U.S. patent application number 16/483146 was filed with the patent office on 2020-07-23 for hydrogel using, as substrate, hyaluronic acid derivative modified with gallol group and use thereof.
The applicant listed for this patent is AMTIXBIO CO., LTD. Invention is credited to Jung Ho Cho, Seung Woo Cho, Jong-Seung Lee, Jung-Seung Lee.
Application Number | 20200230288 16/483146 |
Document ID | / |
Family ID | 63040885 |
Filed Date | 2020-07-23 |
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United States Patent
Application |
20200230288 |
Kind Code |
A1 |
Cho; Seung Woo ; et
al. |
July 23, 2020 |
HYDROGEL USING, AS SUBSTRATE, HYALURONIC ACID DERIVATIVE MODIFIED
WITH GALLOL GROUP AND USE THEREOF
Abstract
The present invention provides a hydrogel platform using, as a
substrate, hyaluronic acid (HA) conjugated to a pyrogallol (PG)
moiety. The HA-PG conjugate of the present invention can be rapidly
crosslinked by two different methods, in each of which an oxidizing
agent is used or a pH is adjusted. The hydrogel of the present
invention can not only have excellent biocompatibility, but also
can have efficiently controlled physical characteristics such as a
crosslinking rate, elasticity, and adhesive strength, depending on
each crosslinking method. On the basis of such excellent stability
and functionality, the hydrogel of the present invention can be
used in various fields including drug delivery, biopharmaceutical
materials such as a wound healing agent or anti-adhesive agent,
medicines, and cosmetic products.
Inventors: |
Cho; Seung Woo; (Seoul,
KR) ; Lee; Jung-Seung; (Seoul, KR) ; Cho; Jung
Ho; (Seoul, KR) ; Lee; Jong-Seung; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMTIXBIO CO., LTD |
Seoul |
|
KR |
|
|
Family ID: |
63040885 |
Appl. No.: |
16/483146 |
Filed: |
February 2, 2018 |
PCT Filed: |
February 2, 2018 |
PCT NO: |
PCT/KR2018/001473 |
371 Date: |
August 2, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1808 20130101;
A61K 47/36 20130101; C08J 3/24 20130101; A61L 27/20 20130101; A61K
38/1891 20130101; A61P 17/02 20180101; C08B 37/0072 20130101; A61L
27/54 20130101; A61L 27/52 20130101; C08J 3/075 20130101; C08J
2305/08 20130101; C08L 5/08 20130101 |
International
Class: |
A61L 27/20 20060101
A61L027/20; A61K 47/36 20060101 A61K047/36; A61L 27/52 20060101
A61L027/52; A61L 27/54 20060101 A61L027/54; A61P 17/02 20060101
A61P017/02; A61K 38/18 20060101 A61K038/18; C08B 37/08 20060101
C08B037/08; C08J 3/24 20060101 C08J003/24; C08J 3/075 20060101
C08J003/075 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 2, 2017 |
KR |
10-2017-0014855 |
Feb 2, 2017 |
KR |
10-2017-0014856 |
Claims
1. A method for preparing a hyaluronic acid hydrogel, comprising: a
step of crosslinking hyaluronic acid derivatives, each of which is
modified with a pyrogallol group, wherein in the hyaluronic acid
derivative, hyaluronic acid has been modified with a pyrogallol
group.
2. The method according to claim 1, wherein the hyaluronic acid
derivative is represented by the following Formula 1: ##STR00010##
(in the above Formula 1, R.sub.1 is a hydroxyl group or
##STR00011## and n is an integer of 1 to 1,000).
3. The method according to claim 1 of 2, wherein in the
crosslinking step, crosslinking is carried out by adding an
oxidizing agent or a pH adjusting agent.
4. The method according to claim 3, wherein the oxidizing agent is
any one selected from the group consisting of sodium periodate,
hydrogen peroxide, horseradish peroxidase, and tyrosinase.
5. The method according to claim 3, wherein in the crosslinking
step carried out by adding the oxidizing agent, crosslinking
represented by the following Formula 2 is formed: ##STR00012## (in
the above Formula 2, HA' represents hyaluronic acid in which the
carboxyl group is substituted with an amide group).
6. The method according to claim 3, wherein the pH adjusting agent
is any one selected from the group consisting of sodium hydroxide,
lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium
hydroxide, magnesium hydroxide, calcium hydroxide, strontium
hydroxide, and barium hydroxide.
7. The method according to claim 3, wherein in the crosslinking
step carried out by adding the pH adjusting agent, crosslinking
represented by the following Formula 3 is formed: ##STR00013## (in
the above Formula 3, HA' represents hyaluronic acid in which the
carboxyl group is substituted with an amide group).
8. A hyaluronic acid hydrogel prepared by crosslinking hyaluronic
acid derivatives, each of which is represented by the following
Formula 1, wherein crosslinking represented by the following
Formula 2 is formed: ##STR00014## ##STR00015## (in the above
Formula 1, R.sub.1 is a hydroxyl group or and n is an integer of 1
to 1,000) ##STR00016## (in the above Formula 2, HA' represents
hyaluronic acid in which the carboxyl group is substituted with an
amide group).
9. The hyaluronic acid hydrogel according to claim 8, wherein the
hyaluronic acid derivative has a molecular weight of 10,000 Da to
2,000,000 Da.
10. The hyaluronic acid hydrogel according to claim 8, wherein the
hyaluronic acid derivative has a gallol group substitution rate of
0.1% to 50%.
11. A hyaluronic acid hydrogel prepared by crosslinking hyaluronic
acid derivatives, each of which is represented by the following
Formula 1, wherein crosslinking represented by the following
Formula 3 is formed: ##STR00017## (in the above Formula 1, R.sub.1
is a hydroxyl group or ##STR00018## and n is an integer of 1 to
1,000) ##STR00019## (in the above Formula 3, HA' represents
hyaluronic acid in which the carboxyl group is substituted with an
amide group).
12. The hyaluronic acid hydrogel according to claim 11, wherein the
hyaluronic acid derivative has a molecular weight of 10,000 Da to
2,000,000 Da.
13. The hyaluronic acid hydrogel according to claim 11, wherein the
hyaluronic acid derivative has a gallol group substitution rate of
0.1% to 50%.
14. A scaffold for tissue engineering, comprising: the hyaluronic
acid hydrogel according to claim 8.
15. A drug delivery carrier, comprising: the hyaluronic acid
hydrogel according to claim 8.
16. The drug delivery carrier according to claim 5, wherein the
drug is an antibody, an antibody fragment, a nucleic acid including
DNA, RNA, or siRNA, a peptide, a gene, a protein, a stem cell, or a
chemical compound.
17. A filler composition, comprising: the hyaluronic acid hydrogel
according to claim 8.
18. An adhesion barrier composition, comprising: the hyaluronic
acid hydrogel according to claim 8.
19. A wound dressing composition, comprising: the hyaluronic acid
hydrogel according to claim 8.
20. A hyaluronic acid derivative which has the following Formula 1
and is modified with a pyrogallol group: ##STR00020## (in the above
Formula 1, R.sub.1 is a hydroxyl group or ##STR00021## and n is an
integer of 1 to 1,000).
21. A filler composition, comprising: a hyaluronic acid derivative
modified with a pyrogallol group.
22. The filler composition according to claim 21, wherein the
hyaluronic acid derivative is represented by the following Formula
I: ##STR00022## (in the above Formula 1, R.sub.1 is a hydroxyl
group or ##STR00023## and n is an integer of 1 to 1,000).
23. The filler composition according to claim 22, wherein the
hyaluronic acid derivative has a molecular weight of 10,000 Da to
2,000,000 Da.
24. The filler composition according to claim 22, wherein the
hyaluronic acid derivative has a gallol group substitution rate of
1% to 20%.
25. The filler composition according to claim 22, wherein the
hyaluronic acid derivative is contained in an amount of 0.1% (w/v)
to 10% (w/v) with respect to the entire filler composition.
26. The filler composition according to claim 21, wherein the
composition is in a liquid state ex vivo, and forms a gelated state
in vivo without a crosslinking agent.
27. The filler composition according to claim 21, wherein the
composition is injected into any one site selected from the group
consisting of a tear trough region, a glabellar frown line region,
an eye-rim region, a forehead region, a nasal bridge region, a
nasolabial line region, a marionette line region, and a neck
wrinkle region.
28. The filler composition according to claim 21, further
comprising: any one cell growth factor selected from the group
consisting of fibroblast growth factor (FGF), epithelial cell
growth factor (EGF), keratinocyte growth factor (KGF), transforming
growth factor alpha (TGF-.alpha.), transforming growth factor beta
(TGF-.beta.), granulocyte colony stimulating factor (GCSF),
insulin-like growth factor (IGF), vascular endothelial growth
factor (VEGF), hepatocyte growth factor (HGF), platelet-derived
growth factor-BB (PDGF-BB), brain-derived neurotrophic factor
(BDNF), and glial cell-derived neurotrophic factor (GDNF).
29. The filler composition of claim 21, further comprising: any one
component selected from the group consisting of a local anesthetic,
an antioxidant, a vitamin, and combinations thereof.
Description
TECHNICAL FIELD
[0001] The present invention relates to a hydrogel based on a
hyaluronic acid derivative modified with a gallol group, and a use
thereof.
BACKGROUND ART
[0002] Interest in functional biomaterials is increasing as markets
for medical and biotech industries, cosmetics industry, and the
like are rapidly expanding. In particular, development of
biocompatible materials using natural polymers for which stability
is ensured, rather than chemically synthesized polymers which may
cause toxicity or side effects, is becoming more popular.
[0003] Various researches and developments have been conducted on
hyaluronic acid as a biocompatible material. Hyaluronic acid is a
bio-derived polymer which has little side effects when applied to
the living body, and is hydrophilic due to a chemical structure of
the sugar contained therein. In addition, due to containing a lot
of moisture, hyaluronic acid is known to have a physical buffering
effect and a lubricating effect on friction in the joints, and to
be involved in flexibility of the skin. In addition, hyaluronic
acid has protection characteristics against bacterial invasion from
the outside and is biodegraded by hyaluronidase in a living body
when transplanted into the living body. Hyaluronic acid is utilized
as an important material for drug delivery systems by causing the
hyaluronic acid to be bound to various drugs. In particular, since
approval by the US Food and Drug Administration, hyaluronic acid
has been extensively utilized as a medical biomaterial, a material
of a scaffold for tissue engineering, and a polymer for drug
delivery. In addition, hyaluronic acid is abundantly present in
several different layers of the skin, and has complex functions
such as a function to supply moisture, a function to assist with
tissue of extracellular matrix, a function to act as a filling
material, and a function to be involved in tissue regeneration
mechanism. However, with aging, amounts of hyaluronic acid,
collagen, elastin, and other matrix polymers present in the skin
decrease. For example, repeated exposure to ultraviolet rays from
the sun causes dermal cells to not only decrease their hyaluronic
acid production, but also to have an increased degradation rate of
hyaluronic acid. Loss of this material results in wrinkles, holes,
moisture loss, and/or other undesirable conditions that contribute
to aging. Therefore, as one of methods for improving skin
condition, a filler composition containing hyaluronic acid as a
main component is widely used.
