HYDROGEL
Field of the Invention
The present invention relates to a hydrogel composed of polysaccharide derivatives
5 having a longterm stability by virtue of formation of a specific aggregate structure and a
manufacturing method thereof.
Background Art
Chemically modified polysaccharides are used widely for foods, daily use articles,
cosmetics, etc., as well as in medical field, by virtue of their hydrogel forming property,
10 biocompatibility, and so on. Among them, hydrogel composed of polysaccharide derivatives
is expected to be widely applied in the medical field, such as for wound dressing to keep the
wound site in wet state, filling material for tissue restoration, postsurgical adhesion barrier,
medium for cell culture, carrier for drug delivery system, etc.
Among the hydrogel for medical use, injectable gel having fluidity is desired as the
15 safe and easy-to-handle hydrogel. Injectable gel having fluidity may be injected into a body
through a syringe and thc like and has a potential to be a medical material for less invasive
endoscopic surgery.
These hydrogels should have a constant quality for a long term during transportation
and storage after manufacturing. However, there is no known method to maintain the
20 stability for some gel composed of polysaccharide derivatives known so far.
In the case that the hydrogel is injected through a syringe, it is desirable that the
similar quality to the injectable solution for medical use be secured. For the injectable
solution for medical use, the number of insoluble particles of a diameter of 10 tlm or more is
specified by US Pharmacopeia, European Pharmacopeia and Japanese Pharmacopeia. Also
I
from a viewpoint of safety, it is required that the particles of a diameter of 10 μm or more be
removed by filtration. However, it was difficult to remove insoluble particles from the
injectable gel composed of polysaccharide derivatives known so far, because its solubility to
water is low or its viscosity is high,
5 Japanese Patent Application Laid-Open Publication No. 2006 -296916 describes an
antiadhesive material composed of a product of a reaction between hyaluronic acid and
phosphatidyl ethanolamine. International Publication W02007/015579 describes an
adhesion barrier composed of a product of a reaction between carboxymethylcellulose and
phosphatidyl ethanolamine . International Publication WO1996/037519 describes an
10 adhesion barrier composed of a hyaluronic acid derivative. International Publication
W02010/016611 describes a highly viscoelastic hydrogel obtained by dissolving the product
of the reaction between carboxymetbylcellulose and phosphatidyl ethanolamine to
physiological saline.
However, longterm storage stability is not considered in any of the documents and
15 there is no description or suggestion regarding the gel with high storage stability. In
addition, there is no description or suggestion regarding the presence of foreign matters to be
referred to later and the method to reduce them.
USP 6869938 describes a technique to filter a solution containing
carboxymethylcellulose and polyethylene oxide using a filter during the process to prepare an
20 aqueous adhesion barrier, although this is not a hydrogel. However, the filter used in this
specification has a pore diameter of 30 [Lm or more, which is much larger than the fi iter to be
mentioned later. Therefore, foreign matters with a diameter of several μm cannot be
removed.
Japanese Patent Application Laid-Open Publication No, 200418750 describes that
an aqueous solution of a hyahuonic acid derivative prepared by bonding "a nucleophilic
reagent which may bond to carboxylic group" to hyahuonic acid was able to pass a porous
filter (pore diameter 0.45 μm) by an alkaline treatment of the reaction solution, although it
was not able to pass the filter before the alkaline treatment. However, this technique is not
5 applicable to unstable compounds because this technique is performed under an alkaline
condition. In addition, the alkaline solution should be neutralized in order to obtain an
aqueous solution with a neutral pH. Furthermore, the specification neither describes nor
suggests the enhancement of filterability by the heat treatment disclosed in the present
specification.
10 Summary of the Invention
The problem to be solved by the present invention is to provide a hydrogel
composed of polysaccharide derivatives having a long-term stability.
As a result of the extensive investigation for the above-mentioned purpose, the
present inventors have found that a hydrogel having a specific aggregate structure has a
15 long-term stability and a good filterability and completed the present invention.
Thus, the present invention provides a hydrogel composed of a polyanionic
polysaccharide derivative having a hydrophobic group introduced by covalent bonding and a
salt solution with a salt concentration of 50 mM or more and 200 mM or less, the hydrogel
having an aggregate structure with an average particle diameter of 100 to 2000 nm in the
20 diluted solution.
Furthermore, the present invention provides a method for manufacturing the
hydrogel, specifically a method for manufacturing the hydrogel comprising the steps of
preparing a mixture containing a polyanionic polysaccharide derivative having a hydrophobic
group introduced and a salt solution having a salt concentration of 50 mM or more and 200
mM or less and subjecting the mixture to heat treatment,
The hydrogel of the present invention has a longterm stability. Therefore, it has
become possible to provide an article having a quality stable for a long term,
Furthermore, the hydrogel of the present invention may be filtered using a filter.
5 Therefore, it has become possible to provide a hydrogel having a high safety by virtue of
removal of foreign matters.
Furthermore, the hydrogel of the present invention is characterized in that it can be
diluted in a solution state while maintaining the aggregate structure, because the liydrogel is
formed by aggregation based on hydrophobic interaction, whereas the conventional hydrogel
10 such as acrylamide gel, agarose, gelatin, etc. has a characteristic that the shape does not
change unless temperature or pH is changed. Therefore, the aggregate structure of the
hydrogel of the present invention may be determined by DLS measurement of the particle
diameter of the aggregate in the diluted solution. As a result, it has been found that the
longterm stability and filterability depend on the particle diameter of the aggregate.
