a temperature-
controlled and/or alkali-controlled process such as the
pH of the aqueous polysaccharide solution is decreased
progressively and uniformly.
The hydrolysis of the chemical substance within
the aqueous polysaccharide solution generates enough
acidic by-products to overall acidify uniformly the
aqueous polysaccharide solution.
The chemical substance includes in an ester,
acid anhydride and lactone compounds.
The chemical substance includes acetic
anhydride, maleic anhydride, succinic anhydride and
butyrolactone.
The chemical substance includes beta-diesters
or water-soluble low-molecular weight polyesters.
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The monolithic ionic polysaccharide hydrogel of
the present invention may be characterized by a three-
dimensional moulding and formation of the said hydrogel
into specific shapes such as beads, rods, membranes and
blocks.
The monolithic ionic polysaccharide hydrogel of
the present invention may be processed such as being
combined with other materials (textiles, foams,
sponges) to form a composite or complex structures.
The monolithic ionic polysaccharide hydrogel of
the present invention may have incorporated therein
therapeutic substances such as antiviral, antifungal,
antibacterial or steroidal and non-steroidal anti-
inflammatory agents, growth factors and hormones.
The monolithic ionic polysaccharide hydrogel of
the present invention may be implanted in animals or
humans for delivering drugs, polypeptides or cells,
reconstructing and replacing epithelial, connective,
muscular or neural tissues.
The monolithic ionic polysaccharide hydrogel of
the present invention may include living animal or
human cells from connective tissues are encapsulated
for forming biohybrid systems, culturing and
engineering biological tissues.
BRIEF DBSCRIPTION OF THE DRAWINGS
Fig. lA shows the chemical structure of the N-
acetylated-D-glucosamine linked units (N-ACE-GLU)
within the Chitin polysaccharide;
Fig. lB shows the chemical structure of the
units linked in the polysaccharide chain, one N-deace-
tylated-D-glucosamine unit (N-DEACE-GLU) and one N-ace-
tylated-D-glucosamine unit (N-ACE-GLU);
Figs. 2A and 2B shows the possible chemical
units in a hyaluronic acid polysaccharide, one N-acety-
CA 02219399 1997-10-24
lated-D-glucosamine unit (N-ACE-GLU) and one Glucuronic
acid (GLU. AC.) unit, one N-deacetylated-D-glucosamine
unit (N-DEACE-GLU) and one Glucuronic acid unit (GLU.
AC.);
Fig. 3A shows the alpha-L-Guluronic acid unit
and 1,4-linked beta-D-Mannuronic acid unit that can be
found in Alginate polysaccharidesi
Fig. 3B shows the 1,4-linked-alpha-D-Galac-
turonic acid units of the pectin polysaccharidesi
Fig. 4 shows an example of the potential chemi-
cal substances (amides) that can be hydrolyzed in an
acidic polysaccharide aqueous solution for producing
alkaline moleculesi and
Fig. 5 shows an example of the potential chemi-
cal substances (acid anhydride, ester) that can be
hydrolyzed in an alkaline polysaccharide aqueous solu-
tion for producing acidic molecules.
DET~TrRn DESCRIPTION OF THE INVENTION
In the present invention, a general principle
is proposed for gelling ionic polysaccharides namely
carboxyl-containing and/or amino-containing polysaccha-
rides into three-dimensionally-shaped monolithic mas-
sive hydrogels. In one of the embodiment of the inven-
tion, D-glucos-amine/N-deacetylated-D-glucosamine con-
taining Chitin and Chitosan derivatives were gelled by
a control-led and progressive induction of an uniform
and continuous 3D network of hydrogen bonds. The intro-
duction of a hydrolyzable chemical substance into an
acidic polysaccharide solution as well as the acid- and
temperature-catalyzed hydrolyzing of the said chemical
substances result into an uniform in si tu pH increase
allowing the bulk-gelation of the (polybasic) polysac-
charide into an one-piece hydrogel. In a symmetrical
way, the second major embodiment contains the introduc-
CA 02219399 1997-10-24
tion of a hydrolyzable chemical substance into an alka-
line polysaccharide solution, the alkali- and tempera-
ture-catalyzed hydrolysis of the said chemical sub-
stance which result into an uniform in si tu pH decrease
allowing the gelation of the (polyacid) polysaccharide
into an one-piece hydrogel. This is specially devoted
to carboxyl-containing polysaccharides, e.g. D-man-
nuronic acid, L-guluronic acid, D-galacturonic acid and
D-glucuronic acid, etc., such as the Alginate, Pectin
or Hyarulonic Acid based polysaccharides.
