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Delivery of Dual Drug Loaded Lipid Based Nanoparticles across the
Blood−Brain Barrier Impart Enhanced Neuroprotection in a
Rotenone Induced Mouse Model of Parkinson’s Disease
Paromita Kundu,
†
Manasi Das,
†
Kalpalata Tripathy,
‡
and Sanjeeb K Sahoo*
,†
†
Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India
‡
Department of Pathology, Shri Ramachandra Bhanj Medical College, Cuttack 753007, India
*
SSupporting Information
ABSTRACT: Parkinson’s disease (PD) is the most widespread form of
dementia where there is an age related degeneration of dopaminergic neurons in
the substantia nigra region of the brain. Accumulation of α-synuclein (αS)
protein aggregate, mitochondrial dysfunction, oxidative stress, and neuronal cell
death are the pathological hallmarks of PD. In this context, amalgamation of
curcumin and piperine having profound cognitive properties, and antioxidant
activity seems beneficial. However, the blood−brain barrier (BBB) is the major
impediment for delivery of neurotherapeutics to the brain. The present study
involves formulation of curcumin and piperine coloaded glyceryl monooleate
(GMO) nanoparticles coated with various surfactants with a view to enhance
the bioavailability of curcumin and penetration of both drugs to the brain tissue
crossing the BBB and to enhance the anti-parkinsonism effect of both drugs in a
single platform. In vitro results demonstrated augmented inhibition of αS
protein into oligomers and fibrils, reduced rotenone induced toxicity, oxidative
stress, and apoptosis, and activation of autophagic pathway by dual drug loaded NPs compared to native counterpart. Further, in
vivo studies revealed that our formulated dual drug loaded NPs were able to cross BBB, rescued the rotenone induced motor
coordination impairment, and restrained dopaminergic neuronal degeneration in a PD mouse model.
KEYWORDS: Parkinson’s disease, α-synuclein, blood−brain barrier, lipid based nanoparticles, curcumin, piperine
Parkinson’s disease (PD) is the second most prevalent
neurodegenerative disorder characterized by progressive
loss of dopaminergic neurons of substantia nigra and
subsequent deprivation of dopamine in the basal ganglia.
1
The PD patient encounters severe physical impairment, and the
major cardinal symptoms of the disease include resting tremors,
bradykinesia, postural instability, and muscular rigidity.
1
Numerous etiological causes have been linked to PD, including
genetic mutations and environmental toxins, but the main
reason for cell death remains obscure. Abnormal accumulations
of aggregated α-synuclein (αS) proteins, high load of oxidative
stress, mitochondrial dysfunction, and impaired apoptosis
machinery are some of the potential unifying factors in the
etiopathogenesis of the disease.
2,3
Treatment of PD has been a
major challenge to the neurologist because treatment strategies
have been mostly symptomatic and the mainstay of treatment
aims at dopamine replacement therapy using drugs such as
levodopa, dopamine receptor agonist, or anticholinergic agents
for managing the motor disability.
4
However, the drug induced
side effects and inability to prevent neurodegeneration of
dopaminergic neurons with long-term treatment curtails the
therapeutic implementation of above conventional agents
clinically.
5,6
Further, treatment of specific aspects of cognition
along with disabling motor fluctuations and dyskinesia are still
at par. The current limitations of conventional medicine have
led us in search for a more holistic approach that can prevent
neurodegeneration, modulate multiple molecular events and
symptomatic features, and also impart minimal side effects for
the effective PD treatment.
In recent years, herbal drugs have received considerable
interest because of the broad spectrum of pharmacological
properties that can be explored for the clinical management of
PD. Studies have strongly indicated that the herbal drug
curcumin effectively counteracts the molecular events of PD
including oxidative stress, deregulated mitochondrial function,
aberrant apoptosis events, and αS aggregation into oligomers
and fibrils in vitro and also restores motor impairment and
attenuates loss of dopaminergic neurons in animal models of
PD.
7−9
Though curcumin is a potent drug with high
therapeutic value, its clinical efficacy in various in vivo studies
is marred because of its poor aqueous solubility, rapid
metabolism, and inadequate tissue absorption, which severely
curtails its bioavailability.
10
To this end, use of adjuvants like
piperine to improve the bioavailability of curcumin has been
Received: July 12, 2016
Accepted: September 19, 2016
Research Article
pubs.acs.org/chemneuro
© XXXX American Chemical Society ADOI: 10.1021/acschemneuro.6b00207
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
well explored through the oral route of administration by
diverse research groups.
11−13
Shoba et al. have explored
piperine as a bioavailability enhancer (inhibiting hepatic and
intestinal glucoronidation processes) to improve the bioavail-
ability of curcumin in preclinical studies and studies conducted
on human volunteers.
14
Further, mounting evidence also
narrates the neuroprotective effect of piperine by counteracting
the high load of oxidative stress and mitochondrial dysfunction
and attenuating apoptosis in diverse in vitro and in vivo PD
disease models.
15,16
Thus, amalgamating the therapeutic
potential of curcumin and piperine in a combination approach,
in which piperine will help to alleviate the bioavailability of
curcumin at the same time both drugs will exhibit anti-
parkinsonism effects by modulating the above unifying factors
associated with the etiopathogenesis of the disease, represents a
rational strategy.
However, delivery of both the drugs at a salutary level to
exert therapeutic efficacy to the brain tissue simultaneously to
achieve an additive or synergistic effect is a challenging task
because of their inability to cross the highly selective blood−
brain barrier (BBB) separating the central nervous system
(CNS) from systemic circulation. Challenges associated with
drug delivery to the CNS have fostered the development of
various nanotechnology based delivery vehicles to enhance the
transport of drugs from blood to the brain.
17,18
Therefore, use
of nanodelivery systems to deliver multiple therapeutic agents
by crossing the BBB at the same time to improve the
limitations associated with conventional regimens seems
promising for effective PD management. Among the various
nanoparticle (NP) regimes available, lipid based NPs have
received much attention as a potent carrier system for CNS
delivery because of their lipophilic nature, biocompatibility,
biodegradability, and size in the nanometer range (10−200
nm), which allows them to readily cross the tight endothelial
cells of BBB.
19,20
Further, lipid based NPs also demonstrate
high drug loading efficiencies, controlled drug release profiles,
improved drug bioavailability, and augmented tissue distribu-
tion.
21
In this context, glycerylmonooleate (GMO), a self-
assembling amphiphilic biocompatible lipid, has gained special
interest to formulate lipid based nanoparticles because of the
ability to enhance the bioavailability of encapsulated drugs.
22−24
Recently, we have developed a GMO based nanoformulation
for delivery of an anticancer drug thereby emphasizing the
scope of such GMO based NPs for CNS delivery.
25
Further,
use of surfactants like Pluronic F-68 and vitamin E−D-α-
tocopherol poly(ethylene glycol) 1000 succinate (vitamin E−
TPGS) coated NPs has proven to be highly effective in
delivering drugs across the BBB.
26−28
With the above concept, the rationale of the present study
was to formulate curcumin and piperine loaded GMO based
dual drug loaded NPs blended with surfactants such as Pluronic
F-68 and vitamin E−TPGS with a view to enhance the
bioavailability of curcumin and penetration of both the drugs to
the brain tissue crossing the BBB and to achieve the combined
anti-parkinsonism effects of both drugs in a single platform.