15 In the present invention, a phrase "having an aggregate structure with an average
particle diameter of 100 to 2000 nm in the diluted solution" means that the particle diameter
of the aggregate in the diluted solution measured by DLS is 100 to 2000 rim.
Hereinafter, the structural difference between the conventional hydrogel and the
hydrogel of the present invention will be discussed using schematic diagrams.
20 The left-hand side of Fig. I is a schematic diagram of the conventional hydrogel.
The right-hand side of Fig. 1 shows a state after dilution for the purpose of analysis. The
network structure does not change by dilution due to the hydrogel's property that the shape
does not change unless temperature or pH changes. The hydrogel cannot be filtered at all.
The left-hand side of Fig. 2 is a schematic diagram of a gel-like solution formed by
4
physical entanglement of the molecular chain. The right-hand side of Fig, 2 shows a state
after dilution for the purpose of analysis. The network structure has been disintegrated by
dilution and the gel has become a solution state without formation of aggregate. Such
gel-like solution has a problem of long-term stability.
5 The left-hand side of Fig. 3 is a schematic diagram of the hydrogel similar to the
hydrogel of the present invention in that it has been formed by aggregation based on
hydrophobic interaction, although the aggregate structure is not homogeneous. Such
aggregate structure may be determined by DLS measurement of the particle diameter of the
aggregate in the diluted solution. The right-hand side of Fig. 3 shows a state after dilution
10 for the purpose of analysis. Particle diameter of the aggregate in the diluted solution is not
homogeneous.
The left-hand side of Fig. 4 is a schematic diagram of the hydrogel of the present
invention. The aggregate structure of such hydrogel is homogeneous and stable, thereby
providing the excellent filterability and long-term stability, The right-hand side of Fig. 4
15 shows a state after dilution for the purpose of analysis of the aggregate structure. Particle
diameter of the aggregate is homogeneous.
Brief Description of the Drawings
Fig. I is a schematic diagram showing the structure of the conventional hydrogel
obtained by crosslinking based on chemical bond.
20 Fig. 2 is a schematic diagram showing the structure of the conventional hydrogel
formed by physical entanglement of the molecular chain.
Fig. 3 is a schematic diagram showing the structure of the conventional hydrogel
formed by aggregation based on hydrophobic interaction.
Fig. 4 is a schematic diagram showing the structure of the hydrogel of the present
invention having a homogeneous aggregate structure formed by hydrophobic interaction.
Fig. 5 shows the viscosity change (angular velocity 10 rad/sec) after the storage for
one month at 40°C in Example 3 and Comparative Example 2.
Fig. 6 shows the result of viscoelasticity measurement before and after filtration in
5 Example 4.
Fig. 7 shows the result of viscoelasticity measurement before and after filtration in
Comparative Example 3.
Fig. 8 shows the change of the number of insoluble particles (per I ml of sample)
before and after filtration in Example 7.
10 Fig. 9 shows the result of DLS measurement in Reference Example 1.
Best Mode for Carrying Out the Invention
[Polysaccharide derivatives]
In the hydrogel of the present invention and the manufacturing method thereof,
polysaccharides used as the raw material for the polysaccharide derivatives are not
15 particularly limited so long as they are polyanionic polysaccharide. Examples include
natural polysaccharides such as hyaluronic acid, alginic acid, pectin, polygalacturonic acid,
etc.; carboxyalkyl polysaccharides such as carboxymethylpullulan, carboxymethylchitin,
ca rboxymethylchitosan, carboxymethylmannan, carboxymethylstarch, carboxymethyldextran,
carboxyethylcellulose, carboxymethylcellulose, etc.; oxidized polysaccharides such as
20 oxidized cellulose, oxidized starch, etc.; and sulfate group-containing polysaccharides such as
chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, etc. Among them,
carboxymethylcellulose and hyaluronic acid are preferable, carboxymethylcellulose being
especially preferable.
Although weight average molecular weight of the raw material polysaccharides is
6
not particularly limited, it is preferably 50,000 Da or more and 1,000,000 Da or less, more
preferably 50,000 Da or more and 500,000 Da or less.
Although cations forming a salt with these polyanionic polysaccharides are not
particularly limited, proton and metal ions, specifically metal ions such as sodium, potassium,
5 lithium, calcium, magnesium, etc. and organic cations such as organic ammonium are
exemplified. Among them, sodium salt of carboxymethylcellulose is preferable by virtue of
its intended effect.
Although the hydrophobic group to be introduced to the polyanionic polysaccharides
is not particularly limited, one having a phosphate ester structure is preferable, one having a
10 phosphatidyl group represented by the following formula (I) being more preferable.
H
0 --C-R2
II II
0
'Cok
I Ox
15 (1)
In formula (1), X is hydrogen atom or alkaline metal. R' and R2 are each
independently a linear or branched chain alkyl group or alkenyl group having 9 to 21 carbon
atoms.
An example of the preferable polysaccharide derivatives is one having the repeat
20 unit of the following formula (2).
CH2ORS
0 R3
0
II 1
(2)
7
In formula (2), R3, R4 and R5 are each independently selected from the group consisting of the
following formula (1-a), (l-b) and (1-c).
