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O H F OH
H
O
O O
H
STA
O
O O
Self-hydrolysis
OH
pH = 7.4 H H O
O
TA O
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Supramolecular nanofibers of triamcinolone acetonide for uveitis therapy Xingyi Li1, Yuqin Wang1, Chengbiao Yang2, Shuai Shi1, Ling Jin1, Zichao Luo1, Jing Yu1, Zhaoliang Zhang1, Zhimou Yang2, Hao Chen1* Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x
10
Supramolecular nanofibers of prodrugs hold
15
20
25
30
advantages for drug release due to its high drug
potential for the treatment of eye diseases.
payload, sustained and constant drug release
1. Introduction
behaviors, and stimuli responsiveness. In this study,
40
Molecular hydrogels,1-6 formed by the self-assembly
we reported on a supramolecular hydrogel mainly
of small molecules,7-11 have shown great potential in
formed by a clinically used drug of triamcinolone
tissue
acetonide (TA). Such hydrogel could only be
preparation,20,
prepared via a ester bond hydrolysis process from
their application in controlled drug release attracts
its prodrug of succinated triamcinolone acetonide
45
engineering,12-15 21
sensing,16-19
materials
and controlled release.22-26 Recently,
extensive research interests and many of them are
(STA). The resulting hydrogel could constantly
promising for practical applications.27,
release TA in the in vitro drug release experiment.
application in controlled drug release, molecular
The TA hydrogel possessed an excellent transscleral
hydrogels can serve as carriers to physically entrap
penetration ability, as evaluated by the in vitro
therapeutic agents. The self-assembled nano-structures
transscleral transport study. The developed TA
50
28
For their
in molecular hydrogels can increase the solubility of
hydrogel also exhibited great ocular compatibility
hydrophobic therapeutic agents that will be released
of rats, as indicated by the optical coherence
from the gels via passive diffusion or degradation of
tomography (OCT) images, HE observation, and
the gels.8,
glial fibrillary acidic protein (GFAP) and vimentin
derivatives of therapeutic agents as hydrogelators.29-31
immuno-staining assays of the retinas. Our TA
55
9, 16
An alternative approach is to use
Molecular hydrogels of therapeutic agents are carrier-
hydrogel showed a decreased efficacy to inhibit
free drug delivery systems with very high and
ocular inflammation in the rat's experiment
designable drug loading capacities. Several anti-cancer
autoimmune uveitis (EAU) model compared to the
and anti-inflammatory drugs have been conjugated
commercial 35
properties of the hydrogel indicated its great
without
TA
causing
suspension
®
(Transton ),
complications
such
as
with peptides to confer such carrier-free drug delivery
but high
intraocular pressure and cataract. These promising
60
systems. Besides, molecular hydrogels of therapeutic agents exhibit little or no burst release phenomenon. A very recent report by Stupp group showed that
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1
Nanoscale Accepted Manuscript
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
5
Page 3 of 11
Nanoscale View Article Online
DOI: 10.1039/C4NR04761C
molecular hydrogel of a dexamethasone derivative
Bedford, MA, USA) was used for the preparation of
could release dexamethasone more sustainably than a
solution.
molecular
2.2 Preparation of succinated triamcinolone
hydrogel
with
physically
entrapped
32
dexamethasone. For
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
Triamcinolone acetonide (TA, 1.3g, 3mmol) and
therapeutic agents with fewer modifications or no
succinic anhydride (0.9g, 8.9mmol) were co-dissolved
modifications will be beneficial, because they can be
in pyridine (20mL) and stirred at room temperature for
synthesized more easily and in large scale. For example,
3h. The organic solvent was removed by a rotary
15
only
applications,
FDA-approved
hydrogelators
molecular
hydrogel
50
minutes vigorous stirring, the flask was placed in a
itself. Recently, we reported on a molecular hydrogel
refrigerator. Finally, the fine crystals were collected
mainly formed by taxol itself that could inhibit tumors
through centrifugation and then lyophilized for further
growth and prevent tumors metastasis. The hydrogel of
applications. 55
STA was dissolved in the PBS buffer solution (pH =
process or pH adjustment could not be applied to
7.4) at a final concentration of 20 mg/mL. The gel
prepare it. The result, in combine with other examples
would form after about 35h at 37oC. We then
of molecular hydrogels of hydrophobic compounds,
monitored the release profile of TA from the TA 60
2, 23, 30
of hydrophobic therapeutic agents
of TA from TA suspension (Transton®) was detected
.
