Drug-bearing supramolecular filament hydrogels as anti-inflammatory agents

Article abstract of DOI:10.7150/thno.19404

We report here on the covalent conversion of the anti-inflammatory agent ketoprofen into self-assembling prodrugs that enable the effective purification of ketoprofen enantiomers, the improved selectivity and potency of ketoprofen, as well as the formatio

Full text of DOI:10.7150/thno.19404

Theranostics 2017, Vol. 7, Issue 7
2003
Ivyspring
International Publisher
Theranostics
2017; 7(7): 2003-2014. doi: 10.7150/thno.19404
Research Paper
Drug-Bearing Supramolecular Filament Hydrogels as
Anti-Inflammatory Agents
Zhipeng Chen1,2,4*, Lei Xing3*, Qin Fan1,2*, Andrew G. Cheetham4, Ran Lin4, Barbara Holt4, Liwen Chen4,


Yanyu Xiao3 and Honggang Cui4,5,6
1. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China;
2. Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China;
3. State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
4. Department of Chemical and Biomolecular Engineering and Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, MD 21218, USA;
5. Department of Oncology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;
6. Center for Nanomedicine, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 North Broadway, Baltimore, MD 21231, USA.
* These authors contributed equally to this work
 Corresponding authors: cpuyanyuxiao@163.com, hcui6@jhu.edu
©
Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2017.01.30; Accepted: 2017.03.03; Published: 2017.05.12
Abstract
We report here on the covalent conversion of the anti-inflammatory agent ketoprofen into
self-assembling prodrugs that enable the effective purification of ketoprofen enantiomers, the
improved selectivity and potency of ketoprofen, as well as the formation of one-component
drug-bearing supramolecular hydrogels. We found that the ketoprofen hydrogelator could exhibit
much-enhanced selectivity for cyclooxygenase 2 (COX-2) over COX-1, reduce the concentration
of inflammatory cytokines (IL-1 and TNFα), and induce apoptosis in fibroblast-like synoviocytes
while maintaining biocompatibility with healthy chondrocytes. In addition, these anti-inflammatory
agent-containing hydrogels demonstrated the ability to retain the therapeutic within a joint cavity
after intra-articular injection, exhibiting a slow, steady release into the plasma. We believe that
upon further optimization these drug-based injectable supramolecular hydrogels could provide the
basis for a local treatment strategy for rheumatoid arthritis and similar conditions.
Key words: supramolecular hydrogel, self-assembly, drug delivery, peptide–drug conjugate, rheumatoid arthritis.
Introduction
The chemical modification of drugs to bestow
additional beneficial properties is a widely explored
bioconjugation strategy, offering the potential for
improved targeting, greater bioavailability, enhanced
efficacy, and reduced side-effects1-4. Peptide
conjugation is one adopted approach that takes
advantage of the multitude of peptides’ biological
roles, providing highly specific ligands that target
particular cell receptors and sequences that can
improve cellular uptake and avoid drug resistance
mechanisms5-8. Furthermore, the conjugation of
peptides that can lead to potential supramolecular
assembly behavior has become a notable extension of
this over the last decade9-17. In doing so, the drug
becomes part of a larger nanostructure with its own
distinct physicochemical properties, one that can be
tailored toward
a
particular route of drug
administration—local or systemic4.
For drugs that can act directly at disease sites,
local administration is the preferred mode of delivery
since it can offer several distinct advantages over
systemic delivery18-22
.
These benefits include
increased bioavailability, reduced systemic exposure
that limits off-target effects and associated adverse
events, and
a
lower total drug cost23-27
.
Anti-inflammatory drugs are well placed to benefit
from local delivery strategies since their continued
use can lead to significant side effects, including
gastrointestinal and kidney problems28, 29. While their
use may be an acceptable short-term risk for acute
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conditions, the long-term relief of localized chronic
diseases would benefit greatly from alternative
approaches. One method is the use of in situ
hydrogels, either polymer or supramolecular-based,
as localized drug depots30-38. The study of these
systems has attracted increasing attention due to their
abundant advantages in drug administration,
including ease of preparation, biocompatibility,
convenience and improved patient compliance, high
China). All other chemical reagents and solvents were
used as received from commercial sources without
further purification.
3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazoliu
m bromide (MTT) was purchased from Sigma (St.
Louis, MO, USA). TNF-α detection kit was purchased
from Nanjing built Biological Technology Co., Ltd.
(Nanjing, China). BCG vaccine was purchased from
Shanghai Institute of Biological Products Co., Ltd.
(Shanghai, China). Trypsin, fetal calf serum and
DMEM (Gibco®) were purchased from Thermo Fisher
Scientific (USA). Annexin V-FITC/PI Cell Cycle and
Apoptosis Analysis Kit was purchased from Nanjing
Keygen Co., Ltd. (Nanjing, China). Deionized distilled
water was used for the preparation of all solutions,
and the other solvents were HPLC grade.
regional drug concentration and low systemic
39-44
toxicity34,
.