The processing techniques of the present inven-
tion enable the specific formation and fashioning of
3D-shaped monolithic continuous hydrogel materials made
of pure polysaccharides or polysaccharide-based blends.
Monolithic polysaccharide hydrogel materials can be
introduced in a wide range of medical or surgical
applications such as cell, drug or gene delivery sys-
tems, reconstructive or replacement implants and bioen-
gineered tissue scaffolding materials.
Of special interest, numerous methods have been
proposed for forming polysaccharide gels, either by
heating a swollen aqueous mixture of polysaccharide
powder into a semi-continuous particulate gel, by heat-
ing a polysaccharide dispersion, by acidifying an alka-
line solution of polysaccharide, by exposing a polysac-
charide solution to acid anhydride vapors such as gase-
ous carbon dioxide, by diluting with water a solution
of polysaccharide into organic solvent, or by dialyzing
alkaline polysaccharide solutions against various solu-
tions. A beta-1,3-glucan polysaccharide was gelled
thermally through a critical temperature neutralization
by reducing the pH of the alkaline polysaccharide solu-
tion through a specific temperature range where this
reduction does not induce immediately a gelation.
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The method herein described differs from all
the others by the fact that 1~ the gelation can be
technically induced in a very similar manner from ionic
polyacid or polybase polysaccharides, 2) the gellation
is produced in situ through a continuous, progressive
and uniform pH modification within the solution volume
or gel structure, 3) the pH modification and gelation
are ensured by an in situ hydrolysis of an appropriate
hydrolyzable chemical substance or mixture thereof, 4)
the gelation attributed to a continuous uniform network
of hydrogen bonds results in a three-dimensionally-
shaped monolithic massive polysaccharide hydrogel, and
can be obtained conveniently in any molds.
~Polyacid~ polysaccharide refers to the ionic
polysaccharides that contain carboxylic groups and are
currently insoluble in acidic media. ~Polybase~ poly-
saccharide refers to the ionic polysaccharides that
contain essentially amino groups and are currently
insoluble in alkaline media.
A ~continuous uniform~ pH modification is said
of a pH change which occurs similarly and simultane-
ously at every point of the solution or structure. A
continuous uniform pH increase from 4,5 to 6 correspond
to a pH change from 4,5 to 6 that can be observed at
the same level and at the same time at every point of
the solution or structure. In a similar way, a
~continuous uniform~ network of hydrogen bonds is said
of a medium or structure wherein hydrogel bonds are
homogeneously distributed over the medium or structure,
and can be viewed as being present at every point of
the said medium or structure.
A ~hydrolyzable~ chemical substance is defined
by a chemical substance or compound that reacts with
water molecules to give arise to basic or acidic mole-
cules. The initial alkali or acid medium as well as the
CA 02219399 1997-10-24
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temperature are susceptible to initiate, catalyze or
inhibit the hydrolysis reaction.
Herein, ~monolithic~ refers to an one-piece
material with its constitutive elements which form a
homogeneous rigid system
Herein, ~massive~ refers to a solid material
that is bulk-formed, occupies the apparent volume, con-
taining regular distribution and homogeneous porosity
and appears as a compact one-piece material.
Herein, ~three-dimensional shape~ is said of
the gelation method which enables any customization of
the final hydrogel structure in terms of shape, volume
and geometry. The gelation can be performed such as to
fashion the hydrogel by molding in any types of solid
recipients, e.g. plastic, metallic or glass, or by the
processing itself, e.g. dropping in oil.
