Self-assembly of a ibuprofen-peptide conjugate to suppress ocular inflammation

Article abstract of DOI:10.1016/j.nano.2017.09.010

In the present study, we designed and synthesized a hydrogelator comprised of ibuprofen (IPF) and GFFY peptide linked through a cleavable ester bond. We found that the synthesized hydrogelator could spontaneously self-assemble into a hydrogel under a heat

Full text of DOI:10.1016/j.nano.2017.09.010

Self-assembly of a ibuprofen-peptide conjugate to suppress ocular inflamma-
tion
Xinxin Yu, Zhaoliang Zhang, Jing Yu, Hao Chen, Xingyi Li
PII:
DOI:
Reference:
S1549-9634(17)30177-6
doi: 10.1016/j.nano.2017.09.010
NANO 1668
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date:
Revised date:
Accepted date:
31 May 2017
19 September 2017
21 September 2017
Please cite this article as: Yu Xinxin, Zhang Zhaoliang, Yu Jing, Chen Hao,
Li Xingyi, Self-assembly of a ibuprofen-peptide conjugate to suppress ocular in-
flammation, Nanomedicine: Nanotechnology, Biology, and Medicine (2017),
0.1016/j.nano.2017.09.010
doi:
1
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ACCEPTED MANUSCRIPT
Self-assembly of a ibuprofen–peptide conjugate to suppress
ocular inflammation
a
#a
b
a
a*
Xinxin Yu , Zhaoliang Zhang , Jing Yu , Hao Chen , Xingyi Li
a
School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, 270
Xueyuan Road, Wenzhou, 325035, P.R. China.
b
Institute of Biomaterials and Engineering, Wenzhou Medical University, Wenzhou, 325035,
P.R. China.
#
Zhaoliang Zhang's contribution to this paper was equal to that of Xinxin Yu and is co-first
author.
Corresponding author: Xingyi Li, PhD, Associated professor, School of Ophthalmology &
Optometry and Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, P.R
China. Tel/fax: +86 577 88833806 E-mail: lixingyi_1984@163.com
Word count for Abstract: 135
Word count for manuscript: 4703
Number of References: 40
Number of figures: 5
Number of tables: 0
Number of Supplementary online-only files, if any: 2
Acknowledgement
This work was financially supported by grants from the National Natural Science
Foundation of China (31671022), the Key Program for International S&T
Cooperation Projects of China (2015DFA50310), the National Science and
Technology Major Project (2014ZX09303301) and the Science and Technology
Bureau of Wenzhou City (Y20140703 and Y20140141).
1

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Abstract
In present study, we designed and synthesized a hydrogelator comprised of
ibuprofen (IPF) and GFFY peptide linked through a cleavable ester bond. We found
that the synthesized hydrogelator could spontaneously self-assemble into a hydrogel
under a heating–cooling process. When the hydrogel was acted upon by an esterase,
IPF was released in a sustained manner. Moreover, the hydrogel had significantly
elevated anti-inflammatory efficacy, compared with IPF, in RAW264.7 macrophages.
The hydrogel showed good cytocompatibility, as well as excellent ocular
biocompatibility when instilled topically. In vivo results further demonstrated that the
hydrogel had therapeutic efficacy comparable to that of a current treatment, sodium
diclofenac (DIC) eyedrops, in suppressing ocular inflammation in lipopolysaccharide
(
LPS) induced uveitis. Our findings illustrated, for the first time, an effective
approach for developing supramolecular assemblies as anti-inflammatory
ophthalmic therapeutics for eye disorders.
Key words: Self-assembled hydrogel, anti-inflammatory, ocular drug delivery,
endotoxin induced uveitis
2

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BACKGROUND
Low molecular weight hydrogels formed by therapeutic agents and their
derivatives have recently received considerable attention because of their high
drug payloads, as well as their sustained and responsive drug release
1
-10
properties.
In particular, supramolecular hydrogels derived from drug–
peptide conjugates are emerging as new candidates for drug delivery because of
1
1-19
their excellent biocompatibility, biodegradability and thixotropic properties.
A number of agents, including anti-tumor (e.g., paclitaxel, camptothecin and
curcumin), anti-bacterial (e.g., vancomycin) and anti-inflammatory (e.g.,
dexamethasone and triamcinolone) drugs, coupled with peptides, were
successfully shown to act as hydrogelators, forming supramolecular hydrogels
2
0-29
for drug delivery.
