Delivery of Dual Drug Loaded Lipid Based Nanoparticles across the

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Delivery of Dual Drug Loaded Lipid Based Nanoparticles Across 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 ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00207 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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ACS Chemical Neuroscience

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Delivery of Dual Drug Loaded Lipid Based Nanoparticles Across Blood Brain Barrier

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Impart Enhanced Neuroprotection in a Rotenone Induced Mouse Model of Parkinson’s

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Disease

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Paromita Kundu¶, Manasi Das¶, Kalpalata Tripathyǂ, Sanjeeb K Sahoo*, ¶

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¶

Institute of Life Sciences, Nalco Square, Bhubaneswar, India, 751023

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ǂ

Department of Pathology, Shri Ramachandra Bhanj Medical College, Cuttack, India, 753007

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ABSTRACT: Parkinson disease (PD) is the most widespread form of dementia where there

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is an age related degeneration of dopaminergic neurons in the substantia nigra region of the

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brain. Accumulation of α-synuclein (αS) protein aggregate, mitochondrial dysfunction,

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oxidative stress and neuronal cell death are the pathological hallmark of PD. In this context,

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amalgamation of curcumin and piperine having profound cognitive properties and antioxidant

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activity seems beneficial. However, blood brain barrier (BBB) is the major impediment for

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delivery of neurotherapeutics to the brain. The present study involves formulation of

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curcumin and piperine co-loaded glyceryl monooleate (GMO) nanoparticles coated with

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various surfactants with a view to enhance the bioavailability of curcumin, penetration of

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both drugs to the brain tissue crossing the BBB and to enhance the anti-parkinsonism effect

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of both drugs in a single platform. In vitro results demonstrated augmented inhibition of αS

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protein into oligomers and fibrils, reduced rotenone induced toxicity, oxidative stress,

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apoptosis and activation of autophagic pathway by dual drug loaded NPs compared to native

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counterpart. Further, in vivo studies revealed that our formulated dual drug loaded NPs were

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able to cross BBB, rescued the rotenone induced motor coordination impairment and

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restrained dopaminergic neuronal degeneration in a PD mice model. 1 ACS Paragon Plus Environment


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Graphic for the Table of Contents 27 28 29 30 31 32 33 34 35

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KEYWORDS: Parkinson's disease, α-synuclein, blood brain barrier, lipid based nanoparticles, curcumin, piperine

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Parkinson’s

disease

(PD)

is

the

second

most

prevalent

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INTRODUCTION:

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neurodegenerative disorder characterized by progressive loss of dopaminergic neurons of

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substantia nigra and subsequent deprivation of dopamine in the basal ganglia.

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patient’s encounters severe physical impairment and the major cardinal symptoms of the

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disease include resting tremors, bradykinesia, postural instability and muscular rigidity.

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Numerous aetiological causes have been linked to PD, including genetic mutations and

47

environmental toxins, but the main reason of cell death remains obscure. Abnormal

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accumulations of aggregated α-synuclein (αS) proteins, high load of oxidative stress,

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mitochondrial dysfunction and impaired apoptosis machinery are some of the potential

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unifying factor in the aetiopathogenesis of the disease. 2, 3 Treatment of PD has been a major

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challenge to the neurologist because treatment strategies have been mostly symptomatic and 2 ACS Paragon Plus Environment

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The PD

1

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the mainstay of treatment aims at dopamine replacement therapy using drugs such as

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levodopa, dopamine receptor agonist, anti-cholinergic agent etc. for managing the motor

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disability.

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neurodegeneration of dopaminergic neurons with long term treatment curtails the therapeutic

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implementation of above conventional agents clinically.

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aspects of cognition along with disabling motor fluctuations and dyskinesia are still at par.

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The current limitations of conventional medicine has led in search for a more holistic

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approach that can prevent neurodegeneration, modulate multiple molecular events and

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symptomatic features and also impart minimal side effect for the effective PD treatment.

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However, the drug induced side effects and inability to prevent

5, 6

Further, treatment on specific

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In recent years, herbal drugs have received considerable interest because of the broad

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spectrum of pharmacological properties that can be explored for the clinical management of

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PD. Studies have strongly indicated that the herbal drug curcumin effectively counteracts the

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molecular events of PD including oxidative stress, deregulated mitochondrial function,

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aberrant apoptosis event, αS aggregation into oligomers and fibrils in vitro and also restores

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motor impairment and attenuate loss of dopaminergic neurons in animal models of PD.