In this study, we attempted to develop a molecular
by a HPLC (Agilent1200) system.
hydrogel mainly formed by triamcinolone acetonide (TA) itself via an ester bond hydrolysis process. The in
2.4 In vitro transscleral penetration test 65
and then the sclera tissue was carefully removed for the
ocular biocompatibility of TA hydrogel was evaluated.
evaluation of transscleral penetration test of various TA
Moreover, the efficiency to inhibit ocular inflammation
formulations. All these tests were compiled with the
in the rat's experiment autoimmune uveitis (EAU)
Guide for the Care and Use of Laboratory Animals, 70
Institute of Laboratory Animal Resources, and were
(Transton®) were also studied in the Lewis rat.
approved by the Institutional Animal Care and Use
2. Experimental
Committee of Wenzhou Medical University. The
2.1 Materials
obtained sclera tissue was placed between the donor
Triamcinolone acetonide (TA) was provided by Baoman Biotechnology (Shanghai, China). Succecide
40
Six male mature Lewis rats (~250 g) were sacrificed
vitro transscleral penetration ability as well as its
model compared with the commercial TA suspension
35
hydrogel formed after 48h at 37oC by a LCMS-20AD (Shimadzu) system. Meanwhile, the release behaviour
probably the only way to prepare molecular hydrogels
30
2.3 Hydrogel formation and drug release
process, while other methods such as heating-cooling
indicated that hydrolysis process was a unique and
25
evaporator and water (70mL) was added. After 20
(Lanreotide) is the one formed by therapeutic agent
taxol was formed through an ester bond hydrolysis
20
acetonide (STA)
of
the
practical
45
and the receiving compartments of the vertical 75
modified diffusion cell. The donor compartment was
acid (SA) was purchaced from Sigma-Aldrich (USA).
filled with 0.5mL 2.0 wt% TA hydrogel or 2.0 wt% TA
All the materials used in the study were analytic
suspension
reagent degree. Milli-Q grade water (Millipore,
compartment was filled with 5 mL PBS (pH=7.4). The
2 | Journal Name, [year], [vol], 00–00
(Transton®),
while
the
receiving
This journal is © The Royal Society of Chemistry [year]
Nanoscale Accepted Manuscript
5
Nanoscale
Page 4 of 11 View Article Online
DOI: 10.1039/C4NR04761C
total amount of penetration drug as a function with
pressure (IOP) of rats administrated with both these
time was determined by HPLC (Agilent 1200) system.
two TA formulations were determined by using the
2.5 In vivo compatibility test
Tono-Pen XL(Mentor, Norwell, MA). Meanwhile, the
The ocular biocompatibility of TA hydrogel and TA
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
45
suspension (50µL; 2.0 wt% TA) via retrobulbar
clinical evaluation.
injection was evaluated by an optical coherence
2.6 Induction and evaluation of experiment
tomography (OCT) and Histological observation in
autoimmune uveitis (EAU) model Lewis rats were immunized with 10% chloral
Lewis rats. The retinal thickness as well as its morphology of rats after retrobulbar injection of TA
15
were monitored by an ultrahigh resolution spectral
Freund’s
domain OCT. Images were obtained at a capture rate of
tuberculosis H37Ra (Difco Laboratories, Detroit, MI)33.