[0004] As conventional hyaluronic acid-related technologies,
examples of synthesizing crosslinked insoluble hyaluronic acid
derivatives using compounds having two functional groups such as
bisepoxide, bishalide, and formaldehyde have been reported in
several pieces of literature. In particular, U.S. Pat. No.
4,582,865 discloses an example of using divinylsulfone for
crosslinking of hyaluronic acid; U.S. Pat. No. 4,713,448 discloses
a crosslinking reaction using formaldehyde; and U.S. Pat. No.
5,356,883 discloses a synthesis example for a hyaluronic acid
derivative gel whose carboxyl group has been modified with
O-acylurea or N-acylurea using various carbodiimides. However,
hyaluronic acid crosslinked products prepared by the methods in
these patents have low stability against a hyaluronic
acid-degrading enzyme and a high content of unreacted chemicals,
which may cause bio-toxicity. In addition, it is not easy to
control crosslinking or physical properties of these products
depending on an intended use. Thus, there are limitations in
applying such products to various medical materials. Therefore, it
is still required to develop a technique capable of easily
controlling physical properties of a hyaluronic acid hydrogel while
maintaining excellent biocompatibility thereof.
[0005] Accordingly, in order to solve these problems, the present
inventors have continually made efforts to develop a technique
capable of improving functionality of hyaluronic acid which is a
biocompatible material. As a result, the present inventors have
developed a hydrogel platform technique based on hyaluronic acid
modified with a pyrogallol group, and have completed the present
invention on the basis of this technique.
Technical Problem
[0006] Accordingly, an object of the present invention is to
provide a hyaluronic acid derivative prepared by modifying
hyaluronic acid with a pyrogallol group and a method for preparing
the same.
[0007] Another object of the present invention is to provide a
method for preparing a hyaluronic acid derivative hydrogel,
comprising a step of crosslinking the hyaluronic acid
derivatives.
[0008] Yet another object of the present invention is to provide a
hyaluronic acid derivative hydrogel having a structure in which the
hyaluronic acid derivatives are crosslinked.
[0009] Still yet another object of the present invention is to
provide a drug delivery carrier or drug delivery system (DDS) using
the hyaluronic acid derivative hydrogel.
[0010] Still yet another object of the present invention is to
provide a medical material such as a scaffold for tissue
engineering, using the hyaluronic acid derivative hydrogel.
[0011] Still yet another object of the present invention is to
provide a wound dressing or adhesion barrier based on the
hyaluronic acid derivative.
[0012] Still yet another object of the present invention is to
provide a filler composition comprising the hyaluronic acid
derivative hydrogel.
[0013] Still yet another object of the present invention is to
provide a method for improving skin wrinkles, comprising a step of
injecting the filler composition into or under the skin of an
individual.
[0014] However, the technical problems to be solved by the present
invention are not limited to the above-mentioned problems, and
other problems not mentioned can be clearly understood by those
skilled in the art from the following description.
Solution to Problem
[0015] In order to achieve the objects of the present invention as
described above, the present invention provides a method for
preparing a hyaluronic acid hydrogel, the method comprising a step
of crosslinking hyaluronic acid derivatives, each of which is
modified with a gallol group, wherein the hyaluronic acid
derivative has been modified with a gallol group due to a reaction
between hyaluronic acid and 5'-hydroxydopamine.
[0016] In an embodiment of the present invention, the hyaluronic
acid derivative may be represented by the following Formula 1,
wherein the hyaluronic acid derivative may have a molecular weight
of 10,000 Da to 2,000,000 Da, and may have a gallol group
substitution rate of about 0.1% to 50%.
##STR00001##
[0017] (In the above Formula 1, R.sub.1 is a hydroxyl group or
##STR00002##
and n is an integer of 1 to 1000).
[0018] In another embodiment of the present invention, in the
crosslinking step, crosslinking may be carried out by adding an
oxidizing agent or a pH adjusting agent, in which the oxidizing
agent may be sodium periodate, hydrogen peroxide, horseradish
peroxidase, or tyrosinase, and the pH adjusting agent may be sodium
hydroxide, lithium hydroxide, potassium hydroxide, rubidium
hydroxide, cesium hydroxide, magnesium hydroxide, calcium
hydroxide, strontium hydroxide, or barium hydroxide.
[0019] In yet another embodiment of the present invention, in the
crosslinking step carried out by adding the oxidizing agent,
crosslinking represented by the following Formula 2 may be formed
between the hyaluronic acid derivatives.
##STR00003##
[0020] (In the above Formula 2, HA' represents hyaluronic acid in
which the carboxyl group is substituted with an amide group.)
[0021] As still yet another embodiment of the present invention, in
the crosslinking step carried out by adding the pH adjusting agent,
crosslinking represented by the following Formula 3 may be formed
between the hyaluronic acid derivatives.
##STR00004##
[0022] (In the above Formula 3, HA' represents hyaluronic acid in
which a carboxy group is substituted with an amide group.)
[0023] In addition, the present invention provides a hyaluronic
acid hydrogel prepared by crosslinking hyaluronic acid derivatives,
each of which is represented by the above Formula 1, for example, a
hyaluronic acid hydrogel in which crosslinking represented by the
above Formula 2 or Formula 3 is formed between the hyaluronic acid
derivatives; and a carrier for delivery of a bioactive substance,
comprising the hyaluronic acid hydrogel. From this viewpoint, in an
embodiment of the present invention, the carrier includes, but is
not limited to, an antibody, an antibody fragment, a protein, a
peptide, a polypeptide, a small molecule chemical compound, DNA
and/or RNA, siRNA, a gene, and stem cells including adult stem
cells, mesenchymal stem cells, or induced pluripotent stem cells
(iPSCs). From this viewpoint, in another embodiment of the present
invention, the carrier for delivery of a bioactive substance
provides sustained release of the bioactive substance in vivo and
ex vivo.
[0024] In addition, the present invention provides a hyaluronic
acid hydrogel prepared by crosslinking hyaluronic acid derivatives,
each of which is represented by the above Formula 1, for example, a
hyaluronic acid hydrogel in which crosslinking represented by the
above Formula 2 or Formula 3 is formed between the hyaluronic acid
derivatives; and a scaffold for tissue engineering, comprising the
hyaluronic acid hydrogel.
[0025] In addition, the present invention provides a filler
composition, comprising a hyaluronic acid derivative modified with
a gallol group or a hyaluronic acid derivative hydrogel prepared by
crosslinking the hyaluronic acid derivatives.
[0026] In an embodiment of the present invention according to this
purpose, the hyaluronic acid derivative may have a molecular weight
of 10,000 Da to 2,000,000 Da; and the hyaluronic acid derivative
may have a pyrogallol group substitution rate of 0.1% to 50%,
preferably 1% to 30%, and more preferably 2% to 20%. In another
embodiment of the present invention, the hyaluronic acid derivative
may be contained in an amount of 0.1% (w/v) to 15% (w/v) with
respect to the entire filler composition.
[0027] In yet another embodiment of the present invention, the
filler composition may be in a liquid state ex vivo and may form a
gelated state in vivo without a crosslinking agent. In still yet
another embodiment of the present invention, the filler composition
may be injected into any one site selected from the group
consisting of a tear trough region, a glabellar frown line region,
an eye-rim region, a forehead region, a nasal bridge region, a
nasolabial line region, a marionette line region, and a neck
wrinkle region. In still yet another embodiment of the present
invention, the filler composition may further comprise any one cell
growth factor selected from the group consisting of fibroblast
growth factor (FGF), epithelial cell growth factor (EGF),
keratinocyte growth factor (KGF), transforming growth factor alpha
(TGF-a), transforming growth factor beta (TGF-.beta.), granulocyte
colony stimulating factor (GCSF), insulin-like growth factor (IGF),
vascular endothelial growth factor (VEGF), hepatocyte growth factor
(HGF), platelet-derived growth factor-BB (PDGF-BB), brain-derived
neurotrophic factor (BDNF), and glial cell-derived neurotrophic
factor (GDNF). In still yet another embodiment of the present
invention, the filler composition may further comprise any one
component selected from the group consisting of a local anesthetic,
an antioxidant, a vitamin, and combinations thereof.
[0028] In addition, the present invention provides a method for
preparing a filler composition, comprising a step of introducing a
pyrogallol group into a skeleton of glucuronic acid in hyaluronic
acid, to prepare a hyaluronic acid derivative; and a step of adding
the hyaluronic acid derivative to an aqueous medium and performing
mixing.
[0029] In addition, the present invention provides a method for
improving skin wrinkles, comprising a step of injecting the filler
composition into or under the skin of an individual.
[0030] In addition, the present invention provides a wound healing
agent or adhesion barrier, comprising a hyaluronic acid derivative
modified with a gallol group or a hyaluronic acid derivative
hydrogel prepared by crosslinking the hyaluronic acid
derivatives.
Advantageous Effects of Invention
[0031] The technique for preparing a hyaluronic acid hydrogel
according to the present invention is based on a hyaluronic acid
derivative modified with a gallol group, and includes a step of
crosslinking the hyaluronic acid derivatives under an appropriate
oxidizing or specific pH condition. The present invention makes it
possible to effectively control physical properties of a hydrogel
such as crosslinking rate, elasticity, and adhesive strength
depending on each crosslinking method, while allowing the hydrogel
to have excellent biocompatibility.
[0032] Thus, the present invention can be utilized for various
fields such as the medical field and the cosmetics field.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIG. 1 illustrates a process of synthesizing a hyaluronic
acid derivative according to the present invention.
[0034] FIG. 2 illustrates results obtained by analyzing, with (a)
FT-IR or (b) H-NMR, a structure of the hyaluronic acid derivative
according to the present invention.
[0035] FIG. 3 illustrates schematic views showing appearances of
hyaluronic acid hydrogels and changes in crosslinking thereof,
depending on different crosslinking methods.
[0036] FIG. 4 illustrates (a) a result obtained by analyzing, with
UV-vis, changes in chemical structure which occur in the process of
preparing a hyaluronic acid hydrogel using NaIO.sub.4, and (b) a
crosslinking mechanism based on the result.
[0037] FIG. 5 illustrates (a) a result obtained by analyzing, with
UV-vis, changes in chemical structure which occur in the process of
preparing a hyaluronic acid hydrogel using NaOH, and (b) a
crosslinking mechanism based on the result.
[0038] FIG. 6 illustrates results obtained by identifying rates at
which the hyaluronic acid hydrogels according to the present
invention are formed. (a) illustrates results obtained by visually
identifying hydrogel formation over time, and (b) illustrates
results obtained by identifying changes in elastic modulus of the
hydrogels over time.