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Though curcumin is a potent drug with high therapeutic value, yet its clinical efficacy in

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various in vivo studies is marred because of its poor aqueous solubility, rapid metabolism and

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inadequate tissue absorption, which severely curtails its bioavailability. 10 To this end, use of

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adjuvants like piperine to improve the bioavailability of curcumin has been well explored

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through oral route of administration by diverse research groups.

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explored piperine as a bioavailability enhancer (inhibiting hepatic and intestinal

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glucoronidation process) to improve the bioavailability of curcumin in preclinical studies and

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studies conducted on human volunteers.

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neuroprotective effect of piperine by counteracting the high load of oxidative stress,

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mitochondrial dysfunction, and attenuating apoptosis in diverse in vitro and in vivo PD

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11-13

7-9

Shoba et al. have

Further, mounting evidence also narrates the

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disease model. 15, 16 Thus, amalgamating the therapeutic potential of curcumin and piperine in

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a combinational approach, in which piperine will help to alleviate the bioavailability of

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curcumin at the same time both the drugs will exhibit anti-parkinsonism effect by modulating

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above unifying factors associated with the aetiopathogenesis of the disease, represents a

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rational strategy.

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However, delivery of both the drugs at a salutary level to exert therapeutic efficacy to

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the brain tissue simultaneously to achieve an additive or synergistic effect is a challenging

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task because of their inability to cross the highly selective blood-brain barrier (BBB)

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separating the central nervous system (CNS) from systemic circulation. Challenges

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associated with drug delivery to the CNS have fostered in the development of various

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nanotechnology based delivery vehicle to enhance the transport of drugs from blood to the

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brain.

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crossing BBB at the same time to improve the limitations associated with conventional

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regimens seems promising for effective PD management. Among the various nanoparticles

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(NPs) regimes available, lipid based NPs has received much attention as potent carrier system

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for CNS delivery because of its lipophilic nature, biocompatibility, biodegradability, and

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having size in the nanometer range (10-200 nm), which allows them to readily cross the tight

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endothelial cells of BBB.

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efficiencies, controlled drug release profile, improved drug bioavailability and augmented

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tissue distribution.

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amphiphilic biocompatible lipid has gained special interest to formulate lipid based

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nanoparticles because of the ability to enhance the bioavailability of encapsulated drugs. 22-24

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Recently, we have developed a GMO based nanoformulation for delivery of anticancer drug

17, 18

Therefore, use of nanodelivery systems to deliver multiple therapeutic agents by

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19, 20

Further, lipid based NPs also demonstrates high drug loading

In this context, glycerylmonooleate (GMO) a self-assembling

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thereby emphasizing the scope of such GMO based NPs for CNS delivery.

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surfactants like Pluronic F-68 and Vitamin E D-α-Tocopherol polyethylene glycol 1000 4 ACS Paragon Plus Environment

Further, use of

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succinate (vitamin E-TPGS) coated NPs has proven to be highly effective in delivering drugs

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across the BBB. 26-28

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With the above concept, the rationale of the present study was to formulate curcumin

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and piperine loaded GMO based dual drug loaded NPs blended with surfactants such as

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Pluronic F-68 and vitamin E-TPGS with a view to enhance the bioavailability of curcumin,

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penetration of both the drugs to the brain tissue crossing the BBB and to achieve the

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combinational anti-parkinsonism effect of both drugs in a single platform. Our results

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demonstrated that curcumin when used in combination with piperine in nanoformulation

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inhibited the aggregation of αS protein into oligomers and fibrils in vitro and also reduced

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rotenone induced cell death in PC12 cells via decreasing the oxidative stress, apoptosis and

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enhancing the autophagic activity. Further, our dual drug loaded NPs enhanced the oral

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bioavailability of curcumin and also efficiently crossed the BBB to deliver the drugs into the

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brain tissue and eventually rescued rotenone induced motor coordination impairment and

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restrained dopaminergic neuronal degeneration in a PD mice model.

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RESULTS AND DISCUSSION

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Increase in incidence of PD at an alarming rate have raised major public health concern and is

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expected to become a major cause of disability worldwide.