Furthermore, hydrogels can be
designed with the ability to gel in response to
physiological stimuli45-47 such as temperature48-50
,
,
55
pH43, 51-53, physical changes in biomaterials54,
56-59
enzymatic
reactions60-62
modification33,
or
chemical
.
Herein, we report on the design and preparation
of a series of self-assembling prodrugs (SAPD)
derived from anti-inflammatory agents that are
capable of forming hydrogels under the physiological
conditions. This concept of SAPD design has attracted
much recent attention for medical applications due to
their inherent biocompatibility and biodegradability,
Cells and animals
Fibroblast-like synovial (FLS) cells were
separated from the joint synovial tissues in rats with
adjuvant-induced arthritis. New Zealand white
rabbits were obtained from the Experimental Animal
Center (Nanjing University of Chinese Medicine,
China) and Sprague Dawley rats were purchased
from the Animal Center of Nanjing University of
Chinese Medicine (Nanjing, China). All animals
received care in compliance with the guidelines
outlined in the Guide for the Care and Use of
Laboratory Animals. The procedures were approved
by the Ethical and Research Committee of the Nanjing
University of Chinese Medicine.
as well as the simplicity of the system design2-4, 57, 63-71
We, therefore, hypothesized that a hydrogel-forming
SAPD that incorporates non-steroidal anti-
.
a
inflammatory drug (NSAID) into its structure could
have great potential for the localized delivery of the
NSAID (Figure 1a). The studied amphiphilic
peptide–drug conjugates were synthesized through
the conjugation of the hydrophobic NSAID
(R/S)-ketoprofen (Ket) to a short peptide with overall
hydrophilicity, separating the resulting epimers via
HPLC. The chirality of the Ket molecule was found to
have a substantial impact on the assembled filament
morphology, and their efficacy as anti-inflammatory
agents, showing excellent selectivity for the inhibition
of cyclooxygenase-2 (COX-2) and inducing apoptosis
in fibroblast-like synoviocytes with no associated
chondrotoxicity. Furthermore, their potential utility as
a localized NSAID depot for rheumatoid arthritis
treatment was assessed through injection of the
(S)-Ket-conjugate hydrogels into the articular cavity
of a knee joint (rat), demonstrating the slow and
sustained release of the drug through diffusion and
degradation of the SAPD.
Preparation of Ketoprofen-N-hydroxysuccini-
mide ester (Ket-NHS)
N-Hydroxysuccinimide (115 mg, 1.0 mmol) and
Ketoprofen (230 mg, 1.0 mmol) were dissolved in
chloroform (10 mL), and N,N′-diisopropyl-
carbodiimide (DIC, 152 mg, 1.2 mmol) was added.
After stirring for 2 h at room temperature, the mixture
was filtered to remove precipitated urea, and the
solvent evaporated under reduced pressure.
Recrystallization from ethanol was performed to give
the pure Ket-NHS ester.
Solid-phase peptide synthesis (SPPS) of
Ket-conjugates
All conjugates were prepared by solid-phase
peptide synthesis (SPPS) using
a 2-chlorotrityl
Materials and Methods
chloride resin (100–200 mesh and 0.3–0.8 mmol/g)
and Fmoc-protected L- or D-amino acids with
appropriate side chain protection—tert-butyl group
for acids (e.g. Fmoc-L-Glu(tBu)-OH). The resin was
initially bubbled with N2 in dry dichloromethane
(DCM) for 20 min, after which the swollen resin was
washed with dry N,N-dimethylformamide (DMF) (3 ×
Materials
Amino
acids
were
purchased
from
Ampebiochem Co. Ltd (Chengdu, China) and
Ketoprofen was obtained from energy-chemical Co.
Ltd (Shang’hai, China). Uranyl acetate (>99.9%) was
obtained from Xi'an Dingtian Chemical Co. Ltd (Xi'an,
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Theranostics 2017, Vol. 7, Issue 7
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3 mL). Next, the first amino acid was loaded onto
resin via its C-terminal carboxylate group by bubbling
the resin in a DMF solution of Fmoc-protected amino
acid (2 equiv.) and N,N-diisopropylethylamine
(DIPEA) for 0.5 h. After washing with DMF (3 × 3
mL), the resin was bubbled with the blocking solution
mg of the appropriate Ket-conjugate was dissolved in
0.5 mL of alkaline water (pH 9.0, room temperature)
with the aid of sonication. Careful adjustment of the
solution pH to 7.0 with 1 M HCl afforded a stable
hydrogel within seconds.