[0039] FIG. 7 illustrates results obtained by identifying rigidity
and elasticity of the hyaluronic acid hydrogels according to the
present invention. (a) illustrates results obtained by identifying
changes in elastic modulus of the hydrogels at 0.1 to 1 Hz, and (b)
illustrates results obtained by comparing elastic modulus and tan 6
(G''/G') of the hydrogels.
[0040] FIG. 8 illustrates results obtained by identifying changes
in rigidity and elasticity of the NaIO.sub.4-crosslinked hyaluronic
acid hydrogels depending on molecular weights and concentrations of
hyaluronic acid derivatives. (a) illustrates results obtained by
identifying changes in elastic modulus of the hydrogels at 0.1 to 1
Hz, and (b) illustrates results obtained by comparing elastic
modulus and tan 6 (G''/G') of the hydrogels.
[0041] FIG. 9 illustrates results obtained by identifying adhesive
strength of the hyaluronic acid hydrogels according to the present
invention. (a) illustrates a result obtained by measuring, with a
rheological instrument, changes in force applied depending on a
distance between plates, and (b) illustrates a result obtained by
quantifying and evaluating adhesive strength.
[0042] FIG. 10 illustrates results obtained by identifying swelling
and degradation patterns of the hyaluronic acid hydrogels according
to the present invention, and such results were obtained by
quantifying and evaluating swelling ratio and degradation ratio of
the hyaluronic acid hydrogels prepared by using hyaluronic acid
derivatives which have been synthesized by adding 0.5.times. (a) or
1.0.times. (b) of 5-hydroxydopamine relative to a molar
concentration of hyaluronic acid.
[0043] FIG. 11 illustrates results obtained by identifying whether
the hyaluronic acid hydrogels according to the present invention
cause cytotoxicity and inflammatory response.
[0044] (a) and (b) respectively illustrate live/dead staining
(scale bar=100 .mu.m) (a) and viability (b) of hADSCs at days 1, 3,
and 7 after encapsulation of the hADSCs in order to evaluate
toxicity of HA-PG hydrogels. (c) illustrates a result obtained by
performing an enzyme-linked immunoadsorption assay for
quantification of TNF-.alpha. secreted from macrophages (RAW 264.7)
when co-cultured with a NaIO.sub.4 or NaOH solution-crosslinked
HA-PG hydrogel (n=4,**p<0.01 vs LPS group).
[0045] FIG. 12 illustrates results obtained by identifying in vivo
compatibility of the hyaluronic acid hydrogels according to the
present invention. (a) illustrates shapes of HA-PG hydrogels (DS
8%) recovered from a subcutaneous region of a mouse on days 0, 7,
28, and 84 after being transplanted into the subcutaneous region.
(b) illustrates in vivo biodegradation profiles of the HA-PG
hydrogels. (c) and (d) respectively illustrate H&E staining (c)
and toluidine blue staining (d) of the hydrogels recovered together
with adjacent tissue on day 28 after transplantation (scale bar=300
.mu.m).
[0046] FIG. 13 illustrates results obtained by identifying in vivo
degradation patterns of the hyaluronic acid hydrogels according to
the present invention in vivo. (a) illustrates results obtained by
visually identifying changes in volume of the hydrogels and (b)
illustrates results obtained by identifying weights of the
remaining hydrogels, after the hydrogels are subcutaneously
transplanted into mice.
[0047] FIG. 14 illustrates results obtained by identifying a
possibility of utilizing, as a drug delivery preparation, a
hydrogel prepared by using NaIO.sub.4. (a) illustrates results
obtained by identifying formation and presence of microparticles
before or after freeze-drying, and (b) illustrates a result
obtained by identifying released amounts of BSA which has been
encapsulated in the microparticles.
[0048] FIG. 15 illustrates results obtained by identifying a
possibility of utilizing, as a drug delivery preparation, a
hydrogel prepared by using NaIO.sub.4. (a) illustrates a
crosslinking reaction using NaIO.sub.4, (b) illustrates a
microscope photograph showing formation of HA-PG microparticles
(scale bar=50 .mu.m), and (c) illustrates a cumulative amount of
VEGF released from a bulk HA-PG hydrogel or HA-PG microparticles
(PBS, 37.degree. C.) (n=4).
[0049] FIG. 16 illustrates VEGF delivery and therapeutic effects of
a hydrogel prepared by using NaIO.sub.4 in a hindlimb ischemic
mouse model. (a) illustrates photographs showing results obtained
by intramuscularly injecting HA-PG microparticles containing VEGF
and making an observation at day 0 (day of drug injection) and day
28, (b) illustrates a numerical representation of the results (n=4
to 5), (c) illustrates results of H&E staining and MT staining,
using normal mouse tissue as a control (scale bar=100 .mu.m), (d)
illustrates a result obtained by quantifying fibrotic area in an
ischemic leg (n=12, **p<0.01 vs PBS group, ##p<0.01 vs HA-PG
group, and @p<0.05 vs VEGF group), and (e) illustrates
photographs of ischemic leg muscle which has been immunostained for
.alpha.-SMA (arteriole formation) and vWF (capillary formation).
Black arrows indicate formation of arteriole (.alpha.-SMA positive)
and capillary (vWF positive) in ischemic tissue, respectively
(scale bar=100 .mu.m). (f) illustrates the number of .alpha.-SMA
positively stained lumens and vWF positive capillaries in ischemic
muscle (n=18.20, *p<0.05, and **p<0.01 vs PBS group,
##p<0.01 vs HA-PG group, and @p<0.01 vs VEGF group).
[0050] FIG. 17 illustrates preparation of a tissue-adhesive HA-PG
hydrogel using NaOH-mediated crosslinking. (a) illustrates
formation of a hyaluronic acid derivative hydrogel by NaOH-mediated
crosslinking. (b) illustrates a result obtained by comparing
adhesive strength between a hyaluronic acid derivative hydrogel
crosslinked with NaOH and a hyaluronic acid derivative hydrogel
crosslinked with NaIO.sub.4. (c) illustrates a result obtained by
comparing mean detachment strength between the respective HA-PG
hydrogels (n=3, **p<0.01 vs NaIO.sub.4 group).
[0051] In FIG. 18, (a) briefly illustrates adhesion chemistry of a
NaOH-induced HA-PG hydrogel, and (b) illustrates photographs
showing direct adhesion of the hydrogel to various mouse organs
(heart, kidney, and liver).
[0052] In FIG. 19, (a) illustrates a result obtained by applying a
NaOH-induced HA-PG hydrogel on a mouse liver surface and performing
H&E staining of the hydrogel adhered on the surface of liver
tissue after 7 days (scale bar=500 .mu.m). (b) illustrates a result
obtained by transplanting human adipose tissue-derived stem cells
(hADSCs) onto the mouse liver using HA-PG crosslinked with NaOH,
and then representing together immunostaining of original liver
tissue and the hADSCs in the hydrogel structure of the present
invention. Cells which had been labeled with Dil prior to
transplantation were detected (left). Immunostaining was performed
for CD44 and cell nuclei were counter-stained with DAPI (right)
(scale bar=500 .mu.m).
[0053] FIG. 20 illustrates gelation of an HA-PG solution according
to the present invention by oxidizing power in the body, in which
FIG. 20(a) illustrates injection of the HA-PG solution and gelation
thereof by oxidizing power in the body; and FIG. 20(b) illustrates
a result obtained by visually identifying the skin of a mouse which
contains a hydrogel formed in the body.
[0054] FIG. 21 illustrates results obtained by injecting the HA-PG
solution according to the present invention into the skin, and then
identifying whether the hydrogel formed in the body is maintained
in the body, in which FIG. 5(a) illustrates results obtained by
visually identifying the hydrogel formed in the body (weeks 0, 10,
and 24); and FIG. 5(b) illustrates a result obtained by measuring
changes in weight of the hydrogel formed in the body for about 6
months.
[0055] FIG. 22 illustrates results obtained by identifying
conditions for formation of an HA-PG hydrogel of the HA-PG solution
according to the present invention and for injection thereof into
the body, in which FIG. 22(a) illustrates a schematic view showing
a process of forming the HA-PG hydrogel; and FIG. 22(b) illustrates
a result obtained by comparing a needle size which is injectable
into the body with conventional filler products (Megafill,
Perlane).
[0056] FIG. 23 illustrates results obtained by injecting the HA-PG
solution according to the present invention into the skin, and then
identifying wrinkle-improving effects before and after the
injection, in which FIG. 23(a) illustrates a result obtained by
causing the wrinkled skin to be made into replicas and making a
comparison; and FIG. 23(b) illustrates results obtained by
quantifying the area, length, and depth of the wrinkles and making
a comparison.
[0057] FIG. 24 illustrates results obtained by making a comparison
with conventional filler products (Megafill, Perlane) in terms of
ability to be maintained in the body, in which FIG. 24(a)
illustrates results obtained by injecting, into the skin, the HA-PG
solutions according to the present invention (200 KDa, 1 MDa), the
Megafill product, and the Perlane product, and then visually
identifying the hydrogels formed in the body (days 0, 14, 28, 56,
84, 168, and 252); and FIG. 24(b) illustrates a result obtained by
measuring changes in weight of the hydrogels formed in the body for
about 9 months.
[0058] FIG. 25 illustrates results obtained by making a comparison
with conventional filler products (Megafill, Perlane) in terms of
ability to be maintained in the body, in which FIG. 25(a)
illustrates results obtained by injecting, into the skin, the HA-PG
solutions according to the present invention (200 KDa, 1 MDa), the
Megafill product, and the Perlane product, and then visually
identifying the hydrogels formed in the body (days 0, 14, 28, 56,
84, and 168); and FIG. 25(b) illustrates a result obtained by
measuring changes in volume of the hydrogels formed in the body for
about 9 months.
[0059] FIG. 26 illustrates results obtained by identifying adhesive
strength in the body of the hydrogel formed in the body, in which
FIG. 26(a) illustrates a result for the Perlane product; and FIG.
26(b) illustrates results for the HA-PG solutions (200 KDa, 1
MDa).
[0060] FIG. 27 illustrates results obtained by making a comparison
with conventional filler products (Megafill, Perlane) in terms of
injectability, and such results were obtained by identifying
changes in extrusion force while injecting the HA-PG solutions
according to the present invention (200 KDa, 1 MDa), the Megafill
product, and the Perlane product using injection needles of various
sizes (21 G, 25 G, 29 G, 30 G).
[0061] FIG. 28 illustrates results obtained by making a comparison
with conventional filler products (Megafill, Perlane) in terms of
injectability, in which FIG. 28(a) illustrates a result obtained by
identifying changes in extrusion force while injecting the HA-PG
solutions according to the present invention (200 KDa, 1 MDa), the
Megafill product, and the Perlane product using an injection needle
of 30 G; and FIG. 28(b) illustrates a result obtained by
identifying changes in extrusion force while injecting the HA-PG
solutions according to the present invention (200 KDa, 1 MDa), the
Megafill product, and the Perlane product using an injection needle
of 29 G.