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disease, an effective treatment strategy for preventing or curing of the disease is a call of the

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hour. Further, BBB the bottle neck for the delivery of neurotherapeutics to the brain has been

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a major limiting factor for the treatment of PD. 30 From the last few years, attention has been

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turning towards the use of various dietary antioxidants for the treatment of PD because of

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their potential neuroprotective properties. However, since most of these antioxidant

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compounds do not cross the BBB, an ample salutary level does not reach to the brain to exert

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considerable pharmacological effect. Despite copious research on CNS drug delivery

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strategies, a very few of them has reached a phase of safe and effective human application.

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Given the enormity of the


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For the pace of innovation, the field of nanotechnology has opened new avenues and

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prospects for delivering therapeutic payload crossing the BBB. 31 To this end, with an aim to

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achieve a combinational therapeutic strategy enabling delivery of high therapeutic payload

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crossing BBB at disease site in a sustained manner and facilitating symptomatic and

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neuroprotective effect, in the present investigation, we have formulated (curcumin and

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piperine) loaded lipid based nanoformulation blended with surfactants and studied its

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therapeutic efficacy for PD treatment in cellular model, PC12 cells. The above cells responds

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towards Nerve Growth Factor (NGF) that convert the cells from proliferating chromaffin-like

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cells to non-dividing sympathetic-neuron-like cells with electrical excitability and sensitivity

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towards neurotoxin rotenone in inducing neuronal degeneration, make it a convenient in vitro

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model to study causes and possible treatments for PD.

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formulated dual drug loaded NPs was also studied in a rotenone induced mouse PD model.

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Further, the efficacy of our

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Preparation and Characterization of (Curcumin and/or Piperine) Loaded NPs.

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In the present study, we have formulated a GMO based NPs surface coated with surfactants

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F-68 and vitamin E TPGS. Previously, our group has shown that GMO could be

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advantageous in helping the NPs to cross the BBB. 24 Further, use of surfactants like F-68 and

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vitamin E TPGS has proven to be highly effective in delivering drug across the BBB.

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Recently, Kulkarni et al. has explored this strategy where surface coating of their polymeric

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NPs by TPGS have dramatically influenced the NPs to deliver drug across the gastrointestinal

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barrier and BBB.

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where surface coating of poly(lactic-co-glycolic acid) (PLGA) NPs by surfactants like

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poloxamer enabled the delivery of drugs into the brain.

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suggest that due to lipophilic nature, lipid based nanoparticle has a natural tendency to cross

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BBB and its small particle size facilitates effective reticuloendothelial system (RES) escape,

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thereby increasing the chance of contact with BBB and for the drug to be taken up by the

27

26

Similar kind of studies have also been conducted by Gelperina et al.

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Further, numerous investigations


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brain. 19, 20 Our formulated NPs also showed a size in the range of 93 ± 11 nm as evident from

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Dynamic light scattering (DLS) analysis (Figure 1A) with negative zeta potential -30.9 ± 0.88

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mV. The nanometer size of the formulated lipid NPs was further authenticated by

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Transmission electron microscope (TEM) analysis (Figure 1B), which showed that the NPs

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were of ~ 60 nm size. Size of NPs as measured by DLS was higher than the size observed in

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TEM analysis, which may be attributed to the state of NPs used for measurement. DLS

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analysis measures the hydrodynamic diameter of particles (consisted of particle core along

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with solvent layer attached to the particle) present in a liquid suspension whereas, TEM

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analysis measures the area of core of dry particle. 33 Similar pattern of size difference of NPs

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measured through TEM and DLS was also evident in our previous study.

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and smooth surface of dual drug loaded NPs was confirmed by Atomic force microscopy

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(AFM) analysis (Figure 1C). Both curcumin and piperine was efficiently loaded in NPs,

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achieving an encapsulation efficiency of ~ 65 % (for both drugs) in single or in dual

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nanoformulation as evident from High performance liquid chromatography (HPLC) analysis.

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Sustained delivery of the entrapped drug from the NPs represents an important therapeutic

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advantage, as it lowers the frequency of dosing. In vitro release kinetics study suggested

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sustained release of both curcumin and piperine from dual drug loaded NPs over a period of 8

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days (Figure 1D). Both the drugs exhibited a biphasic release pattern with an initial burst

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(desorption, diffusion and dissolution of drug present at the surface) followed by sustained

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drug release.

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In vitro Inhibition of

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Spherical shape

α-Synuclein Aggregation into Oligomers and Fibrils.

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Aggregation of αS is a key pathological feature of PD and studies suggest that oligomeric and

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protofibrillar structures of αS are the toxic entities that sabotage intracellular organelle

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function, induces oxidative stress thereby leading to neuronal cell death.