5
-CH2COOX (1--b)
10
0
In formula (1-b) and formula (l-c), X is hydrogen atom or alkaline metal. In
formula (l-c), R6 and R7 are each independently a linear or branched chain alkyl group or
15 alkenyl group having 9 to 21 carbon atoms.
Although the equivalency of the substituent represented by formula (1-a) and the
equivalency of the substituent represented by formula (lab) are not particularly limited, it is
preferable that the equivalency allows the polyanionic polysaccharide derivatives to dissolve
in a salt solution containing 50 mM or more and 200 mM or less of sodium chloride, The
20 equivalency of the substituent represented by formula (l-c) is preferably 0.25 to 5.0
mol%/sugar residue.
X in formula (1-b) and formula (1-c) is hydrogen atom or alkaline metal, preferably
alkaline metal from a viewpoint of solubility. More preferably, X is sodium considering the
safety in the living body.
R6 and R7 in formula (1-c) are each independently a linear or branched chain alkyl
group or alkenyl group having 9 to 21 carbon atoms, preferably an alkenyl group having 9 to
21 carbon atoms from a viewpoint of solubility, more preferably an alkenyl group having 17
carbon atoms.
5 [Hydrogel from polysaccharide derivatives]
The above-mentioned polysaccharide derivatives may be used to form a hydrogel by
mixing them with a salt solution under a specific condition followed by heating (Hereinafter,
such hydrogel may be briefly denoted as "hydrogel").
The salt solution to be used has a salt concentration of 50 mM or more and 200 mM
10 or less, preferably is a salt solution containing 50 mM or more and 200 mM or less of sodium
chloride, more preferably is a buffer solution containing 120 mM or more and 180 mM or less
of sodium chloride with a pH adjusted to 6.5 or more and 77.5 or less, most preferably is a
phosphate-buffered physiological saline. Although the solubility of macromolecules
decreases with increase of salt strength due to the salting-out effect, it is possible to obtain
15 the necessary strength by dissolving the polysaccharide derivatives to a phosphate-buffered
physiological saline using the method of the present invention.
As for the concentration of the polysaccharide derivatives, hydrogel having
appropriate viscoelasticity may be obtained with the polysaccharide derivatives used in the
present invention in the amount of 0.1 to 5.0 weight parts, preferably 03 to 3.0 weight parts
20 relative to 100 weight parts of water.
While usually any hydrogel may be obtained with the desired gel strength by
increasing the polymer concentration, the hydrogel of the present invention may be obtained
with a sufficient gel viscoelasticity even at a polymer concentration as low as 5 wt% or less
relative to water. Specifically preferable physical properties of the hydrogel are represented
9
by the viscoelasticity such that the hydrogel does not flow down when the vessel containing
the hydrogel is tilted, Such hydrogel readily deforms when touched with a spatula and the
like and may be readily applied to the affected area. Such hydrogel may also be injected
using a device having a cannula such as a syringe.
5 As for the preferable viscoelasticity of the hydrogel, absolute viscosity when
measured with a dynamic viscoelasticity measurement apparatus called rheometer under
conditions of temperature of 37°C and angular velocity of 10 rad/sec is 0.5 to 100 Pa-sec,
more preferably 2 to 30 Pa-sec. Although this range is suitable to simultaneously satisfy the
handling property as the injectable gel and the retaining property in the body, this range may
10 be changed as needed depending on the purpose of usage.
As for the aggregate particle diameter of the hydrogel of the present invention, the
average particle diameter measured using a dynamic light scattering photometer under
conditions of temperature of 20°C, measurement wavelength of 532.0 nm, concentration of
the polysaccharide derivative of 03 wt%, and detection angle of 90.0° is preferably 100 to
15 2000 nm, more preferably 120 to 1500 nm, even more preferably 150 to 1200 urn, most
preferably 200 to 1000 nrn. This range is suitable to satisfy the handling property as the
injectable gel and the retaining property in the body, as well as the longterm stability and
filterability.
Heating temperature for manufacturing the hydrogel of the present invention is 70°C
20 or higher and 1440°C or lower, preferably 100°C or higher and 135°C or lower.
Although the heating time for manufacturing the hydrogel of the present invention is
not particularly limited so long as it is longer than the shortest time required for the hydrogel
to become filterable depending on the heating temperature, it is preferably 1 minute or longer
and 3 hours or shorter, considering the thermal decomposition of the polysaccharide
10
derivatives. Although the particle diameter distribution of the aggregate of such hydrogel
shifts toward the smaller direction as the heat treatment proceeds, it will converge in a certain
range over time.
Although the pressure during heating for manufacturing the hydrogel of the present
5 invention is not particularly limited, so long as water does not reach the boiling point
depending on the heating temperature, it is preferably atmospheric pressure to 10 atm.
The hydrogel of the present invention is filterable. More specifically, the hydrogel
may be filtered with a porous filter having a pore diameter of 5 lzm or more. Presumably
this is because the average particle diameter of the aggregate of the polysaccharide derivative
10 molecules in the hydrogel becomes smaller by heat treatment,
On the other hand, it has been confirmed that the average molecular weight of the
polysaccharide derivatives in the hydrogel of the present invention did not decrease even by
heat treatment.
Although the manufacturing method of the present invention is a method for
15 manufacturing a hydrogel comprising the steps of preparing a mixture containing a
polyanionic polysaccharide derivative having a hydrophobic group introduced and a salt
solution with a salt concentration of 50 mM or more and 200 mM or less and subjecting this
mixture to heat treatment, it is preferable that the manufacturing method of the present
invention further comprise a step of filtering the mixture with a porous filter having a pore
20 diameter of 5μm or more after the heat treatment.