[0062] FIG. 29 illustrates results obtained by making a comparison
with conventional filler products (Megafill, Perlane) in terms of
injectability, in which FIG. 29(a) illustrates a result obtained by
comparing break loose forces while injecting the HA-PG solutions
according to the present invention (200 KDa, 1 MDa), the Megafill
product, and the Perlane product using injection needles of various
sizes (21 G, 25 G, 29 G, 30 G); and FIG. 29(b) illustrates a result
obtained by comparing dynamic glide forces while injecting the
HA-PG solutions according to the present invention (200 KDa, 1
MDa), the Megafill product, and the Perlane product using injection
needles of various sizes (21 G, 25 G, 29 G, 30 G).
[0063] FIG. 30 illustrates results obtained by injecting, into the
skin, the HA-PG solutions according to the present invention in
which epithelial cell growth factor (20 ng/ml, 1 .mu.g/ml, 20
.mu.g/ml) has been encapsulated, and then identifying
wrinkle-improving effects before and after the injection, in which
FIG. 30(a) illustrates results obtained by causing the wrinkled
skin to be made into replicas and making a comparison; and FIG.
30(b) illustrates results obtained by quantifying the area, length,
and depth of the wrinkles and making a comparison.
[0064] FIG. 31 illustrates results obtained by injecting, into the
skin, the HA-PG solutions according to the present invention in
which epithelial cell growth factor (20 ng/ml, 1 .mu.g/ml, 20
.mu.g/ml) has been encapsulated, performing H&E staining of OCT
frozen sections thereof, and performing histopathological
examination.
[0065] FIG. 32 illustrates results obtained by comparing skin
tissue-regenerating effects over time (1 month) among a
conventional filler product (Perlane), the HA-PG solution in which
epithelial cell growth factor has not been encapsulated, and the
HA-PG solution according to the present invention in which
epithelial cell growth factor (10 .mu.g/ml) has been encapsulated,
and such results were obtained by performing H&E staining of
OCT frozen sections thereof and performing histopathological
examination.
[0066] FIG. 33 illustrates results obtained by comparing skin
tissue-regenerating effects over time (1 month) among a
conventional filler product (Perlane), the HA-PG solution in which
epithelial cell growth factor has not been encapsulated, and the
HA-PG solution according to the present invention in which
epithelial cell growth factor (10 .mu.g/ml) has been encapsulated,
and such results were obtained by performing Masson's trichrome
(MT) staining of OCT frozen sections thereof and performing
histopathological examination.
[0067] FIG. 34 illustrates results obtained by applying the HA-PG
solution according to the present invention to a wound site, and
then visually identifying whether a hydrogel is formed at the wound
site, and thus the hydrogel is adhered thereto.
[0068] FIG. 35 illustrates a view briefly showing a process of
formulating the HA-PG solution according to the present invention
into a powder form.
[0069] FIG. 36 illustrates results obtained by injecting, into the
skin, the HA-PG solution according to the present invention which
has been re-solubilized from a freeze-dried powder form thereof,
and then visually identifying a hydrogel formed in the body.
[0070] FIG. 37 illustrates results obtained by identifying storage
stability of the HA-PG solution according to the present invention,
and such results were obtained by identifying whether or not the
HA-PG solution is gelated in a storage container while storing the
HA-PG solution at room temperature (25.degree. C.) or in a
refrigerated state (4.degree. C.).
[0071] FIG. 38 illustrates results obtained by identifying storage
stability of the HA-PG solution according to the present invention.
Nitrogen gas was injected into a storage container to block contact
between the HA-PG solution and oxygen, and then the HA-PG solution
was stored in a refrigerated state (4.degree. C.) for 3 days; on
the other hand, the HA-PG solution was stored in a frozen state
(-80.degree. C.) for 10 days. Then, these solutions were injected
into the skin, and hydrogels formed in the body were visually
identified, so that the results were obtained.
DETAILED DESCRIPTION OF INVENTION
[0072] Hereinafter, the present invention will be described in
detail.
[0073] The present invention provides a method for preparing a
hyaluronic acid hydrogel, the method comprising a step of
crosslinking hyaluronic acid derivatives, each of which is modified
with a gallol group, wherein the hyaluronic acid derivative has
been modified with a gallol group due to a reaction between
hyaluronic acid and 5'-hydroxydopamine.
[0074] The term "hyaluronic acid (HA)" as used herein refers to a
high molecular linear polysaccharide which contains, as a repeating
unit, a disaccharide in which D-glucuronic acid (GlcA) and
N-acetyl-D-glucosamine (GlcNAc) are linked via .beta.
1,3-glycosidic bond, and includes both hyaluronic acid and salts
thereof. For the salts, a sodium salt, a potassium salt, a
magnesium salt, a calcium salt, an aluminum salt, and the like are
exemplified, but are not limited thereto.
[0075] The disaccharide repeating unit of hyaluronic acid may be
represented by the following Formula 4, and may be 1 to 1,000, but
is not limited thereto.
##STR00005##
[0076] Hyaluronic acid is found in ocular vitreous humor, joint
synovial fluid, chicken comb, and the like, and is known as a
highly biocompatible biomaterial. Despite high applicability of a
hyaluronic acid hydrogel to biomaterials, limited mechanical
properties due to a natural polymer itself make it difficult for
the hyaluronic acid hydrogel to be applied to biomaterials (for
example, drug delivery carrier, scaffold for tissue engineering).
Thus, the present inventors have introduced a gallol group having a
high oxidizing ability into hyaluronic acid to prepare a hyaluronic
acid derivative (Preparation Example 1), and have crosslinked such
hyaluronic acid derivatives under an appropriate oxidizing or
specific pH (pH 8 to 9) condition to prepare a hydrogel
(Preparation Example 2). Accordingly, the present invention has
technical significance in that physical properties such as
crosslinking rate, elasticity, and adhesive strength of a hydrogel
can be efficiently controlled (Examples 1 and 3).
[0077] The term "hyaluronic acid derivative" or "hyaluronic
acid-pyrogallol conjugate" as used herein is interpreted as
including both hyaluronic acid or a salt thereof in which a gallol
group is introduced into a skeleton of glucuronic acid in the
hyaluronic acid or the salt thereof.
[0078] As an embodiment, the hyaluronic acid derivative may be
prepared by a reaction between a terminal of the disaccharide
repeating unit of the above Formula 4, specifically, a carboxyl
group thereof and 5'-hydroxydopamine. The hyaluronic acid
derivative prepared by the reaction contains at least one repeating
unit represented by the following Formula 5, and may be represented
by the following Formula 1.
##STR00006##
[0079] (In the above Formula 1, R.sub.1 is a hydroxyl group or
##STR00007##
and n is an integer of 1 to 1,000.)
[0080] In addition, the hyaluronic acid derivative may have a
molecular weight of 10,000 Da to 2,000,000 Da, and the hyaluronic
acid derivative may have a gallol group substitution rate of 0.1%
to 50%, but the pyrogallol group substitution rate is not limited
thereto.
[0081] The "substitution rate" means that a specific functional
group in hyaluronic acid or a salt thereof is replaced or modified
with a gallol group. The rate of being substituted with the gallol
group is defined as a rate of repeating units into which the gallol
group has been introduced in the entire hyaluronic acid repeating
units, and may be represented, by definition, as a numerical value
from more than 0 to equal to or less than 1, a numerical value from
more than 0% and equal to or less than 100%, or a numerical value
from more than 0% by mol to equal to or less than 100% by mol.
[0082] The term "hydrogel" as used herein means a three-dimensional
structure of a hydrophilic polymer retaining a sufficient amount of
moisture. For the purpose of the present invention, the hydrogel
indicates a hydrogel formed of hyaluronic acid derivatives, each of
which is modified with a gallol group.
[0083] A process of preparing the hyaluronic acid hydrogel may be
carried out by a crosslinking reaction between the hyaluronic acid
derivatives. For the crosslinking reaction, the process may further
include a step of mixing the hyaluronic acid derivatives with PBS
and the like to prepare a hyaluronic acid hydrogel precursor
solution. Such crosslinking may be performed by chemical
crosslinking caused by UV irradiation, physical crosslinking, or
biological crosslinking, to form a hydrogel. Here, the chemical
crosslinking caused by UV irradiation includes photo-crosslinking,
crosslinking utilizing a reactive crosslinker, or the like. The
biological crosslinking includes crosslinking utilizing a binding
force between heparin and growth factor, crosslinking using
complementary bonding of DNA or the like, and the like. The
physical crosslinking includes crosslinking by hydrogen bonding,
crosslinking by hydrophobic interaction, crosslinking utilizing
electrostatic interaction, or the like. Preferably, crosslinking
may be performed by adding an oxidizing agent or a pH adjusting
agent. The oxidizing agent may be sodium periodate, hydrogen
peroxide, horseradish peroxidase, or tyrosinase, and the pH
adjusting agent may be sodium hydroxide, lithium hydroxide,
potassium hydroxide, rubidium hydroxide, cesium hydroxide,
magnesium hydroxide, calcium hydroxide, strontium hydroxide, or
barium hydroxide. However, the oxidizing agent and the pH adjusting
agent are not limited thereto.
[0084] In an embodiment, in a case where the oxidizing agent is
added and crosslinking is performed, crosslinking represented by
the following Formula 2 is formed between the hyaluronic acid
derivatives. In Formula 2, HA' represents hyaluronic acid in which
the carboxyl group is substituted with an amide group.
##STR00008##
[0085] In another embodiment, in a case where the pH adjusting
agent is added and crosslinking is performed, crosslinking
represented by the following Formula 3 is formed between the
hyaluronic acid derivatives. In Formula 3, HA' represents
hyaluronic acid in which the carboxyl group is substituted with an
amide group.
##STR00009##
[0086] In an embodiment of the present invention, in the step of
crosslinking hyaluronic acid derivatives, each of which is modified
with a gallol group, a hyaluronic acid hydrogel was respectively
prepared by using sodium periodate, which is an oxidizing agent, or
sodium hydroxide, which is a pH adjusting agent (see Preparation
Example 2). As a result, it was possible to identify that the
hyaluronic acid hydrogels prepared according to the respective
crosslinking methods exhibit remarkable differences in physical
properties such as crosslinking rate, rigidity, elasticity,
adhesive strength, and degradation pattern, together with excellent
biocompatibility (see Examples 1 to 3).
[0087] In an embodiment of the present invention, there is provided
a filler composition comprising the hyaluronic acid derivative or
hyaluronic acid hydrogel of the present invention. The filler
composition of the present invention is provided in a liquid or
solution state in which hyaluronic acid derivatives into each of
which a gallol group has been introduced are mixed in an aqueous
medium. In particular, in a case of being injected into the body,
such a solution, although not containing a crosslinking component,
can form a hydrogel only with oxidizing power in the body without
interpersonal deviation, thereby serving to replenish skin tissue
and to retain moisture in the skin. In an embodiment, the aqueous
medium is a phosphate buffered saline (PBS), and the hyaluronic
acid derivative may be contained in an amount of preferably 0.1%
(w/v) to 20.0% (w/v), and more preferably 0.3% (w/v) to 10.0%
(w/v), with respect to the entire filler composition. However,
various modifications or alterations may be made depending on
aqueous media and content ratios in a filler composition which are
well known in the art.