Transmission electron microscopy
(16:3:1 of DCM/MeOH/DIPEA) for 0.5
h
to
All produced nanostructures were visualized by
transmission electron microscopy (TEM) using the
following representative procedure. The sample to be
imaged was deposited onto a copper grid (300 mesh)
coated with a carbon membrane and then stained
with 2 % uranyl acetate for 1 min. A piece of filter
paper was used to blot most of the solution way,
resulting in a thin liquid film. The grid was then
allowed to dry before characterization. After air
deactivate the unreacted sites. The resins were then
treated with 20 % piperidine (in DMF) for 0.5 h to
remove the protecting group, followed by coupling of
the next Fmoc-protected amino acid (2 equiv.) in the
sequence to the free amino group on the resin. A
mixture of DIPEA and O-benzotriazole-N,N,N’,N’-
-tetramethyluronium hexafluorophosphate (HBTU) (2
equiv.) was used to effect coupling. These two steps
were repeated to elongate the peptide chain until the
final sequence was obtained. The resin was washed
with DMF for 3–5 times after each step. Next, the
terminal Fmoc group of the peptide was removed,
and Ketoprofen attached through reaction with the
Ket-NHS ester. Finally, the drug–peptide conjugate
was cleaved with TFA (10 mL) for 2 h, followed by
trituration into diethyl ether. The resulting crude
products were purified by reverse phase HPLC, using
a slow gradient (see below for conditions) and careful
fractionation. Collected fractions were analyzed for
epimeric purity, with appropriate fractions combined
and lyophilized. The purity was confirmed by HPLC
(Figure 1(b) and (c)) and the identity verified by
LC-MS analysis (Figure S1).
drying, the sample was observed using
a
TEM/H-7650 (Hitachi, Japan) operating at 80 kV.
Images were captured with a CCD/MT280B camera.
COX inhibitor screening assay
COX inhibition by (S)-Ket and (S)-Ket-conjugate
hydrogelators was determined using
a
COX
Fluorescent Inhibitor Screening Assay Kit. Each
compound was tested against COX-1 (ovine) and
COX-2 (human recombinant) enzymes separately in
96-well black assay plates. COX activity was
monitored through the action of its peroxidase
component on the reaction between PGG2 and ADHP
(10-acetyl-3,7-dihydroxyphenoxazine),
which
produces the highly fluorescent compound resorufin.
The resorufin production was quantified using a
multimode detector with an excitation wavelength in
the 530–540 nm range and an emission range of
585–595 nm. All compounds were assayed in
triplicate. After combining 10 μL of inhibitor, 10 μL of
Heme solution, 10 μL of the fluorometric substrate, 10
μL of enzyme (either COX-1 or COX-2), and 150 μL of
assay buffer, 10 μL of arachidonic acid solution was
added to initiate the reaction. The plate was analyzed
exactly two minutes after incubation at room
temperature. In this experiment, the blank data
without enzyme and inhibitors, and the control data
without inhibitors were also measured.
Reverse phase HPLC protocols
Preparative HPLC was performed using a
Varian ProStar Model 325 HPLC (Agilent
Technologies, Santa Clara, CA) equipped with a
fraction collector. Preparative separations utilized a
Varian PLRP-S column (150 × 25 mm, 100 Å, 10 µm),
with a mobile phase comprising 0.1% (v/v) aqueous
NH4OH and acetonitrile (20:80 0.01 min, 45:55 25.00
min, 20:80 26.00 min, stop 30 min) at a flow rate of 25
mL/min.
Analytical HPLC was performed on a C18
analytical column (4.6 mm × 250 mm, 5 μm; Hanbon
Sci. & Tech. Huanan, China) coupled with a C18
guard cartridge (4.6 mm × 10 mm, 5 μm; Hedera),
maintained at 30 °C. The mobile phase comprised
acetonitrile and 0.1 % aqueous TFA, and a gradient
method was employed for the analysis (34:66 0.01 min,
41:59 25.00 min, 34:66 25.01 min, stop 30 min). The
mobile phase was filtered through a 0.45 μm filter and
delivered at a flow rate of 1.0 mL/min. The Ket
absorbance was monitored at 254 nm.
In vitro FLS cell toxicity assay
The cytotoxicity of the (S)-Ket-conjugates
towards FLS cells was quantified by an MTT assay
using
FLS
cells
obtained
directly
from
arthritis-inflicted joints of a rat animal model. FLS
cells were incubated at 37 °C in MEM media
containing 10 % fetal calf serum under an atmosphere
of 5 % CO2 in air. For the cell toxicity assay, FLS Cells
were pre-cultured in 96-well cell culture plates at
1.0×105/well in 100 µl medium and grown overnight.
The cells were then incubated with various
General procedure for hydrogel preparation
To prepare hydrogels of L- or D-VEVE-(S)-Ket, 4
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Theranostics 2017, Vol. 7, Issue 7
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concentrations of L-VEVE-(S)-Ket, D-VEVE-(S)-Ket
and (S)-Ket for 48 h. After the incubation times, 20 µl
of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) solution in PBS (pH 7.4)
was added to each well. After the cells had been
collagenase for 4 hours in an incubator at 37 °C with 5
% CO2. The isolated chondrocytes were cultured to
confluence in 75 cm3 flasks at 37 °C in a humidified
atmosphere containing 5 % CO2 for 5 days. The
culture medium was Dulbecco’s Modified Eagle’s
Medium supplemented with 10 % fetal calf serum.
incubated for another
4 h, the medium was
withdrawn and 150 µl DMSO was added to each well
to dissolve the MTT formazan crystals. The
absorbance of each well was then measured at 490 nm
in a microplate reader. Cell viability was calculated as
a percentage of viable cells after treated with drugs
compared with the untreated cells. The IC50 values of
these hydrogelators were read from their activity
curves, which were measured with six different
concentrations of the hydrogelators.
