Nanoparticle-encapsulated P2X7 receptor antagonist in a pH-sensitive polymer as a potential local drug delivery system to acidic inflammatory environments

Article abstract of DOI:10.1016/j.bmcl.2015.08.004

We have developed nanoparticles of anti-inflammatory P2X₇ receptor antagonist encapsulated in a pH-sensitive polymer, poly(tetrahydropyran-2-yl methacrylate) (poly(THPMA)), as a potential local drug delivery system to target to acidic inflammatory environments, in which P2X₇ receptors are implicated in the pathology of inflammation via the activation of immune cells. The nanoparticles were prepared using single emulsion methods, also their size and shape were confirmed by microscopy and spectroscopy, etc. The profiles of the pH-dependent degradation, release of antagonist and biological activities were investigated. The nanoparticles that encapsulated the 3,5-dichloropyridine derivative (2) with poly(THPMA), were observed to be more slowly cleaved than the blank nanoparticles. Moreover, the free P2X₇ receptor antagonists potently inhibited the receptor activation, whereas the nanoparticles of the 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA) exhibited much lower P2X₇ antagonistic activity through sustained encapsulation. Thus, the nanoparticles of the 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA) may be utilized to develop a pH-sensitive local drug delivery system for controlled release of anti-inflammatory therapeutics in acidic physiological environments.

Full text of DOI:10.1016/j.bmcl.2015.08.004

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Bioorganic & Medicinal Chemistry Letters 25 (2015) 4197–4202
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Nanoparticle-encapsulated P2X7 receptor antagonist
in a pH-sensitive polymer as a potential local drug delivery
system to acidic inflammatory environments
Sun-Mi Lee a, Joong-Heui Cho b, So-Deok Lee c, Yong-Chul Kim a,c,
⇑
a Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
b New Drug Development Center (NDDC), Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), 80 Cheombok-ro, Dong-gu, Daegu 701-310, Republic of Korea
c School of Life Sciences, Gwangju Institute of Science and Technology (GIST), 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju 500-712, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed nanoparticles of anti-inflammatory P2X7 receptor antagonist encapsulated in a pH-
sensitive polymer, poly(tetrahydropyran-2-yl methacrylate) (poly(THPMA)), as a potential local drug
delivery system to target to acidic inflammatory environments, in which P2X7 receptors are implicated
in the pathology of inflammation via the activation of immune cells. The nanoparticles were prepared
using single emulsion methods, also their size and shape were confirmed by microscopy and spec-
troscopy, etc. The profiles of the pH-dependent degradation, release of antagonist and biological activities
were investigated. The nanoparticles that encapsulated the 3,5-dichloropyridine derivative (2) with poly
(THPMA), were observed to be more slowly cleaved than the blank nanoparticles. Moreover, the free P2X7
receptor antagonists potently inhibited the receptor activation, whereas the nanoparticles of the 3,5-
dichloropyridine derivative (2) encapsulated poly(THPMA) exhibited much lower P2X7 antagonistic
activity through sustained encapsulation. Thus, the nanoparticles of the 3,5-dichloropyridine derivative
(2) encapsulated poly(THPMA) may be utilized to develop a pH-sensitive local drug delivery system
for controlled release of anti-inflammatory therapeutics in acidic physiological environments.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 4 May 2015
Revised 30 July 2015
Accepted 4 August 2015
Available online 7 August 2015
Keywords:
P2X7 receptor antagonists
Nanoparticles
Encapsulation
pH sensitive polymer
Poly(tetrahydropyran-2-yl methacrylate)
(poly(THPMA))
The purinergic P2X7 receptor is a ligand-gated ion channel, that
is, activated by ATP binding. The P2X7 receptor is widely expressed
in cells of the central and peripheral nervous systems, including
microglia, macrophages, and the retina.1 The P2X7 receptor has
been reported to be involved in inflammation and chronic pain
through the activation of immune cells, suggesting that the recep-
tor is a promising therapeutic target of these diseases.2 The target
validation of the P2X7 receptor has been established by studies on
P2X7-knockout animals, which exhibited resistance to inflamma-
tion and neuropathic pain.3 For spinal cord injuries, the potential
utilization of P2X7 receptor antagonists as a novel therapeutic
approach was confirmed by a report describing the increased level
of P2X7 receptor expression and ATP concentration in the region of
injury and the striking recovery of the injury following treatment
with a P2X7 receptor antagonist, oxidized ATP.2 Additionally, sig-
nificant attention has been given to the close relationship between
the P2X7 receptor and joint inflammation, as occurs in rheumatoid
arthritis, because the activation of the P2X7 receptor mediates IL-
1b release, which is an important cytokine in joint inflammatory
diseases.4 Therefore, the etiological factors of rheumatoid arthritis
for inflammation affect the targeting factors involving P2X7
receptors.
Hence, P2X7 receptor antagonists have been extensively devel-
oped for the treatment of inflammation, neurodegeneration and
chronic pain based on the pathological functions of the receptor
upon activation by a high concentration of ATP in acidic inflamma-
tory environments.5 KN-62 (1) is one of the early P2X7 receptor
antagonists and is a potent hP2X7 receptor antagonist, with an
IC50 value of 51 nM.6 Despite the potent antagonistic effect of
KN-62 (1) on the P2X7 receptor, this antagonist exhibits several
disadvantages such as poor water solubility7 and noncompetitive
antagonistic activity8 that limit its development as a drug. Our
group has developed a 3,5-dichloropyridine derivative (2) as a
novel P2X7 receptor antagonist with an IC50 value of 13 nM for
the hP2X7 receptor in ethidium bromide (EtBr) uptake assays.
Moreover, this novel P2X7 receptor antagonist exhibits potent inhi-
bitory activity in the release of the proinflammatory cytokine IL-1b,
⇑
Corresponding author. Tel.: +82 62 715 2502 (O)/2558 (Lab); fax: +82 62 715
iNOS/COX-2 expression and NO production in LPS/IFN-
c/BzATP-
2484.
stimulated THP-1 cells, suggesting that the 3,5-dichloropyridine
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
http://dx.doi.org/10.1016/j.bmcl.2015.08.004
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