More recently, Xu et al. described a hydrogelator,
composed of a D-amino acid and a non-steroidal anti-inflammatory drug
(NSAID), which had enhanced selectivity as a cyclooxygenase-2 (COX-2)
3
inhibitor. In addition, we recently demonstrated that the covalent conjugation
of ibuprofen (IPF) with a simple peptide sequence, through an amide bond,
produced a hydrogel-forming substance. Unfortunately, the conjugate had much
3
0
lower pharmacological activity than the parent drug. This undesired
shortcoming significantly limited the in vivo applications of the conjugate.
Therefore, it would be beneficial to design a drug–peptide conjugate that not
only would self-assemble to form a hydrogel, but would also retain the
biological activity of the native drug.
Considering the exceptional anti-inflammatory activity of native IPF, we choose
to release this drug from the hydrogel via an enzymatic reaction. Drawing from
1, 4,
previous studies reporting enzymatic disassembly of supramolecular hydrogelators,
2
1
we designed and synthesized a hydrogelator composed of IPF and a GFFY peptide,
linked through a cleavable ester bond. Introduction of the aromatic compound
hydroxybenzoic acid (HYD) as an enzyme cleavable linkage not only facilitated
self-assembly of the compound, forming a hydrogel, through π–π stacking and
3

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hydrophobic interactions, but also enabled restoration of biologically active IPF by
esterase catalyzed hydrolysis. We further investigated the intraocular biocompatibility
and in vivo anti-inflammatory efficacy of the resulting hydrogel in a rabbit model.
METHODS
Materials
HYD and IPF were from J&K Scientific (Shanghai, China). Lipopolysaccharide
(
LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Diclofenac sodium
eyedrops (0.1 wt% DIC; Shenyang Xingqi Pharmaceutical Co., Ltd, Shenyang, China)
was from the Eye Hospital of Wenzhou Medical University. N-Fmoc-protected amino
acids
HBTU) were from GL Biochem (Shanghai, China) Ltd. All other chemicals were
analytical grade.
and
O-Benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluorophosphate
(
Synthesis and characterization of IPF–HYD
Briefly, IPF (1.0 g, 4.85 mmol) was first dissolved into SOCl (15 mL) and
2
refluxed for 3 h. Next, unreacted SOCl was removed under vacuum to yield a yellow
2
liquid, containing the acyl chloride. Then, hydroxybenzoic acid (HYD; 0.67 g, 4.85
mmol) and pyridine (5 mL) were added to the acyl chloride in dry acetone (10 mL) at
0
°C and the solution stirred at room temperature for 5 h. Finally, the mixture was
filtered and the organic solvent removed by evaporation. The residue was purified by
flash chromatography on silica gel, eluted with ethyl acetate:petroleum ether 1:3–1:2
(v/v) to obtain the corresponding product at 75% yield. The final product was
1
characterized by H-NMR.
Synthesis of IPF–HYD–GFFY hydrogelator
Briefly, the IPF–HYD–GFFY hydrogelator was synthesized by classic solid-phase
peptide synthesis using 2-chlorotrityl chloride resin and N-Fmoc-protected amino
acids. The 2-chlorotrityl chloride resin was first swelled in dry dichloromethane
(DCM) for 20 min and then the first amino acid was loaded onto the resin, as a
solution of Fmoc-protected amino acid (1.5 equiv) and N,N-diisopropylethylamine
(DIEA; 2 equivalents) in DMF, for 2 h. After washing the column 5 times with DCM,
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the unreactive sites of the resin were blocked for 10 min with a blocking solution
(
DCM:MeOH:DIEA = 17:2:1). The Fmoc protecting group was then removed by
adding 20% piperidine in DMF, followed by coupling the Fmoc-protected amino acid
2 equivalents) to the free amino group on the resin using DIEA (2 equivalents) and
(
HBTU (1 equivalents) as the coupling agent, in DMF, for 2 h. These two steps were
repeated to elongate the peptide chain. Finally, IPF–HYD was coupled to the peptide
by adding DIEA (2 equivalents) and HBTU (1 equivalents), in DMF, for 2 h. The
resulting IPF–HYD–GFFY hydrogelator was cleaved from the resin with washing
solution (TFA: TIS: H O = 95:2.5:2.5; v/v/v). The crude product was purified by
2
1
reversed phase HPLC, characterized by H-NMR (Fig. 1) and MS, and lyophilized for
further use.