The hydrogel of the present invention may be applied to an adhesion barrier, medical
use other than the adhesion barrier (wound dressing, filling material, medium for cell culture,
carrier for drug delivery system, etc.), daily use products such as hair care products and
moisturizing agent for skin, cosmetic use, and so on.
11
Examples
(1) The materials used for the Examples and Comparative Examples are as follows.
(i) Sodium carboxymethylcellulose (CMCNa: made by Dai-ichi Kogyo Seiyaku Co., Ltd.);
P603A (Degree of substitution of hydroxyl group to carboxymethyl group 0.7, weight average
5 molecular weight 250,000 Da), PRS (Degree of substitution of hydroxyl group to
carboxymethyl group 0.8, weight average molecular weight 230,000 Da), PM250-L (Degree
of substitution of hydroxyl group to carboxymethyl group 0.7, viscosity average molecular
weight 540,000 Da)
( ) Tetrahydrofuran (THF: made by Wako Pure Chemical Industries, Ltd.)
10 (iii) L- a- dio leoylp hop ha tidylethanolamine (DOPE: made by NOF Corporation)
(iv) Methyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC•HC1: made by
Osaka Synthetic Chemical Laboratories, Inc.)
(v) 3,4-dihydro-3..hydroxy.4-oxo.1,2,3.benzotriazine (DHBT: made by Wako Pure Chemical
Industries, Ltd,)
15 (vi) N-hydroxysuccinimide (HOSu: made by Wako Pure Chemical Industries, Ltd.)
(vii) Phosphate-buffered physiological saline;
Sodium chloride (NaCl: made by Wako Pure Chemical Industries, Ltd.) 136.9 mM, Sodium
dihydrogenphosphate (NaH2PO4-2H20: made by Wako Pure Chemical Industries, Ltd.) 2.92
mM, Sodium hydrogenphosphate hydrate (Na2HPO4.12H20: made by Wako Pure Chemical
20 Industries, Ltd.) 9.02 mM
(viii) Phosphate buffer;
Sodium dihydrogenphosphate 2.92 mM, Sodium hydrogenphosphate hydrate 9.02 nrM
(ix) HEPES buffer;
2-[4-(2-hydroxyethyl)-1--piperazinyl]ethanesulfonic acid (HEPES: made by GIBCO) 50 mM
12
(2) Measurement of phosphatidylethanolamine content in polysaccharide derivatives
Content of phospnatidylethaiolamine in polysaccharide derivatives was calculated
from integral ratio of the peaks of 'H--NMR spectra.
(3) Longterm stability test of the hydrogel
5 Accelerated test was performed at 40°C. Viscosity of the hydrogel was measured
just after preparation and after storage at 40°C using a viscoelasticity measurement apparatus
RFSIII (made by TA Instruments, Japan).
(4) Measurement of viscosity before and after filtration
The hydrogel filled in a syringe with a PVDF filter (pore diameter 5 μm) attached at
10 the end was filtered by manual pushing at 25°C. Viscosity was measured using a
viscoelasticity measurement apparatus RFSIII (made by TA Instruments, Japan) before and
after filtration.
(5) Measurement of particle diameter by dynamic light scattering method
Particle diameter distribution was measured using a fiber optical dynamic light
15 scattering photometer FDLS 3000 (made by Otsuka Electronics Co., Ltd,) under the
following conditions.
Measurement conditions: Measurement temperature 20°C, Measurement wavelength 532.0
nm, Measurement concentration 0.3 wt%, Detection angle 90.0°, Number of integration
cycles 100, Pretreatment: filtered with a PVDF filter (pore diameter 5 μm)
20 (6) Measurement of sugar concentration before and after filtration
The hydrogel filled in a syringe with a PVDF filter (pore diameter 5μm) attached at
the end was filtered by manual pushing at 25°C. Sugar concentration was measured by
sulfuric acid anthrone method before and after filtration.
(7) Measurement of the number of insoluble particles before and after filtration
.13
The hydrogel filled in a syringe with a PVDF filter (pore diameter 5μm) attached at
the end was filtered by mamtal pushing at 25°C. The number of insoluble particles was
measured using an automatic particle counter for liquid System 9703-150 (made by HIAC)
before and after filtration.
5 (8) Measurement of molecular weight of polysaccharide derivatives
CMCNa was dissolved in 100 mM sodium chloride aqueous solution at a
concentration of 0.02 to 0.05 wt% and its intrinsic viscosity was measured using an
Ubbelohde viscometer to calculate the viscosity average molecular weight.
[Example 1]
10 (1) Synthesis of raw material
To a mixed solvent of water and tetrahydrofuran 1:1 (volume ratio), 3000 mg of
CMCNa (PRS) was dissolved, To this solution was added 349 mg (0.50 mmol) of DOPE,
143 mg (0.74 mmol) of WSC•HC1, and 324 mg (1.98 mmol) of DHBT. After the reaction
was completed, the polysaccharide derivative was obtained by filtering out a precipitate
15 formed by adding the reaction mixture to ethanol. The solid obtained was washed with
ethanol and dried under reduced pressure. Degree of substitution of the polysaccharide
derivative obtained with DOPE was 1.47 mol%/sugar residiue.