The presence of (polyacid) carboxyl or
(polybase) amino groups which are relatively reactive
with a large number of organic functions renders such
ionic polysaccharides easily cross-linkable through
chemical pathways. Physical gelation can also originate
from hydrogen bonds, either intra-chain or inter-
chains, involving these carboxyl or amino groups and
thus lead to water-insoluble polymeric materials. There
exist few available processing methods for the forma-
tion of polysaccharide hydrogels that are physically
cross-linked into dense continuous networks and fash-
ioned in massive pieces with controlled pores and
shapes. In particular, the processing and building of
monolithic massive hydrogels, one-piece non-particulate
hydrogels, that could be modulated in composition, per-
formances and properties and molded as required by
medical or surgical end-uses would represent a major
advance for polysaccharide-based biomaterials. Physical
cross-linking by forming three-dimensional networks of
CA 02219399 1997-10-24
- 16 -
hydrogen bonds involving amino or carboxyl groups and
the oxygen may help in building the expected monolithic
hydrogels.
In one preferred embodiment, amino-containing
(polybase) polysaccharides are dissolved in acidic
aqueous media such as acetic acid, hydrochloride acid,
ascorbic acid, etc. to form a clear polysaccharide
solution ranging from 0.1 to 10%, preferentially from
1.0 to 5.0% w/v. For example, an acidic solution of
2.0% w/v partially N-deacetylated Chitosan polysaccha-
ride in 0.1 M to 10 M acetic acid solutions, preferen-
tially 0.5 M to 6 M acetic acid solution, has a pH
around 3.5-4.5. The pH of the acidic polysaccharide
solution can be increased to 5.0 to 6.0 by introducing
the necessary amount of an low-concentration alkali,
e.g. a 1.0% sodium or potassium hydroxide, but not to a
pH level superior to the gelation pH. The gelation pH
of an acidic 2.0% w/v Chitosan solution was found at
37~C to be around 6.2-6.4.
Dissolution of polybase polysaccharides such
the Chitin or Chitosan may be obtained with organic or
mineral acids such as acetic acid, formic acid, hydro-
chloride acid, sulfuric acid, phosphoric acid, maleic
acid, tartaric acid, ascorbic acid, etc. or a mixture
thereof. The control over, or fine tuning of, the pH of
the acidic aqueous solution can be performed by drop-
ping organic or mineral alkali such as sodium hydrox-
ide, potassium hydroxide or amine in order a pH of the
polybase polysaccharide acidic aqueous solution ranging
from 3.0 to 6.2.
Any water-soluble chemical components can be
added, dissolved and homogenized into the acidic poly-
saccharide solutions if they do not break down the
homogeneity, uniformity or continuity of the polysac-
charide solution or bring non-uniformly or non-progres-
CA 02219399 1997-10-24
sively the pH to a level superior to the gelation pH.
If requested for obtaining a controlled dissolution of
the water-soluble components, the said component may be
dissolved first in an aqueous solution under specific
conditions, then the aqueous solution may be acidified
adequately to dissolve the poly-base polysaccharide.
The introduction of thixotropic agents (0.1-20%
w/v, preferentially 0.5-10%) may help in stabilizing
the polybase polysaccharide aqueous solution as well as
the resulting polysaccharide hydrogels. For example,
thixotropic silica agents have been proven to give more
consistency and integrity to the polybase polysaccha-
ride and to eliminate the prevent shrinking after gela-
tion and washing. Suitable inorganic materials include
silica, alumina, zinc, zirconia and titanium based
inorganics, calcium phosphate, calcium carbonate, apa-
tite and the like.
Even in high proportions, the incorporation of
water-soluble polymers has been demonstrated that does
not prevent the gelation of the polybase polysaccharide
even. Mixtures of Chitosan with water-soluble polymers
with 70% w/w of the said polymer (70:30 polymer/ Chito-
san) gelate similarly to pure Chitosan (100% Chitosan),
while the chemical and physicomechanical properties of
the resulting hydrogel change with the proportion of
the said polymer. Suitable water-soluble polymers
include polyvinyl alcohol, polyvinyl phosphate, poly-
ethylene oxide, polyethylene glycol, polypropylene gly-
col, polyvidone, polyacrylamide, acrylic and meth-
acrylic esters, polysorbates, polyester diols, cellu-
losic polymers, polypeptides, phospholipids and the
like.
In a similar way, water-soluble active agents
may be introduced in the polybase polysaccharide hydro-
gel prior to the gelation and consist in complexants,
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surfactants, tonicity preserving or wetting agents.