Formation of the IPF–HYD–GFFY hydrogel
Briefly, a calculated weight of the IPF–HYD–GFFY hydrogelator was first
suspended into phosphate buffered saline (PBS, pH 7.4) followed by the addition of a
molar equivalent of Na CO , in aqueous solution (1 mM). This produced a transparent
2
3
solution after heating at 80°C. The IPF–HYD–GFFY supramolecular hydrogel
formed spontaneously after cooling this solution to room temperature.
Characterization of the IPF–HYD–GFFY hydrogel
Transmission electron microscope (TEM)
The microstructure of the IPF–HYD–GFFY supramolecular hydrogel was observed
by TEM. The IPF–HYD–GFFY hydrogel samples were placed onto a grid and stained
with 0.5 wt% phosphotungstic acid for TEM observation.
Rheology
A rheology test was performed on a TA AR2000 rheometer (New Castle, Delaware,
USA) using a 40 mm cone-plate. For the dynamic time sweep, the solution of
hydrogelator (0.5 mL; 0.5 wt% IPF–HYD–GFFY) was directly transferred to the
rheometer and the storage modules (G’) as well as loss modules (G’’), as a function of
time, were monitored at 37°C, with a frequency of 1 Hz and strain of 1%. Dynamic
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frequency sweep was performed in the region of 0.1–100 rad/s at a strain of 1% and
dynamic strain sweep was performed at a frequency of 1 Hz.
Circular dichroism (CD) spectroscopy
The CD experiment was performed using 0.01 cm quartz cells in a J-810 CD
spectrometer fitted with a Peltier temperature controller. CD spectra of the
supramolecular hydrogel were recorded between 180 and 260 nm with a 1 nm data
pitch.
In vitro release experiment
Briefly, 0.3 mL 0.5 wt% IPF–HYD–GFFY supramolecular hydrogel was
pre-formed in 5 mL glass bottles and these were placed in an air bath (37°C). Next, 1
mL PBS or PBS containing 20 U/mL porcine liver esterase (pH 7.4) was added for an
in vitro release experiment. At predetermined time points, aliquots of the release
medium were collected for quantitative determination of the released IPF by high
performance liquid chromatography (HPLC) and another 1 mL freshly prepared PBS
(with or without esterase) was added. HPLC analysis was performed on a reversed
phase C18 column (4.6×150 mm, 5μm, ZORBAX Eclipse XDB-C18). The mobile
phase was composed of methanol and 0.1 mol/L monobasic potassium phosphate
(adjusted to pH 3.5 with phosphoric acid) (75/25; v/v) at flow rate of 1.0 mL/min,
with peaks in the eluent detected by a diode array detector (DAD) at 270 nm.
In vitro cytotoxicity assay
Cytotoxicity of the IPF–HYD–GFFY supramolecular hydrogel was evaluated in
vitro using the MTT assay. The mouse fibroblast cell line (L-929 cells) and human
corneal epithelial cell line (HCEC cells) are widely used to screen chemical toxicity
of compounds. Therefore, cytotoxicity of the IPF–HYD–GFFY supramolecular
hydrogel for both L-929 and HCEC cells was assessed. Briefly, the L-929 or HCEC
4
cells were seeded in a 96-wells plate at a density of 1 × 10 cells/well with 100 µL
cell culture medium (RPMI1640 containing 10% FBS) and incubated overnight. Then,
1
00 µL medium containing different IPF–HYD–GFFY hydrogel concentrations, from
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0
–1000 μM, was added and, after 24 h incubation, cell viability was detected by the
MTT assay.
In vitro anti-inflammatory activity test
RAW264.7 cells were seeded into 35 mm culture dishes overnight, and then
different amounts of IPF or IPF–HYD–GFFY supramolecular hydrogel were added
for a 24 h incubation. After that, LPS was added to the cells at a final concentration of
0
.5 μg/mL. After 16 h incubation, total cellular proteins were extracted using RIPA
lysis buffer, separated by 10% SDS-PAGE and transferred to a PVDF membrane. The
membrane was blocked in PBST with 5% skimmed milk and incubated with primary
antibody overnight at 4°C. Then, the membrane was washed and incubated with the
appropriate HRP-linked secondary antibody for 2 h at room temperature. Protein
bands were visualized with a gel imaging system (ChemiDoc™ Imaging Systems,
Bio-Rad, Hercules, CA, USA) using enhanced chemiluminescence (ECL) substrates
from CWBIO (Beijing, China). Primary antibodies used were: iNOS (1:2000, Cell
Signaling Technology, Beverly, MA, USA), COX-2 (1:2000, Cell Signaling
Technology) and GAPDH (1:2000, Santa Cruz Biotechnology, Heidelberg, Germany).