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evidences has shown the potentiality of polyphenols and alkaloids towards inhibition of αS 7 ACS Paragon Plus Environment

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A series of


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fibrillation.

In relation to this, the putative role of curcumin in inhibiting αS protein

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aggregation, thereby protecting neuronal cell death in PD has been documented.

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recent studies have shown the protective effect of piperine (natural alkaloids) against

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neuronal injury in PD. 15, 38 However, the effect of piperine on αS protein aggregation has not

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yet been explored. Therefore in the present study, we have explored the effect of curcumin,

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piperine and combination of both the drugs on αS protein aggregation into oligomers and

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fibrils using AFM analysis (Figure 2). Photomicrographs of αS clearly depict the formation

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of oligomers or long and thin fibrillar structure following incubation of αS protein for 24 hrs

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and 6 days respectively (Figure 2 and Supporting information, Figure S1A, B). Importantly,

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co-incubation of αS with curcumin and piperine (native or nanoformulations) both in single

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as well as in combination resulted in formation of aggregates of smaller size as compared to

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αS oligomers formed without any drug treatment (Figure 2A and Supporting information

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Figure S1A). The efficiency of disruption into smaller size was higher for drug loaded NPs

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than respective native drugs. Noteworthy, combination of curcumin and piperine elucidated

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more profound inhibition of oligomeric aggregation compared to single counterpart and dual

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drug loaded NPs exhibited still more inhibitory effect. Further, anti-fibrillar activity of the

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above formulations was also elucidated from AFM images (Figure 2B and Supporting

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information, Figure S1B), showing disruption of long fibrillar structure to small oligomeric

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morphology and more or less it was found that dual drug loaded NPs inhibits the fibril

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formation profoundly than other formulations. In a recent study conducted by Ahsan et al. the

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anti-aggregation property of native curcumin have been well documented and similar kind of

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results was observed, thus corroborating our findings. 37 Importantly, our results also indicate

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the anti-aggregation property of piperine alone (in native or in NPs) however, the mechanism

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by which piperine inhibits the aggregation of αS protein remains to be elucidated. The

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inhibitory effect of piperine and curcumin on αS aggregation was further validated by


37

Further,

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Thioflavin T (ThT) binding assays. Our result demonstrates a higher ThT fluorescence signal

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in case of oligomeric and fibrillar form of αS. However, co-treatment with curcumin or

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piperine or combination of both drugs (native or NPs) resulted in lower fluorescence

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intensity, suggesting the successful inhibition of oligomerization and fibrillation event

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(Supporting information, Figure S1C). Noteworthy, combinational drug treatment resulted in

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more profound inhibition and dual drug loaded NPs exhibited significant inhibitory effect

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than other treatments. In a recent study, Ahmad et al. have documented that curcumin can

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completely inhibit oligomerization and fibrillation by performing ThT assay, thus

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substantiating our observation with curcumin. 39

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Cellular Uptake Study. Cellular uptake of the drug loaded NPs are essential for

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attaining ample drug level to elicit a substantial therapeutic response. In this regard, exploring

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the intrinsic fluorescence property of curcumin, an in vitro cellular uptake analysis was

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performed using fluorescence spectrophotometer and confocal microscopy. The quantitative

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uptake study by fluorescence spectrophotometer clearly reveals significant uptake of

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curcumin nanoparticles (CNPs) (~ 8 fold higher uptake at 1hr and 2 hrs time point, ~ 16 fold

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higher uptakes at 4 hrs time point) compared to native curcumin in PC12 cells (Figure 3A). In

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a similar study, Wang et al. demonstrated a time dependent enhanced uptake of rhodamine B

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loaded PLGA NPs in MG-63 cancer cells.

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comparison to native curcumin was further evident from confocal microscopy results and

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substantiates the observation of quantitative uptake study (Figure 3B). The higher uptake of

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CNPs may be realized from the fact that, drug loaded NPs enters the cells by endocytic

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pathway, while internalization of free drugs occurs through passive diffusion process and

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reaches saturation after reaching certain concentration inside the cells.

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enhanced uptake efficiency of CNPs may also have resulted due to the fact that, drug loaded

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nanoformulations can prevent endolysosomal degradation of drug, thereby increasing its

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Enhanced intracellular uptake of CNPs in


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Additionally, the


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concentration to several folds.