Our results demonstrated that curcumin when used in
combination with piperine in nanoformulation inhibited the
aggregation of αS protein into oligomers and fibrils in vitro and
also reduced rotenone induced cell death in PC12 cells via
decreasing the oxidative stress and apoptosis and enhancing the
autophagic activity. Further, our dual drug loaded NPs
enhanced the oral bioavailability of curcumin and also
efficiently crossed the BBB to deliver the drugs into the brain
tissue and eventually rescued rotenone induced motor
coordination impairment and restrained dopaminergic neuronal
degeneration in a PD mouse model.
■RESULTS AND DISCUSSION
Increase in incidence of PD at an alarming rate has raised major
public health concern, and PD is expected to become a major
cause of disability worldwide.
29
Given the enormity of the
disease, an effective treatment strategy for preventing or curing
of the disease is a call of the hour. Further, the BBB, the
bottleneck for the delivery of neurotherapeutics to the brain,
has been a major limiting factor for the treatment of PD.
30
From the past few years, attention has been turning toward the
use of various dietary antioxidants for the treatment of PD
because of their potential neuroprotective properties. However,
since most of these antioxidant compounds do not cross the
BBB, an ample salutary level does not reach the brain to exert
considerable pharmacological effect. Despite copious research
on CNS drug delivery strategies, very few of them has reached a
phase of safe and effective human application. For the pace of
innovation, the field of nanotechnology has opened new
avenues and prospects for delivering therapeutic payload
crossing the BBB.
31
To this end, with an aim to achieve a
combination therapeutic strategy enabling delivery of high
therapeutic payload crossing the BBB at the disease site in a
sustained manner and facilitating symptomatic and neuro-
protective effect, in the present investigation, we have
formulated a curcumin and piperine loaded lipid based
nanoformulation blended with surfactants and studied its
therapeutic efficacy for PD treatment in cellular model, PC12
cells. The above cells’response toward nerve growth factor
(NGF) that converts the cells from proliferating chromaffin-like
cells to nondividing sympathetic-neuron-like cells with
electrical excitability and sensitivity toward neurotoxin
rotenone in inducing neuronal degeneration make it a
convenient in vitro model to study causes and possible
treatments for PD.
32
Further, the efficacy of our formulated
dual drug loaded NPs was also studied in a rotenone induced
mouse PD model.
Preparation and Characterization of NPs Loaded with
Curcumin, Piperine, or Both. In the present study, we have
formulated GMO based NPs surface coated with surfactants F-
68 and vitamin E−TPGS. Previously, our group has shown that
GMO could be advantageous in helping NPs to cross the
BBB.
24
Further, use of surfactants like F-68 and vitamin E−
TPGS has proven to be highly effective in delivering drugs
across the BBB.
26
Recently, Kulkarni et al. has explored this
strategy where surface coating of their polymeric NPs by TPGS
has dramatically influenced the NPs to deliver drug across the
gastrointestinal barrier and BBB.
27
Similar kinds of studies have
also been conducted by Gelperina et al. where surface coating
of poly(lactic-co-glycolic acid) (PLGA) NPs by surfactants like
poloxamer enabled the delivery of drugs into the brain.
28
Further, numerous investigations suggest that due to their
lipophilic nature, lipid based nanoparticles have a natural
tendency to cross the BBB and their small particle size
facilitates effective reticuloendothelial system (RES) escape,
thereby increasing the chance of contact with the BBB for the
drug to be taken up by the brain.
19,20
Our formulated NPs also
showed a size in the range of 93 ±11 nm as evident from
dynamic light scattering (DLS) analysis (Figure 1A) with
negative ζpotential, −30.9 ±0.88 mV. The nanometer size of
the formulated lipid NPs was further authenticated by
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B
transmission electron microscope (TEM) analysis (Figure 1B),
which showed that the NPs were ∼60 nm size. Size of NPs as
measured by DLS was higher than the size observed in TEM
analysis, which may be attributed to the state of NPs used for
measurement. DLS analysis measures the hydrodynamic
diameter of particles (consisting of particle core along with
solvent layer attached to the particle) present in a liquid
suspension, whereas TEM analysis measures the area of the
core of a dry particle.
33
Similar pattern of size difference of NPs
measured through TEM and DLS was also evident in our
previous study.
34
Spherical shape and smooth surface of dual
drug loaded NPs was confirmed by atomic force microscopy
(AFM) analysis (Figure 1C). Both curcumin and piperine were
efficiently loaded in NPs, achieving an encapsulation efficiency
of ∼65% (for both drugs) in single or in dual nanoformulation
as evident from high performance liquid chromatography
(HPLC) analysis. Sustained delivery of the entrapped drug
from the NPs represents an important therapeutic advantage,
because it lowers the frequency of dosing. An in vitro release
kinetics study suggested sustained release of both curcumin and
piperine from dual drug loaded NPs over a period of 8 days
(Figure 1D). Both drugs exhibited a biphasic release pattern
with an initial burst (desorption, diffusion, and dissolution of
drug present at the surface) followed by sustained drug release.
In Vitro Inhibition of α-Synuclein Aggregation into
Oligomers and Fibrils. Aggregation of αSisakey
pathological feature of PD and studies suggest that oligomeric
and protofibrillar structures of αS are the toxic entities that
sabotage intracellular organelle function and induce oxidative
stress thereby leading to neuronal cell death.
35
A body of
evidence has shown the potentiality of polyphenols and
alkaloids toward inhibition of αSfibrillation.
36
In relation to
this, the putative role of curcumin in inhibiting αS protein
aggregation, thereby protecting neuronal cells from death in
PD, has been documented.
37
Further, recent studies have
shown the protective effect of piperine (a natural alkaloid)
against neuronal injury in PD.
15,38
However, the effect of
piperine on αS protein aggregation has not yet been explored.
Therefore, in the present study, we have explored the effect of
curcumin, piperine, and combination of both the drugs on αS
protein aggregation into oligomers and fibrils using AFM
analysis (Figure 2). Photomicrographs of αS clearly depict the
formation of oligomers or long and thin fibrillar structure
following incubation of αS protein for 24 h and 6 days,
respectively (Figure 2 and Supporting Information, Figure
S1A,B). Importantly, co-incubation of αS with curcumin and
piperine (native or nanoformulations) both alone and in
combination resulted in formation of aggregates of smaller size
compared to αS oligomers formed without any drug treatment
(Figure 2A and Supporting Information, Figure S1A). The
efficiency of disruption into smaller size was higher for drug
loaded NPs than respective native drugs. Noteworthy,
combination of curcumin and piperine elucidated more
profound inhibition of oligomeric aggregation compared to
single counterpart, and dual drug loaded NPs exhibited still
more inhibitory effect. Further, antifibrillar activity of the above
formulations was also elucidated from AFM images (Figure 2B
and Supporting Information, Figure S1B), showing disruption
of long fibrillar structure to small oligomeric morphology, and
more or less it was found that dual drug loaded NPs inhibit the
fibril formation more profoundly than other formulations. In a
recent study conducted by Ahsan et al., the antiaggregation
property of native curcumin has been well documented and
similar results were observed, thus corroborating our findings.