(2) Preparation of hydrogel
To 1980 mg of phosphate-buffered physiological saline, 20 mg of the polysaccharide
20 derivative synthesized in (1) was dissolved to prepare a solution at a concentration of 1.0
wt%. The mixture was heated for 20 minutes at 121°C.
(3) Measurement of viscosity and average particle diameter of the hydrogel
Viscosity of the sample prepared in (2) was measured at an angular velocity of 10
rad/sec. Absolute viscosity was about 7.1 Pa-sec, showing that a hydrogel with high
14
viscosity was prepared. This hydrogel is referred to as Sample 1, Average particle
diameter of a diluted solution of this sample was measured to be 580 nm.
[Example 2]
(1) Synthesis of raw material
5 To a mixed solvent of water and tetrahydrofuran 1: 1 (volume ratio), 3000 mg of
CMCNa (PM250-L) was dissolved. To this solution was added 384 mg (0.52 mmol) of
DOPE, 99 mg (0.52 mmol) of WSC•HC1, and 337 mg (2.06 mmol) of DHBT. After the
reaction was completed, the polysaccharide derivative was obtained by filtering out a
precipitate formed by adding the reaction mixture to ethanol. The solid obtained was
10 washed with ethanol and dried under reduced pressure. Degree of substitution of the
polysaccharide derivative obtained with DOPE was 0.76 mol%/sugar residue.
(2) Preparation of hydrogel
To 1980 mg of phosphate-buffered physiological saline, 20 mg of the polysaccharide
derivatives synthesized in (1) was dissolved to prepare a solution at a concentration of 1.0
15 wt%. The mixture was heated for 20 minutes at 121°C.
(3) Measurement of viscosity and average particle diameter
Viscosity of the sample prepared in (2) was measured at an angular velocity of 10
rad/sec. Absolute viscosity was about 13.1 Pa-sec, showing that a hydrogel with high
viscosity was prepared. This hydrogel is referred to as Sample 2. Average particle
20 diameter of a diluted solution of this sample was measured to be 1789 inn.
[Comparative Example 1]
(1) Raw material
The polysaccharide derivatives synthesized in Example 1 and Example 2 were used.
(2) Preparation of hydrogel (prepared using water as a solvent)
15
To 1980 mg of water, 20 mg each of the polysaccharide derivatives synthesized in
Example I and Example 2 was dissolved to prepare a solution at a concentration of 1.0 wt%,
The mixture was heated for 20 minutes at 121°C.
(3) Measurement of viscosity and average particle diameter
5 Viscosity of the samples prepared was measured at an angular velocity of 10 rad/sec.
Absolute viscosity was 0,1 Pa-sec or less for the sample made using the polysaccharide
derivative synthesized in Example 1 and about 0,3 Pa-sec for the sample made using the
polysaccharide derivative synthesized in Example 2, showing that neither samples were
gelled. Measurement of average particle diameter for the diluted solutions of these samples
10 showed that the scattering intensity was insufficient and that the aggregate was not formed.
[Example 3]
(1) Raw material
The polysaccharide derivatives synthesized in Example 1 and Example 2 were used.
(2) Preparation of hydrogel
15 To 1980 mg of phospliate-buffered physiological saline, 20 mg each of the
polysaccharide derivatives synthesized in Example 1 and Example 2 was dissolved to prepare
a solution at a concentration of 1.0 wt%. The mixture was heated for 20 minutes at 121°C.
(3) Longterm stability test
Viscosity of the samples prepared in (2) was measured just after preparation and
20 after storage at 40°C for one month. Absolute viscosity of both samples measured at an
angular velocity of 10 rad/sec was not changed. Results are shown in Fig. 5.
[Comparative Example 2]
(1) Raw material
To a mixed solvent of water and tetrahydrofuran 1:1 (volume ratio), 3000 mg of
16
CMCNa (PM250-L) was dissolved, To this solution was added 351 mg (0.47 mmol) of
DOPE, 399 mng (2.08 minor) of WSC'HCI, and 239 mg (2.08 mmol) of HOSu, After the
reaction was completed, the polysaccharide derivative was obtained by filtering out a
precipitate formed by adding the reaction mixture to ethanol. The solid obtained was
5 washed with ethanol and dried under reduced pressure. Degree of substitution of the
polysaccharide derivative obtained with DOPE was 2.39 mol%/sugar residue. In addition, a
polysaccharide derivative having a degree of substitution with DOPE of 2.79 mol%/sugar
residue was similarly synthesized.
(2) Preparation of hydrogel (prepared using water as a solvent)
10 To 1980 mg of water, 20 mg of the polysaccharide derivatives synthesized in (1) was
dissolved to prepares solution at a concentration of 1.0 wt%. The mixture was heated for 20
minutes at 121°C.
(3) Longterm stability test and measurement of average particle diameter
Viscoelasticity of the samples prepared in (2) was measured just after preparation
15 and after storage at 440°C for one month. Absolute viscosity measured at an angular velocity
of 10 rad/sec was significantly changed, The results with Sample 3, which was a hydrogel
prepared using the polysaccharide derivative having a degree of substitution with DOPE of
2,39 mol%/sugar residue, and Sample 4, which was a hydrogel prepared using the
polysaccharide derivative having a degree of substitution with DOPE of 2.79 mol%/sugar
20 residue, are shown in Fig. 5. Measurement of average particle diameter for the diluted
solutions of these samples just after preparation showed that the scattering intensity was
insufficient and that the aggregate was not formed.