Chondrocyte biocompatibility test
Chondrocytes were seeded onto a 96-well plate
(1 × 104 cells/well) in 100 μL of DMEM medium with
10 % FBS. After 18 hours of incubation at 37 °C and 5
% CO2, the chondrocytes were attached to the bottom
of the 96-well plate. The medium was then replaced
by another 100 μL of growth medium that contained
serial diluents of Ket and hydrogelators at
concentrations in the 0.016–0.71 mg/ml range and the
chondrocytes were incubated at 37 °C and 5 % CO2 for
an additional 72 h. Chondrocyte proliferation was
determined by the addition of 20 μL of MTT (5
mg/mL) and incubating at 37 °C for another 4 h in the
dark, the media were withdrawn and 150 µl DMSO
was added to each well to dissolve the MTT formazan
crystals. The absorbance of each well was then
measured at 490 nm using a Microplate Reader
(TECAN Austria GmbH). Chondrocytes without
treatment were used as a control. All experiments
were conducted in triplicate.
Determination of IL-1 and TNF-α
FLS cells were pre-cultured in 96-well cell
culture plates at 1×105/well in 100 µl in MEM media
containing 10 % fetal calf serum at 37 °C under an
atmosphere of 5 % CO2 in air and grown overnight.
The cells were then incubated with L-VEVE-(S)-Ket,
D-VEVE-(S)-Ket, and (S)-Ket (0.355 mg/ml) for 48 h.
Cell supernatants were analyzed by ELISA to evaluate
the levels of IL-1 and IFN-γ. Cells were acquired on a
flow cytometer, and data were analyzed using the
FlowJo software. Production of IFN-γ was analyzed in
CD11cþ gated population.
Determination of the in vitro release profile
Apoptosis detection
200 μL of (S)-Ket-conjugate hydrogels and
(S)-Ket-conjugate solution and (S)-Ket were prepared,
all with concentrations of 4 mg/ml of (S)-Ket or
FLS cells were seeded at a density of 1 × 106 on
each well of a 24-well plate and allowed to grow
overnight. Cells were treated with L-VEVE-(S)-Ket,
D-VEVE-(S)-Ket, and (S)-Ket at the concentration of
0.016-0.71 mg/ml for 48 h, followed by trypsinization.
The cells were repeatedly washed and centrifuged at
1800 rpm for 3 min, and the supernatants were
discarded. Cell density was determined. The cells
were then re-suspended in annexin-binding buffer at
∼1×106 cells/mL, preparing a sufficient volume for
100 μL per assay. To 100 μL of cell suspension were
added 5 μL of Alexa Fluor 488 annexin V and 5 μL of
100 μg/mL PI working solution, after which the
solution was incubated for 15 min at room
temperature. After the incubation period, 400 μL of
Annexin binding buffer was added to each sample,
followed by gentle mixing in an ice-bath. The samples
were immediately analyzed using flow cytometry.
(S)-Ket-conjugate.
Solutions
were
dialyzed
(Spectra/Por® membrane: MWCO = 800–1000) for 3
days against PBS buffer solution. With the addition of
PBS buffer (200 μL, pH 7.4) onto the surface of
hydrogels, the gels were incubated at 37 °C for 24
hours. The release solutions (200 μL) were taken and
refreshed at a predetermined time, which were
analyzed by analytical HPLC at 260 nm to determine
the
quantities
of
released
Ket-conjugate
hydrogelators.
In vivo hydrogel release experimental protocol
Eighteen male Sprague Dawley rats (230–240 g)
were randomly divided into 3 groups, and then the
rats were administered 200 μL of (S)-Ket solution,
L-VEVE-(S)-Ket hydrogel and D-VEVE-(S)-Ket
hydrogel via IA injection.