37
Importantly, our results also indicate the antiaggregation
properties of piperine alone (native or in NPs); however, the
mechanism by which piperine inhibits the aggregation of αS
protein remains to be elucidated. The inhibitory effect of
piperine and curcumin on αS aggregation was further validated
by thioflavin T (ThT) binding assays. Our results demonstrate
a higher ThT fluorescence signal in the case of oligomeric and
fibrillar forms of αS. However, cotreatment with curcumin or
piperine or combination of both drugs (native or NPs) resulted
in lower fluorescence intensity, suggesting the successful
inhibition of oligomerization and fibrillation events (Supporting
Information, Figure S1C). Noteworthy, combination drug
treatment resulted in more profound inhibition, and dual
drug loaded NPs exhibited significantly greater inhibitory effect
than other treatments. In a recent study, Ahmad et al. have
documented that curcumin can completely inhibit oligomeriza-
tion and fibrillation by performing ThT assay, thus substantiat-
ing our observation with curcumin.
39
Cellular Uptake Study. Cellular uptake of the drug loaded
NPs is essential for attaining ample drug level to elicit a
substantial therapeutic response. In this regard, exploring the
intrinsic fluorescence property of curcumin, an in vitro cellular
uptake analysis was performed using fluorescence spectropho-
tometer and confocal microscopy. The quantitative uptake
study by fluorescence spectrophotometer clearly reveals
significant uptake of curcumin nanoparticles (CNPs) (∼8
fold higher uptake at 1 and 2 h time point, ∼16-fold higher
uptakes at 4 h time point) compared to native curcumin in
PC12 cells (Figure 3A). In a similar study, Wang et al.
demonstrated a time dependent enhanced uptake of rhodamine
B loaded PLGA NPs in MG-63 cancer cells.
40
Enhanced
intracellular uptake of CNPs in comparison to native curcumin
Figure 1. Physicochemical characterization of dual drug loaded NPs.
(A) Size of curcumin and piperine loaded NPs (CPNPs) as measured
by dynamic laser light scattering. (B) Transmission electron
micrograph of CPNPs, depicting that the formulated particles are in
the nanometer size range. Inset shows a higher magnification of the
particle. (C) AFM image of CPNPs, depicting their smooth and
spherical topology. All experiments were performed in triplicate, and a
representative image has been provided. (D) In vitro release kinetics of
curcumin and piperine from dual drug loaded NPs as percent of drug
release. Data represented as mean ±SEM (n= 3).
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C
was further evident from confocal microscopy results,
substantiating the observation from the quantitative uptake
study (Figure 3B). The higher uptake of CNPs may be realized
from the fact that drug loaded NPs enter the cells by the
endocytic pathway, while internalization of free drugs occurs
through the passive diffusion process and reaches saturation
after reaching certain concentration inside the cells.
41
Addi-
tionally, the enhanced uptake efficiency of CNPs may also have
resulted because drug loaded nanoformulations can prevent
endolysosomal degradation of drug, thereby increasing its
concentration several fold.
33
In comparison, free drug in
solution is vulnerable toward lysosomal degradation and as a
result may not be sufficiently accumulated inside the cells.
42
In Vitro Cellular Cytotoxicity Assay. Previous studies
have reported that, exposure of dopaminergic cells such as
PC12 cells or SH-S5Y cells to neurotoxin rotenone (a
mitochondrial complex I inhibitor) causes oxidative damage
and promotes the accumulation and aggregation of αS protein
thereby accurately imitating many aspects of PD pathogenesis
and cell death. Herbal drugs like curcumin and piperine have
shown promising results in protecting the dopaminergic cells
from rotenone induced cytotoxicity.
38,43
However, the
therapeutic potential of combination of both the drugs in
protecting cells from rotenone induced toxicity has not been
explored to date. Therefore, to assess the effect of curcumin or
piperine or combination of both drugs on rotenone induced
toxicity, cell viability study by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was performed in
PC12 cells. The cytotoxicity study with rotenone reveals a dose
dependency with 2 μg/mL of rotenone causing approximately
50% cell death (Supporting Information, Figure S2). Further, to
test the protective effect of curcumin, piperine, or both for
rotenone induced toxicity, we exposed the PC12 cells to
rotenone (2 μg/mL) along with various concentrations of
curcumin, piperine, or both, native or in nanoformulations. All
the drug treatments (native or in NPs) protected against
rotenone induced cell death to a substantial extent in a dose
dependent manner, and drug loaded NPs exhibited superior
protection compared to native counterparts (Figure 4).
Importantly combination of curcumin and piperine in nano-
particles revealed more profound protection against rotenone
induced toxicity than other formulations. The superior
protective efficacy of drug loaded NPs in comparison to
respective free drug may be attributed to the enhanced cellular
Figure 2. Atomic force microscopy analysis to study the aggregation of α-synuclein (αS) protein. Representative AFM images of αS, showing
inhibitory effect of different treatments on the formation of (A) oligomers and (B) fibrils. The reaction mixture containing 10 μMαS, 50 mM
sodium phosphate buffer, pH 7, 20% ethanol, and 10 μM FeCl3were treated with 7.5 μg/mL of native curcumin (CN), native piperine (PN), and
combination of native curcumin and native piperine (CPN) or equivalent concentration of curcumin NPs (CNPs), piperine NPs (PNPs), and
curcumin and piperine NPs (CPNPs), or an equivalent amount of void NPs (VNPs) incubated at room temperature under continuous shaking
overnight (for oligomer formation) and 6 days (for fibril formation). Experiment has been performed in triplicate, and representative image has been
provided.
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D
internalization and sustained drug release properties exhibited
by NPs.
Measurement of Intracellular Oxidative Stress. Accu-
mulating evidence advocates that oxidative damage and
mitochondrial impairment contribute to the cascade of
processes leading to degeneration of dopaminergic neurons in
PD.
44,45
In this context, diverse studies suggest a decreased
level of potent antioxidant gluathione (GSH) (directly
quenches reactive hydroxyl free radicals) in the substantia
nigra of PD patients, thus portraying the putative role of this
antioxidant in PD pathogenesis.
46
Therefore, in the present
study, we explored the effect of curcumin and piperine either
alone or in combination in restoring GSH level. Results depict a
significant reduction in cellular GSH level in rotenone treated
PC12 cells compared to untreated control cells (Supporting
Information, Figure S3A). A study conducted by Sharma et al.
documented the inhibitory effect of rotenone induced GSH in
vivo, thus corroborating our observation.
47
Importantly,
administration of antioxidants like curcumin and piperine
(native or NPs; alone or in combination) attenuated GSH
depletion induced by rotenone. Note that, combination drug
treatment (curcumin plus piperine), native and NPs, resulted in
more profound inhibition, and dual drug loaded NPs exhibited
superior effect to other treatments. The enhanced restoration of
GSH in combination drug treatment may have resulted from
the combined antioxidant properties exhibited by both
curcumin and piperine.
48,49
GSH synthesis and utilization is
directly regulated by Nrf2,
50
and curcumin is known to up
regulate Nrf2 against oxidative stress.
51
Since we have observed
an increase GSH level on treatment with curcumin in dual drug
loaded NPs (Supporting Information,FigureS3A),we
anticipate that it might have occurred because of activated
Nrf2. Lipid peroxidation is a key feature in the pathogenesis of
PD where reactive oxygen species (ROS) readily attack the
polyunsaturated fatty acid of membrane lipids resulting in
significant neuronal cell damage.
52
Therefore, alleviating lipid
peroxidation with potent antioxidants like curcumin and
piperine may seem to be clinically beneficial.