Quantitative analysis of protein levels was performed using Image J Software
(National Institutes of Health, Bethesda, MD, USA). Protein levels were normalized
to that of GAPDH and these values were standardized to those in samples from
corresponding groups treated only with LPS, without hydrogel or IPF.
Intraocular biocompatibility test
Animals were provided by Wenzhou Medical University Laboratory Animal Center.
Japanese big-eared white rabbits (weight, 2.0–2.5 kg; age, 6–8 weeks) were raised
under a 12 h light and 12 h dark cycle and were supplied with food and water ad
libitum. All animal experiments were approved by the Ethical Committee of Wenzhou
Medical University. Six rabbits were used for the intraocular biocompatibility test. In
this test, the right eyes were instilled with 50 μL 0.3 wt% IPF–HYD–GFFY
supramolecular hydrogel 4 times per day for 3 successive days and the left eyes were
instilled with normal saline (NS) as a control. On 1, 3 and 7 days post instillation, the
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clinical signs, including hyperemia, conjunctiva edema and anterior chamber
abnormalities, were monitored by a slit-lamp microscopy. In addition, fluorescein
staining was used to check the integrity of the corneal epithelium. The corneal
thickness, as well as corneal endothelial cell density, were measured by the Optovue
iVue (Optovue Inc., Fremont, CA, USA) and a noncontact specular microscope
(Topcon SP-3000P, Topcon Corporation, Tokyo, Japan), respectively, during the
entire course of the experiment. Histological observation by H&E staining was
employed to visualize morphological and microstructural changes in corneal tissues.
Endotoxin-induced uveitis (EIU) model and treatment
3
1
Similar to a previous study , the EIU model was induced by direct intravitreal
injection of 100 ng LPS in the rabbit eye with a 1 mL insulin syringe (30G).
Twenty-four rabbits were randomly divided into three groups (n = 8). After induction
by LPS, the right eyes were immediately instilled with either 0.3 wt% IPF–HYD–
GFFY supramolecular hydrogel (0.0735 wt% IPF) or the clinical product 0.1 wt%
DIC eyedrops, each at 4 times per day. The left eyes were untreated and served as
controls. Twenty-four hours later, the clinical signs of an inflammatory response were
evaluated, in a blinded manner, by two experienced investigators. After this
evaluation, the rabbits were immediately sacrificed and aqueous humor samples were
collected for quantitative analysis of cell infiltration, performed using a
hemocytometer under a light microscope with 100× magnification. In addition,
measurement of leakage, based on tumor necrosis factor-α (TNF-α) and interleukin 6
(
IL-6) levels, was assessed by ELISA. The whole eyeballs were excised and
immersed into 10% formalin for fixation overnight, followed by paraffin embedding.
The eyes were then sectioned (5 μm) and stained with hematoxylin and eosin (H&E)
for histopathological observations.
Statistical analysis
The data were subjected to one-way analysis of variance using the Origin 7.5
software package (Northampton, MA, USA). Statistical significance was defined at a
probability level (p< 0.05).
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RESULTS
Preparation and characterization of hydrogel
As a follow-up to previous studies, we successfully constructed a
hydrogelator composed of IPF and GFFY peptide linked by a cleavable ester
bond (Fig. S1–S2). We then tested its gelation ability. As shown in Fig. 2A, a
transparent hydrogel formed spontaneously under a heating–cooling process
with a minimum gelation concentration (MGC) of 0.25 wt%. The TEM image
showed that the hydrogel had a typical filamentous structure with filament
diameters of 15–30 nm (Fig. 2A). The rheological test (Figs. 2B and S3
)
indicated that the elastic network, formed by the nanofibrils, made the hydrogel
relatively mechanically strong. CD spectroscopy, revealing the secondary
structure of peptides packing in the fibrils of the hydrogel, exhibited a negative
band at 208 nm and positive bands in the vicinity of 188 nm, corresponding to
3
2
the characteristic spectrum of a β-sheet structure (Fig. 2C). This result agreed
with the finding by Yang et al., who demonstrated that the low molecular
weight hydrogel derived from GFFY peptide, with different capping groups at
3
3
the N-terminus, exhibited a typical β-sheet structure.