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S.-M. Lee et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4197–4202
derivative (2) may be a suitable anti-inflammatory agent targeting
the P2X7 receptor.9
Among the P2X7-receptor-related diseases, rheumatoid arthritis
is a systemic inflammatory disorder that affects several tissues and
organs but principally attacks synovial joints. Although rheuma-
toid arthritis is an autoimmune disease, one of the major players
in this disease is IL-1b, which is released following activation of
the P2X7 receptor.10 The region of joint inflammation is known to
be an acidic environment because rheumatoid arthritis evokes
the activation of a metabolic byproduct,11 which results in an
imbalance between the increased metabolic activity and insuffi-
cient vascular supply, leading to the induction of anaerobic glycol-
ysis to form lactate and H+.12 Hence, the pH of inflammatory
arthritic tissue is observed to be approximately 5.1 compared with
a pH of 7.4 for normal tissues in a physiological environment.13
Due to this unique pathophysiological feature of inflammation,
as occurs in rheumatoid arthritis, our study aimed at targeted ther-
apy using encapsulated agents, that is, novel P2X7 receptor antag-
onists that have been previously identified by our group as anti-
inflammatory drug candidates, in pH-sensitive polymers for selec-
tive degradation in the acidic environment of the inflammatory
region.14 The pH-sensitive biodegradable polymers, poly(tetrahy-
dropyran-2-yl methacrylate) (poly(THPMA)),15 poly[2-(N,N-di-
ethylamino)ethyl methacrylate] (PDMAEMA)16 and poly(b-amino
ester) derivatives,17 have been developed in several studies.
Among these polymers, we selected poly(THPMA) as the pH-sensi-
tive polymer because the major degradation product under acidic
conditions, poly(methacrylic acid) (PMAA), is nontoxic and water
soluble.15
Figure 1. Antagonists of the P2X7 receptor. KN-62 (1) is a potent hP2X7 receptor
antagonist, with an IC50 value of 51 nM.6 The 3,5-dichloropyridine derivative (2) is a
novel P2X7 receptor antagonist, with an IC50 value of 13 nM for hP2X7 receptors in
an ethidium bromide uptake assay developed by our group.9
In this study, we report nanoparticle-encapsulated P2X7 recep-
tor antagonist in a pH-sensitive polymer as a potential local drug
delivery system. We describe the preparation of the nanoparticles,
which encapsulate anti-inflammatory drug candidate, as well as
the dissolution and release of the drugs under acidic conditions
(pH 5.1 to 6). We prepared nanoparticles using 3,5-dichloropy-
ridine derivative (2) as a P2X7 receptor antagonists. For clarifica-
tion of the development of this passive targeting drug delivery
system for an acidic inflammatory environment, we compared
the weight change of the nanoparticles and the in vitro release of
encapsulated agent at pH 5.1 and pH 7.4, respectively. The biolog-
ical activity was also examined. A SRB assay was performed to
assess the cytotoxicity in hP2X7-expressing HEK-293 cell line and
validate the antagonistic effect on the P2X7 receptor using an
EtBr uptake assay and hP2X7-expressing HEK-293 cell. These
nanoparticles represent a potential local drug delivery system to
acidic inflammatory environments with more selective and effec-
tive release at lower pH. Based on these pathological features, we
propose a novel type of nanoparticle for passive targeting to min-
imize the side effects of anti-inflammatory drugs and to overcome
the inherent properties of the drug, such as pharmacokinetic prob-
lems (Fig. 1).
Figure 2. Scheme of 3,5-dichloropyridine derivative (2) and poly(THPMA).
(2) and poly(THPMA) were dissolved in dichloromethane. The mix-
ture was added to aqueous solution of polyvinyl alcohol (PVA) and
subsequently emulsified using a probe sonicator at 80 W for 2 min
to form primary emulsion that was soon added to the second aque-
ous PVA solution, followed by stirring at room temperature for 4 h
until evaporation of the organic solvent was complete. The result-
ing nanoparticles were collected by centrifugation at 13,000 rpm
for 20 min at 4 °C, re-suspended in cold water, and centrifuged
again to remove excess PVA. Finally, the nanoparticles were lyophi-
lized to yield a white, fluffy powder. Moreover, the blank nanopar-
ticles were prepared by single emulsion method same as poly
(THPMA) nanoparticles.
To confirm of characterization of P2X7 receptor antagonist-en-
capsulated nanoparticles, first, we determined particle size which
is directly mean diameter by photon correlation spectroscopy
(PCS) using water solvent dispersion, and also, surface and mor-
phology analyzed by means of scanning electron microscopy
(SEM) examination. To quantify the amount of drug loaded in
nanoparticles, calibration curves for each drug were determined
using UV spectroscopy at the 250 nm wavelength (the detailed
procedure was described in notes and the calibration curves are
provided in the Supporting materials).19 We measured the weight
loss of the blank or poly(THPMA) nanoparticles encapsulated
3,5-dichloropyridine derivative (2) at pH values corresponding to
We synthesized 3,5-dichloropyridine derivative (2) as following
below scheme (Fig. 2). 3,5-Dichlroro-4-iodopyridine and hydrazine
hydrate were refluxed and then amide coupling reaction was
occurred.9
Poly(tetrahydropyran-2-yl
methacrylate)
(poly
(THPMA)), a pH-sensitive polymer, was polymerized by radical
reaction using AIBN from tetrahydro-2H-pyran-2-yl methacrylate,
monomer of poly(THPMA), which is synthesized from esterifica-
tion of methacrylic acid with 3,4-dihydro-2H-pyran by p-toluene-
sulfonic acid monohydrate.15 After synthesis of antagonist and
polymer, we confirmed structure by 1H NMR, mass spectrometry
and molecular weight was measured by GPC, these data were
shown in notes.18 Encapsulated nanoparticle and blank nanoparti-
cle were prepared by single emulsion method. Single emulsion
method is given sonicated power only once at oil/water condition
for fabricating micelle. First of all, 3,5-dichloropyridine derivative