15
Our results
depict an augmented lipid peroxidation (as evident from
increased formation of thiobarbituric acid reactive substances
(TBARS) a by product of lipid peroxidation) in cells treated
with rotenone, and cotreatment with curcumin or piperine
(alone or in combination), native or in NPs, resulted in
substantial reduction of lipid peroxidation (Supporting
Information, Figure S3B). Noteworthy, combination drug
treatment in NPs exhibited a superior effect to other treatments
following enhanced cellular internalization and synergistic
antioxidant activity imparted by both drugs.
Induction of Autophagy−Lysosome Function. The
autophagy−lysosome pathway (ALP) is a vital mechanism for
the removal of abnormal and aggregated proteins, and ample of
evidence suggests an impairment in this pathway, which
thereby aggravates disease progression.
53
We therefore, studied
the expression of LC3 II (a marker of autophagosome
formation) and Lamp2 (lysosomal marker) through Western
blot analysis following different treatments (Figure 5A). Results
indicate a remarkable enhancement in expression of LC3 II and
Lamp2 protein following cotreatment with curcumin and
piperine (alone or incombination), native or in NPs.
Importantly, dual drug loaded NPs exhibited augmented
expression of both the proteins compared to other treatments,
suggesting the potentiality of dual drug loaded NPs in
activation of ALP. The restoration of autophagy activity is
further authenticated by immunofluorescence analysis of LC3 II
in transiently transfected PC12 cells with RFP-LC3 followed by
different treatments in combination with rotenone. Result
shows few puncta formed, that is, conversion of cytosolic LC3-I
to membrane bound LC3-II, in rotenone treated cells.
However, a significant increase in RFP-LC3 puncta was
Figure 3. In vitro cellular uptake study. (A) Quantitative cellular
uptake study of CN and CNPs in PC12 cells for different time periods
by fluorescence spectrophotometer (ex = 420 nm, em = 525 nm).
Data are presented as mean ±SEM (n= 3). ***p< 0.001 CNPs in
comparison to CN. (B) Qualitative cellular uptake analysis of CN and
CNPs in PC12 cells at 2 h time point by confocal microscope
equipped with FITC filter (ex = 488 nm, em = 525 nm) and with PI
filter (ex = 535 nm, em = 617 nm). Experiment has been performed in
triplicate, and representative image has been provided.
Figure 4. Cytotoxicity study to assess the protective effect of different
treatments on rotenone induced cytotoxicity in PC12 cells for 48 h by
MTT assay. Cells were co-incubated with various concentrations of
the drug with 2 μg/mL rotenone (R),and cellular viability was
determined. Results are presented as mean ±SEM (n= 4). p< 0.05 is
considered significant. ###,pcorresponds to rotenone vs control and
*p,**p,or***pcorresponds to different treatments vs rotenone: (1)
CN; (2) CNPs; (3) PN; (4) PNPs; (5) CPN; (60) CPNPs; (7)
VNPs.
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E
observed in rotenone treated PC12 cells coadministered with
curcumin and piperine (both alone and in combination), native
or in NPs. Importantly, dual drug loaded NPs exhibited higher
puncta formation compared to other treatments thereby
suggesting the profound autophagic inducing activity in the
case of dual drug loaded NPs (Figure 5B). Accumulating
evidence strongly suggests αS protein as a major structural
component of Lewy bodies (the pathological hallmark of PD)
and narrates the crucial role of this protein in pathogenesis of
PD.
35
Further, Wu et al. recently reported an increased
expression of αS on rotenone treatment in cultured PC12 cells,
indicating impairment in protein degradation.
54
We thus
investigated the effect of curcumin and piperine on αS protein
in PC12 cells. Our results showed increased accumulation of αS
protein in rotenone treated cells (Figure 5A). Importantly,
coadministration with curcumin and piperine (alone or in
combination), native or in NPs, effectively inhibited the
rotenone induced accrual of αS with dual drug loaded NPs
eliciting more profound effects (Figure 5A). In a recent study
by Jiang et al. curcumin efficiently reduced accumulation of
A53T αS protein by recovering the macroautophagy process,
thereby suggesting the therapeutic role of the drug in
ameliorating the neurodegenerative pathology of PD.
55
Since,
both curcumin and piperine are known to induce autophagy,
enhanced autophagic activity following a combinational drug
treatment, both native and NPs, might have resulted due to the
synergistic action of both the drugs.
56
To further confirm the
protective role of autophagy against rotenone induced
cytotoxicity, cells were exposed to an autophagy inhibitor, 3-
methyladenine (3MA), along with rotenone and different
concentrations of curcumin and piperine in combination
(native or in NPs) for 48 h. Results showed that on addition
of 3MA the drugs could not protect the cells from rotenone
induced cytotoxicity clearly indicating that autophagy plays a
crucial role in protecting the PC12 cells from cell death (Figure
5C).
Modulation of Apoptosis. Apoptosis is known to play a
crucial role in the loss of neurons in PD, and deregulated
mitochondrial function (a characteristic feature of PD)
significantly implicates induction of apoptotic response.
57
Therefore, in our study, we have examined a panel of
antiapoptotic and proapoptotic proteins related to mitochon-
drial function. In the present study, we have examined the effect
of curcumin and piperine on these proteins by Western
blotting, and results indicate a substantial decrease in the
protein ratio of Bcl-2 to BAX (Figure 6A,B) and an increase in
cleaved caspase 3 and cleaved PARP expression in rotenone
treated PC12 cells, thus marking the prevalence of apoptosis
(Figure 6C,D). However, an increase in Bcl-2/BAX ratio and
decrease in cleaved caspase 3 and cleaved PARP expression was
observed in rotenone exposed cells coadministered with
curcumin and piperine in combination (native or in NPs)
compared to only rotenone treatment, thus suggesting the role
of drugs in combination toward eliciting a survival mechanism.
Importantly, dual drug loaded NPs exhibited more profound
effect compared to other treatments following enhanced
cellular internalization. The study of apoptosis was further
validated through flow cytometry, where clear induction of
apoptosis (both early as well as late apoptosis) was observed in
rotenone treated cells as compared to control group. However,
when treated in combination with curcumin and piperine both
native and in nanoformulation cells were protected from
apoptosis induced by rotenone (Figure 6E,F) suggesting the
activation of cell survival pathway.
Enhanced Plasma Bioavailability and Brain Biodis-
tribution of Dual Drug Loaded NPs. Poor bioavailability of
curcumin is a major impediment toward therapeutic success of
this novel molecule in preclinical settings. To this end, Shoba et
al. have explored piperine as a bioavailability enhancer and
documented superior enhancement in the bioavailability of
curcumin in preclinical studies and studies conducted on
human volunteers.
14
Further numerous researchers have
Figure 5. (A) Western blot analysis was performed to investigate the
expression of LC3 II, Lamp 2, and α-synuclein proteins following
different treatments in PC12 cells. Cells were cotreated with R (2 μg/
mL) along with 2 μg/mL of CN, PN, CPN, and CNPs, PNPs, and
CPNPs for 48 h. (1) Control; (2) R; (3) R + CN; (4) R + CNPs; (5)
R + PN; (6) R + PNPs; (7) R + CPN; (8) R + CPNPs. (B) Study of
autophagy in PC12 cells through confocal microscope. In brief, cells
were transfected with RFP-LC3 plasmid and exposed to above
treatments for 48 h. Cells exhibiting RFP-LC3 puncta (indicator of
autophagosome formation) were observed under confocal microscope.