[Example 4]
(1) Synthesis of raw material
1l
To a mixed solvent of water and tetrahydrofuran 1:1 (volume ratio), 3000 mg of
CMCNa (P603A) was dissolved. To this solution was added 384 mg (0.52 mmol) of DOPE,
247 mg (1.29 mmol) of WSC•HC1, and 337 mg (2.06 mmol) of DHBT, After the reaction
was completed, the polysaccharide derivative was obtained by filtering out a precipitate
5 formed by adding the reaction mixture to ethanol. The solid obtained was washed with
ethanol and dried under reduced pressure. Degree of substitution of the polysaccharide
derivative obtained with DOPE was 1.30 mol%/sugar residue.
(2) Preparation of hydrogel
To 1980 mg of phosphate-buffered physiological saline, 20 mg of the polysaccharide
10 derivative synthesized in (1) was dissolved to prepare a solution at a concentration of 1.0
wt%. The mixture was heated for 5 minutes at 121°C.
(3) Measurement of viscosity before and after filtration and average particle diameter
Viscoelasticity of the sample prepared in (2) was measured before and after filtration.
There was no change in viscoelasticity after filtration, Results are shown in Fig, 6 and
15 Table 1. Average particle diameter of the diluted solution of this sample was measured to be
446 nm.
[Comparative Example 3]
(1) Raw material
The polysaccharide derivative synthesized in Example 4 was used.
20 (2) Preparation of hydrogel (prepared without heat treatment)
To 1980 mg of phosphate-buffered saline, 20 mg of the above-mentioned
polysaccharide derivative was dissolved to prepare a hydrogel at a concentration of 1.0 wt%.
(3) Measurement of viscosity before and after filtration and average particle diameter
Viscoelasticity of the sample prepared in (2) was measured before and after filtration,
'18
There was a significant change in viscoelasticity before and after filtration. Results are
shown in rig. 7 and Table
Average particle diameter of the diluted solution of this sample was measured to be
2820 nm.
5 [Table 1]
Change in viscoelasticity before and after filtration in Example 4 and Comparative Example 3
(Angular velocity 10 rad/sec)
Example 4 Comparative Example 3
Eta* [Pa-sec] tan A Eta* [Pa-sec] tan A
Before filtration 7.63 0.04 1.45 0.98
After filtration 7.20 0.04 2.49 0.19
[Example 5]
(1) Raw material
10 CMCNa (P603A) was used.
(2) Preparation of hydrogel diluted solution
To 1980 mg of phosphate-buffered physiological saline, 20 mg of CMCNa (P603A)
was dissolved to prepare a solution at a concentration of 1.0 wt%. This solution was divided
into two aliquots, one with a heat treatment at 121°C for 20 minutes and one without the heat
15 treatment. Each solution was diluted with 100 mM sodium chloride aqueous solution to
prepare solutions at a concentration of 0.05 to 0.02 wt%.
(3) Measurement of molecular weight of polysaccharide derivatives
Viscosity average molecular weight was measured from intrinsic viscosity for the
samples with and without heat treatment. The viscosity average molecular weight of the
20 sample with heat treatment and the sample without heat treatment was 105,000 Da and
115,000Da, respectively. Since the viscosity average molecular weight of these samples is
similar, it was confirmed that the molecular weight of the polysaccharide derivatives did not
19
change with heat treatment.
[Example 6]
(1) Synthesis of raw material
To a mixed solvent of water and tetrahydrofuran 1:1 (volume ratio), 3000 mg of
5 CMCNa (PRS) was dissolved. To this solution was added 349 mg (0.47 mmol) of DOPE, 99
mg (0.52 mmol) of WSC•HC1, and 337 mg (2.06 mmol) of DHBT. After the reaction was
completed, the polysaccharide derivative was obtained by filtering out a precipitate formed
by adding the reaction mixture to ethanol. The solid obtained was washed with ethanol and
dried under reduced pressure. Degree of substitution of the polysaccharide derivative
10 obtained with DOPE was 1.57 m.ol%/sugar residue, Similarly, a polysaccharide derivative
having a degree of substitution of 1.07 mol%/sugar was synthesized.
(2) Preparation of hydrogel
To 1980 mg of phosphate-buffered physiological saline, 20 mg of the polysaccharide
derivatives synthesized in (1) was dissolved to prepare a solution at a concentration of 1.0
15 wt%. The mixture was heated for 5 minutes at 121°C and filtered with a PVDF filter (pore
diameter 5μm).
(3) Measurement of sugar concentration before and after filtration and average particle
diameter
Sugar concentration of the samples prepared in (2) was measured before and after
20 filtration. There was no change in sugar concentration before and after filtration. Results
are shown in Table 2. Average particle diameter of the diluted solution of these samples was
224 nrn for the sample with degree of substitution of DOPE of 1.07 mol%/sugar residue and
648nm for the sample with degree of substitution of 1.57 mol%.
[Table 2]
20
Change of sugar concentration before and after filtration in Example 6
PE Degree of snh'titution
[mol%/sugar]
Sugar concentration
after 'filtration/before filtration
1.07 1.03
1.57 0.97
[Example 7]
(1) Hydrogel
The samples prepared in Example 6 were used.