48 frames per second using a light source with a
The rats were monitored daily by a slit-lamp for
Mycobacterium
clinical signs of EAU, and the severity was graded
were anesthetized
mydriasis
no inflammation; grade 1: mild iris vessel engorgement
with 0.25% (w/v) tropicamide. Thereafter, the animals
and minimal retinal vasculitis; grade 2: mild retinal
were placed horizontally in front of the slit lamp
vasculitis and anterior chamber cells; grade 3, fibrous
followed
by
the
60
exudates at the pupil margin and moderate retinal
forth movement of slit lamp. After 1, 7 and 14days of
vasculitis; and grade 4, severe retinal vaculitis and
drug injection, the whole eye ball were enucleated,
retroiridal hypopyon. After 10days of injection, the rats
fixed by 10% paraformaldehyde, dehydrated through a
were separately treated with (1) saline solution (50µL);
graded alcohol series alcohol solution and embedded in
(2) commercial TA suspension (Transton®, 50µL; 2.0 65
wt%) and (3) TA hydrogel (50µL; 2.0 wt%) via
staining). For immunocytochemistry of Vimentin and
retrobulbar injection. Ocular examination and clinical
GFAP,
(anti-GFAP
scores were preformed by a slit-lamp with an
antibody and anti-Vimentin antibody, 1:200, Abcam,
experienced doctor. After 17days, rat eyes were
Hong Kong, China ) were made in 1% BSA containing
enucleated, snap frozen, and then cut into 10µm thick
primary
antibody
solutions
0.1% Triton X-100 and incubated for overnight at 4 C.
70
cryosections
as
described
previously24.
The
To visualize the nucleus, the sections were incubated
cryosections were then mounted onto superfrost plus
with hoechst (1:1000, sigma, MO, USA) at room
glass slides (Fisher Scientific, TX, USA), and fixed
temperature for 5 min. After the washing, coverslips
with ice-cold acetone for 15 min. The sections were
were applied on the sections with anti-fade mounting
blocked with 1% BSA at room temperature for 2 h, and
medium and fluorescent images were captured using a
40
containing
from 0 to 4 according to the previous study. Grade 0:
o
35
55
adjuvant
100nm and a 3µm depth resolution. The animals
paraffin wax for histopathological observation (H£E
30
hydrate solution followed by subcutaneous injection of 100µL of IRBP peptide emulsified with a complete
and retinal images were acquired with the back and
25
50
hydrogel and TA suspension at various time points
center wavelength of 840nm, a broad wavelength of
20
eyes of rats were monitored daily by a slit-lamp for
75
incubated with goat anti-rat IL-6 antibody (1:200,
inverted fluorescence microscope (Nikon Eclipse Ti,
R&D, MN, UAS) and rabbit anti-rat IL-17 antibody
Japan).
(1:200,
During the whole experiment, the intraocular This journal is © The Royal Society of Chemistry [year]
Abcam,
Hong Kong,
China) at room
temperature for 4 hour. After washing with PBS, The Journal Name, [year], [vol], 00–00 | 3
Nanoscale Accepted Manuscript
5
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DOI: 10.1039/C4NR04761C
sections were incubated with donkey anti-rabbit alexa
example, a gel in Fig.1). LC-MS results indicated that
fluor 555 (1:1000, Abcam, Hong Kong, China) and
the hydrogel was formed by the ester bond hydrolysis
donkey anti-goat alexa fluor 488 (1:1000, Abcam,
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
wt% STA, the minimum hydrolysis percentage of STA
followed by washing with PBS. To visualize the
for hydrogel formation was about 56%. It took about
nucleus, the sections were incubated with hoechst
35h for the solution (pH 7.4) to form a gel at 37oC. The
(1:1000, sigma, MO, USA) at room temperature for 5
hydrolysis percentage of STA kept increasing in the gel
sections
with
anti-fade
fluorescent images were captured using an inverted
percentage of STA in the resulting gels could not reach
fluorescence microscope (Nikon Eclipse Ti, Japan).
100%. This property might hinder its practical
3. Results and discussion
applications because the ratio of the TA and STA in gel
and
medium
characterization
of TA
50
It is well-known that steroids have excellent self-
TA at higher pH values in the presence of polymer
assembly abilities. They have been used to prepare
additives, because the polymer additives could stabilize
17, 32
many self-assembled nano-carriers and hydrogels
.
This information stimulated us to test whether steroids
the nanofibers totally composed of hydrophobic 55
TA2.7.15.23-26. The resulting gel was stable for more than
themselves can form molecular hydrogels or not.