In vitro release study
We monitored the release profile of IPF from the hydrogel in PBS (pH 7.4)
and PBS containing 20 U/ml esterase at 37°C (Fig. S4). As shown in Fig. 2D
,
the hydrogel displayed a constant release of IPF in esterase solution during a
period of 24 h, but released minimal amounts of IPF in PBS lacking esterase.
This confirmed that IPF release from the hydrogel was enzymatically
controlled.
In vitro cytotoxicity assay
Because cell compatibility is a very import property for new biomaterials
being developed for tissue engineering and drug delivery applications, we
examined cytotoxicity of the hydrogel for L-929 and HCEC cells, after a 24 h
incubation. The hydrogel showed negligible cytotoxicity for both cell types at
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concentrations up to 800 μM (Fig. S5). Thus, it might be suitable for potential
drug delivery applications.
In vitro anti-inflammatory activity
To evaluate the pharmacological activity of IPF in the hydrogel, we
examined changes in levels of COX-2 and iNOS in RAW264.7 cells treated
with 0.5 μg/mL LPS (Fig. 3). Incubation of cells with either native IPF or the
hydrogel alone produced only trace amounts of COX-2 and iNOS, while
significant levels of COX-2 and iNOS were released after treatment with 0.5
μg/mL LPS alone. In the LPS-stimulated cells, increased concentrations of IPF
or hydrogel in the medium significantly supressed expression of COX-2 and
iNOS, in a dose-dependent manner (Fig. 3). More important, the highest dose
of hydrogel (500 μM) inhibited both COX-2 and iNOS expression more
effectively than did the native drug. This indicated that the hydrogel had
enhanced pharmacological activity over IPF alone. These observations
suggested that the hydrogel we designed, without compromising the biological
activity of IPF, is a potentially promising candidate for anti-inflammatory
therapy.
Ocular biocompatibility
To evaluate the in vivo biocompatibility of the hydrogel, we examined
irritation in rabbit eyes after successive treatment with normal saline and
hydrogel for 3 days. The eyes treated with both normal saline and hydrogel
exhibited a normal clinical appearance, with no apparent changes in corneal
transparency or damage to corneal epithelium, as shown by green fluorescence
staining (Fig. 4A). Corneal thickness and mean endothelial cell counts were not
significantly changed during the entire period of the experiment (Fig. 4B).
Consistent with the clinical observations, histological observations showed a
normal corneal architecture and thickness and no inflammatory cell infiltration
or edema after hydrogel treatment (Fig. 4C).
In vivo anti-inflammatory efficacy
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Beside the in vitro pharmacological activity and in vivo biocompatibility
assessments, we further evaluated the in vivo therapeutic efficacy of the
hydrogel in LPS-induced uveitis, with the clinically product diclofenac (0.1 wt%
DIC) eyedrops as a reference treatment. Fig. 5A shows representative
photographs of the eyes, at 24 h after various treatments. The figure clearly
illustrates that the eyes treated with only normal saline exhibited a severe
3
4-36
inflammatory response, with abundant exudation in the anterior chamber.
In
contrast, the eyes treated with either 0.1 wt% DIC eye drops or the hydrogel
formulation had significantly attenuated inflammation in the anterior chamber,
with minimal exudation. Inflammatory cell counts in the aqueous humor
suggested that the cellular infiltration in fellow eyes from each group were
5
5
significantly elevated, to (102 ± 33) × 10 and (94 ± 34) × 10 cells/mL,
respectively (Fig. 5B). This indicated a breakdown of the blood–aqueous
3
4, 35
barrier, enabling influx of inflammatory cells into the anterior chamber
. In
eyes treated with 0.1 wt% DIC eye drops or the hydrogel, cellular infiltration in
5
the aqueous humor was significantly decreased to (41 ± 11) × 10 and (40 ± 17)
5
×
10 cells/mL, respectively (p < 0.05). This result strongly suggested that the
hydrogel exhibited a therapeutic efficacy comparable to that of the clinical
product, DIC eye drops, in suppressing the inflammatory response. We next
assessed IL-6 and TNF-α levels in the aqueous humor (Figs. 5C and D).