[0088] The term "filler" as used here means an injectable material
that replenishes skin tissue, such as by injecting a biocompatible
material into or under the skin to improve wrinkles and restore
cosmetic volume. At present, as materials for preparing fillers
which have been approved by FDA or MFDS, collagen, hyaluronic acid,
calcium hydroxyapatite, polylactic acid, and the like are
mentioned. Among these, hyaluronic acid is a material similar to a
human body constituent and can be used without skin reaction test;
and hyaluronic acid has a characteristic of attracting 214 water
molecules per molecule, thereby effectively retaining moisture in
the skin. Thus, hyaluronic acid currently accounts for about 90% of
the filler market. Due to use of a hyaluronic acid derivative into
which a gallol group having high oxidizing power has been
introduced, the filler composition according to the present
invention can form a hydrogel only with oxidizing power in the body
without addition of a crosslinking agent so that biocompatibility
is enhanced, and the above-mentioned hydrogel formed in the body
can maintain its shape in a transparent state for a long period of
time (at least 6 months). Thus, the filler composition has
technical significance in that it shows an excellent
wrinkle-improving effect according to the characteristics of
hyaluronic acid as described above, has excellent adhesive strength
and stability in the body as compared with conventional filler
products, and can be stably injected into a target site regardless
of extrusion force (Examples 2 and 3).
[0089] In addition, the filler composition of the present invention
may be formulated into a powder form, and more specifically into a
freeze-dried powder form, to provide ease of use and storage
stability. On the other hand, the above-mentioned filler
composition requires a pretreatment step in which it is dissolved
in an aqueous medium such as PBS and solubilized before being
injected into the skin. However, it is also possible to directly
apply a filler composition, which has been made into a solution,
depending on storage and formulation conditions thereof.
[0090] In another embodiment, in order to impart an effective
skin-regenerating effect, the filler composition of the present
invention may further comprise a cell growth factor or a vitamin.
The cell growth factor collectively refers to a polypeptide that
facilitates cell division, growth, and differentiation, and may be
preferably selected from the group consisting of fibroblast growth
factor (FGF), epithelial cell growth factor (EGF), keratinocyte
growth factor (KGF), transforming growth factor alpha
(TGF-.alpha.), transforming growth factor beta (TGF-.beta.),
granulocyte colony stimulating factor (GCSF), insulin-like growth
factor (IGF), vascular endothelial growth factor (VEGF), hepatocyte
growth factor (HGF), platelet-derived growth factor-BB (PDGF-BB),
brain-derived neurotrophic factor (BDNF), and glial cell-derived
neurotrophic factor (GDNF). The cell growth factor may be contained
in an amount of 20 ng/ml to 20 .mu.g/ml, but is not limited
thereto.
[0091] In yet another embodiment, the filler composition of the
present invention may further comprise a local anesthetic to
alleviate pain during an injection process. The local anesthetic
may be selected from the group consisting of ambucaine, amolanone,
amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine,
bupivacaine, butacaine, butamben, butanilicaine, butethamine,
butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine,
cyclomethicaine, dibucaine, dimethisoquine, dimethocaine,
diferodone, dycyclonine, ecgonidine, ecgonine, ethyl chloride,
etidocaine, beta-eucaine, euprocine, fenalcomine, fomocaine,
hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate,
leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine,
meprylcaine, metabutoxycaine, methyl chloride, myrtecaine,
naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine,
phenacaine, phenol, piperocaine, piridocaine, polidocanol,
pramoxine, prilocaine, procaine, propanocaine, proparacaine,
propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine,
bupivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine,
zolamine, and salts thereof The anesthetic may be contained in an
amount of preferably 0.1% by weight to 5.0% by weight, and more
preferably 0.2% by weight 1.0% by weight, with respect to a weight
of the entire filler composition, but the amount is not limited
thereto.
[0092] In another embodiment, the filler composition of the present
invention may further comprise an antioxidant in order to prevent
oxidation and degradation of a hydrogel produced by being gelated
in the body. The antioxidant may be selected from the group
consisting of polyol, mannitol, and sorbitol. The antioxidant may
be contained in an amount of preferably 0.1% by weight to 5.0% by
weight, and more preferably 0.2% by weight 1.0% by weight, with
respect to a weight of the entire filler composition, but the
amount is not limited thereto.
[0093] In an aspect of the present invention, the present invention
provides a method for preparing a filler composition, comprising a
step of introducing a gallol group into a skeleton of glucuronic
acid in hyaluronic acid, to prepare a hyaluronic acid derivative;
and a step of adding the hyaluronic acid derivative to an aqueous
medium and performing mixing.
[0094] In the present invention, the step of adding the hyaluronic
acid derivative to an aqueous medium and performing mixing may be
performed preferably in a state in which contact with oxygen and
other oxidizing sources is blocked (for example, by injection of
nitrogen gas) under a condition at about 4.degree. C. to 28.degree.
C. and/or at a pH of 4 to 8, in order to prevent gelation, before
injection, of the filler composition which is in a liquid
state.
[0095] In an embodiment, the aqueous medium is a phosphate buffered
saline (PBS), and the hyaluronic acid derivative may be the above
Formula 1 produced by a reaction between hyaluronic acid and
5'-hydroxydopamine. The filler composition of the present invention
may be prepared by adding the afore-mentioned cell growth factor,
local anesthetic, antioxidant, vitamin, and combinations
thereof.
[0096] In another aspect of the present invention, there is
provided a method for improving skin wrinkles, comprising a step of
injecting the filler composition into or under the skin of an
individual. In the present invention, a target site to which the
filler composition is applied may be any site of the body such as
the individual's face, neck, ear, chest, hips, arms, and hands, and
may preferably be any one site which is a wrinkled region in the
skin and is selected from the group consisting of a tear trough
region, a glabellar frown line region, an eye-rim region, a
forehead region, a nasal bridge region, a nasolabial line region, a
marionette line region, and a neck wrinkle region. However, the
site is not limited thereto. In addition, in the present invention,
the term "individual" means a subject requiring improvement of skin
wrinkles, and more specifically includes all of humans, non-human
primates, and the like.
[0097] In particular, the filler composition of the present
invention can be easily injected into a target site regardless of
extrusion force. In the injecting step, the filler composition can
be injected into or under the skin by using needles or cannulas of
various sizes. As a method of injecting the filler, for example, a
serial puncture method in which multiple injections are made at a
small amount each; a linear threading method in which a needle is
caused to go forward by about 10 mm, and then injections are made
at a small amount each while being withdrawn backward; a fanning
method in which a needle is once inserted, and then the linear
threading method is repeatedly performed in the same manner with
slightly twisted angles without removing the inserted needle; and
the like may be used.
[0098] In yet another aspect of the present invention, there is
provided a hyaluronic acid hydrogel prepared by crosslinking
hyaluronic acid derivatives, each of which is represented by the
above Formula 1, in which crosslinking represented by the above
Formula 2 is formed between the hyaluronic acid derivatives; and a
drug delivery carrier, a drug delivery system, or a scaffold for
tissue engineering, comprising the hyaluronic acid hydrogel.
[0099] In addition, in still yet another aspect of the present
invention, there is provided a hyaluronic acid hydrogel prepared by
crosslinking hyaluronic acid derivatives, each of which is
represented by the above Formula 1, in which crosslinking
represented by the above Formula 3 is formed between the hyaluronic
acid derivatives; and a drug delivery carrier, a drug delivery
system, or a scaffold for tissue engineering, comprising the
hyaluronic acid hydrogel.
[0100] The hyaluronic acid hydrogel of the present invention can be
used as an artificial extracellular matrix which is an effective
skeleton for drug delivery, and has technical significance in that
a nano- or micro-unit microparticle form thereof can be implemented
due to superior oxidizing ability of the hyaluronic acid derivative
modified with a gallol group. The drug is not particularly limited,
and may preferably include, but is not limited to, a chemical
substance, a small molecule, a peptide, an antibody, an antibody
fragment, a nucleic acid including DNA, RNA, or siRNA, a protein, a
gene, a virus, a bacterium, an antibacterial agent, an antifungal
agent, an anticancer agent, an anti-inflammatory agent, a mixture
thereof, and the like.
[0101] In addition, the hyaluronic acid hydrogel of the present
invention can be used as a scaffold for tissue engineering based on
excellent elasticity and adhesive strength. The tissue engineering
means that cells or stem cells isolated from the patient's tissue
are cultured in a scaffold to prepare a cell-scaffold complex, and
then the prepared cell-scaffold complex is transplanted again into
the living body. The hyaluronic acid hydrogel can be implemented
with a scaffold similar to a biological tissue, in order to
optimize regeneration of biological tissues and organs. Therefore,
the hyaluronic acid hydrogel can be used for a gene therapeutic
agent or a cell therapeutic agent.
[0102] In addition, the hydrogel of the present invention can be
also used as cosmetics, and medical materials such as a wound
healing agent, a wound covering material, an adhesion barrier, or a
dental matrix. However, uses of the hydrogel of the present
invention are not limited thereto.
[0103] Hereinafter, preferred examples will be described in order
to facilitate understanding of the present invention. However, the
following examples are provided only for the purpose of easier
understanding of the present invention, and the scope of the
present invention is not limited by the following examples.
PREPARATION EXAMPLES
Preparation Example 1
Preparation of Hyaluronic Acid Derivative Modified with Gallol
Group
[0104] As illustrated in FIG. 1, a hyaluronic acid derivative
modified with a gallol group according to the present invention was
prepared. Specifically, hyaluronic acid (MW 200K, Lifecore
Biomedical, LLC, IL, USA) was completely dissolved in water (TDW),
and 1 equivalent of N-hydroxysuccinimide (NHS, Sigma-Aldrich, Inc.,
St. Louis, Mo., USA) and 1.5 equivalent of
1-(3-dimethylaminopropyl)-3-ehtylcarbodiimide hydrochloride (EDC,
Thermo Scientific, Rockford, Ill., USA) were added thereto. After
30 minutes, 1 equivalent of 5'-hydroxydopamine (Sigma-Aldrich,
Inc.) was added thereto as a PG moiety and the resultant was
allowed to react at pH 4 to 4.5 for 24 hours. Reactants having two
different molar ratios were used in the preparation of HA-PG
conjugates (HA:EDC:NHS:PG=1:1.5:1:1 or 2:1.5:1:1). Then, EDC, NHS,
and 5'-hydroxydopamine were removed by dialysis based on PBS and
water (Cellu/Sep (tradename), dialysis membrane (6.8 kDa cut-off,
Membrane Filtration Products Inc., Seguin, Tex., USA)), and the
solvent was evaporated through freeze-drying to prepare the
hyaluronic acid derivative of the present invention. In order to
identify synthesis of the hyaluronic acid derivative, analysis was
performed with Fourier transform-infrared spectroscopy (FT-IR)
(Vertex 70, Bruker, Billerica, Mass., USA) and proton nuclear
magnetic resonance (H-NMR) (Bruker 400 MHz, Bruker). As a result,
as illustrated in FIG. 2(a), it was possible to identify a newly
formed amide bond through a strong peak at a wavenumber region of
about 1,580 cm.sup.-1 to 1,700 cm.sup.-1, and as illustrated in
FIG. 2(b), it was possible to identify structures of the aromatic
benzene ring and --CH.sub.2CH.sub.2-- in 5'-hydroxydopamine,
through peaks in the vicinity of 6.5 ppm and 3 ppm, respectively.