6 months at room temperature (20-25oC). Meanwhile,
Therefore, we designed and synthesized succinated TA
if we mixed STA (90 molar percentage) and TA (10
(STA in Scheme 1) as the precursor of the possible
molar percentage) with equal molar of succinic acid in
gelator. The STA could be obtained in one simple step
PBS solution (total weight concentration was 2.0 wt%),
of chemical reaction with a high yield (Fig. S-1), which
30
system might vary batch to batch. Such shortcoming might be overcome by totally converting the STA to
hydrogel
25
and it was about 68.5% after 14 days at 37oC (Fig.1A). One shortcoming of our system was that the hydrolysis
Preparation
mounting
45
and
3.1
20
of STA. For the gel from a PBS solution containing 2.0
Hong Kong, China) at room temperature for 1 h,
min. After washing, coverslips were applied on the
15
40
60
the resulting suspension could not form a gel by the
guaranteed its large scale preparation. Moreover, its
heating-cooling
water solubility was high up to 4.5 wt% (45mg/mL in
observations indicated that the hydrolysis process
phosphate buffer saline (PBS, pH 7.4)).
might be the only way to form the hydrogel of TA.
O
H F
O
OH
F O
OH
O
H O
pH = 7.4
65
O
STA
O
methods.
These
O
H
(viscosity
O
TA
by rheology. As shown in Fig.1B, both the storage modulus (elastisity or G’) and the loss modulus
OH H
sonication
We then characterized the hydrogel at 48 h time point
OH
Self-hydrolysis
O O
H
H
or
35
exhibited
weak
frequency
bigger than that of G” and it was >1,000 Pa, suggesting
cheme 1 Chemical structures of succinated triamcinolone
hydrogelation process via an ester bond hydrolysis process
G”)
dependences. The value of G’ was at least 5 times
S
acetonide (STA) and triamcinolone acetonide (TA) and
or
70
the presence of a high elastic three dimensional network in the gel. We observed fibrils in the hydrogel by transmission electronic microscopy (TEM, Fig.1C).
We indeed observed a hydrogel formation from PBS
The fibrils were about 20-50 nm in diameter and longer
solutions containing more than 1.15 wt% of STA (for
than 1 µm. These fibrils entangled with each other to
4 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]
Nanoscale Accepted Manuscript
5
Nanoscale
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DOI: 10.1039/C4NR04761C
form larger fibers. We then monitored the release
the gel, and D) Drug release profile of TA from the 2.0 wt% TA
profile of TA from either 2.0 wt% TA suspension
suspension (Transton®) and 2.0 wt% TA gel (a gel at 48 h time
(Transton®) or 2.0 wt% TA hydrogel as function with
35
3.2 In vitro transscleral penetration test
time at 37oC (Fig.1D). We observed a constant release
In order to investigate the potential application of
rate of TA from TA hydrogel during the 12 h
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
our developed TA hydrogel for posterior eye diseases
experimental period (about 55µg/mL per-hour). There
therapy, we compared its transscleral penetration
was about 42% percentage of total TA got released 40
from the TA hydrogel in the period of 12 h study.
15
could
strengthen
the
formulations was performed by using rat sclera. As
supramolecular
presented in Fig.2, the higher accumulative amounts of
hydrogels and slow down the release of drug
TA transported across the sclera were observed for the
molecules4.20.24.29. We therefore imaged that the 45
addition of polymer additives to our gels might prolong
The accumulative amount of transported TA from
suspension, it was clearly observed that TA was
donor compartments of the TA hydrogel group was
sustained release during the whole experimental period
about 25 times than that of the Transton® group at 6 h
(about 1.5µg/mL per-hour). Only about 15% of total 50
TA was released from TA suspension at 12h time point.