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S.-M. Lee et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4197–4202
4199
acidic inflammatory tissue (pH 5.1, 100 mM acetate buffer) and the
normal physiological condition (pH 7.4, 100 mM HEPES buffer) to
check of pH-dependent degradation of the nanoparticles. The
nanoparticles were dispersed in each buffer at a concentration of
2 mg/mL. The suspensions were incubated at 37 °C with mixing
for 4 days. At 0, 24, 48 and 96 h, 5 mL of the suspension were col-
lected. The nanoparticles were re-suspended in water and lyophi-
lized. The lyophilized nanoparticles were collected and weighed. At
a regular interval, we measured the amount of released P2X7
antagonist following degradation of the poly(THPMA) nanoparti-
cles encapsulating 3,5-dichloropyridine derivative (2) at pH 5.1,
as an acidic inflammatory condition, and at pH 7.4 for 8 days.
These release experiment was performed for elucidating the
amount of the drug accumulation via in vitro system as a function
of different pH.
The poly(THPMA) molecular weight and the poly-dispersity
regarding the type of polymer structure were determined to be
28,276 and 1.371, respectively. The synthesized polymer was
determined to be a more uniform structure based on the poly-dis-
persity value near 1. The overall size of each nanoparticle ranged
from 300 to 400 nm, which lies within the nanoscale range
(Table 1). The size of the poly(THPMA) nanoparticles without drug
encapsulation was 340 nm. The poly(THPMA) nanoparticles encap-
sulating the 3,5-dichloropyridine derivative (2), which used for the
single emulsion method, were slightly larger (363 nm) than the
blank nanoparticles. The percentages of the loaded drug are sum-
marized in Table 1. In general, 3,5-dichloropyridine derivative (2)
was encapsulated more than 64.6% in the poly(THPMA) nanoparti-
cles. We confirmed that each nanoparticle possessed a globular
shape with a smooth surface without distorted holes, as shown
in Figure 3.
Using the blank poly(THPMA) and 3,5-dichloropyridine
derivative (2) encapsulated poly(THPMA) nanoparticles, the rate
of degradation and morphological change of the nanoparticles
were studied at pH 5.1 and 7.4, which correlate with an acidic
inflammatory
and
normal
physiological
environment,
respectively. In Figure 4a, morphological changes of
3,5-dichloropyridine derivative (2) encapsulated poly(THPMA)
nano-particles for 0 to 2 days at different pH values (pH 7.4
and pH 5.1). At the point of 1 day, each different pH manner
had almost same degradation rate, but time goes by, the rate of
degradation was faster in pH 5.1 than normal condition pH 7.4.
Acidic environments cause the nanoparticles to degrade and
release the encapsulated drug more rapidly than under normal
pH conditions for similar processing times. After 2 days of incuba-
tion, the rate of degradation of the blank poly(THPMA) (80%) was
observed to be more rapid than that of the 3,5-dichloropyridine
derivative (2) encapsulated poly(THPMA) (50%) at pH 5.1
(Fig. 4b and c). The degradation rates of 3,5-dichloropyridine
derivative (2) encapsulated poly(THPMA) at pH 5.1 and pH 7.4