Suitable agents include beta-cyclodextrin and its
derivatives, sorbitan esters, lecithin, sodium chlo-
ride, potassium chloride, sorbitol, mannitol and the
like.
The incorporation of water-soluble therapeutic
or bioactive agents such as the anti-inflammatory,
antibacterial, antifungal or antiviral agents, growth
factors or other hormones and their derivatives and
synthetic analogs. Suitable therapeutic or bioactive
agents may include progesterone, norgestrel, estradiol,
norethisterone, testosterone, prednisolone, cortisone,
dexamethasone, gentamycin, erythromycin, penicillin,
neomycin, norfloxacin, methicillin, amphotericin,
natamycin, doxorubicin, bleomycin, cisplatin, 5-fluor-
ouracil, naltrexon, phenobarbitone, chlorpromazine,
methadone, diazepam and the like.
The viscosity of the (amino-containing) poly-
base polysaccharide solution will increase with
increasing pH levels in the said solution. This is due
to the interconversion of ammonium salts of the D-glu-
cosamine units of the polysaccharide to free amines,
and results in a more water insoluble form of the poly-
saccharide. Such an interconversion is an equilibrium
process, time-dependent and reversible. This explains
that the polybase polysaccharide hydrogel would weaken
if placed in acidic media.
The in si tu increase to a certain level of the
pH within the polybase polysaccharide aqueous solution
would logically result in a water-insoluble polysaccha-
ride. As a consequence, an uniform and continuous in
situ increase of the pH within the polybase polysaccha-
ride solution may result in a uniform and continuous
gelation, or bulk-gelation, of the polybase polysaccha-
CA 02219399 1997-10-24
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ride, thus producing a monolithic massive polybase
polysaccharide gel.
To obtain such an in si tu pH increase and bulk-
gelation, the method promotes the addition and dissolu-
tion of a hydrolyzable chemical substance within thepolybase polysaccharide aqueous solution, and the
homogenization of the said aqueous mixture. The time-
dependent, temperature-controlled hydrolysis in situ of
the hydrolyzable chemical substance in the said aqueous
solution, would result in a pH increase uniformly, con-
tinuously and progressively toward the occurrence of a
water-insoluble polybase polysaccharide, the monolithic
massive polysaccharide hydrogel.
Suitable hydrolyzable chemical substances for
gelling polybase polysaccharide contains amides or ure-
thanized groups, and may include urea, thiourea,
selenourea, ureids, cyclic ureids, guanadine, carbamic
acid, cyanuric acid, urethanized products and the like.
The equilibrium reaction of the hydrolysis of
some products may be controlled by a degassing if one
resulting products is gaseous, for example, such the
carbon dioxide formed during the hydrolysis of urea. By
eliminating the gaseous carbon dioxide, the equilibrium
is displaced to favor the hydrolysis reaction, the
ammonium production in situ which accelerates the gela-
tion of the polybase polysaccharide. Degassing also
occurs naturally during the hydrolysis of urea within
the polybase polysaccharide aqueous solution.
The second embodiment concerns the carboxyl-
containing polysaccharides (polyacid) which dissolve inalkaline aqueous media to form clear polysaccharide
solutions ranging from 0.1 to 10%, preferentially from
1.0 to 5.0% w/v.
The interconversion of carboxylate salts of the
polyacid units of the polysaccharide to free carboxyls
CA 02219399 1997-10-24
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results in a more water insoluble form of the polysac-
charide. Thus an in si tu decrease to a certain level of
the pH within the polyacid polysaccharide aqueous solu-
tion would logically result in a gelated polysaccha-
ride. As a consequence, an uniform and continuous insi tu decrease of the pH within the polyacid polysaccha-
ride solution may result in an uniform and continuous
gelation, or bulk-gelation, of the polyacid polysaccha-
ride, thus producing a monolithic massive polyacid
polysaccharide gel.
To obtain such an in si tu pH decrease and bulk-
gelation, the method promotes the dissolution and
homogenization of a hydrolyzable chemical substance
within the polyacid polysaccharide aqueous solution.