time point. This might be explained by that more TA was released from TA hydrogel than that from TA
As compared with TA hydrogel system, TA suspension
hydrogel at same time point (Fig.1D), yet resulting in
showed more sustained release behaviour of TA, 25
TA hydrogel group as compared with the Transton® group (p
Nanoscale Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: X. Li, Y. Wang, C. Yang, S. Shi, L. Jin, Z. Luo, J. Yu, Z. Zhang, Z. Yang and H. Chen, Nanoscale, 2014, DOI: 10.1039/C4NR04761C.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/nanoscale
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
O H F OH
H
O
O O
H
STA
O
O O
Self-hydrolysis
OH
pH = 7.4 H H O
O
TA O
Nanoscale Accepted Manuscript
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View Article Online
H F OH O OH
Nanoscale
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DOI: 10.1039/C4NR04761C
Cite this: DOI: 10.1039/c0xx00000x
ARTICLE TYPE
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Supramolecular nanofibers of triamcinolone acetonide for uveitis therapy Xingyi Li1, Yuqin Wang1, Chengbiao Yang2, Shuai Shi1, Ling Jin1, Zichao Luo1, Jing Yu1, Zhaoliang Zhang1, Zhimou Yang2, Hao Chen1* Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x
10
Supramolecular nanofibers of prodrugs hold
15
20
25
30
advantages for drug release due to its high drug
potential for the treatment of eye diseases.
payload, sustained and constant drug release
1. Introduction
behaviors, and stimuli responsiveness. In this study,
40
Molecular hydrogels,1-6 formed by the self-assembly
we reported on a supramolecular hydrogel mainly
of small molecules,7-11 have shown great potential in
formed by a clinically used drug of triamcinolone
tissue
acetonide (TA). Such hydrogel could only be
preparation,20,
prepared via a ester bond hydrolysis process from
their application in controlled drug release attracts
its prodrug of succinated triamcinolone acetonide
45
engineering,12-15 21
sensing,16-19
materials
and controlled release.22-26 Recently,
extensive research interests and many of them are
(STA). The resulting hydrogel could constantly
promising for practical applications.27,
release TA in the in vitro drug release experiment.
application in controlled drug release, molecular
The TA hydrogel possessed an excellent transscleral
hydrogels can serve as carriers to physically entrap
penetration ability, as evaluated by the in vitro
therapeutic agents. The self-assembled nano-structures
transscleral transport study. The developed TA
50
28
For their
in molecular hydrogels can increase the solubility of
hydrogel also exhibited great ocular compatibility
hydrophobic therapeutic agents that will be released
of rats, as indicated by the optical coherence
from the gels via passive diffusion or degradation of
tomography (OCT) images, HE observation, and
the gels.8,
glial fibrillary acidic protein (GFAP) and vimentin
derivatives of therapeutic agents as hydrogelators.29-31
immuno-staining assays of the retinas. Our TA
55
9, 16
An alternative approach is to use
Molecular hydrogels of therapeutic agents are carrier-
hydrogel showed a decreased efficacy to inhibit
free drug delivery systems with very high and
ocular inflammation in the rat's experiment
designable drug loading capacities. Several anti-cancer
autoimmune uveitis (EAU) model compared to the
and anti-inflammatory drugs have been conjugated
commercial 35
properties of the hydrogel indicated its great
without
TA
causing
suspension
®
(Transton ),
complications
such
as
with peptides to confer such carrier-free drug delivery
but high
intraocular pressure and cataract. These promising
60
systems. Besides, molecular hydrogels of therapeutic agents exhibit little or no burst release phenomenon. A very recent report by Stupp group showed that
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1
Nanoscale Accepted Manuscript
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
5
Page 3 of 11
Nanoscale View Article Online
DOI: 10.1039/C4NR04761C
molecular hydrogel of a dexamethasone derivative
Bedford, MA, USA) was used for the preparation of
could release dexamethasone more sustainably than a
solution.
molecular
2.2 Preparation of succinated triamcinolone
hydrogel
with
physically
entrapped
32
dexamethasone. For
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
Triamcinolone acetonide (TA, 1.3g, 3mmol) and
therapeutic agents with fewer modifications or no
succinic anhydride (0.9g, 8.9mmol) were co-dissolved
modifications will be beneficial, because they can be
in pyridine (20mL) and stirred at room temperature for
synthesized more easily and in large scale. For example,
3h. The organic solvent was removed by a rotary
15
only
applications,
FDA-approved
hydrogelators
molecular
hydrogel
50
minutes vigorous stirring, the flask was placed in a
itself. Recently, we reported on a molecular hydrogel
refrigerator. Finally, the fine crystals were collected
mainly formed by taxol itself that could inhibit tumors
through centrifugation and then lyophilized for further
growth and prevent tumors metastasis. The hydrogel of
applications. 55
STA was dissolved in the PBS buffer solution (pH =
process or pH adjustment could not be applied to
7.4) at a final concentration of 20 mg/mL. The gel
prepare it. The result, in combine with other examples
would form after about 35h at 37oC. We then
of molecular hydrogels of hydrophobic compounds,
monitored the release profile of TA from the TA 60
2, 23, 30
of hydrophobic therapeutic agents
of TA from TA suspension (Transton®) was detected
.
