New formulation of old aspirin for better delivery

Article abstract of DOI:10.1039/c5cc07316b

For better use of cyclooxygenase dependent anti-inflammatory properties and mitochondrial activities of aspirin, new hydrophobic analogues of aspirin were developed and successfully encapsulated in polymeric nanoparticles (NPs). In vivo anti-inflammatory effects of these NPs using a mouse model demonstrated unique properties of an optimized aspirin analogue to inhibit production of pro-inflammatory and enrichment of anti-inflammatory cytokines.

Full text of DOI:10.1039/c5cc07316b

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DOI: 10.1039/C5CC07316B
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New formulation of old aspirin for better delivery†
Akil. A. Kalathil,a‡ Anil Kumar,a‡ Bhabatosh Banik,a‡ Timothy A. Ruiter,a Rakesh K. Pathak,a and
Shanta Dhara
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
For better use of cyclooxygenase dependent anti-inflammatory neuro-degeneration.
properties and mitochondrial activities of aspirin, new
Current knowledge8 and clinical data9 indicate that aspirin
hydrophobic analogues of aspirin were developed and successfully can be an attractive addition to treatment regiments for
encapsulated in polymeric nanoparticles (NPs). Anti-inflammatory neurodegenerative diseases. By acknowledging the fact that
effects of these NPs in vivo using a mouse model demonstrated although few of the nonsteroidal anti-inflammatory drugs such
unique properties of an optimized hydrophobic aspirin analogue as aspirin can get access to the brain tissue by crossing the
to inhibit production of pro-inflammatory and enrichment of anti- tight junctions of the blood brain barrier (BBB), but plasma
inflammatory cytokines.
protein binding activity of this class of molecule limits the
Conditions that include neuro-inflammation, oxidative effectiveness of such uptake,10 we hypothesized that new
stress, and mitochondrial injury play different roles in
O
Average Diameter (nm)
from three experiments
NT-(Asp)2-NP
100
T-(Asp)2-NP
(C)
(A)
(B) 300
the prognosis of neurodegenerative diseases such as
stroke, Alzheimer’s disease, Parkinson’s disease (PD),
Huntington’s disease, and amyotrophic lateral sclerosis.1
Although these diseases demonstrate different
pathologies, inflammation and oxidative stress are the
common players.2 Degradation in mitochondrial health
also plays an integral role in overall damage during
neuro-degeneration.3 Anti-inflammatory substances
such as aspirin and mitochondria-acting antioxidant
coenzyme Q10 are described to have potential
neuroprotective roles in these diseases.4 Aspirin or
acetylsalicylic acid can potentially have a number of
roles in neurodegenerative diseases: (i) platelet
inhibition through acetylation and prevention of new
clots from developing,5 (ii) aspirin can play roles in PD by
suppressing formation of dopamine quinone,6 (iii)
cyclooxygenase (COX)-independent effect of aspirin on
Ca2+ signaling7 for mitochondrial dysfunction related
O
O
O
250
200
150
O
O
O
O
O
80
60
40
20
0
50
O
0
1
Ps
T
Ps
T
Ps
T
Ps
-N
) 4
10
100
-N
-N
-N
) 2
Oc-[G1]-(Asp)2
)
2
)
4
[Oc-[G1]-(Asp)2] (µM)
-(Asp
T
-(Asp
-(Asp
-(Asp
N
N
O
Average Zeta Potential (mV)
from three experiments
O
Aspirin
NT-(Asp)4-NP T-(Asp)4-NP
120
80
60
40
20
0
O
O
O
O
O
80
40
0
Lipophilic Tail
O
O
O
O
O
O
O
O
O
-20
-40
O
O
Ps
T
Ps
T
Ps
T
Ps
-N
) 4
0.1
1
10
100
-N
-N
-N
) 2
O
)
2
)
4
O
O
[Oc-[G2]-(Asp)4] (µM)
-(Asp
-(Asp
-(Asp
-(Asp
O
T
Oc-[G2]-(Asp)4
N
N
Fig. 1. (A) Structure of two newly constructed hydrophobic aspirin
derivatives. (B) Analyses of physicochemical properties of these NPs
derived from hydrophobic Oc-[G1]-(Asp)2 and Oc-[G2]-(Asp)4 for better
delivery. (C) Cytotoxic properties of these NPs in RAW 264.7
macrophages as determined by the MTT assay.
hydrophobic analogues of aspirin can be extremely important
as aspirin lacks properties required for well formulation in a
nanoparticle (NP) system for better delivery. Further more,
gastric toxicity arising from non-specific platelet inhibition by
aspirin is a major problem11 and one of the solutions can be
slow-release of aspirin at low dosage.12 Thus, NPs with
controlled release properties can provide beneficial
manipulation towards pharmacological formulation for aspirin.