In comparison, free drug in solution is vulnerable towards

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lysosomal degradation and as a result may not be sufficiently accumulated inside the cells. 42

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In vitro Cellular Cytotoxicity Assay. Previous studies have reported that, exposure

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of dopaminergic cells such as PC12 cells, SH-S5Y cells to neurotoxin rotenone (a

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mitochondrial Complex I inhibitor) causes oxidative damage, promote the accumulation and

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aggregation of αS protein thereby accurately imitating many aspect of PD pathogenesis and

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cell death. Herbal drugs like curcumin and piperine have shown promising results in

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protecting the dopaminergic cells from rotenone induced cytotoxicity.

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therapeutic potential of combination of both the drugs in protecting rotenone induced toxicity

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has not been explored till date. Therefore, to assess the effect of curcumin or piperine or

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combination of both drugs on rotenone induced toxicity, cell viability study by 3-(4,5-

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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed in PC12

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cells. The cytotoxicity study with rotenone reveals a dose dependency with 2 µg/ml of

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rotenone causing approximately 50 % of cell death (Supporting information, Figure S2).

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Further, to test the protective effect of curcumin or/and piperine on rotenone induced toxicity,

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we exposed the PC12 cells with rotenone (2 µg/ml) along with various concentration of

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curcumin, piperine or combination of both the drugs in native or in nanoformulations. All the

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drug treatments (in native or in NPs) protected against rotenone induced cells death to a

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substantial extent in a dose dependent manner and drug loaded NPs exhibited superlative

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protection compared to native counterparts (Figure 4). Importantly combination of curcumin

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and piperine in nanoparticles revealed profound protection against rotenone induced toxicity

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than other formulations. The superior protective efficacy of drug loaded NPs in comparison

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to respective free drug may be attributed to the enhanced cellular internalization and

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sustained drug release property exhibited by NPs.


38, 43

However,

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Measurement of Intracellular Oxidative Stress. Accumulating evidence advocates

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that oxidative damage and mitochondrial impairment contributes to the cascade of processes

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leading to degeneration of dopaminergic neurons in PD.

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suggest that the decreased level of potent antioxidant Gluathione (GSH) (directly quenches

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reactive hydroxyl free radicals) in the substantia nigra of PD patients, thus portraying the

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putative role of this antioxidant in PD pathogenesis.

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explored the effect of curcumin and piperine either alone or in combination in restoring GSH

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level. Result depicts a significant reduction in cellular GSH level in rotenone treated PC12

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cells compared to untreated control cells (Supporting information, Figure S3A). Study

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conducted by Sharma et al. documented the inhibitory effect of rotenone induced GSH in

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vivo, thus corroborating our observation.

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curcumin and piperine (native or NPs) (single or in combination) attenuated GSH depletion

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induced by rotenone. Note to mention that, combinational drug treatment (curcumin plus

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piperine) in native and NPs resulted in more profound inhibition and dual drug loaded NPs

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exhibited superior effect than other treatments. The enhanced restoration of GSH in

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combinational drug treatment may have resulted owing to the combined antioxidant property

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exhibited by both curcumin and piperine.

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regulated by Nrf2

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Since we have observed an increase GSH level on treatment with curcumin in dual drug

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loaded NPs (Supporting information, Figure S3A) we anticipate that it might have occurred

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because of an activated Nrf2. Lipid peroxidation is a key feature in the pathogenesis of PD

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where reactive oxygen species (ROS) readily attacks the polyunsaturated fatty acid of

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membrane lipids resulting in significant neuronal cells damage.

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peroxidation with potent antioxidants like curcumin and piperine may seem to be clinically

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beneficial.

15

50

47

46

44, 45

In this context diverse studies

Therefore in the present study, we

Importantly, administration of antioxidants like

48, 49

GSH synthesis and utilization is directly

and curcumin is known to up regulates Nrf2 against oxidative stress.

52

51

Therefore alleviating lipid

Our result depicts an augmented lipid peroxidation (as evident from increased



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formation of Thiobarbituric acid reactive substances (TBARS) a by product of lipid

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peroxidation) in cells treated with rotenone and co-treatment with curcumin and/or piperine

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(single or in combination) in native or in NPs resulted in substantial reduction of lipid

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peroxidation (Supporting information, Figure S3B). Noteworthy, combinational drug

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treatment in NPs exhibited a paramount effect that other treatments following enhanced

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cellular internalization and synergistic antioxidant activity imparted by both the drugs.