Experiment has been performed in triplicate, and representative image
has been provided. (C) Investigating the effect of autophagy inhibition
toward protective effect of different treatments on rotenone induced
cytotoxicity in PC12 cells. In brief, cells were exposed to R (2 μg/mL)
or 10 mM 3MA and R (2 μg/mL) or coadministered with 10 mM
3MA, R (2 μg/mL), and different concentrations of curcumin and
piperine native or in NPs for 48 h, and cell viability was assessed by
MTT assay. Data presented as mean ±SEM (n= 4). p< 0.05 is
considered significant. **por ***pcorresponds to treatment with
inhibitor and drug treatment vs with only drug treatment.
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exploited novel nanodelivery systems to increase bioavailability
of curcumin.
58
In the present investigation, we explored
piperine along with curcumin in lipid based NPs to enhance
the bioavailability of curcumin in plasma to achieve a
therapeutic dose in the brain to combat PD. Further, lipid
based NPs may also aid toward crossing BBB (owing to
lipophilic nature and nanometer size) to deliver the payload in
an enhanced way at the site of action. Our in vivo results
indicate low bioavailability (in plasma) (Figure 7A) and low
biodistribution (in brain) (Figure 7B) of native curcumin
administered orally, at all the time points studied. Further, the
amount of curcumin decreased in a time dependent manner.
The lower bioavailability of native curcumin may have resulted
due to poor aqueous solubility, degradation under alkaline
conditions, limited gastrointestinal absorption, and presystemic
transformation to glucornides in the liver leading to faster
elimination.
10
Low bioavailability of native curcumin in plasma
(0.06 μg/mL) of rat (500 mg/kg orally administered curcumin)
was documented by Yang et al. thus, corroborating our
observation with native curcumin.
59
Importantly, coadministra-
tion of curcumin with piperine (native or in NPs) resulted in
significant enhanced bioavailability and distribution of
curcumin in brain tissue compared to only curcumin treatment.
The enhanced bioavailability of curcumin might have resulted
due to the inhibitory effect of piperine on hepatic and intestinal
glucoronidation process (that causes curcumin metabolic
degradation).
14
Further, encapsulation of drug in NPs
significantly enhanced the bioavailability of curcumin in plasma
as well as its distribution in brain tissue compared to respective
native counterpart. The improved efficacy in drug loaded NPs
might have resulted due to better solubility of curcumin in
nanoformulation and BBB crossing ability of lipid based NPs.
In accordance with our observation, in a recent study
Ramalingam et al. have shown the improved oral bioavailability
and brain biodistribution of curcumin loaded with N-trimethyl
chitosan coated solid lipid NPs compared to native drug.
60
Note that dual drug loaded NPs elucidated superior
bioavailability and brain biodistribution of curcumin than
other treatments that could be due to combined approach of
using piperine and lipid based NPs. Because our drug loaded
NPs exhibited prolonged plasma retention, augmented
bioavailability, and enhanced brain tissue distribution of
curcumin, we anticipated a substantial delivery of our dual
drug loaded NPs to the substatia nigra region of brain. To
endorse the above view, uptake of NPs in substantia nigra of
brain was evaluated by confocal microscopy (Figure 7C). The
confocal microscopy images of the mid brain section clearly
indicate the presence of CNPs or CPNPs in the substantia
nigra region as evident from green fluorescence of curcumin,
thus narrating the putative role of our lipid based NPs in
Figure 6. Study of apoptosis. (A) Representative immunoblots of Bcl2 and BAX expression and (B) quantification of Bcl2/BAX. (C) Representative
immunoblot of apoptotic protein caspase 3. (D) Representative immunoblot of apoptotic protein PARP. (1) control; (2) R; (3) R + CPN; (4) R +
CPNPs. (E) Analysis of apoptosis by flow cytometry to study the protective effect of CPN or CPNPs against rotenone induced apoptosis in PC12
cells for 48 h. Apoptosis percentage was analyzed by annexin V-PE and 7-AAD staining. Experiment has been performed in triplicate, and
representative image has been provided. (F) Bar diagram depicting total percentage of apoptotic cells. p< 0.05 is considered significant. ###p
corresponds to rotenone vs control, and ***pcorresponds to different treatment vs rotenone.
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crossing BBB to deliver the therapeutic payload efficiently. NPs
have shown remarkable potential as a brain targeting system
compared to native drugs. In relation to this, in a recent study
Kakkar et al. have shown the brain targeting and BBB crossing
ability of lipid based NPs to deliver curcumin to the brain, thus
substantiating our observation of enhanced brain targeting with
lipid based NPs.
61
Restoration of Functional Deficits in a Rotenone
Induced Mouse Model of PD. Resting tremor, bradykinesia,
muscular rigidity, and postural instability are the cardinal
manifestations of PD. Therefore, a therapeutic molecule that
imparts symptomatic relief in PD may be considered beneficial.
In the present study, curcumin and piperine have shown
substantial therapeutic benefits in preclinical testing by
modulating various molecular and biochemical aspects of
pathogenesis of PD; therefore, we next focused on exploring
the utility of curcumin and piperine loaded NPs in providing
functional relief in a rotenone induced mouse model of PD.
62
This model exhibits key symptomatic features of PD (impaired
motor balance and coordination) as evident from rotarod
motor performance study in which mice treated with rotenone
spent less time on the rod compared to untreated control
(Figure 8A). Importantly, rotenone induced mice coadminis-
tered with curcumin and piperine in combination (native or
NPs) showed significant extension of time spent on the rod,
with dual drug loaded NPs exhibiting more significant effect
than native counterpart. These results suggest the potent role of
drug combination, specifically dual drug loaded NPs in
ameliorating motor dysfunction in rotenone induced PD
model. The motor coordination restoration observed with
dual drug combination in an enhanced way can be explained by
considering the putative role of both curcumin and piperine in
modulating various molecular and biochemical aspects of the
pathogenesis of PD in vitro observed in the present
investigation. Further, the superior efficacy of dual drug loaded
NPs over native drug combination may have resulted following
enhanced plasma bioavailability, superior brain targeting by
crossing BBB, increased delivery to substantia nigra of brain,
and augmented cellular internalization. In a recent study, da
Rocha Lindner et al. have demonstrated the superior protective
efficacy of resveratrol (RVT)-loaded polysorbate 80 (PS80)-
coated poly(lactide) nanoparticles compared to native resver-
atrol in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced
PD model, thus suggesting the advantage of NPs over native
drug in neuroprotection.
63
Degeneration of dopaminergic
neurons is another cardinal sign of pathogenesis of PD. We
therefore further tried to evaluate the effect of curcumin and
piperine in protecting against the degeneration of dopaminergic
neurons in rotenone induced PD mice. Because the gold
standard marker in the identification of dopaminergic neurons
is tyrosine hydroxylase (TH), the rate limiting enzyme in
dopamine synthesis, we studied the presence of TH positive
neurons to mark the presence of dopaminergic neuronal cells.
16
Immunohistochemistry study of the substantia nigra region
clearly indicates low density of TH positive neurons in
rotenone treated mice compared to untreated control (Figure
8B,C). Noteworthy, coadministration of curcumin and piperine
(native or NPs) resulted in higher density of TH positive
Figure 7. Pharmacokinetics of curcumin in mice (A) plasma and (B) brain tissue after single oral administration of CN, CNPs, CPN, and CPNPs at
a dose of 100 mg/kg body weight. Values are presented as mean ±SEM (n= 3). *p< 0.05, **p< 0.01, or ***p< 0.001 for CN vs CPNPs; #p,##p,
or ###pcorresponds to CPN vs CPNPs. (C) Histological sections of the substantia nigra region of brain tissues (marked by immunoexpression for
TH, red) showing efficient accumulation of curcumin (green) after 2 h oral treatment, and representative image has been provided (n= 3). Insets are
the higher magnification of area showing the presence of CNPs and CPNPs (white arrow).