In this study, we attempted to develop a molecular
by a HPLC (Agilent1200) system.
hydrogel mainly formed by triamcinolone acetonide (TA) itself via an ester bond hydrolysis process. The in
2.4 In vitro transscleral penetration test 65
and then the sclera tissue was carefully removed for the
ocular biocompatibility of TA hydrogel was evaluated.
evaluation of transscleral penetration test of various TA
Moreover, the efficiency to inhibit ocular inflammation
formulations. All these tests were compiled with the
in the rat's experiment autoimmune uveitis (EAU)
Guide for the Care and Use of Laboratory Animals, 70
Institute of Laboratory Animal Resources, and were
(Transton®) were also studied in the Lewis rat.
approved by the Institutional Animal Care and Use
2. Experimental
Committee of Wenzhou Medical University. The
2.1 Materials
obtained sclera tissue was placed between the donor
Triamcinolone acetonide (TA) was provided by Baoman Biotechnology (Shanghai, China). Succecide
40
Six male mature Lewis rats (~250 g) were sacrificed
vitro transscleral penetration ability as well as its
model compared with the commercial TA suspension
35
hydrogel formed after 48h at 37oC by a LCMS-20AD (Shimadzu) system. Meanwhile, the release behaviour
probably the only way to prepare molecular hydrogels
30
2.3 Hydrogel formation and drug release
process, while other methods such as heating-cooling
indicated that hydrolysis process was a unique and
25
evaporator and water (70mL) was added. After 20
(Lanreotide) is the one formed by therapeutic agent
taxol was formed through an ester bond hydrolysis
20
acetonide (STA)
of
the
practical
45
and the receiving compartments of the vertical 75
modified diffusion cell. The donor compartment was
acid (SA) was purchaced from Sigma-Aldrich (USA).
filled with 0.5mL 2.0 wt% TA hydrogel or 2.0 wt% TA
All the materials used in the study were analytic
suspension
reagent degree. Milli-Q grade water (Millipore,
compartment was filled with 5 mL PBS (pH=7.4). The
2 | Journal Name, [year], [vol], 00–00
(Transton®),
while
the
receiving
This journal is © The Royal Society of Chemistry [year]
Nanoscale Accepted Manuscript
5
Nanoscale
Page 4 of 11 View Article Online
DOI: 10.1039/C4NR04761C
total amount of penetration drug as a function with
pressure (IOP) of rats administrated with both these
time was determined by HPLC (Agilent 1200) system.
two TA formulations were determined by using the
2.5 In vivo compatibility test
Tono-Pen XL(Mentor, Norwell, MA). Meanwhile, the
The ocular biocompatibility of TA hydrogel and TA
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
45
suspension (50µL; 2.0 wt% TA) via retrobulbar
clinical evaluation.
injection was evaluated by an optical coherence
2.6 Induction and evaluation of experiment
tomography (OCT) and Histological observation in
autoimmune uveitis (EAU) model Lewis rats were immunized with 10% chloral
Lewis rats. The retinal thickness as well as its morphology of rats after retrobulbar injection of TA
15
were monitored by an ultrahigh resolution spectral
Freund’s
domain OCT. Images were obtained at a capture rate of
tuberculosis H37Ra (Difco Laboratories, Detroit, MI)33.