The dynamic behavior of the Ket was monitored
at predetermined intervals (0.5, 1, 2, 3, 4, 6, 8 and 24
hours, and 2, 4, 8, 16, 24 and 32 days) post-injection by
LC-MS/MS. The rats were anesthetized with ether. A
heparinized capillary was then inserted into
eyeground veins to obtain 0.3 mL blood in a plastic
centrifuge tube. The plasma samples were collected
Chondrocyte isolation and culture
Normal articular cartilage harvested from two
New Zealand White rabbits was washed three times
with phosphate-buffered solution (PBS) containing 1
% penicillin and streptomycin. The cartilage slices
were digested with 0.25 % trypsin at 37 °C for 30 min,
thoroughly washed, and treated with 0.2 % type II
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after centrifugation at 4,000 rpm for 10 min and
immediately stored at −20 °C until analysis. Next, 200
μl methanol containing an internal standard
(chlorzoxazone, 50 ng/ml) was added to the 50 μl
plasma samples to precipitate protein, centrifuged at
12,000 rpm for 10 min, and 20 μl of supernatant was
assayed by LC-MS/MS.
one-dimensional nanostructure formation, the
tetrapeptide sequence Val-Glu-Val-Glu (VEVE) was
chosen
as
this
possesses
alternating
hydrophobic–hydrophilic side-chain functionalities.
This particular peptide sequence can, when
conjugated to a larger hydrophobic moiety, form
nanobelt structures and even undergo hydrogel
formation73-76. L- or D-amino acids were utilized to
fully probe the influence that the stereochemistry has
on the self-assembly behavior and to evaluate any
correlation between the structure and activity of
NSAID hydrogelators77. By conjugating the racemic
(R/S)-Ket to the enantiomerically pure L- or D-VEVE
peptides, two sets of epimeric conjugates are formed:
L-VEVE-(S)-Ket and L-VEVE-(R)-Ket (Figure 1a), and
D-VEVE-(S)-Ket and D-VEVE-(R)-Ket. Surprisingly,
after conjugation, both sets can be separated by HPLC
purification, as indicated by a difference in retention
time in their respective HPLC traces (Figure 1b–c). In
contrast, the racemic mixtures of (R/S)-Ketoprofen
have identical retention times (data not shown) and
thus cannot be purified by the HPLC techniques. The
separation of the racemic mixture after peptide
conjugation is noteworthy, likely due to a difference
in binding affinity to the HPLC column’s stationary
phase as a result of the peptide conjugation.
LC-MS/MS quantitation of Ket plasma
concentrations
Plasma samples collected from the rats were
analyzed
by
quantitative
LC-MS/MS.
The
LC-MS/MS equipment consisted of two LC-20AD
pumps, an SIL-20ACHT auto-sampler, a CTO-20AC
column oven (Shimadzu Corporation, Japan) and a
4000 Qtrap® mass spectrometer (AB SCIEX, USA),
equipped with a Turbo ion source. Analyst software
(Version 1.5.1) and Multi Quant software (Version
1.5.1) were used for data acquisition and analysis,
respectively. LC separation was performed on a
Agilent Extend C18 column (30 mm × 4.6 mm, 1.8 μm)
using gradient elution with a mobile phase of
methanol and 5 mM aqueous ammonium acetate
(10/90 0.01 min, 10/90 0.5 min, 90/10 2 min, 90/10 4.5
min, 10/90 5.0 min, stop 6 min, v/v) at a flow rate of
1.0 mL/min. The column temperature was
maintained at 40 °C and the auto-sampler
temperature at 4 °C. The mass spectrometer was
operated in the negative ionization mode. The
injection volume was 10 μL. The ion spray voltage
was set to −4500 V, and the ion source temperature
was set at 450 °C. The curtain gas and collision gas
were 35.0 psi and set at medium level, respectively.
Ion source gas 1 and ion source gas 2 were both set at
40 psi. The declustering potential (DP), entrance
potential (EP), collision energy (CE) and collision cell
exit potential (CXP) for ketoprofen were −20, −10, −11
and −15 V, respectively. Detection of the ions was
performed in the multiple reaction monitoring (MRM)
mode, monitoring the transition of the m/z 253
precursor ion to the m/z 209 product ion for
ketoprofen, and the m/z 709 precursor ion to the m/z
132 product ion for the Ket-conjugates. The standard,
chlorzoxazone, was monitored using the m/z 285
precursor ion to the m/z 193 product ion transition.
Self-assembly characterization
The self-assembly of all the Ket-conjugates was
initiated by directly dissolving each molecule into an
aqueous solution to reach
a
predetermined
concentration. After aging for at least 24 h,
transmission electron microscopy (TEM) imaging was
conducted
characteristics of the respective Ket-conjugate
assemblies. We found that assembly of
to
examine
the
morphological
L-VEVE-(S)-Ket afforded large, rigid, multi-strand
nanoribbons with average widths of 49 ± 10 nm, with
the constituent strands averaging 4.6 ± 0.5 nm in
width (Figure 2a). These filamentous structures also
possessed a tendency to align up to form parallel
bundles. In contrast, the associated epimer
L-VEVE-(R)-Ket
gave
a
more
filament-like
morphology with average widths of 12 ± 1.7 nm
(Figure 2b). A similar difference was observed for the
D-peptide
conjugates.
D-VEVE-(R)-Ket,
the
Results and Discussion
enantiomer of L-VEVE-(S)-Ket, formed multi-strand
intertwined nanoribbons (ribbon widths of 22 ± 5 nm
and strand widths of 4.8 ± 0.9 nm) (Figure 2d).