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H
neurons, with dual drug loaded NPs exhibiting more intense
effect in protecting against rotenone induced degeneration of
dopaminergic neurons.
■CONCLUSIONS
In the present study, we have formulated a dual drug (curcumin
and piperine) loaded lipid based nanoformulation and studied
its anti-parkinsonism effect through various in vitro and in vivo
studies. Our present data demonstrate the neuroprotective
effect of our dual drug loaded NPs by inhibiting the aggregation
of αS protein, reducing the cytotoxicity and oxidative stress
induced by rotenone, activation of autophagy mediated protein
degradation, and induction of antiapoptotic events. However, a
detailed investigation on the primary target of these drugs to
ameliorate PD pathogenesis is warranted in near future.
Further, the drug loaded NPs also significantly reversed the
neurobehavioral abnormalities and neuronal degeneration in
the substantia nigra in a PD mouse model. The better
therapeutic effect of curcumin and piperine in dual drug loaded
NPs may be due to improved bioavailability of curcumin, ability
to cross BBB, and synergistic effect exhibited by both the drugs.
Thus, the present study suggests the potential of our dual drug
loaded NPs in ameliorating Parkinson’s pathogenesis in clinical
settings.
■METHODS
Materials. CUR-500, containing curcumin (>95%), was purchased
from UNICO Pharmaceuticals (Ludhiana, India). GMO was
purchased from Eastman (Tennessee, USA). Sodium chloride and
piperine were obtained from MP Biomedicals (Illkirch, France).
Acetonitrile was purchased from Spectrochem, India. Dimethyl
sulfoxide (DMSO), methanol, ethanol, and acetic acid were procured
from E-merk (Mumbai, India). Hematoxylin was obtained from
Thermo Fisher Scientific, Mumbai, India. Lipofectamine 2000
transfection reagent; nerve growth factor (NGF mouse protein,
native, 7S subunit) was purchased from Invitrogen Corp. (CA, USA).
Skimmed milk powder was procured from Himedia Laboratories Pvt.
Ltd., Mumbai, India. mRFP-LC3 (plasmid no. 21075) was obtained
from Addgene Inc. (MA, USA). Sodium deoxycholate, ethylene glycol-
bis(2-amino ethyl ether)-N,N,N,N-tetraacetic acid (EGTA), ethylene
diamine tetra-acetic acid (EDTA), 5,5′-dithiobis-2-nitrobenzoic acid
(DTNB), poly(L-lysine), vitamin E D-α-tocopherol poly(ethylene
glycol) 1000 succinate (vitamin E−TPGS), 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), Pluronic F-68, poly-
(ethylene glycol) (PEG)-10 000, phenylmethylsulfonyl fluoride
(PMSF), sodium orthovanadate (NaVO4), β-glycerophosphate,
protease inhibitor cocktail, thiobarbituric acid (TBA), butylated
hydroxytoluene (BHT), L-glutathione reduced, thioflavin T, sodium
dodecyl sulfate (SDS), and 4′,6-diamidino-2-phenylindole (DAPI)
were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other
chemicals used were purchased from Sigma-Aldrich (St. Louis, MO,
USA) and used without further purification.
Preparation of Drug Loaded Lipid Based NPs. Dual drug
loaded NPs were formulated by following our previous published
protocol with few modifications.
25
Briefly, 50 mg of curcumin and 50
mg of piperine was dispersed in the fluid phase of GMO (500 μLat40
°C) and vortexed. This mixture was subjected to emulsification with
10 mL of Pluronic F-68 solution (5% w/v) by sonication using a
microtip probe sonicator (VC 505, Vibracell Sonics, MA, USA) set at
an amplitude of 30% for 2 min in an ice bath. The resultant solution
was further emulsified with 10 mL of vitamin E−TPGS (5% w/v) as
mentioned above. The emulsion obtained was centrifuged at 1000 rpm
for 1 min to remove the unentrapped curcumin/piperine, followed by
addition of PEG-10000 (20 mg/mL) as a lyoprotectant with constant
vortexing for 5 min.
64
Finally the emulsion was lyophilized for 6 days
(−50 °C and <0.05 mbar, Labconco Free Zone 12, Labconco
Corporation, Kansas, USA) to obtain the lyophilized powder for
further use. Single drug loaded NPs (curcumin or piperine) were also
formulated following this protocol.
Physicochemical Characterization of Dual Drug Loaded
NPs. The particle size and ζpotential of the NPs was measured by
Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK) using our
previously published protocol.
65
Size and surface topology of the NPs
was further assessed by TEM and AFM, respectively, following our
previously published protocol.
34
Entrapment efficiency of curcumin
and piperine in drug loaded NPs was estimated by reverse phase
isocratic mode of RP-HPLC (Waters 600, Waters Co., MA, USA).
Briefly, ∼1 mg/mL of curcumin NPs (CNPs)/piperine NPs (PNPs)
or (curcumin and piperine) loaded NPs (CPNPs) was dissolved in
ACN, sonicated for 2 min at an amplitude of 30% in an ice bath, and
the supernatant was collected following centrifugation. Curcumin
concentration was measured using the mobile phase ACN/sodium
acetate buffer (20 mM, pH 3.0)/methanol in a ratio of 6:1:3 at a
wavelength of 420 nm, and piperine was measured using the mobile
phase ACN/potassium dihydrogen phosphate (25 mM, pH 4.5) in a
ratio of 6.5:3.5 at a wavelength of 345 nm.
66,67
The amount of drug in
the NPs was obtained from the peak area correlated to a standard
curve prepared under identical conditions. In vitro release kinetics of
(curcumin and piperine) loaded NPs were performed in PBS as per
Figure 8. Effect of different drug treatments on rotenone induced (A)
behavioral deficit in mice as assessed by rotarod and (B) representative
image of TH positive neurons in the substantia nigra region of the
brain following different treatments has been provided (black arrow).
(C) TH positive neurons count per field to study the dopaminergic
neuronal cell death in the substantia nigra. Data are presented as mean
±SEM (n= 4). p< 0.05 is considered significant. ###pcorresponds to
rotenone vs control and *por ***pcorresponds to different treatment
vs rotenone and •••pcorresponds to CPN vs CPNPs. Experiment has
been performed with n= 4 animals.
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I
previously published protocol.
25
All analysis was performed in
triplicate.
α-Synuclein Aggregation Assay. α-Synuclein aggregation
analysis was performed following the protocol of Danzer et al.