From the above results, it was found that 5'-hydroxydopamine is
introduced into the hyaluronic acid derivative of the present
invention due to an amide bond formed between the carboxyl group of
hyaluronic acid and the amine group of 5'-hydroxydopamine. In order
to calculate a degree of substitution of gallol group with respect
to the HA skeleton, the HA-PG conjugate was dissolved in PBS (pH 5)
at 1 mg/ml and absorbance of the solution was measured at 283 nm
(UV-visible spectrophotometer) (Cary 100 UV-vis, Varian Inc., Palo
Alto, Calif., USA)). Percentage of carboxy groups substituted with
PG in the HA was calculated using a standard curve obtained through
serial dilution of a 5-hydroxydopamine solution (from 1 mg/mL
concentration).
Preparation Example 2
Preparation of Hyaluronic Acid Hydrogel
[0105] The hyaluronic acid derivatives of Preparation Example 1
were crosslinked to prepare a hyaluronic acid hydrogel, in which
each crosslinking method using sodium periodate (NaIO.sub.4) as an
oxidizing agent or sodium hydroxide (NaOH) as a pH adjusting agent
(pH 8 to 9) was employed. Specifically, the hyaluronic acid
derivatives were dissolved in PBS (1% (w/v), 2% (w/v)), and then
crosslinking was allowed to proceed while performing mixing with
4.5 mg/ml of NaIO.sub.4 or 0.08 M NaOH at a volumetric ratio of
1.5:1 to 4:1 relative to the hyaluronic acid derivative solution.
As illustrated in FIG. 3, through each of these crosslinking
methods, a light brown-colored hyaluronic acid hydrogel or a
blue-colored hyaluronic acid hydrogel was prepared.
[0106] In order to specifically identify crosslinking of the
hyaluronic acid hydrogel, analysis with ultraviolet-visible
spectroscopy (UV-vis) was performed. In a case of using NaIO.sub.4,
as illustrated in FIG. 4, it was possible to identify that a
wavelength region of 350 to 400 nm changes over time (FIG. 4(a)),
meaning instantaneous formation and decrease of radicals, which are
intermediate products, in an oxidation process; and it was found
that biphenol is then formed by radical polymerization (FIG. 4(b)).
In addition, in a case of using NaOH, as illustrated in FIG. 5, it
was possible to identify that a wavelength region of 600 nm changes
over time (FIG. 5(a)); from this result, it was found that a charge
transfer complex and benzotropolone are formed by [5+2]
tautomerization in an oxidation process (FIG. 5(b)).
EXAMPLES
Example 1
Changes in Physical Properties of Hyaluronic Acid Hydrogel
Depending on Crosslinking Method
[0107] In the present example, changes in physical properties of
hyaluronic acid hydrogels, depending on difference in crosslinking
method in Preparation Example 2, were compared. On the other hand,
in the hydrogel preparation step, a molar concentration ratio of
hyaluronic acid to 5'-hydroxydopamine (0.5.times.
(HA:EDC:NHS:5'-hydroxydopamine=2:1.5:1:1), 1.times.
(HA:EDC:NHS:5'-hydroxydopamine=1:1.5:1:1)) could be used to adjust
a rate of being substituted with a gallol group to 4% to 5%
(0.5.times.) or 8% to 9% (1.times.) (not shown), and changes in
physical properties of hydrogels depending on a degree of
substitution of 5'-hydroxydopamine were also compared.
Specifically, hydrogel formation and changes in elastic modulus
over time were compared depending on crosslinking methods; and
elasticity, adhesive strength, swelling, and degradation patterns
depending on the crosslinking method and the degree of substitution
of 5'-hydroxydopamine were respectively compared and analyzed.
[0108] 1-1. Comparison of Formation Rate of Hyaluronic Acid
Hydrogel
[0109] As illustrated in FIG. 6(a), it was possible to identify
that in a case of using NaIO.sub.4, a light brown-colored hydrogel
is instantly formed through an immediate crosslinking reaction; on
the other hand, in a case of using NaOH, a blue-colored hydrogel is
formed relatively slowly. In addition, storage moduli (G') and loss
moduli (G'') were measured over time, and the measured results were
compared with those for the hydrogel (HA-CA(NaIO.sub.4)) obtained
by crosslinking hyaluronic acid derivatives, into each of which a
catechol group had been introduced, using NaIO.sub.4. As a result,
as illustrated in FIG. 6(b), all hydrogels were stably formed as
the oxidation proceeded (G' >G''). In particular, when a time
point at which the hydrogel is formed is identified through a time
point at which a storage modulus curve and a loss modulus curve
come into contact with each other, it was found that about 2 to 3
minutes is needed in a case of being crosslinked using NaOH, and
about 30 seconds is needed for the HA-CA (NaIO.sub.4); on the other
hand, crosslinking proceeds so quickly that measurement cannot be
made, in a case of being crosslinked using NaIO.sub.4.
[0110] A viscoelastic coefficient of hyaluronic acid was analyzed
by measuring storage modulus (G') and loss modulus (G'') within a
frequency sweep mode in a frequency range of 0.1 to 1 Hz.
Elasticity of hyaluronic acid was calculated by dividing a storage
coefficient by a loss coefficient at 1 Hz (n=45). Gelation kinetics
of the HA-PG were measured with a rheometer (MCR 102, Anton Paar,
Va., USA) in a time sweep mode at a controlled strain and frequency
of 10% and 1 Hz, respectively. Two oxidizing agents (NaIO.sub.4 and
NaOH) were added 30 minutes after the initial measurement of G1 and
G''. Adhesiveness of the hydrogel was measured by recording
detachment stress of the completely crosslinked hydrogel between a
probe and a substrate plate in a rheometer (MCR 102, Anton Paar)
(n=3). A pulling speed for the probe was 5 .mu.m/sec.
[0111] 1-2. Comparison of Elasticity and Adhesive Strength
[0112] As illustrated in FIG. 7(a), despite differences in the
crosslinking method or the degree of substitution of
5'-hydroxydopamine, in all cases, storage modulus (G') was measured
to be higher at a certain level than loss modulus (G''), which made
it possible to identify that the hydrogels were stably formed. In
addition, elastic modulus representing rigidity of hydrogel and tan
6 (G''/G') representing a degree of elasticity were calculated. As
a result, as illustrated in FIG. 7(b), in a case of using
NaIO.sub.4, better rigidity of hydrogel was exhibited and all
hydrogels had excellent elastic force even at low substitution
rates; on the other hand, in a case of using NaOH, better elastic
force was obtained as the substitution rate increased. In addition,
it was found that in a case of the same crosslinking method, these
respective excellent physical properties are also improved as a
substitution rate of 5'-hydroxydopamine increases
(0.5.times.<1.times.). In addition, based on the above results,
differences in physical properties of the hydrogels crosslinked
with NaIO.sub.4, depending on molecular weights (40, 200, 500 kDa)
and concentrations (1% (w/v), 2% (w/v)) of the hyaluronic acid
derivatives, were identified. As a result, as illustrated in FIG.
8, in all cases, the storage modulus (G') was measured to be higher
at a certain level than the loss modulus (G''), which made it
possible to identify that the hydrogels were stably formed (FIG.
8(a)); and it was found that the hydrogel exhibits further improved
rigidity and elastic force as hyaluronic acid has an increased
molecular weight and has a higher concentration (FIG. 8(b)). In
addition, in order to evaluate adhesive strength of the hydrogels,
each of the hydrogels was bridged between respective plates of a
rheological instrument (Bohlin Advanced Rheometer, Malvern
Instruments, Worcestershire, UK) and force applied to the
instrument was measured while increasing spacing between the
plates. The above results were compared with those for the hydrogel
(HA-CA(NaIO.sub.4)) obtained by crosslinking hyaluronic acid
derivatives, into each of which a catechol group had been
introduced, using NaIO.sub.4. As a result, as illustrated in FIG.
9, the hydrogel formed by using NaOH and the HA-CA (NaIO.sub.4)
exhibited superior adhesive strength, whereas almost no adhesive
strength was observed in the hydrogel formed by using
NaIO.sub.4.
[0113] 1-3. Comparison of Swelling and Degradation Patterns
[0114] As illustrated in FIG. 10, slightly different swelling and
degradation patterns were exhibited depending on the crosslinking
method and the degree of substitution of 5'-hydroxydopamine.
Swelling characteristics were evaluated by measuring a weight of
the remaining hydrogel at specific time points (days 0, 1, 2, 3, 5,
7, 14, and 28). Specifically, the HA-PG was incubated at 37.degree.
C. in PBS and a swelling rate was calculated by the following
expression: (Wt-Wi)/Wi.times.100, where Wt is a weight of the
hydrogel at each time point, and Wi is a weight of the hydrogel at
an initial time point (n=4 to 5). In order to investigate a
degradation profile, the HA-PG hydrogel which had been swollen for
3 days was treated with hyaluronidase (5 units/mL, Sigma) and a
weight of remaining hyaluronic acid was measured at respective time
points (n=4 to 7, 0, 2, 5, 12, 24, 48, and 72 hours). In a case of
using NaIO.sub.4 rather than NaOH, a low swelling rate was
exhibited and degradation proceeded faster. For the same
crosslinking method, in a case where a substitution rate of
5'-hydroxydopamine increases, such swelling rate and degradation
rate tended to decrease. These results, taken together, indicate
that even though hyaluronic acid derivatives having the same
structure are used, hydrogels are crosslinked in different bonding
forms depending on the crosslinking method (Preparation Example 2),
which leads to changes in inherent physical properties such as
rigidity, elasticity, adhesive strength, swelling, and degradation.
In addition, on the contrary, it was found that even though the
same crosslinking method is adopted, the above physical properties
were also changed depending on a structure of the hyaluronic acid
derivative.