D-VEVE-(S)-Ket, the enantiomer of L-VEVE-(R)-Ket,
formed the more filament-like structures with average
widths of 11 ± 1.7 nm (Figure 2c).
This difference in morphology—ribbons/
bundles vs. individual filaments—may arise from the
inherent chirality of the Ketoprofen molecule
Ket-conjugate synthesis
The NSAID-based hydrogelators utilized in this
study were created through the connection of racemic
(R/S)-Ketoprofen to the backbone of small
tetrapeptides using standard solid-phase Fmoc
peptide synthesis protocols72 (Figure 1a). To provide
the necessary intermolecular interactions for
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Theranostics 2017, Vol. 7, Issue 7
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matching with the chirality of the chosen amino acids.
The projection of the methyl group at the chiral
carbon relative to the amino acid side chains likely has
a substantial impact on the molecular assembly
pathway and internal packing. For the enantiomeric
L-VEVE-(S)- Ket and D-VEVE-(R)-Ket conjugates, the
methyl group of Ket is projected into the same plane
as the hydrophobic isopropyl groups of the Val
side-chains and so would contribute to the
hydrophobic interactions (Figure 2e). For the
corresponding
epimers
L-VEVE-(R)-Ket
and
D-VEVE-(S)-Ket, however, the methyl group projects
into the same plane as the hydrophilic carboxylic acid
groups of the Glu side chains. This hydrophobic
group in the predominantly hydrophilic plane may
disrupt the packing that would prevent extensive
lateral associations and thus give
filament-like appearance.
a narrower
Figure 1. Synthesis of Ketoprofen–peptide conjugates. (a) Representative example of the synthetic procedure, showing the separation of the epimers l-VEVE-(S)-Ket
and l-VEVE-(R)-Ket via HPLC purification. (b) and (c) HPLC chromatograms of the separated epimers of the l-VEVE and d-VEVE Ketoprofen conjugates, respectively.
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Theranostics 2017, Vol. 7, Issue 7
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Figure 2. Self-assembly of the ketoprofen–peptide conjugates. (a)–(d) Representative transmission electron micrographs of l-VEVE-(S)-Ket (a), l-VEVE-(R)-Ket (b),
d-VEVE-(S)-Ket (c), and d-VEVE-(R)-Ket (d). All scale bars are 200 nm. The stereochemical relationships between the four conjugates are also indicated. (e) Newman
projections of the four conjugates showing the Ketoprofen methyl alignment relative to the hydrophobic Val and hydrophilic Glu side chains. View shown is along the
peptide axis from the Ketoprofen α-carbon, extending to the first Glu residue.
the inhibition of COX-2, especially for the intended
local delivery application. The IC50 values of
Inhibition of COX enzymes
(S)-Ketoprofen is a commonly used drug for the
management of arthritic conditions such as
rheumatoid arthritis (RA), with its (R) form
possessing little bioactivity. One mode of action for
(S)-Ket is the inhibition of the cyclooxygenase
enzymes COX-1 and COX-2. However, it is the
inhibition of COX-1 that can lead to gastrointestinal
ulceration, and so it is advantageous if the conjugate
can show greater selectivity for inhibition of COX-2
over COX-1. We, therefore, performed in vitro
inhibition assays for both COX-1 and COX-2 enzymes
to evaluate the efficacies of the NSAID-containing
SAPDs (Figure 3). The calculated IC50 values of
L-VEVE-(S)-Ket and D-VEVE-(S)-Ket against COX-2
were 4.8 and 5.8 μM, respectively. Since the IC50 of
(S)-Ket against COX-2 was found to be 2.2 μM, these
IC50 values for the two conjugates are reasonable for
L-VEVE(S)-Ket and D-VEVE-(S)-Ket against COX-1
enzyme were found to be 20.3 and 27.7 μM,
respectively, both much higher than the IC50 value of
5.1 μM for (S)-Ket. However, the reduction in
Ketoprofen efficacy against both COX-1 and COX-2 is
compensated by the much-improved selectivities
against COX-2. In both designs of employing natural
and non-natural amino acids, L-VEVE-(S)-Ket and
D-VEVE-(S)-Ket exhibited excellent selectivities of S =
4.2 and 4.8, respectively, toward COX-2, similar to
those observed for other NSAID hydrogelators78-80
.
Undoubtedly, the attachment of the small L- or
D-peptides to Ket greatly impedes its binding to
COX-1, which should reduce any associated adverse
gastrointestinal and renal effects.
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Theranostics 2017, Vol. 7, Issue 7
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among which interleukin-1 (IL-1) and tumor necrosis
factor α (TNFα) play central roles 86. Addressing the
issue of FLS-mediated tissue damage in RA is,
therefore, a viable strategy for the prevention or
limitation of further joint degradation.
To evaluate if our (S)-Ket conjugates can affect
the proliferation of FLS cells, we incubated FLS cells
obtained from rabbits with arthritic joints with the
conjugates and assessed their proliferation after 48 h.