68
In
brief, stock solution of purified αS (Sigma-Aldrich, St. Louis, MO,
USA) was prepared in distilled water, and subsequent dilutions were
made in 50 mM sodium phosphate buffer (pH 7). Oligomeric forms of
αS were generated by dissolving 10 μM of the protein in reaction
buffer (50 mM sodium phosphate buffer, containing 20% ethanol and
10 μM of FeCl3) at room temperature under continuous shaking with
overnight incubation while predominantly fibrillar forms of αS were
generated by following the same condition with the incubation time
increased up to 6 days. Further, to evaluate the inhibitory effect of
curcumin and piperine on αS aggregation, 10 μM protein dissolved in
reaction buffer was co-incubated with 7.5 μg/mL of curcumin or
piperine (alone or in combination) both in native and in nano-
formulations and kept shaking overnight (oligomer study) or 6 days
(fibrillar study). Following the incubation period, the morphology of
αS with different treatments was visualized by AFM analysis. For this,
∼10 μL aliquot of each sample was applied onto a freshly cleaved mica
surface and left to dry at room temperature for 10 min. Samples were
then rinsed with Milli Q water and dried under nitrogen flow. Samples
were imaged in contact mode set at a frequency of 13 kHz and
scanned at a speed of 1 Hz. Topographic images were analyzed, and
height distribution plot was generated using JPK data processing
software. Experiment was performed in triplicate, and representative
images have been provided.
Cell Culture. Rat PC12 cell line was obtained from National
Centre for Cell Sciences Cell Repository, Pune, India. Cells were
cultured in RPMI media supplemented with 10% heat inactivated
horse serum, 5% fetal bovine serum (FBS), 1% L-glutamine, and 1%
penicillin−streptomycin (Himedia Laboratories Pvt. Ltd., Mumbai,
India) and maintained at 37 °C in a 5% CO2atmosphere incubator
(Hera Cell, Thermo scientific, Waltham, USA). All chemicals for cell
culture were purchased from PAN Biotech (GmbH, Germany) unless
otherwise mentioned. All the cellular experiments were performed in
differentiated PC12 cells, obtained by culturing the cells in RPMI
media containing NGF (50 ng/mL) and 1% FBS for 3 days and used
subsequently for further experiments.
Cellular Uptake Study. Quantitative and qualitative cellular
uptake of native curcumin and curcumin loaded NPs in PC12 cells was
evaluated by fluorescence spectrophotometer and confocal micros-
copy, respectively, following our previously published protocol.
25
For
quantitative cellular uptake study, 1 ×105PC12 cells seeded in poly(L-
lysine) coated 12 well plates (Corning Inc., NY, USA) were treated
with 2 μg/mL native curcumin or equivalent concentration of
curcumin loaded NPs for different time points, and intracellular
concentration of curcumin was quantified using fluorescence
spectrophotometer (ex 420 nm, em 525 nm). Experiments were
performed in triplicate. For qualitative cellular uptake study, cells were
exposed to above drug treatments for 2 h and counterstained with
propidium iodide for nuclear staining. Images were visualized in
confocal laser scanning microscope (Leica TCS SP5, Leica Micro-
systems GmbH, Germany) equipped with an argon laser with an FITC
filter (ex 488 nm, em 525 nm) and PI filter (ex 535 nm, em 617 nm).
The images were processed using Leica Application Suite software.
Experiment was performed in triplicate, and representative image has
been provided.
In Vitro Cell Viability Assay. Cell viability was analyzed using
MTT based colorimetric assay as described in our previously published
protocol.
69
Briefly, PC12 cells seeded at a density of 5 ×103cells per
well in a poly(L-lysine) coated 96 well plate (Corning Inc., NY, USA)
were treated with different concentrations of rotenone (2 μg/mL) or
cotreated with rotenone (2 μg/mL) along with different concen-
trations of curcumin or piperine (alone or in combination), native as
well as in NPs, for 48 h. Cells treated with only media were used as
control for the experiment. At the end of the incubation period, cell
viability was assessed by MTT assay. Data represented as mean ±SEM
(n= 4).
Western Blot Analysis. Western blot analysis of different proteins
in PC12 cells following various treatments was carried out following
previously published protocol.
34
Briefly, PC12 cells (1 ×106cells)
seeded in poly(L-lysine) coated T-25 flask were exposed to rotenone
(2 μg/mL) or coadministered rotenone (2 μg/mL) and curcumin or
piperine (2 μg/mL, alone or in combination), both native and in
nanoformulations. After 48 h of treatment, the cells were harvested
and washed with PBS, and whole cell lysate was prepared with
radioimmunoprecipitation assay buffer (RIPA) buffer. Western blot
analysis of various proteins were performed using specific primary
antibodies recognizing LC3 (Novus Biologicals, Colorado, USA), α-
synuclein, Lamp2, β-actin (Santa Cruz Biotechnology, Inc., CA, USA),
BAX, Bcl-2, PARP, or caspase 3 (Cell Signaling Technology, Inc., MA,
USA) and their respective secondary antibodies. The band intensity
was measured by ImageJ software.
Fluorescence Imaging of Red Fluorescent Protein (RFP) LC3
for Autophagy Study. The RFP-LC3 transfected PC12 cells were
generated by transfecting pRFP-LC3 plasmid into subconfluent PC12
cells using Lipofectamine 2000 transfection kit.
70
Briefly, PC12 cells at
density of 1 ×106were seeded in 60 ×15 mm2petridish (Corning,
NY, USA) for overnight attachment. Next day, 8 μg of plasmid DNA
was mixed with 20 μL of Lipofectamine 2000 reagent in serum free
RPMI media, incubated for 20 min at room temperature, and then
added to the cells. After 8 h of incubation, the transfection medium
was replaced with fresh culture medium. Next day, RFP-LC3
transfected PC12 cells were seeded at a density of 1 ×105cells on
poly(L-lysine) coated coverslips. The cells were then differentiated
with 50 ng/mL NGF media for 3 days. The cells were then treated for
48 h with rotenone (2 μg/mL) or coadministered rotenone (2 μg/
mL) with curcumin or piperine (2 μg/mL, alone or in combination)
both native and in nanoformulations. After 48 h, the cells were washed
with PBS and fixed with 4% paraformaldehyde. Cells were again
washed with PBS, incubated with DAPI to stain the nucleus, and
finally mounted with aqueous mounting media (Vector Laboratories,
California, USA) and observed under confocal microscopy (Leica TCS
SP5, Leica Microsystems, Germany) for the formation of RFP-LC3
puncta, a primary marker of autophagosome formation. Further, to
determine the effect of impaired autophagosome pathway on cell
cytotoxicity, cellular viability was assessed in PC12 cells treated with
3MA (autophagosome inhibitor). In brief, 5 ×103PC12 cells seeded
in 96 well plates were treated with 3MA (10 mM), rotenone (2 μg/
mL), and different concentrations of curcumin and piperine in
combination (native or in NPs) for 48 h, and cell viability was assessed
by MTT assay as mentioned before.
Apoptosis Study. Apoptotic cell death in PC12 cells following
different treatments was studied by flow cytometry following a
previously published protocol.
34
Briefly, 3 ×105PC12 cells seeded in
six well plates were treated with rotenone (2 μg/mL) or
coadministered rotenone (2 μg/mL) with curcumin and piperine (2
μg/mL in combination) native and in nanoformulations for 48 h. Cells
treated with only media served as control for the experiment. After the
incubation period, cells were washed three times with PBS and
processed for apoptosis analysis using Annexin V-PE and 7-
aminoactinomycin D (7-AAD) (BD FACSCalibur Flow Cytometer,
BD Biosciences, CA, USA) in FL2-H and FL3-H channel, respectively,
using FlowJo software.
In Vivo Study. Male Balb/c mice and male C57BL/6 mice were
used for different in vivo experiments with the approval of the
Institutional Animal Ethics committee of the Institute of Life Sciences,
Bhubaneswar. The animals were housed at a constant temperature and
relative humidity with alternating 12-h cycles of light and dark. Mice
were housed in standard laboratory cages and had free access to food
and water throughout the study period. For the pharmacokinetics
study, animals were fasted overnight before dosing.