48 frames per second using a light source with a
The rats were monitored daily by a slit-lamp for
Mycobacterium
clinical signs of EAU, and the severity was graded
were anesthetized
mydriasis
no inflammation; grade 1: mild iris vessel engorgement
with 0.25% (w/v) tropicamide. Thereafter, the animals
and minimal retinal vasculitis; grade 2: mild retinal
were placed horizontally in front of the slit lamp
vasculitis and anterior chamber cells; grade 3, fibrous
followed
by
the
60
exudates at the pupil margin and moderate retinal
forth movement of slit lamp. After 1, 7 and 14days of
vasculitis; and grade 4, severe retinal vaculitis and
drug injection, the whole eye ball were enucleated,
retroiridal hypopyon. After 10days of injection, the rats
fixed by 10% paraformaldehyde, dehydrated through a
were separately treated with (1) saline solution (50µL);
graded alcohol series alcohol solution and embedded in
(2) commercial TA suspension (Transton®, 50µL; 2.0 65
wt%) and (3) TA hydrogel (50µL; 2.0 wt%) via
staining). For immunocytochemistry of Vimentin and
retrobulbar injection. Ocular examination and clinical
GFAP,
(anti-GFAP
scores were preformed by a slit-lamp with an
antibody and anti-Vimentin antibody, 1:200, Abcam,
experienced doctor. After 17days, rat eyes were
Hong Kong, China ) were made in 1% BSA containing
enucleated, snap frozen, and then cut into 10µm thick
primary
antibody
solutions
0.1% Triton X-100 and incubated for overnight at 4 C.
70
cryosections
as
described
previously24.
The
To visualize the nucleus, the sections were incubated
cryosections were then mounted onto superfrost plus
with hoechst (1:1000, sigma, MO, USA) at room
glass slides (Fisher Scientific, TX, USA), and fixed
temperature for 5 min. After the washing, coverslips
with ice-cold acetone for 15 min. The sections were
were applied on the sections with anti-fade mounting
blocked with 1% BSA at room temperature for 2 h, and
medium and fluorescent images were captured using a
40
containing
from 0 to 4 according to the previous study. Grade 0:
o
35
55
adjuvant
100nm and a 3µm depth resolution. The animals
paraffin wax for histopathological observation (H£E
30
hydrate solution followed by subcutaneous injection of 100µL of IRBP peptide emulsified with a complete
and retinal images were acquired with the back and
25
50
hydrogel and TA suspension at various time points
center wavelength of 840nm, a broad wavelength of
20
eyes of rats were monitored daily by a slit-lamp for
75
incubated with goat anti-rat IL-6 antibody (1:200,
inverted fluorescence microscope (Nikon Eclipse Ti,
R&D, MN, UAS) and rabbit anti-rat IL-17 antibody
Japan).
(1:200,
During the whole experiment, the intraocular This journal is © The Royal Society of Chemistry [year]
Abcam,
Hong Kong,
China) at room
temperature for 4 hour. After washing with PBS, The Journal Name, [year], [vol], 00–00 | 3
Nanoscale Accepted Manuscript
5
Page 5 of 11
Nanoscale View Article Online
DOI: 10.1039/C4NR04761C
sections were incubated with donkey anti-rabbit alexa
example, a gel in Fig.1). LC-MS results indicated that
fluor 555 (1:1000, Abcam, Hong Kong, China) and
the hydrogel was formed by the ester bond hydrolysis
donkey anti-goat alexa fluor 488 (1:1000, Abcam,
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
wt% STA, the minimum hydrolysis percentage of STA
followed by washing with PBS. To visualize the
for hydrogel formation was about 56%. It took about
nucleus, the sections were incubated with hoechst
35h for the solution (pH 7.4) to form a gel at 37oC. The
(1:1000, sigma, MO, USA) at room temperature for 5
hydrolysis percentage of STA kept increasing in the gel
sections
with
anti-fade
fluorescent images were captured using an inverted
percentage of STA in the resulting gels could not reach
fluorescence microscope (Nikon Eclipse Ti, Japan).
100%. This property might hinder its practical
3. Results and discussion
applications because the ratio of the TA and STA in gel
and
medium
characterization
of TA
50
It is well-known that steroids have excellent self-
TA at higher pH values in the presence of polymer
assembly abilities. They have been used to prepare
additives, because the polymer additives could stabilize
17, 32
many self-assembled nano-carriers and hydrogels
.
This information stimulated us to test whether steroids
the nanofibers totally composed of hydrophobic 55
TA2.7.15.23-26. The resulting gel was stable for more than
themselves can form molecular hydrogels or not.