Additionally, hydrophobic aspirin analogues will help in
improving pharmacokinetic (PK) parameters of the generic
drug13 as incorporation of new derivatives into a NP system
can increase the blood circulation time of the drug when
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administered by intravenous (i.v.) route in contrast to usual (4) (Figures S7-S12) for conjugation of two aspirin molecules.
View Article Online
DOI: 10.1039/C5CC07316B
aspirin administration.
This dendron Oc-[G1]-(OH)2 was reacted with aspirin chloride
Here we report construction and optimization of new (Figures S13-S15) to generate a hydrophobic dendron Oc-[G1]-
aspirin analogues for their formulation in biodegradable NPs (Asp)2 containing two molecules of aspirin linked through
with properties which will allow slow controlled delivery of cleavable ester bonds (Figure 1A, Figure S16-S18). Our efforts
aspirin molecules in the vicinity of the target tissue and in to encapsulate Oc-[G1]-(Asp)2 in PLGA-b-PEG-TPP polymer to
particular in the mitochondria for possible applications in generate T-(Asp)2-NPs and in PLGA-b-PEG-OH polymer to yield
neuro-degenerative diseases. As
investigated utilities of these new aspirin-NP formulations in the NPs (Table S1), however the diameter of both T/NT-(Asp)2-
mice model of inflammation. NPs were ~200 nm (Table S1, Figure 1B) which disqualify these
a disease model, we NT-(Asp)2-NPs resulted in high loading of the dendron inside
Prior to the synthesis of new aspirin derivatives, we first NPs to be suitable for mitochondrial association as our
assessed whether generic aspirin can be incorporated in the previous studies indicated that NP size below 100 nm is
hydrophobic core of biodegradable polymeric NPs. As we required for these properties.14-15 Next, we increased the
would like to target conditions such as mitochondrial number of dendron arms further to increase hydrophobicity of
dysfunctions associated with oxidative stress, impaired Ca2+ the dendron and constructed Oc-[G2]-(OH)4 (Figures S19-S24)
signaling, inflammatory processes demonstrated by brain cells and further conjugation of aspirin resulted in Oc-[G2]-(Asp)4
during neurodegenerative processes, we selected
a
(Figure 1A, Figures S25-27) with four aspirin molecules.
biodegradable
poly(lactic-co-glycolic
acid)-block- Incorporation of Oc-[G2]-(Asp)4 into NPs to generate T-(Asp)4-
polyethyleneglycol
PEG) functionalized with
terminal triphenylphosphonium
cation (TPP) with significant
mitochondrial
properties previously reported
by us.14,15 In our continuing
effort to evaluate the potential
of the targeted NPs (T-NPs)
derived from this PLGA-b-PEG-
TPP polymer to deliver payload
that can work by accessing
(PLGA-b-
O
(D)
T-(Asp) -NPs
(A)
(B)
O
a
O
O
Aspirin
O
O
NT-(Asp) -NPs
4
O
OH
O
O
4
x
n
y
Oc-[G2]-(Asp)4
O
100
80
60
40
20
0
PLGA-b-PEG-OH
O
Lipophilic Tail
O
O
O
O
O
O
NT-(Asp)4-NP
association
O
O
O
O
O
O
O
O
O
O
y
P
x O
O
Br-
z
Oc-[G2]-(Asp)4
O
O
O
O
PLGA-b-PEG-TPP
O
0
4
8
12 48 96
Time (h)
Oc-[G2]-(Asp)4
T-(Asp)4-NP
NT-(Asp)4-NP
(C)
70
35
0
20
15
10
5
80
100
80
60
40
20
0
T-(Asp)4-NP
60
40
20
0
unique
targets
at
the
mitochondria, we first evaluated
whether aspirin (Asp) can be
incorporated in the T/NT-NPs.