Example 2
Analysis for Cytotoxicity and Biocompatibility
[0115] In the present example, cytotoxicity and biocompatibility of
the hyaluronic acid hydrogel of Preparation Example 2 were
evaluated. First, in order to identify whether the hydrogel causes
toxicity and inflammation response in 3D cell culture, human
adipose-derived stem cells (hADSCs) (1.0.times.10.sup.6 cells per
100 .mu.L of hydrogel) were cultured in the hydrogel, and the
LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, Calif.,
USA) was used to perform live/dead staining at respective time
points (days 1, 3, and 7) according to the manufacturer's
instructions. Stained cells were observed with a confocal
microscope (LSM 880, Carl Zeiss, Oberkochen, Germany), and
viability was quantified by counting viable and dead cells from a
fluorescence image (n=4 to 5). The hADSCs were obtained from ATCC
(ATCC, Manassas, Va. USA) and were cultured in the Mesenchymal Stem
Cell Basal Medium (ATCC) supplemented with Growth Kit-low serum
(ATCC) and 1% penicillin/streptomycin (Invitrogen).
[0116] In addition, immune cells (Raw 264.7) were co-cultured in a
hydrogel using a Transwell system (permeable supports with 3.0
.mu.m pores, Corning, New York, N.Y., USA), and then an amount of
tumor necrosis factor (TNF-.alpha.) secreted by inflammation
response was measured using enzyme-linked immunosorbent assay
(ELISA). Raw 264.7 cells were incubated overnight, and then seeded
on a 96-well plate (2.0.times.10.sup.4 cells per well). Then, 50
.mu.l of hydrogel was loaded through an upper inserting portion of
the Transwell, and then additional incubation was performed for 24
hours. An amount of TNF-.alpha. in the medium collected from the
co-culture was quantitated using a TNF-.alpha. enzyme-linked
immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis,
Minn., USA).
[0117] In order to evaluate in vivo biocompatibility of the HA-PG
hydrogel, 100 .mu.l of the HA-PG hydrogel (final concentration of
2% [w/v]) formed by using NaIO.sub.4 or NaOH was subcutaneously
transplanted into ICR mice. Before the transplantation, the mice
(5-week-old male, OrientBio, Seongnam, Korea) were anesthetized
with ketamine (100 mg/kg, Yuhan, Seoul, Korea) and xylazine (20
mg/kg, Bayer Korea, Ansan, Korea). For tissue analysis, hyaluronic
acid structures were recovered with adjacent tissues at
predetermined time points (days 0, 7, 14, 28, and 84). The
recovered hyaluronic acid was fixed in 4% paraformaldehyde (Sigma)
for 2 days, embedded in OCT compound (Leica Biosystems, Wetzlar,
Hesse, Germany), and then cut into 6 .mu.m thickness. The cut
samples from the in vivo experiment were stained with H&E to
evaluate hydrogel maintenance. Toluidine blue staining was
performed to investigate an immune response after the
transplantation of the HA-PG hydrogel. In vivo degradation of the
HA-PG hydrogel was evaluated by measuring a remnant weight of the
recovered hydrogel structure at each time point (n=3 to 5).
[0118] As a result, as illustrated in FIGS. 11 and 12, neither of
the two types of hydrogels depending on the crosslinking method
exhibited cytotoxicity up to 7 days after the culture (FIGS. 11(a)
and 11(b)). TNF-.alpha. which had been increased due to LPS was
detected only in a small amount in a case where treatment with the
above-mentioned two hydrogel forms is performed, and this was not
largely different from the control (NT) in which no treatment had
been performed (FIG. 11(c)). In addition, as illustrated in FIG.
12, even in a case where the hydrogel is transplanted into mice,
specific inflammatory findings were not observed, and it was
possible to identify that a structure thereof is well maintained
without proliferation of inflammation-related cells such as
macrophages in the vicinity of the transplanted hydrogel (FIGS.
12(c) and 12(d)).
Example 3
Analysis for Degradation Pattern In Vivo
[0119] In the present example, hyaluronic acid hydrogels having a
difference in the crosslinking method (NaIO.sub.4/NaOH) and the
molar ratio (0.5.times., 1.times.) of the reacted
5'-hydroxydopamine was subcutaneously transplanted into mice. On
the day of transplantation, and at weeks 1, 4, and 12 after the
transplantation, the mice were sacrificed and respective hydrogels
were collected. For these hydrogels, a degree of swelling was
visually identified, and a remnant amount in vivo was calculated,
thereby analyzing degradation pattern in vivo and the like.
[0120] As a result, as illustrated in FIG. 13, it was possible to
identify that the hydrogel obtained by performing crosslinking
using NaIO.sub.4 has a smaller degree of swelling, and it was found
that such a hydrogel exhibits a faster degradation in vivo. In
addition, for the same crosslinking method, in a case where a
substitution rate of 5'-hydroxydopamine (gallol group) increases, a
degradation rate tends to decrease.
[0121] The results of Examples 2 and 3 suggest that the hyaluronic
acid hydrogel according to the present invention can be utilized,
for example, in the biomaterial field such as a drug delivery
carrier and a scaffold for tissue engineering. In particular,
considering the inherent physical properties of Example 1 and the
like, the hydrogel formed by using NaIO.sub.4 which exhibits rapid
crosslinking rate and in vivo degradation pattern can be utilized
as a drug delivery carrier in the form of fine particles, and the
hydrogel formed by using NaOH which exhibits excellent adhesive
strength and a slow in vivo degradation pattern can be utilized as
a scaffold for tissue engineering and the like.
Example 4
Utilization as Drug Delivery System through Formation of
Microparticles
[0122] 4-1. Sustained Release of Protein using HA-PG Hydrogel
Microparticles
[0123] In the present example, as an embodiment of the hydrogel
formed by using NaIO.sub.4, a drug delivery preparation in the form
of microparticles having a nano- or micro-unit diameter was
prepared and efficacy thereof was identified. First, an oil/water
emulsion method was used to induce formation of an emulsion of the
hyaluronic acid derivative solution (HA-PG) of Preparation Example
2, and an oxidizing agent (NaIO.sub.4) was added to the solution.
Then, formation and presence of microparticles were identified
before and after freeze-drying. In addition, the HA-PG solution was
mixed with a protein (bovine serum albumin, BSA), and the mixture
was made into an emulsion form. Then, an oxidizing agent
(NaIO.sub.4) was added thereto and crosslinking was performed,
thereby preparing microparticles in which BSA was encapsulated. A
protein release pattern of the microparticles was identified over
14 days. As a result, as illustrated in FIG. 14, microparticles
containing, as a component, a hyaluronic acid hydrogel were
produced through the above-mentioned preparation process, and were
maintained unchanged even after undergoing the freeze-drying
process (FIG. 14(a)). In addition, it was possible to identify that
encapsulated proteins are hardly released in an HA-PG bulk
hydrogel, whereas proteins are released at a constant rate in the
HA-PG particle (FIG. 14(b)). Thus, the hydrogel formed by using
NaIO.sub.4 in the form of microparticles can be utilized as a drug
delivery preparation.
[0124] 4-2. Sustained Release of Antibody using HA-PG Hydrogel
Microparticles
[0125] First, as a method for sustained and controlled delivery of
growth factors for therapeutic application, HA-PG hydrogel
microparticles were prepared by an oil/water emulsion method using
ultrafast gel-formation with NaIO.sub.4-mediated crosslinking (FIG.
15(a)). HA-PG microparticles can be prepared by simple addition of
the HA-PG solution to a water-in-oil emulsion. Rapid chemical
crosslinking of the HA-PG emulsion by NaIO.sub.4 in an oil phase
resulted in production of a large number of submicron HA particles
within a few minutes. The produced HA-PG microparticles exhibited a
spherical shape with an average diameter of 8.8 .mu.m (.+-.3.9
.mu.m) (FIG. 15(b)). Interestingly, vascular endothelial growth
factors (VEGFs) encapsulated in the HA-PG microparticles were
slowly released for 60 days, whereas VEGFs were hardly released
from the hydrogel structure for the bulk HA-PG (FIG. 15(c)). In a
previous study conducted by the present inventors, the present
inventors have found that growth factors encapsulated in a
catechol-modified alginate hydrogel are hardly released due to
strong binding of the proteins to oxidized catechol during a
gelation process. Like the catechol group, a PG group may also
induce strong binding of the growth factors to the hydrogel
structure. However, it appears that due to a remarkably increased
surface area of growth factors for release, the HA-PG formed as
fine particles can release VEGFs.
[0126] HA-PG microparticles into which VEGFs (Peprotech, Rocky
Hill, N.J., USA) are incorporated were applied to facilitate
therapeutic angiogenesis in peripheral vascular diseases.
Intramuscular injection of VEGFs contained in HA-PG microparticles
(VEGF loading dose: 6 .mu.g per mouse) showed a remarkably improved
therapeutic effect in a limb ischemic mouse model prepared by
resection and ligation of femoral artery. Mice (balb/c, 6-week-old
female) were obtained from OrientBio Inc. After 4 weeks of
injection, there was no leg cut or tissue necrosis in the group
treated with the VEGF-containing HA-PG microparticles. On the other
hand, only 20% of the group treated with PBS or only HA-PG without
VEGFs exhibited improvement in ischemic legs (FIG. 16(a)). Single
bolus administration of VEGFs at the same dose (6 .mu.f per mouse)
showed slight improvement in ischemic legs; however, 50% of limb
ischemic mice still exhibited leg loss or tissue necrosis (FIG.
16(b)). In addition, histological analysis identified by H&E
and Masson's trichrome (MT) staining showed minimal muscle damage
and fibrosis formation in an HA-PG/VEGF group of the ischemic mouse
model (FIGS. 16(c) and 16(d)). In addition, immunohistochemical
analysis using .alpha.-smooth muscle actin (.alpha.-SMA) and von
Willebrand factor (vWF) for arteriole staining and capillary
staining, respectively, showed that arteriole and capillary are
remarkably increased in the HA-PG/VEGF group as compared with
controls (PBS, HA-PG, and VEGF) (FIGS. 16(e) and 16(f)). These
results show substantial improvement in angiogenesis. NaOH-mediated
crosslinking was used to prepare an adhesive hydrogel scaffold for
non-invasive and injection-free cell transplantation (FIG. 17(a)).
From previous studies, it has been identified that
catechol-modified polymers exhibit strong tissue adhesive strength
through high binding affinity to various nucleophiles of oxidized
catechol in proteins.
Example 5
Identification of Gelation of Hyaluronic Acid Filler Composition
using Oxidizing Power in Body and Wrinkle-Improving Effect
Thereof
[0127] 5-1. Formation and Maintenance of Hydrogels using Oxidizing
Power in Body
[0128] The HA-PG solution prepared in Preparation Example 1 was
injected subcutaneously into mice. Then, it was identified whether
or not gelation of the injected HA-PG solution can proceed in the
body without addition of a crosslinking agent. In addition, changes
in the weight of the formed HA-PG hydrogel were measured, under a
condition in the body, for about 6 months, to identify whether the
formed HA-PG hydrogel can be maintained in the body for a long
time.