Aqueous humor levels of the cytokines IL-6 and TNF-α in eyes treated with 0.1
wt% DIC eye drops or the hydrogel were much lower than in the fellow eyes (p
<
0.05). Consistent with the clinical observations, histological analysis
demonstrated that treatment with either 0.1 wt% DIC eye drops or the hydrogel
formulation attenuated neutrophil infiltration and protein exudation in the
anterior chamber, compared with in corresponding control, untreated, eyes (Fig.
S6). Based on these results, we propose that IPF–HYD–GFFY supramolecular
hydrogel is a potential therapeutic alternative for ocular inflammation.
DISCUSSION
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In the past three decades, diphenylalanine (FF) has been widely investigated
as one of the simplest and most common recognition motifs for molecular
1
4, 20, 29, 30
self-assembly
. Generally, efficient hydrogelation of FF-based
oligopeptides require substitution at the N-terminus with many aromatic groups.
This can facilitate self-assembly of the oligopeptides to form supramolecular
4, 5, 33
hydrogels, via π–π stacking and hydrophobic interactions
. More recently,
Xu et al. introduced a variety of prodrug supramolecular hydrogels produced by
conjugation of NSAIDs to the N-termini of FF-based peptides, using amide
linkages. However, such conjugates had significantly lower pharmacological
3
activity than the native drugs . Herein, we introduced an enzymatically
cleavable linkage, HYD, to construct a hydrogelator composed of IPF and a
GFFY peptide for topical treatment of anterior uveitis (Fig. S2). In contrast to
conventional physical encapsulation approaches, this supramolecular hydrogel
not only can enable restoration of the biological activity of the drug by esterase
catalyzed hydrolysis, but can also significantly alleviate concerns associated
with drug–carrier issues. Results of the in vitro anti-inflammatory test indicated
that the supramolecular hydrogel exhibited greater anti-inflammatory efficacy
than the parent drug, as demonstrated by decreased COX-2 and iNOS levels. A
similar phenomenon was widely demonstrated in self-assembled hydrogels
containing anti-cancer drugs. In these, the self-assembled nanostructure of the
drugs facilitated their uptake by cancer cells and, hence, increased their
3
, 5, 20, 29
pharmacological efficacy
. Topically instilled in the rabbit eye, the
supramolecular hydrogel displayed satisfactory ocular biocompatibility,
without causing any side effects. Taking into the consideration that rabbit eyes
are considered more susceptible to foreign substances than those of humans,
this suggested that the hydrogel would be suitable for further clinical
applications.
To date, many therapeutic strategies, including steroidal drugs and NSAIDs,
31, 35, 37, 38
have been used for anterior uveitis
. Although they have lower
anti-inflammatory activities than steroidal drugs, the NSAIDs also have lower
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risks of the severe side effects associated with steroids (e.g., cataracts and
glaucoma). IPF, as one type of NSAID, has lower anti-inflammatory efficacy
than DIC, which has significantly limited its usefulness for treating anterior
uveitis. Among the limitations of eyedrop formulations are rapid drug clearance
and poor bioavailability after topical instillation, necessitating frequent
administration to maintain effective therapeutic drug concentrations. This not
only produces variable drug concentration profiles, but also increases the risk of
potential toxic side effects. Prodrug supramolecular hydrogels derived from
drug–peptides, acting as "drug self-delivery" systems, might be alternative,
more promising, ocular drug delivery systems because they would not involve
drug carriers or prolonged drug retention at the corneal surface. In our study,
we compared the therapeutic efficacy of 0.3 wt% IFP–HYD–GFFY hydrogel
(
0.0735 wt% IPF) with that of the clinical product 0.1 wt% DIC eyedrops in the
uveitis model. Based on our findings, treatment with either 0.1 wt% DIC
eyedrops or the supramolecular hydrogel significantly attenuated the
inflammatory response (e.g., redness, exudation) in the anterior chamber (Fig.