0
Ps
y-N
Ps
-N
Ps Ps
-N
y-N
) 4
Ps
Ps
Ps Ps
-N
y-N
) 4
Ps
-N
Ps
-N
) 4
Ps
Ps
-N
) 4
-N
-N
y-N
t
)
4
t
t
)
4
t
)
4
)
4
-Emp -(Asp -Emp -(Asp
-Emp
-(Asp -Emp -(Asp
T
-(Asp
-(Asp
NT
-(Asp
-(Asp
T
T
T
T
T
NT
NT
20 nm
20 nm
NT
NT
NT
Nanoprecipitation
targeted PLGA-b-PEG-OH
polymer (Figures S1, S2) or
targeted PLGA-b-PEG-TPP
of
non-
Fig. 2. (A) Structure of Oc-[G2]-(Asp)4. (B) Synthesis of T/NT-(Asp)4-NPs from different polymers and
Oc-[G2]-(Asp)4. (C) Diameter, zeta potential, percent loading, %EE, and TEM of T/NT-(Asp)4-NPs. (D)
Release kinetics of Oc-[G2]-(Asp)4 from T and NT-NPs.
NP and NT-(Asp)4-NPs indicated sizes below 100 nm and highly
polymer (Figures S3, S4, S5) in presence of aspirin afforded low
encapsulation efficiency (EE) and percent loading of aspirin
inside NT/T-Asp-NPs (Figure S6). Poor encapsulation of aspirin
inside the hydrophobic core arises from hydrophilic properties
of aspirin. Thus, we hypothesized that construction of
hydrophobic analogues which can release aspirin by taking
advantages of the hydrolytic agents present in the cellular
milieu can be attractive strategy for better delivery of aspirin
at the target with improved PK and biodistribution (bioD)
properties when administered in vivo.
Analyses of the properties required for incorporation of
aspirin inside hydrophobic core and to increase therapeutic
efficacy prompted us to explore the possibility of use of a
hydrophobic dendritic platform as the number of aspirin
moieties required can easily be tuned.16 The dendritic
structure plays important roles in finding optimized structure
for aspirin analogue.16b We first developed a first generation
[G1] hydrophobic biodegradable dendron with an octyl (Oc)
chain connected to two available –OH moieties Oc-[G1]-(OH)2
positive surface for the T-NPs (Table S2, Figure 1B).
Comparison of NP sizes from these two dendrons indicated
that Oc-[G2]-(Asp)4 will be a more appropriate derivative for
aspirin delivery. Further, cytotoxicity of T/NT-(Asp)2-NPs and
T/NT-(Asp)4-NPs in RAW 264.7 macrophages indicated that the
NPs derived from Oc-[G1]-(Asp)2 are relatively more toxic to
the cells whereas the NPs from Oc-[G2]-(Asp)4 did not
demonstrate any such toxicity up to 100 µM (Figure 1C). One
possible reason for the toxicity of Oc-[G1]-(Asp)2-NPs might be
their larger size compared to the Oc-[G1]-(Asp)4 NPs.
Transmission electron microscopy (TEM) image analysis of
T/NT-(Asp)2-NPs indicated the presence of aggregates with
different surface properties (Figure S28). These surface
properties may be responsible for the higher toxicity of the
T/NT-(Asp)2-NPs.17 Further studies are required to understand
the exact mechanism of cytotoxicity of T/NT-(Asp)2-NPs. Based
on the size and toxicity of the NPs, we decided to use Oc-[G2]-
(Asp)4 for delivery of aspirin using NP platform.
2 | J. Name., 2012, 00, 1-3
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Nanoprecipitation was carried out using 20% feed of Oc- around 3 h after administration.19 In our studVieiesw AfroticllleoOwnilninge
DOI: 10.1039/C5CC07316B
[G2]-(Asp)4 (Figure 2A) with PLGA-b-PEG-TPP polymer to result similar protocol in C57BL/6 or BALB/c (albino) mice, we
in T-(Asp)4-NPs or with PLGA-b-PEG-OH polymer to produce observed enhanced levels of TNF-α and IL-6 in the serum after
NT-(Asp)4-NPs (Figure 2B). Control T/NT-Empty-NPs were also intraperitoneal administration of 100 µg of LPS per animal and
prepared. Dynamic light scattering (DLS) studies indicated that the levels peaked at 1.5 and 3 h for TNF-α and IL-6,
these NPs have diameter below 100 nm; T-NPs demonstrated respectively (Figure S29, data shown for C57BL/6 mice). Next,
high positively charged surface, and the NT-NPs were 8 week old BALB/c male mice were administered with saline or
negatively charged (Figure 2C). Determination of percent Oc- aspirin (20 mg/kg) or NT-(Asp)4-NP (20 mg/kg with respect to
[G2]-(Asp)4
loading
by
high
performance
liquid aspirin) or T-(Asp)4-NP (20 mg/kg with respect to aspirin)
chromatography (HPLC) indicated high loadings of 17±2% for (Table S3 for characterization of these NPs) by i.v. injections
NT and 16.6±0.6% for T NPs, respectively (Figure 2C). TEM and after 12 h, these animals were subsequently treated with
based analyses of the NPs further supported the diameter and intraperitoneally injected LPS for 1.5 h and 3 h (Figure 3A).