Surprisingly, we found little significant difference in
the extent of FLS proliferation between those treated
with L-VEVE-(S)-Ket or D-VEVE-(S)-Ket and those of
the control group (Ket) (Figure 4a), with all three
groups exhibiting IC50 values in the 0.1–0.3 mg/ml
range. Additionally, compared with the untreated
control group, the FLS cells produced trace amounts
of IL-1 (Figure 4b) and a comparatively lower amount
of TNFα (Figure 4c) after treatment with Ket,
L-VEVE-(S)-Ket, or D-VEVE-(S)-Ket. Furthermore, an
assay to determine the extent of apoptosis in the FLS
cells upon treatment with Ket, L-VEVE-(S)-Ket, and
D-VEVE-Ket (all at 0.4 mg/ml Ket), showed that all
three induced a greater degree of cell death than
untreated groups (Figure 4d). A notable aspect is that
the concentration of (S)-Ket required to induce
significant apoptosis is in the millimolar range, a
concentration that would not only be difficult to attain
within the articular cavity through systemic delivery
but would also result in severe side-effects due to the
dosage required to do so. By converting the drug into
a hydrogelator, it thus becomes possible to deliver the
drug at a concentration that would be able to reduce
the FLS population within the synovial pannus and
limit further damage, without the risk of significant
side-effects.
Figure 3. Inhibition (IC50) of COX-1 and COX-2 by (S)-Ket, l-VEVE-(S)-Ket
and d-VEVE-(S)-Ket. The selectivity, S, is defined as the ratio of the IC50 values
towards COX-1 and COX-2.
Inhibitory activity against
fibroblast-like-synoviocytes
In RA, fibroblast-like synoviocytes (FLS) play a
significant role in the inflammation response and
subsequent damage to the cartilage and bone of the
81,
joint
82, and constitute a major component of a
synovial pannus that forms within the joint. In this
pannus, the FLS have transitioned to a more
aggressive phenotype in response to the initial
inflammatory conditions and no longer undergo
83
apoptosis
.
These
cytokines,
FLS
cells
chemokines
produce
and
pro-inflammatory
proteases 84, that invade and destroy cartilage and
bone 85. Inflammatory cytokines have been implicated
as important mediators of joint destruction in RA,
Figure 4. In vitro studies of FLS toxicity. (a) Cytotoxicity assay of FLS cells treated with (S)-Ket (, IC50 = 159 μg/mL), l-VEVE-(S)-Ket (, IC50 = 223 μg/mL), and
d-VEVE-(S)-Ket (, IC50 = 278 μg/mL) for 48 h. The effect on IL-1 (b) and TNFα (c) production by FLS cells treated with (S)-Ket, l-VEVE-(S)-Ket, and d-VEVE-Ket.
(d) Induction of apoptosis on FLS cells treated with Ket, l-VEVE-(S)-Ket, and d-VEVE-(S)-Ket. All experiments were performed in triplicate (n = 3). Asterisks (*)
denote statistically significant differences calculated by one-way ANOVA test, *p < 0.05, **p < 0.01.
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Theranostics 2017, Vol. 7, Issue 7
2011
Due to potential chondrotoxicity, the two
Ket-conjugates were further investigated for
biocompatibility through incubation with primary
rabbit chondrocytes for 72 h at 37 °C (Figure S2).
Encouragingly, no toxic effect could be seen, with the
chondrocytes maintaining an almost 100% viability in
the presence of either conjugate, even at the higher
concentration of 0.71 mg/mL. This result indicates
that they are chondrocyte compatible and would be
suitable as locally administrable therapeutics.
different manner87. The hydrogels were then assessed
for their ability to act as a long-term drug depot.
Accordingly, we incubated 200 μL of both L- and
D-VEVE-(S)-Ket-conjugate hydrogels (4 mg/ml) at 37
°C for 40 d with 200 μL of PBS buffer solution (pH 7.4)
in separate dialysis bags, which were refreshed and
monitored at predetermined time points (Figure 5b).
(S)-Ket solution and L-VEVE-(S)-Ket-conjugate
solution (assembled but not gelled) were used as
controls. As expected, the hydrogels exhibited the
slowest release kinetics with 100% of the
(S)-Ket-conjugate released only after 30 d had elapsed.
Dynamic light scattering of the released hydrogel
solution exhibits little difference with that of the
non-gelled (S)-Ket-conjugate solution (data not
shown), suggesting the conjugates are released as a
mixture of nanostructures and small aggregates. In
sharp contrast, drug release from the (S)-Ket solution
and L-VEVE-(S)-Ket-conjugate solution releases
within a few hours.