The time-dependent, temperature-controlled in situ
hydrolysis within the resulting homogeneous aqueous
solution would lead to a pH decrease uniformly, con-
tinuously and progressively toward the occurrence of a
water-insoluble polyacid polysaccharide, the said mono-
lithic massive polysaccharide hydrogel.
Typical example consists of an Alginate poly-
saccharide solution ( 2.0% W/V) obtained by dissolving
either an Alginate salts in water or an Align acid in
alkaline aqueous media (e.g. potassium hydroxide 0.01
mM to 100 mM) If necessary, the pH of the alkaline
polysaccharide solution is regulated (decreased) by
dropping the necessary amount of an low-concentration
acid, e.g. a 1.0% acetic acid, but not to a pH level
inferior to the gelation pH.
As for polybase polysaccharides, any water-
soluble chemical components can be added, dissolved and
homogenized into the alkaline polysaccharide solutions
if they do not inhibit or impair the gellation process
or the hydrogel structure. If requested for obtaining a
controlled dissolution of the water-soluble components,
CA 02219399 1997-10-24
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the said component may be dissolved first in an aqueous
solution under specific conditions, then the aqueous
solution may be basified adequately to dissolve the
polyacid polysaccharide.
As for polybase polysaccharides, the introduc-
tion of thixotropic agents (0.1-20% W/V, preferentially
0.5-10%) may help in stabilizing the polyacid polysac-
charide aqueous solution as well as the resulting poly-
saccharide hydrogels. Suitable inorganic materials
include silica, alumina, zinc, zirconia and titanium
based inorganics, calcium phosphate, calcium carbonate,
apatite and the like.
As for polybase polysaccharides, water-soluble
polymers has been demonstrated that does not prevent
the gelation of the polyacid polysaccharide and may be
mixed to the polyacid polysaccharide aqueous solution.
Suitable water-soluble polymers include polyvinyl alco-
hol, polyvinyl phosphate, polyethylene oxide, polyeth-
ylene glycol, polypropylene glycol, polyvidone, poly-
acrylamide, acrylic and methacrylic esters, polysor-
bates, polyester diols, cellulosic polymers, polypep-
tides, phospholipids and the like.
In a similar way, water-soluble active agents
may be introduced in the polyacid polysaccharide hydro-
gel prior to the gelation and consist in complexants,surfactants, tonicity preserving or wetting agents.
Suitable agents include beta-cyclodextrin and its
derivatives, sorbitan esters, lecithin, sodium chlo-
ride, potassium chloride, sorbitol, mannitol and the
like.
Anti-inflammatory, antibacterial, antifungal or
antiviral agents, growth factors or other hormones and
their derivatives and synthetic analogs may be incorpo-
rated as well within the solution. Suitable therapeutic
3 5 or bioactive agents may include progesterone, norg-
CA 02219399 1997-10-24
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estrel, estradiol, norethisterone, testerone, predniso-
lone, cortisone, dexamethasone, gentamycin, erythromy-
cin, penicillin, neomycin, norfloxacin, methicillin,
amphotericin, natamycin, doxorubicin, bleomycin, cis-
platin, 5-fluorouracil, naltrexon, phenobarbitone,
chloro-promazine, methadone, diazepam and the like.
Suitable hydrolyzable chemical substances for
gelling polyacid polysaccharide contains acid anhy-
drides, beta-diesters and esters-containing chains that
hydrolyzed into acids. It may include acetic anhydride,
tartaric anhydride, malonic anhydride, maleic anhy-
dride, polyester diols products and the like.
For both polyacid and polybase polysaccharides,
the gelation is induced through an uniform in situ pH
change by hydrolyzing in situ a selected hydrolyzable
chemical substances. The hydrolysis of the said chemi-
cal substance in aqueous solutions is time-dependent,
temperature-controlled and acid or alkali-catalyzed.
The hydrolysis rate in situ of the introduced hydrolyz-
able chemical substance may be controlled, lowered orincreased, within the range of temperature going from
0~C to 100~C, preferably from 4~C to 80~C. For polyacid
polysaccharides, acid anhydrides hydrolyze quite spon-
taneously and a lowering of the temperature may help in
controlling the hydrolysis rate in situ. For polybase
polysaccharide, amide hydrolysis may be initiated or
stimulated by increasing the temperature.