confirmed that these spherical NPs are homogeneous (Figure Serum samples were isolated from the treated animals and
2C). Studies suggested that aspirin is an antiplatelet agent that pro-inflammatory and anti-inflammatory cytokine levels in the
can be effective as an early treatment in acute ischemic stroke serum samples were evaluated by the enzyme-linked
and aspirin therapy should be used within 48 h of the initiation immunosorbent assay (ELISA). As seen with C57BL/6 mice,
of symptoms.18 This made us realize that although controlled serum TNF-α and IL-6 levels were increased after
release NPs can be invaluable addition to aspirin therapeutic administration of LPS in BALB/c Albino mice following similar
regiments, but the NPs should have release properties where patterns where TNF-α level was peaked at ~1.5 h and
significant portion of aspirin can get released in ~48 h. maximum IL-6 level was found at ~3 h and these levels were
Investigation of release kinetics of aspirin derivative from significantly higher (P<0.001) than only saline treated groups
T/NT-(Asp)4-NPs under physiological conditions of pH 7.4 at 37 (Figure 3B). Preventative treatment with aspirin (20 mg/kg)
°C demonstrated release of ~50% Oc-[G2]-(Asp)4 indicating followed by LPS for 1.5 h, TNF-α level was less than LPS alone,
that these NPs might be suitable for aspirin delivery for but these differences did not reach any statistical significance
neuroprotection (Figure 2D). Aspirin molecules are attached to (LPS vs. aspirin + LPS: non significant for TNF-α, Figure 5B).
Serum samples from the
i.p. injection
of LPS
(A)
i.v. injection of
T/NT-(Asp)4-NPs or aspirin
group of animals treated
with NT-(Asp)4-NPs prior to
LPS treatment for 1.5 h had
significantly lower TNF-α
than only LPS treated group
IL-6, TNF-α, IL-10
determination
1.5 h
12 h
IL-6, TNF-α, IL-10
determination
3 h
(B)
(P
=
0.001-0.01). Most
LPS treatment
for 1.5 h
LPS treatment
LPS treatment
for 1.5 h
LPS treatment
for 3 h
LPS treatment
for 1.5 h
LPS treatment
for 3 h
for 3 h
significantly, TNF-α levels
from the animals treated
with T-(Asp)4-NPs followed
by LPS treatment for 1.5 h
was drastically reduced
compared to only LPS (P <
0.001) (Figure 3B). Serum
TNF-α levels in the T-(Asp)4-
NP treated LPS stimulated
group was significantly lower
than the levels found in the
4000
3000
2000
1000
0
ns
1600
***
***
***
***
12000
8000
4000
0
ns
ns
ns
ns
**
***
ns
***
800
**
***
***
***
0
e
e
n
e
e
n
e
e
n
i
l
n
i
n
i
PS PS PS PS
i
PS PS PS PS
L L L L
PS PS PS PS
i
l
PS PS PS PS
n
i
l
l
PS PS PS PS
PS PS PS PS
L L L L
l
l
L
L
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L
L
L
L
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L
L
L
L
Sa
+
+
+
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+
+
+
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+
+
+
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+
+
+
Sa
+
+
+
Sa
+
+
+
n
n
n
n
n
n
ri
ri
Ps Ps
-N -N
Ps Ps
-N -N
ri
ri
Ps Ps
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Ps Ps
-N -N
ri
ri
i
i
i
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-N -N
Ps Ps
-N -N
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i
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)
4
)
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)
4
) 4
Asp
Asp
)
4
)
4
)
4
)
4
)
4
)
4
)
4
) 4
Asp
Asp
Asp
Asp
-(Asp-(Asp
-(Asp-(Asp
T
-(Asp-(Asp
-(Asp-(Asp
T
-(Asp-(Asp
-(Asp-(Asp
T
T
T
T
T
T
T
T
T
T
N
N
N
N
N
N
Fig. 3. (A) Experimental design for evaluation of anti-inflammatory properties of Oc-[G2]-(Asp)4-NPs
under preventative condition using BALB/c Albino male mice. (B) Pro-inflammatory TNF-α, IL-6 and anti-
inflammatory IL-10 levels in the serum samples of BALB/c Albino mice treated with different constructs
and LPS. ***: P<0.001; **: P = 0.001-0.01; ns: non significant.