In Vivo Bioavailability Study. Male Balb/c mice (4−6 weeks
old), weighing 22 ±10 g, were divided into five groups (n= 3). Group
1, control, was administered 0.5% carboxymethyl cellulose sodium salt
(CMC). Group 2 was administered native curcumin dispersed in 0.5%
CMC at a dose of 100 mg/kg body weight; group 3 was administered
CNPs dispersed in distilled water at an equivalent dose to native
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curcumin (100 mg/kg body weight). Group 4 was administered native
curcumin and piperine (1:1 ratio) dispersed in 0.5% CMC at a dose of
100 mg/kg body weight. Group 5 was administered CPNPs dispersed
in distilled water at an equivalent dose of curcumin and piperine native
of 100 mg/kg body weight. After oral administration by gavage with a
catheter at different time points (0.5, 2, 6, 48 h) blood samples were
collected from retro-orbital plexus into precoated heparin tubes.
Curcumin concentration in plasma was estimated by HPLC as
mentioned before using the protocol of Shaikh et al.
67
To study the
amount of curcumin present in the brain tissue, biodistribution
analysis was carried out at the above time points for all five groups.
Briefly, the brain was dissected out at different time points,
homogenized with PBS (BD-144 Tissue Homogenizer, BD Bioscience,
Haryana, India), and lyophilized. The lyophilized samples were
processed for estimation of curcumin present in brain tissue by
HPLC using previously published protocol.
67
Immunofluorescence. Male Balb/c mice (4−6 weeks old),
weighing 22 ±10 g, were divided into five groups (n=3)as
mentioned above. The mice were administered the previously defined
treatments for 2 h. Following treatment, transcardial perfusion was
performed with 4% paraformaldehyde (pH 7.4) under deep anesthesia
with xylazine and ketamine. After perfusion, the brain was quickly
removed and postfixed in 4% paraformaldehyde solution at 4 °C
overnight. Postfixed mid brain region was trimmed out and embedded
in paraffin, followed by preparation of multiple coronal sections (5
μm) using a microtome. Slides containing paraffin embedded brain
sections were deparaffinized with xylene and rehydrated with ethanol
with concentration gradient of 100−70%, followed by washing with
water. The slides were then boiled in antigen retrieval solution for 20
min and allowed to cool at room temperature. The sections were then
washed with PBS containing 0.1% Tween 20 (PBST) and then
blocked with 2.5% horse serum for 1 h at 37 °C. After blocking,
sections were incubated with a rabbit polyclonal anti-TH antibody
(1:200 dilutions) overnight at 4 °C. After a 10 min rinse in PBST, the
sections were incubated with anti-rabbit IgG secondary antibody Alexa
Fluor 594 conjugate (Invitrogen Corp., CA, USA) for 45 min. Finally
the sections were washed with PBST and mounted with aqueous
mounting media, and fluorescence (green for curcumin and red for
TH positive cells) was observed under confocal microscope (Leica
TCS SP5, Leica Microsystems, GmbH, Germany).
Animal Model. Male, 8−10 week old C57BL/6 mice (25−30 g)
were randomly assigned to five groups (n= 4). Group 1, control, was
administered 0.5% CMC orally as a vehicle once daily for 28 days;
group 2 was administered rotenone suspended in 0.5% CMC orally,
once daily at a dose of 30 mg/kg body weight for 28 days. Group 3
was administered native curcumin and native piperine (1:1 ratio)
orally at a dose of 200 mg/kg body weight, every alternate day, 30 min
before administration of rotenone. Group 4 was administered CPNPs
orally at an equivalent dose of 200 mg/kg body weight, every alternate
day, 30 min before administration of rotenone. Group 5 was
administered void NPs orally at an equivalent dose of 200 mg/kg
body weight every alternate day 30 min before administration of
rotenone. At the end of the experiment, on 29th day, the mice were
subjected to rotarod task for monitoring the behavioral pattern of
mice, and the presence of dopaminergic neurons (TH positive) in
brain tissue was further studied through immunohistochemistry.
Motor Performance Study. The behavior of each mouse was
assessed by the rotarod test, as described by Inden et al.
62
by using
rotarod treadmill (Orchid Scientific and Innovative India Pvt Ltd.,
Nashik, India). In the present study, mice (from all groups of
treatment along with control) were placed on the rod rotating at 20
rpm, and the falling latencies were recorded for up to 250 s. All mice
were tested on 29th day after the initial rotenone administration.
Immunohistochemistry. In brief, transcardial perfusion was
performed with 4% paraformaldehyde (pH 7.4) under deep anesthesia
with xylazine and ketamine. The brain was collected, and multiple
coronal sections (5 μm) were obtained using a microtome. Slides
containing paraffin embedded brain sections were deparaffinized with
xylene and rehydrated with ethanol with concentration gradient of
100−70% followed by washing with water. The slides were then boiled
in antigen retrieval solution for 20 min and were allowed to cool at
room temperature followed by washing with PBST. Sections were then
incubated with 3% hydrogen peroxide for 15 min at room temperature
to remove the endogenous peroxidase activity and then blocked with
2.5% horse serum for 1 h at 37 °C. After blocking for 1 h, sections
were incubated with a rabbit polyclonal anti-TH antibody (1:200
dilutions, Millipore, Darmstadt, Germany) overnight at 4 °C. After a
10 min rinse in PBST, the sections were incubated with biotinylated
secondary universal horse anti-rabbit/mouse IgG (Vectastain Kit;
Vector Laboratories, California, USA) for 45 min, followed by
incubation with avidin−biotin peroxidase complex for 30 min at room
temperature. Sections were washed with PBST and exposed to
diaminobenzidine (DAB), and photographs of the brain sections were
taken using phase contrast microscope (Leica EZ, UK) with 40×
objective, and then TH positive neurons were counted. A certified
human pathologist evaluated all the stained slides.
Statistics. Results are expressed as mean ±SEM or mean ±SD.
Statistical analysis of the data was performed by applying Student’st
test and one way and two way ANOVA using GraphPad Prism
Software, and values of p< 0.05 were indicative of significant
differences.
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.6b00207.
Histogram analysis of oligomers and fibrils obtained from
AFM study, dose dependent cytotoxicity of rotenone in
PC12 cells, and cellular GSH and lipid peroxidation assay
(PDF)
■AUTHOR INFORMATION
Corresponding Author
*Sanjeeb K Sahoo. Phone 91-674-2302094. Fax 91-674-
2300728. E-mail sanjeebsahoo2005@gmail.com.
Author Contributions
P.K, and S.K.S. conceived and designed the project. P.K and
M.D. performed the experiments. P.K., M.D., K.T., and S.K.S.
analyzed the data. P.K., M.D., and S.K.S. contributed to the
preparation of manuscript.
Funding
P.K. acknowledges University Grants Commission (UGC),
New Delhi, India, for providing the award of Junior Research
Fellowship (JRF).
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
S.K.S. and P.K. acknowledge Dr. Rupesh Dash, Scientist at
Institute of Life Sciences, and lab members for experimental
help in autophagy study. S.K.S. and P.K. are also thankful to Dr.
Shantibhusan Senapati, Scientist at Institute of Life Sciences,
and lab members for their help in in vivo studies. Technical help
of Mr. Priyadarshi Ray in AFM and Mr. Madan Mallick for
histological sectioning is acknowledged.
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