The present invention will be more readily un-
derstood by referring to the following examples which
are given to illustrate the invention rather than to
limit its scope.
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EXAMPLBasic formulation and method for processinq monolithic
Chitosan hydlu~els.
Typical experiment is carried out by dissolving
0.2 g of chitosan in 10 ml of aqueous solution of ace-
tic acid (O. 5M). The pH measurements indicate an aver-
age value around 5.0 for a such solution. Urea (8 g) is
then added to previous solution, followed by heating
the resulting solution up to 85~C, in order to acceler-
ate the hydrolysis of urea, and therefore increase thepH level. When the pH level reaches 6.0 (around the
starting point of gelation, pH 6.2-6.4, of a 2% Chito-
san solution at 37~C), the mixture is cooled down to
37~C and maintained at this temperature for 24 hours,
enough time to achieve the gelation process. The
resulting hydrogel is immersed in renewed baths of dis-
tilled water in order to remove the excess in urea and
ammonium salts. A limited but visible shrinkage of the
monolithic massive Chitosan hydrogel is observed ( 1/4-
1/3).
Table 1
Chemical constituentsProportions
Water 10 ml
Acetic Acid, 99.9% 0.30 g
Chitosan 0.2 9
Urea 8 9
Adiusting the PH of the Chitosan solution prior to gelformation
The process of Example II is similar to the one
presented in the Example I, except that the pH of the
acidic Chitosan aqueous solution is increased by adding
drops of a 1.0% potassium or sodium hydroxide solution
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in order to reach a pH level ranging from 5.0 to 6Ø
Once the pH of the polysaccharide aqueous solution is
stabilized at 37-40~C, the urea is added and the mix-
ture is maintained at this temperature until the gela-
tion process is accomplished. The resulting hydrogel isimmersed in renewed baths of distilled water in order
to remove the excess in urea and ammonium salts.
Table 2
Chemical constituents Proportions
Water 10 ml
Acetic Acid, 99.9% 0.30 g
Chitosan 0.2 g
Sodium/Potassium hydroxide s.q. pH 6.0
Urea 49
EXAMPLE III
Processing monolithic Silica particles modified
Chitosan gels
The process of the Example III is the same as
in the Example I, but a fumed silica AEROSIL~ 300 was
added to the acidic Chitosan aqueous solution t5-10%
w/w Chitosan). AEROSIL solid inorganic is coagulated
silicon dioxide with spherical particles of 10-20 nm.
AEROSIL 300 is a fluffy white powder and contains par-
ticles of 7 nm average diameter. A 4.0% AEROSIL 300aqueous dispersion has a pH varying from 3.6 to 4.3.
Once the AEROSIL 300 particles are homogeneously dis-
persed in the Chitosan aqueous solution, the gelation
is initiated as previously described in Example I. The
resulting hydrogel is immersed in renewed baths of dis-
tilled water in order to remove the excess in urea and
ammonium salts. The resulting monolithic massive Chito-
san hydrogel seems to be stabilized structurally, more
compact and does not shrink at all. It seems that the
CA 02219399 1997-10-24
silica particles allow to retain water within the Chi-
tosan hydrogel.
Table 3
Chemical constituents Proportions
Water 10 ml
Acetic Acid, 99.9% 0.30 9
Chitosan 0.2 g
Fumed silica particles, AEROSIL~ 300 0.012 g
Urea 8g
BXAMPLB IV
Monolithic PVA/Chitosan hydrogels with high PVA content
A 0.2 g of 99+% hydrolyzed polyvinyl alcohol is
dissolved in 5 ml of a lM acetic acid solution by heat-
ing to 60~C. When the PVA is dissolved and the PVAsolution is clear, the heating is stopped, then 0.1 g
of medium M.W. Chitosan is added to the PVA aqueous
solution. When the Chitosan is dissolved, the
PVA/Chitosan aqueous mixture is generally a clear vis-
cous mixture. A 5.0 g of urea is added to the said mix-
ture and dissolved by heating which results in a less
viscous mixture. The PVA/Chitosan/urea mixture is
homogenized and maintained at a high temperature (80-
85~C~ for some minutes, then placed in thermal bath at
40~C. The PVA/Chitosan/urea mixture is maintained at
40~C until the urea totally hydrolyzes and progres-
sively induces a complete gelation of the system.