NT-(Asp)4-NP
plus
LPS
treated animals when 1.5 h
time point was considered (P < 0.001) (Figure 3B). Thus, these
results indicated that T-(Asp)4-NPs are considerably more
effective than aspirin or NT-(Asp)4-NPs in inhibiting TNF-α
production upon LPS stimulation in vivo. Preventative
treatment with aspirin, NT-(Asp)4-NPs, or T-(Asp)4-NPs prior to
stimulation with LPS for 3 h did not show any significant
differences in serum TNF-α levels as this cytokine declined by
3 h (Figure 3B). In our experimental conditions, the level of IL-6
in only LPS treated samples was significantly increased from
that in saline treated group at 1.5 h and the level increased
further when LPS treatment was carried out for 3 h. When LPS
treatment for 1.5 h was considered, the IL-6 levels were
the dendron structure with aliphatic ester bonds. Thus, in the
presence of cellular esterases, the dendron scaffold will
release aspirin upon enzymatic hydrolysis.16a Given that the
aliphatic esters are more prone to get hydrolyzed as compared
to aromatic ester, it will preferentially release aspirin rather
than its final metabolite, salicylic acid.
To explore the anti-inflammatory properties of the new
aspirin derivative in NP formulation in vivo, we used mice
stimulated with lipopolysaccharide (LPS). An earlier study
demonstrated that intraperitoneally injected LPS can cause
secretion of significant amounts of tumor necrosis factor alfa
(TNF-α), which peaks around at 1.5 h and interleukin-6 (IL-6) at
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significantly reduced for the groups where preventative
treatments were carried out with aspirin, NT-(Asp)4-NPs, or T-
(Asp)4-NPs (Figure 3B). The IL-6 level in the T-(Asp)4-NP treated
group was lower than the group administered with NT-(Asp)4-
NPs at 1.5 h, however the differences between these two
groups did not reach statistical significance (Figure 3B). These
observations indicated that the targeted NP formulation of Oc-
[G2]-(Asp)4 is as effective as aspirin in preventing LPS induced
IL-6 secretion in vivo. When LPS treatment was carried out for
3 h, only aspirin showed reduced IL-6 levels compared to LPS
alone. Empty targeted or non-targeted NPs without aspirin did
not show any inflammatory or anti-inflammatory responses
under same experimental conditions (Figure S30).
Anti-inflammatory IL-10 determination in the serum
samples demonstrated no significant amounts of this cytokine
at the 1.5 h LPS treated samples. However, when 3 h LPS
treatment period was considered, a significantly higher level of
this anti-inflammatory cytokine was detected in the serum
samples from the animals which were pretreated with T-
(Asp)4-NPs prior to LPS stimulation, no other treated group
showed such a high IL-10 level (Figure 3B). The compelling
properties of T-(Asp)4-NPs in inhibiting production of pro-
inflammatory cytokines and induction of anti-inflammatory IL-
10 indicated that the new formulation of aspirin can be an
attractive candidate for further exploration for potential
activities in inflammation. Detailed mechanistic investigations
will also require to understand the effects of aspirin on the
mitochondria of cells and possible relation of mitochondrial
activity of aspirin with inflammatory properties if there is any.
This work provides first hydrophobic analogue of aspirin
which can be loaded inside polymeric NPs efficiently, thus
overcoming the disadvantages arising from physicochemical
properties of aspirin which do not allow its encapsulation
inside the hydrophobic core of NPs. Conjointly, our findings
highlighted potential abilities of this new hydrophobic aspirin
analogue Oc-[G2]-(Asp)4 encapsulated mitochondria targeted
NP as a possible therapeutic intervention of the central
nervous system inflammation leading to protection against
neurodegenerative diseases with inflammatory symptoms.
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This work was supported, in whole or in part, by
Department of Defense Prostate Cancer Idea award
(W81XWH-12-1-0406), National Institute of Neurological
Disorders and Stroke of National Institutes of Health under
award number R01NS093314, and by the Office of the Vice
President for Research, UGA as a start-up fund.
4 | J. Name., 2012, 00, 1-3
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