after 4 days were observed 70% and 40%, respectively. These
results indicate that the nanoparticles encapsulating the agent
were more slowly degraded, and the release time could be
controlled using the passive targeting drug delivery system.
At a regular interval, we measured the amount of released
P2X7 antagonists by HPLC analysis following degradation of the
3,5-dichloropyridine derivative (2) encapsulated poly (THPMA)
nanoparticles at pH 5.1, as an acidic inflammatory condition,
and at pH 7.4 for
8
days.20 Although nearly 70% of the
nanoparticles were degraded after 4 days at pH 5.1, as illustrated
in Figure 4b and c, which show the weight change of whole
particles from 0 to 4 days, only 20% of the 3,5-dichloropyridine
derivative (2) was released during this period (Fig. 5). The
release of compound (2) was completed in 8 days at pH 5.1,
whereas only 35% of compound (2) was released at pH 7.4 even
after 8 days.
In a cell proliferation assay to evaluate the cytotoxicity, the poly
(THPMA) nanoparticles exhibited of no cytotoxic effect in the
hP2X7-expressing HEK-293 cell line up to 10 lM. The detailed data
Table 1
Characterization of nanoparticles
Nanoparticles
Encapsulation
efficiency (%)
Particle
sizeb (nm)
a
c
Blank poly(THPMA)
Poly(THPMA) encapsulated 3,5-
dichloropyridine derivative (2)
—
340 82
363 67
d
64.6
is shown in Supporting materials.21 Next, the P2X7 receptor antag-
onistic activity of the nanoparticles was determined. As shown in
Figure 6 and 100 nM of each free agent and the poly(THPMA)
nanoparticles, 3,5-dichloropyridine derivative (2) encapsulated
poly(THPMA) and KN-62 (1) as a positive control, were evaluated.22
The free forms of KN-62 (1), 3,5-dichloropyridine derivative (2)
exhibited potent P2X7 receptor antagonistic activity, with 45% and
90% inhibition effect, respectively, at a concentration of 100 nM,
a
Any agent was not loaded in blank poly(THPMA) nanoparticles.
Each nanoparticles were determined particle size by photon correlation spec-
b
troscopy (PCS) at three times.
c
Blank poly(THPMA) nanoparticles had 1.3311 of refractive index and the scat-
tering intensity was 11559 (cps).
d
Poly(THPMA) nanoparticles encapsulated 3,5-dichloropyridine derivative (2)
had 1.3312 of refractive index and the scattering intensity was 11734 (cps).
Figure 3. Measurement of the particle size, surface and morphology analysis for nanoparticle characterization. (a) Blank poly(THPMA) nanoparticles, (b) poly(THPMA)
nanoparticles which encapsulated 3,5-dichloropyridine derivative (2).

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Figure 4. pH-Dependent degradation of the nanoparticles. (a) Morphological changes of 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA) nanoparticles for 0 to
2 days at different pH values (pH 7.4 and pH 5.1). (b and c) Total weight change of blank poly(THPMA) and 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA)
nanoparticles, respectively, at pH 5.1 and pH 7.4.
Figure 5. Percent release of 3,5-dichloropyridine derivative (2) from nanoparticles in a pH-dependent manner as a function of processing time. Every test was extracted more
than 3 times and statistics were shown in error bar.