The PVA/Chitosan system is a 66.7:33.3 (% w/w)
hydrogel. The resulting hydrogel is immersed in renewed
25 baths of distilled water in order to remove the excess
in urea and ammonium salts.
CA 02219399 1997-10-24
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Table 4
Chemical constituents Proportions
Water 5 ml
Acetic Acid, 99.9% 0.30 g
Polyvinyl alcohol 0.20 g
Chitosan 0.10 9
Urea 5.0 9
Example V
Monolithic PVA/Chitosan hydrogels
The processing method of the Example V is
strictly identical to the one described in the Example
IV, except 0.15 g of PVA is dissolved, then 0.15 g of
Chitosan is incorporated such as the resulting
PVA/Chitosan system is a 50:50 (% w/w) hydrogel. The
PVA/Chitosan/urea mixture with 50:50 proportions is the
less viscous system. The resulting hydrogel is immersed
in renewed baths of distilled water in order to remove
the excess in urea and ammonium salts.
Table 5
Chemical constituents Proportions
Water 5 ml
Acetic Acid, 99.9% 0.30 9
Polyvinyl alcohol 0.15 g
Chitosan 0.15 g
Urea 5.0 9
Monolithic PVA/Chitosan gels h~ o~els with low PVA
content
The processing method of the Example VI is
strictly identical to the one described in the Example
IV, except 0.10 g of PVA is dissolved, then 0. 20 g of
CA 02219399 1997-10-24
Chitosan is incorporated such as the resulting
PVA/Chitosan system is a 33.3:66.7 (% w/w) hydrogel.
The resulting hydrogel is immersed in renewed baths of
distilled water in order to remove the excess in urea
5 and ammonium salts.
Table 6
Chemical constituents Proportions
Water 5 ml
Acetic Acid, 99.9% 0.30 g
Polyvinyl alcohol 0.10 9
Chitosan 0.20 9
Urea 5.0 9
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following,
in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be
applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
Example VII
Basic formulation and method for processing Monolithic
Alqinate hy~loyels
Typical experiment was carried out by gelling a
2% (w/w) aqueous solution of alginate. The preparation
consisted on the dissolution of 0.2g of alginate sodium
salt in 10 mL of distilled water. The pH value of such
a solution was in the vicinity of 8.2. This resulting
solution was then cooled down to about 2~C, after
CA 02219399 1997-10-24
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which ~0.2g of a finely ground maleic anhydride was
slowly added and carefully dispersed in the viscous
aqueous solution of alginate. Once a quite homogeneous
dispersion was reached, the mixture was returned back
at room temperature and left for a spontaneous and
progressive hydrolysis of anhydride, which caused a
progressive pH decrease and therefore allowed the gel
formation. In the final step, the alginate gel formed
was washed with a large volume of distilled water in
order to remove any excess of maleic acid.
Table 7
Chemical constituents Proportions
Water 10 ml
Alginate 0.2 g
Maleic anhydride 0.2 g
Example VIII15 Basic formulation and method for processing Monolithic
Pectate or polygelacturonic acid hydroqels
Typical experiment was carried out by gelling a
2% (w/w) aqueous solution of polygalacturonic acid.
The preparation consisted on the dissolution of 0.2g of
Polygalacturonic acid in 10 mL of distilled water. The
pH value of such a solution was in the vicinity of 8.2.
This resulting solution was then cooled down to about
2~C, after which ~0.2g of a finely ground maleic
anhydride was slowly added and carefully dispersed in
the viscous aqueous solution of polygalacturonic acid.
Once a quite homogeneous dispersion was reached, the
mixture was returned back at room temperature and left
for a spontaneous and progressive hydrolysis of
anhydride, which caused a progressive pH decrease and
therefore allowed the gel formation. In the final
step, the polygalacturonic acid gel formed was washed
CA 02219399 1997-10-24
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with a large volume of distilled water in order to
remove any excess of maleic acid.
Table 8
Chemical constituents Proportions
Water 10 ml
Poly9~ ctllronic acid 0.2 9
Maleic anhydride 0.2 9
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following,in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be
applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