5
A). A number of studies indicated that breakdown of the blood–aqueous
humor barrier is the major cause of initiation of inflammatory symptoms in
3
1, 34, 39
EIU
. Recruitment of leukocytes from the peripheral blood to the aqueous
humor was significantly inhibited by treatment with 0.1 wt% DIC eyedrops or
the hydrogel (Fig. 5B). Inflammatory mediators, including nitric oxide (NO),
TNF-α, interleukin-1 (IL-1) and IL-6, play very important roles in progression
3
8-40
of uveitis
. Both the 0.1 wt% DIC eyedrops and hydrogel treatments
decreased IL-6 and TNF-α production dramatically (Figs. 5C and D). These
results confirmed that the therapeutic efficacy of 0.1 wt% DIC eyedrops and the
hydrogel likely resulted from downregulation of inflammatory cytokines.
In summary, we designed and synthesized a hydrogelator composed of IPF
and a GFFY peptide linked by a cleavable ester bond. Under a heating–cooling
protocol, a hydrogel formed spontaneously via intermolecular noncovalent
interactions (e.g., π–π stacking and hydrophobic interactions). The resulting
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hydrogel was cytocompatible and had excellent ocular biocompatibility in vivo.
Although native IPF has lower anti-inflammatory efficacy than DIC, the 0.3 wt%
IFP–HYD–GFFY hydrogel (0.0735 wt% IPF) had comparable therapeutic
efficacy to that of the clinical product 0.1 wt% DIC eyedrops in an
LPS-induced rabbit uveitis model. Both treatments acted by decreasing IL-6
and TNF-α levels in the aqueous humor. Overall, our findings identify a
strategy for constructing an NSAID supramolecular hydrogel as a potential
therapeutic alternative for ocular inflammation.
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Figure 2
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Figure 3
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Figure captions
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Fig. 1. H-NMR spectrum of IPF–HYD–GFFY hydrogelator.
Fig. 2. (A) TEM image of the supramolecular hydrogel (insert image: 0.5 wt%
IPF–HYD–GFFY hydrogel); (B) Dynamic strain sweep of the supramolecular
hydrogel. 0.5 wt% IPF–HYD–GFFY hydrogelator (G’: Storage modulus; G’’:
Loss modulus); (C) CD spectrum of the IPF–HYD–GFFY supramolecular
hydrogel; (D) Cumulative release of IPF from the supramolecular hydrogel
formed by 0.5 wt% IPF–HYD–GFFY hydrogelator in PBS and PBS containing
20 U/mL esterase.
Fig. 3. Effects of various concentrations of IPF and IPF–HYD–GFFY
supramolecular hydrogel on LPS-induced levels of iNOS and COX-2 proteins
in RAW264.7 cells.
Fig. 4. (A) Corneal appearance, anterior chamber abnormalities and fluorescence
staining in eyes from the control and IPF–HYD–GFFY hydrogel groups on d 1 of
treatment; (B) Corneal thickness in the control and IPF–HYD–GFFY hydrogel groups
after 1, 3 and 7 d treatment; (C) Corneal endothelial cell counts in control and IPF–
HYD–GFFY hydrogel groups after 1, 3 and 7 d treatment; (D) H&E histological
analysis of corneal tissue from the control and IPF–HYD–GFFY hydrogel groups
after 1, 3 and 7 d treatment.
Fig. 5. (A) Clinical signs in anterior chambers of normal eyes and eyes from the
control group, 0.1 wt% DIC group and 0.3 wt% IPF–HYD–GFFY
supramolecular hydrogel (0.0735 wt% IPF) group. The blue arrow indicates
exudation; (B) Cell infiltration in control and experimental groups at 24 h post
LPS induction; (C) Aqueous humor IL-6 levels in control and experimental
groups at 24 h post LPS induction; (D) Aqueous humor TNF-α levels in control
and experimental groups at 24 h post lipopolysaccharide induction.
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Graphical Abstract
We have designed and synthesized a hydrogelator composed of ibuprofen (IPF)
and GFFY peptide via a cleavable ester bond linkage. The proposed supramolecular
hydrogel exhibits an enhanced anti-inflammatory activity in in RAW264.7
macrophage and displays comparable therapeutic efficacy with clinically used sodium
diclofenac (DIC) eyedrops to suppress the ocular inflammation in lipopolysaccharide
(
LPS) induced uveitis. This work, for the first time, illustrates an effective approach
for developing supramolecular assemblies as anti-inflammatory ophthalmic
therapeutics for treating eye disorders.
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