In vitro and in vivo drug release
We next evaluated the potential of using the
conjugates as a local delivery platform. Gelation tests
of L-VEVE-(S)-Ket and D-VEVE-(S)-Ket clearly
suggested that the two conjugates were indeed
suitable hydrogelators, forming stable, slightly
opaque hydrogels in water at a concentration of 5 mM
or higher (Figure 5a). The cloudiness of the hydrogels
could stem from the bundling and alignment of the
filaments
which
scatter
light
in
a
Figure 5. Study of the (S)-Ketoprofen-containing conjugates. (a) Images of the self-supporting hydrogels formed by l-VEVE-(S)-Ket and d-VEVE-(S)-Ket (both 5 mM
in water). (b) In vitro release profiles of Ket-based formulations: (S)-Ket solution (), d-VEVE-(S)-Ket prodrug (), l-VEVE-(S)-Ket hydrogel () and d-VEVE-(S)-Ket
hydrogel () (Data points are slightly offset in the x-axis for clarity). (c) Illustration of the proposed intra-articular delivery of an NSAID-containing hydrogel to an
RA-afflicted joint. The hydrogel forms from the entanglement of self-assembled NSAID–peptide conjugate nanostructures. (d) In vivo pharmacokinetic study of
intra-articularly administered (S)-Ket solution (), l-VEVE-(S)-Ket hydrogel () and d-VEVE-(S)-Ket hydrogel ().
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Theranostics 2017, Vol. 7, Issue 7
2012
To evaluate the potential of the hydrogels as a
local drug depot in an in vivo environment, six male
rats were administered 200 μL of L-VEVE-(S)-Ket and
D-VEVE-(S)-Ket hydrogels (containing 400 μg
Ket-conjugate) via intra-articular injection (Figure 5c).
Supramolecular filament hydrogels of similar types
are known to be shear thinning and injectable and can
be delivered using a needle mounted on a syringe.
Following intra-articular injection with a needle, the
dynamic behavior of the hydrogels was monitored at
favor long term retention at the injection site. Overall,
the presented Ket–peptide hydrogelators represent a
highly promising platform upon which to base a new
local therapy for rheumatoid arthritis, and will be
explored through further optimization and in vivo
experiments to assess their efficacy against an RA
model. The ability to deliver a high concentration of
Ketoprofen may not only aid with pain relief and
minimize gastrointestinal side-effects but may also
prevent further progression of the disease through
their inhibitory action against FLS cell proliferation.
This approach could also provide a useful strategy for
reducing the adverse drug reactions of other
therapeutic agents that would benefit from local
administration.
predetermined
intervals
post-injection
using
LC-MS/MS analysis to quantify the Ketoprofen
plasma concentration. It can be clearly seen that both
hydrogels slowly release the (S)-Ket-conjugate over
an extended period, maintaining a near constant
plasma concentration close to 100 ng/mL with no
burst release characteristics (Figure 5d). In contrast,
(S)-Ket solution exhibited rapid release with the
classic burst release profile (Cmax = 10 μg/mL). These
results suggest that these Ket-conjugate hydrogels are
capable of retaining the bulk of the administered
therapeutic within the arterial cavity, thus
Supplementary Material
Supplementary figures.
http://www.thno.org/v07p2003s1.pdf
Acknowledgements
This work was supported by the National
Science Foundation (DMR 1255281), and by the
National Natural Science Foundation of China (No:
81473147, 81274100, 21301191), 2011 Collaborative
Innovation Center of Chinese Medicinal Resources
Industrialization (No: ZDXM-2–3), the Priority
Academic Program Development of Jiangsu Higher
Education Institutions (PAPD), Six talent peaks
project in Jiangsu Province, Outstanding talent
training program of Nanjing University of Chinese
Medicine of Pharmacy.
maintaining
a
high local concentration. That
Ket-containing species are released indicates that the
hydrogel is undergoing slow decay within the cavity,
either through diffusional loss or biological activity
(e.g. proteolysis), releasing the Ket-conjugate into the
surrounding environment at a steady rate. Although
many experimental parameters remain to be tested
and optimized, these release experiments clearly
demonstrate the clinical potential of using these
supramolecular filament hydrogels as local
anti-inflammatory agents to treat rheumatoid
arthritis.
Competing Interests
The authors have declared that no competing
interest exists.
Conclusions
In conclusion,
a
novel supramolecular
anti-inflammatory hydrogel made entirely of a
peptide–NSAID conjugate was designed for the
potential treatment of arthritic diseases via
intra-articular administration, constituting a new
platform for the delivery of therapeutic agents by
biocompatible, target specific and long-acting
hydrogels. Additionally, the incorporation of L- or
D-amino acids make the Ket-conjugates highly
selective for inhibition of COX-2 over COX-1, thereby
minimizing adverse effects associated with inhibition
of the latter. In vitro studies revealed that the
Ket-conjugate supramolecular hydrogels could
benefit RA-afflicted joints by suppressing the levels of
IL-1 and TNF-α through induction of apoptosis in
FLS, and thus may protect RA damaged joints from
further degradation. Furthermore, an in vivo release
study demonstrated that this platform is capable of a
steady and sustained drug release profile that would
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