6 months at room temperature (20-25oC). Meanwhile,
Therefore, we designed and synthesized succinated TA
if we mixed STA (90 molar percentage) and TA (10
(STA in Scheme 1) as the precursor of the possible
molar percentage) with equal molar of succinic acid in
gelator. The STA could be obtained in one simple step
PBS solution (total weight concentration was 2.0 wt%),
of chemical reaction with a high yield (Fig. S-1), which
30
system might vary batch to batch. Such shortcoming might be overcome by totally converting the STA to
hydrogel
25
and it was about 68.5% after 14 days at 37oC (Fig.1A). One shortcoming of our system was that the hydrolysis
Preparation
mounting
45
and
3.1
20
of STA. For the gel from a PBS solution containing 2.0
Hong Kong, China) at room temperature for 1 h,
min. After washing, coverslips were applied on the
15
40
60
the resulting suspension could not form a gel by the
guaranteed its large scale preparation. Moreover, its
heating-cooling
water solubility was high up to 4.5 wt% (45mg/mL in
observations indicated that the hydrolysis process
phosphate buffer saline (PBS, pH 7.4)).
might be the only way to form the hydrogel of TA.
O
H F
O
OH
F O
OH
O
H O
pH = 7.4
65
O
STA
O
methods.
These
O
H
(viscosity
O
TA
by rheology. As shown in Fig.1B, both the storage modulus (elastisity or G’) and the loss modulus
OH H
sonication
We then characterized the hydrogel at 48 h time point
OH
Self-hydrolysis
O O
H
H
or
35
exhibited
weak
frequency
bigger than that of G” and it was >1,000 Pa, suggesting
cheme 1 Chemical structures of succinated triamcinolone
hydrogelation process via an ester bond hydrolysis process
G”)
dependences. The value of G’ was at least 5 times
S
acetonide (STA) and triamcinolone acetonide (TA) and
or
70
the presence of a high elastic three dimensional network in the gel. We observed fibrils in the hydrogel by transmission electronic microscopy (TEM, Fig.1C).
We indeed observed a hydrogel formation from PBS
The fibrils were about 20-50 nm in diameter and longer
solutions containing more than 1.15 wt% of STA (for
than 1 µm. These fibrils entangled with each other to
4 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]
Nanoscale Accepted Manuscript
5
Nanoscale
Page 6 of 11 View Article Online
DOI: 10.1039/C4NR04761C
form larger fibers. We then monitored the release
the gel, and D) Drug release profile of TA from the 2.0 wt% TA
profile of TA from either 2.0 wt% TA suspension
suspension (Transton®) and 2.0 wt% TA gel (a gel at 48 h time
(Transton®) or 2.0 wt% TA hydrogel as function with
35
3.2 In vitro transscleral penetration test
time at 37oC (Fig.1D). We observed a constant release
In order to investigate the potential application of
rate of TA from TA hydrogel during the 12 h
Published on 07 October 2014. Downloaded by University of Edinburgh on 15/10/2014 10:51:31.
10
our developed TA hydrogel for posterior eye diseases
experimental period (about 55µg/mL per-hour). There
therapy, we compared its transscleral penetration
was about 42% percentage of total TA got released 40
from the TA hydrogel in the period of 12 h study.
15
could
strengthen
the
formulations was performed by using rat sclera. As
supramolecular
presented in Fig.2, the higher accumulative amounts of
hydrogels and slow down the release of drug
TA transported across the sclera were observed for the
molecules4.20.24.29. We therefore imaged that the 45
addition of polymer additives to our gels might prolong
The accumulative amount of transported TA from
suspension, it was clearly observed that TA was
donor compartments of the TA hydrogel group was
sustained release during the whole experimental period
about 25 times than that of the Transton® group at 6 h
(about 1.5µg/mL per-hour). Only about 15% of total 50
TA was released from TA suspension at 12h time point.
time point. This might be explained by that more TA was released from TA hydrogel than that from TA
As compared with TA hydrogel system, TA suspension
hydrogel at same time point (Fig.1D), yet resulting in
showed more sustained release behaviour of TA, 25
TA hydrogel group as compared with the Transton® group (p