[0129] As a result, as illustrated in FIGS. 20 and 21, it was found
that the HA-PG solution, which has been injected subcutaneously in
a liquid state, is gelated within 5 minutes to form an HA-PG
hydrogel in the body, and that the formed HA-PG hydrogel is
maintained well in the body in a transparent state for 6 months
without any large difference in weight. In addition, as illustrated
in FIG. 22, in a case where the HA-PG solution is exposed to an
oxidizing condition in the body, quinones are easily formed among
gallol groups having a strong self-oxidizing ability, so that
hyaluronic acid is polymerized and an HA-PG hydrogel is formed.
Accordingly, it was found that unlike filler products (Megafill,
Perlane) which are composed of formulations in a polymerized form,
the filler composition of the present invention can be used as a
formulation in a solution state, which allows for easier injection
into the skin and allows a variety of additional ingredients to be
contained. That is, the HA-PG solution of the present invention
formed a hydrogel in the body without addition of a crosslinking
agent and could be stably maintained therein. From this, it was
possible to identify a possibility of utilizing the HA-PG solution
as a filler composition.
[0130] 5-2. Identification of Wrinkle-Improving Effect
[0131] Based on the results in Example 5-1, a wrinkle-improving
effect of the HA-PG solution was identified. Specifically, hairless
mice were given Calcitriol at 0.2 .mu.g/day each for 5 times a week
over a total of 6 weeks, so that skin wrinkles were induced. Then,
the HA-PG solution was injected subcutaneously into the skin, and
the wrinkled skin before and after the injection was made into
replicas. The area, length, and depth of the wrinkles were measured
using a wrinkle analysis machine, and compared. As a result, as
illustrated in FIG. 23, the area, length, and depth of the wrinkles
before injection of the HA-PG solution were about 6 mm.sup.2, about
36 mm, and about 90 .mu.m, respectively, whereas those after
injection of the HA-PG solution were about 2 mm.sup.2, about 19 mm,
and about 68 .mu.m, respectively. From this, it was possible to
identify that the area, length, and depth of the wrinkles are
remarkably decreased. These results indicate that the HA-PG
solution of the present invention can be utilized as a filler
composition for improving skin wrinkles.
[0132] 5-3. Comparison with Existing Filler Products
[0133] (1) Comparison in Terms of Adhesive Strength and Fixability
in Body
[0134] In order to more specifically identify a possibility of
utilizing the HA-PG solution as a filler, for HA-PG hydrogels based
on hyaluronic acid derivatives having various molecular weights
(200 KDa, 1 MDa) and commercially available conventional filler
products (Megafill, Perlane), maintenance and degradation patterns
thereof in the body were measured for about 9 months and compared.
In addition, for the hydrogels produced by the HA-PG solution of
the present invention and the Perlane product, adhesive strength or
fixability in the body were compared. As a result, as illustrated
in FIGS. 24 and 25, it was possible to identify that for the
Megafill and Perlane products, weight and volume of the hydrogel in
the body tend to decrease from about one month after the day of
injection, whereas for the HA-PG hydrogels, shapes thereof are
maintained for 9 months without large changes in weight and volume.
It was found that the HA-PG hydrogels have an excellent ability to
be maintained in the body. In addition, for the hydrogels, adhesive
strength in the body was identified. As a result, as illustrated in
FIG. 26, it was possible to identify that the Perlane product
exhibits a phenomenon in which the hydrogel produced thereby is not
fixed to the skin and leans to a specific site, whereas the
hydrogel produced by the HA-PG solution is well maintained in the
body by adhering and being fixed to the injection site. Such
excellent adhesive strength in the body is a result of the reaction
between a functional group, such as an amine group and a thiol
group, existing on the skin and the hyaluronic acid derivative of
the present invention (FIG. 22(a)). That is, the HA-PG solution of
the present invention exhibits better stability and adhesiveness in
the body than the existing filler products.
[0135] (2) Comparison of Injectability (Injectability Test)
[0136] In order to specifically identify excellent injectability of
the HA-PG solution, for the HA-PG hydrogels based on hyaluronic
acid derivatives with various molecular weights (200 KDa, 1 MDa)
and commercially available conventional filler products (Megafill,
Perlane), changes in extrusion force depending on injection needle
sizes (21 G, 25 G, 29 G, 30 G) were not only measured using the
Universal Testing Machine (UTM), but also the break loose force,
which is a force required to initially move a syringe, and the
dynamic glide force, which is a force required to maintain motility
of a moving syringe, were quantified and compared. As a result, as
illustrated in FIGS. 27 and 28, Megafill was not extruded in a
small-sized injection needle due to a large particle size, and
extrusion was possible only at 21 G or less. Even in a case of
Perlane, extrusion force in an irregular form was exhibited at 29 G
and 30 G due to a particle size. On the other hand, in a case of
the HA-PG solutions (200 KDa, 1 MDa) of the present invention, it
was possible to identify that effective extrusion is achieved even
with a small force in all sizes of needles including 29 G and 30 G.
In addition, break loose forces and dynamic glide forces were
compared. As a result, as illustrated in FIG. 29, as compared with
Megafill, for which measurement was not possible due to poor
extrusion, and Perlane showing a high force value, the HA-PG
solutions of the present invention (200K, 1M) exhibited a
remarkably low force value. Thus, it was found that the HA-PG
solution of the present invention can be easily injected regardless
of extrusion force for injection, that is, an injection needle
size. These results indicate that the HA-PG solutions of the
present invention can be injected directly to a target site and can
be more stably injected.
[0137] 5-4. Functional Filler Composition Containing Cell Growth
Factor
[0138] In order to identify application as a functional filler, an
HA-PG solution in which epithelial cell growth factors (EGFs; 20
ng/ml, 1 .mu.m/ml, 20 .mu.g/ml) are encapsulated was injected into
the skin of hairless mice in which wrinkles had been induced by the
method of Example 5-2; the wrinkled skin before and after the
injection was made into replicas; and the area, length, and depth
of the wrinkles were measured using a wrinkle analysis machine, and
compared. At one month after the injection of the HA-PG solution in
which the EGFs are encapsulated, the skin tissue was collected and
OCT frozen sections thereof were constructed. Then, through
hematoxylin & eosin (H&E) staining of the OCT frozen
sections, histopathological examination was performed. In addition,
at one month after injection, into the hairless mice, an HA-PG
solution in which EGFs (10 .mu.g/ml) are encapsulated, an HA-PG
solution, and Perlane which is an existing product, differences in
skin tissue regeneration were compared through H&E and Masson's
trichrome (MT) in the same manner as above.
[0139] As a result, as illustrated in FIG. 30, it was possible to
identify that in a case where the HA-PG solution in which EGFs are
encapsulated is injected, regardless of sizes of the formed
wrinkles, the area, length, and depth of the wrinkles are all
remarkably decreased as compared with before the injection. In
addition, as illustrated in FIG. 31, collagen fibers in the dermal
layer of the skin were destroyed to show poor density and irregular
arrangement; on the other hand, as a concentration of the
encapsulated epithelial cell growth factors increased, the collagen
fibers tended to be regularly arranged. From this, it was possible
to identify significant skin-regenerating and wrinkle-improving
effects. In addition, as illustrated in FIGS. 32 and 33, in a case
where the HA-PG solution in which the EGFs are encapsulated is
injected, it was possible to identify that the epidermal layer
thickened due to induction of wrinkles is remarkably thinned as
compared with the other comparative groups (No filler, Perlane,
HA-PG) and that a collagen density of the dermal layer is largely
increased. These results indicate that a cell growth factor for
improving skin wrinkles is applied to the HA-PG solution of the
present invention, and thus the resultant can be utilized as a
functional filler.
Example 6
Preparations for Wound Dressing, Wound Healing Agent, and Adhesion
Barrier
[0140] In order to identify application as a dressing preparation
for wound healing, an HA-PG solution was applied to a wound-induced
animal model in which the dorsal skin of mice was incised to a size
of 1 cm x 1 cm, and then crosslinking was performed with
surrounding active oxygen. Then, formation of a hydrogel at the
wound site, and thus adhesion of the HA-PG solution to the wound
site were identified. As a result, as illustrated in FIG. 34, it
was possible to identify that a hydrogel film is formed within 10
minutes after the HA-PG solution has been applied to the wound
site, from which the HA-PG solution adheres to the wound site.
These results indicate that the HA-PG solution can be utilized as a
new type of wound dressing material which has excellent oxidizing
ability and can be evenly applied to a wound without any additional
additive.
Example 7
Formulation in Powders and Storage Stability Analysis 7-1.
Formulation in Powders
[0141] In order to identify a possibility of formulating a filler
composition in powders, as illustrated in FIG. 35, the HA-PG
solution of Preparation Example 1 was freeze-dried to prepare a
filler composition in a powder form. Thereafter, the filler
composition in a powder form was dissolved in PBS (pH 7) to be
resolubilized, and then subcutaneously injected into mice to
observe whether a hydrogel was formed. As a result, as illustrated
in FIG. 36, it was possible to identify that the filler composition
of the present invention, which has been resolubilized in a
freeze-dried powder state, is crosslinked by oxidizing power in the
body to form a hydrogel as before. These results indicate that the
filler composition of the present invention, which can be
formulated in powders, can provide users with ease of use and ease
of storage.
[0142] 7-2. Storage Stability Analysis
[0143] In order to identify specific storage stability of the
filler composition, it was first identified whether the HA-PG
solution of Preparation Example 1 is gelated in a storage container
while storing the same at room temperature (25.degree. C.) or a
refrigerated (4.degree. C.) state. In addition, nitrogen gas was
injected into the storage container to block contact between the
HA-PG solution and oxygen, and then stored in a refrigerated
(4.degree. C.) state for 3 days; on the other hand, the HA-PG
solution was stored as a frozen (-80.degree. C.) state for 10 days.
Then, these solutions were injected subcutaneously into mice to
observe whether hydrogels were formed. As a result, as illustrated
in FIG. 37, due to high oxidizing power, gelation of the filler
composition of the present invention proceeded within one day at
room temperature and within three days at a refrigerated state. On
the other hand, as illustrated in FIG. 38, it was possible to
identify that under a condition where oxygen is blocked, in a case
of being in a refrigerated state, gelation does not proceed even
after 3 days have lapsed; in a case where the HA-PG solution is
stored in a frozen state and then thawed after 10 days, the HA-PG
solution maintains properties of a solution, and even in a case
where this HA-PG solution is injected into the skin of mice, such
an HA-PG solution is crosslinked due to oxidizing power in the body
to form a hydrogel as before. These results indicate that the
filler composition of the present invention can be stored for a
longer period of time under a condition where oxygen is blocked or
in a frozen state.
[0144] It will be understood by those skilled in the art to which
the present invention belongs that the foregoing description of the
present invention is for illustrative purposes and that various
changes and modifications may be readily made without departing
from the technical spirit or essential features of the present
invention. Therefore, it is to be understood that the
above-described examples are illustrative in all aspects and not
restrictive.
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