5 (2) Measurement of the number of insoluble particles before and after filtration
The number of insoluble particles of a diameter of 10 1-.Lm or more and 25 μm or
more was measured for 1 mL of the samples from (1) before and after filtration. As a result,
the number of insoluble particles of a diameter of 10 μm or more and 25 μm or of the samples
decreased by one-half to one twentieth compared to the control substance. Results are
10 shown in Fig. 8.
[Example 8]
(1) Synthesis of raw material
To a mixed solvent of water and tetrahydrofuran 1:1 (volume ratio), 3000 mg of
CMCNa (PRS) was dissolved. To this solution was added 349 mg (0.50 mmol) of DOPE,
15 143 mg (0.74 mmol) of WSC•HC1, and 324 mg (1.98 mmol) of DHBT. After the reaction
was completed, the polysaccharide derivative was obtained by filtering out a precipitate
formed by adding the reaction mixture to ethanol. The solid obtained was washed with
ethanol and dried under reduced pressure. Degree of substitution of the polysaccharide
derivative obtained with DOPE was 1.44 mol%/sugar residue,
20 (2) Preparation of hydrogel
To 1980 mg of a phosphate buffer containing 200 mM sodium chloride, 20 mg of the
polysaccharide derivative synthesized in (1) was dissolved to prepare a solution at a
21
concentration of 1.0 wt%. The mixture was heated for 5 minutes at 121°C, filtered with a
PVDF filter (pore diamcici 1 μm), and heated again for 20 minutes at 121°C.
(3) Measurement of viscosity before and after filtration and average particle diameter
Viscoelasticity of the sample prepared in (2) was measured before and after filtration.
5 There was no change in absolute viscosity measured at an angular velocity of 10 rad/sec after
filtration. Results are shown in Table 3. Average particle diameter of the diluted solution
of this sample was measured to be 620 nm.
(4) Long-term stability test
Viscoelasticity of the sample prepared in (2) was measured just after preparation and
10 after storage at 40°C for one month. Absolute viscosity measured at an angular velocity of
10 rad/sec was not changed. Results are shown in Table 3.
[Example 9]
(1) Raw material
The polysaccharide derivative synthesized in Example 8 was used.
15 (2) Preparation of hydrogel
To 1980 mg of a HEPES buffer containing 137 mM sodium chloride, 20 mg of the
above-mentioned polysaccharide derivative was dissolved to prepare a solution at a
concentration of 1.0 wt%, The mixture was heated for 5 minutes at 121°C, filtered with a
PVDF filter (pore diameter 5 μm), and heated again for 20 minutes at 121°C.
20 (3) Measurement of viscosity before and after filtration and average particle diameter
Viscoelasticity of the sample prepared in (2) was measured before and after filtration.
There was no change in absolute viscosity measured at an angular velocity of 10 rad/sec after
filtration, Results are shown in Table 3. Average particle diameter of the diluted solution
of this sample was measured to be 887 rms.
(4) Longterm stability test
Viscoelasticity of the sample prepared in (2) was measured just after preparation and
after storage at 40°C for one month. Absolute -viscosity measured at an angular velocity of
10 rad/sec was not changed. Results are shown in Table 3.
5 [Table 3]
Change in viscoelasticity before and after filtration and after storage at 40°C for one month in
Examples 8 and 9 and Comparative Examples 4 and 5 (angular velocity 10 rad/sec)
Eta* [Pa•sec
A
Heating Just after gel
Storage at
verage
Solvent condition re aration 40 C for dpiaarmtiectleer
Before After one month [nm]
filtration filtration
Phosphate
buffer
Example 8 containing 4.8 4.5 4.4 620
200 mM
NaCl
HEPES
buffer
Example 9 containing 121°C, 7.3 6.8 6.5 887
137mM min
NaCl
Comparative Phosphate 0.1 or less
No
aggregate
Example 4 buffer formed
Comparative HEPES
0.2
No
aggregate
Example 5 buffer formed
[Comparative Example 4]
(1) Raw material
10. The polysaccharide derivative synthesized in Example 8 was used,
(2) Preparation of hydrogel (prepared using phosphate buffer as a solvent)
To 1980 mg of phosphate buffer, 20 mg of the above-mentioned polysaccharide
derivative was dissolved to prepare a solution at a concentration of 1.0 wt%. The mixture
was heated for 5 minutes at 121°C, filtered with a PVDF filter (pore diameter 5 μm), and
15 heated again for 20 minutes at 121°C.
23
(3) Measurement of viscosity and average particle diameter
Viscoelasticity of the sample prepared in (2) was measured at an angular velocity of
10 rad/sec. Absolute viscosity was about 0,1 Pa-sec or less, showing that the sample was
not gelled. Measurement of average particle diameter for the diluted solution of this sample
5 showed that the scattering intensity was insufficient and that the aggregate was not formed,
[Comparative Example 5]
(1) Raw material
The polysaccharide derivative synthesized in Example 9 was used.
(2) Preparation of hydrogel (prepared using HEPES buffer as a solvent)
10 To 1980 mg of HEPES buffer, 20 mg of the above mentioned polysaccharide
derivative was dissolved to prepare a solution at a concentration of 1.0 wt%. The mixture
was heated for 5 minutes at 121°C, filtered with a PVDF filter (pore diameter 5 μm), and
heated again for 20 minutes at 121°C.
(3) Measurement of viscosity and average particle diameter
15 Viscoelasticity of the sample prepared in (2) was measured at an angular velocity of
10 rad/sec. Absolute viscosity was about 0.3 Pa-sec, showing that the sample was not gelled.
Measurement of average particle diameter for the diluted solution of this sample showed that
the scattering intensity was insufficient and that the aggregate was not formed.
[Reference Example 1]
20 (1) Raw material
The polysaccharide derivative synthesized in Example 4 was used.
(2) Preparation of hydrogel
To 1980 mg of phosphate-buffered physiological saline, 20 mg of the
above-mentioned polysaccharide derivative was dissolved to prepare a solution at a
2 4
concentration of 1.0 wt%. The mixture was repeatedly heated for 20 minutes at 121°C three
times.
(3) DLS Measurement
DLS measurement was performed for the sample prepared in (2), Results are
5 shown in Fig. 9. Although the particle diameter distribution shifted toward the smaller
direction by a treatment at 121°C for 20 minutes, the change in the particle diameter
distribution became less significant to converge in a certain range even after repeating the
same treatment.
Industrial Applicability
10 The hydrogel of the present invention may be used, for example, as medical articles
such as antiadhesive material, a DDS carrier, etc.

CLAIMS
1. A hydrogel comprising a polyanionic polysaccharide derivative having a
hydrophobic group introduced by covalent bonding and a salt solution having a salt
concentration of 50 mM or more and 200 mM or less, the hydrogel having an aggregate
5 structure with an average particle diameter of 100 to 2000 nm in the diluted solution.
2. The hydrogel according to claim 1, wherein the polyanionic polysaccharide is a
polysaccharide having a carboxymethyl group.
3. The hydrogel according to claim 1, wherein the polyanionic polysaccharide is
carboxymethylcellulose.
10 4. The hydrogel according to any of claims 1 to 3, wherein the hydrophobic group has
a phosphate ester structure.
5. The hydrogel according to any of claims 1 to 3, wherein the hydrophobic group has
15
a phosphatidyl group represented by the following formula (1):
0
o®P6mvo 0
H ox (1)
wherein X is hydrogen atom or alkaline metal; R' and R2 are each independently a linear or
branched chain alkyl group or alkenyl group having 9 to 21 carbon atoms.
20 6. The hydrogel according to any of claims 1 to 5, wherein the manufacturing process
of the hydrogel comprises a heating step at 70°C or higher and 140°C or lower and the
average particle diameter of the aggregate is 120 to 1500 mu.
7. The hydrogel according to any of claims I to 5, wherein the manufacturing process
of the hydrogel comprises a heating step at 100°C or higher and under a pressurized condition
26
at atmospheric pressure to 10 atm and the average particle diameter of the aggregate is 150 to
1200 nm.
8. The hydrogel according to any of claims 1 to 7, wherein the equivalency of the
hydrophobic group is 0.25 to 5.0 mol%/sugar residue.
5 9. The hydrogel according to any of claims 1 to 8, wherein the solvent is a salt solution
containing 50 mM or more and 200 rnM or less of sodium chloride.
10. The hydrogel according to any of claims 1 to 8, wherein the solvent is a buffer
containing 120 mM or more and 180 mM or less of sodium chloride with a pH adjusted to 6.5
or more and 7.5 or less and the average particle diameter of the aggregate is 200 to 1000 nm.
10 11. The hydrogel according to any of claims 1 to 8, wherein the solvent is a
phosphate-buffered physiological saline.
12. The hydrogel according to any of claims I to 11, wherein the concentration of the
polysaccharide derivative is 0.3 to 3.0 wt%.
13. A hydrogel obtained by filtrating the hydrogel according to any of claims 1 to 12
15 with a porous filter having a pore diameter of 5 μm or more.
14. The hydrogel according to claim 13, wherein the number of insoluble particles of a
diameter of 10 μm or more is 3000 or less per 1 mL and/or the number of insoluble particles
of a diameter of 25 μm or more is 300 or less per 1 mL.
15. A method for manufacturing a hydrogel, comprising the steps of preparing a mixture
20 containing a polyanionic polysaccharide derivative having a hydrophobic group introduced
and a salt solution with a salt concentration of 50 mM or more and 200 mM or less and
subjecting this mixture to heat treatment.
16. The manufacturing method according to claim 15, wherein the step of the heat
treatment is a step of treatment at 70 to 140°C and for I minute or longer and 3 hours or
27
shorter,
17. The manufac i i-; method according to claim 15 or 16, wherein the salt solution is
physiological saline.
18. The manufacturing method according to any of claims 15 to 17, comprising a step of
further filtering the hydrogel with a porous filter having a pore diameter of 5 tLrn or more
after the heat treatment.
Dated this 12/11/2012
RANJNA WLI/-ITA-DUTT
OF REMFRY & SAGAR
ATTORNEY FOR TIE APPLICANT[S]
ABSTRACT
A hydrogel composed of a polyanionic polysaccharide derivative having a
hydrophobic group introduced by covalent bonding and a salt solution with a salt
concentration of 50 mM or more and 200 mM or less, the hydrogel having an aggregate
5 structure with an average particle diameter of 100 to 2000 nm in the diluted solution, as well
as a method for manufacturing the hydrogel comprising the steps of preparing a mixture
containing a polyanionic polysaccharide derivative having a hydrophobic group introduced
and a salt solution with a salt concentration of 50 mM or more and 200 mM or less and
subjecting the mixture to heat treatment. The hydrogel of the present invention has a
10 longterm stability and may be filtered using a porous filter having a pore diameter of 5 μm
or more.