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Induction of Autophagy-Lysosome Function. Autophagy-lysosome pathway (ALP)

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is a vital mechanism for the removal of abnormal and aggregated proteins and ample of

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evidences suggest an impairment in this pathway which thereby aggravate the disease

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progression.

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formation) and Lamp2 (lysosomal marker) through western blot analysis following different

287

treatments (Figure 5A). Results indicate a remarkable enhancement in expression of LC3 II

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and Lamp2 protein following co-treatment with curcumin and/or piperine (in single or

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combination) in native or in NPs. Importantly, dual drug loaded NPs exhibited augmented

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expression of both the proteins compared to other treatments, suggesting the potentiality of

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dual drug loaded NPs in activation of ALP. The restoration of autophagy activity is further

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authenticated by immunofluorescence analysis of LC3 II in transiently transfected PC12 cells

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with RFP-LC3 followed by different treatments in combination with rotenone. Result shows

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few puncta formation i.e. conversion of cytosolic LC3-I to membrane bound LC3-II in

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rotenone treated cells. However, a significant increase in RFP-LC3 puncta was observed in

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rotenone treated PC12 cells co-administered with curcumin and/or piperine (both in single as

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well as in combination) in native or in NPs. Importantly, dual drug loaded NPs exhibited

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higher puncta formation compared to other treatments thereby suggesting the profound

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autophagic inducing activity in case of dual drug loaded NPs (Figure 5B). Accumulating

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evidences strongly suggest αS protein as a major structural component of Lewy bodies (the

53

We therefore, studied the expression of LC3 II (a marker of autophagosome


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pathological hallmark of PD) and narrates the crucial role of this protein in pathogenesis of

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PD.

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treatment in cultured PC12 cells, indicating impairment in protein degradation.

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investigated the effect of curcumin and piperine on αS protein in PC12 cells. Our result

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showed increase accumulation of αS protein in rotenone treated cells (Figure 5A).

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Importantly, co-administration with curcumin and/or piperine (single or in combination) in

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native or in NPs effectively inhibited the rotenone induced accrual of αS with dual drug

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loaded NPs eliciting more profound effect (Figure 5A). In a recent study by Jiang et al.

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curcumin efficiently reduced accumulation of A53T αS protein by recovering the

310

macroautophagy process, thereby suggesting the therapeutic role of the drug in ameliorating

311

the neurodegenerative pathology of PD.

312

induce autophagy therefore, an enhanced autophagic activity following a combinational drug

313

treatment both in native as well as NPs might have resulted due to the synergistic action of

314

both the drugs.

315

induced cytotoxicity, cells were exposed to an autophagy inhibitor 3-Methyladenine (3MA)

316

along with rotenone and different concentration of curcumin and piperine in combination (in

317

native or in NPs) for 48 hrs. Results showed that on addition of 3MA the drugs could not

318

protect the cells from rotenone induced cytotoxicity clearly indicating that autophagy plays a

319

crucial role in protecting the PC12 cells from cell death (Figure 5C).

35

Further, Wu et al. recently reported an increased expression of αS on rotenone

56

55

54

We thus

Since, both curcumin and piperine are known to

To further confirm the protective role of autophagy against rotenone

320

Modulation of Apoptosis. Apoptosis is known to play a crucial role in the loss of

321

neurons in PD and deregulated mitochondrial function (a characteristic feature of PD)

322

implicates significantly towards induction of apoptotic response.

323

we have examined a panel of anti-apoptotic and pro-apoptotic proteins related to

324

mitochondrial function. In the present study, we have examined the effect of curcumin and

325

piperine on these proteins by western blotting and result indicates a substantial decrease in 13 ACS Paragon Plus Environment

57

Therefore, in our study,


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

326

the protein ratio of Bcl-2 to BAX (Figure 6A and 6B) and an increase in cleaved caspase 3

327

and cleaved PARP expression in rotenone treated PC12 cells, thus marking the prevalence of

328

apoptosis (Figure 6C and 6D). However, an increase in Bcl-2: BAX ratio and decrease in

329

cleaved caspase 3 and cleaved PARP expression was observed in rotenone exposed cells co-

330

administered with curcumin and piperine in combination (in native or in NPs) compared to

331

only rotenone treatment, thus suggesting the role of drugs in combination towards eliciting a

332

survival mechanism. Importantly, dual drug loaded NPs exhibited more profound effect

333

compared to other treatments following enhanced cellular internalization. The study of

334

apoptosis was further validated through flow cytometry, where clear induction of apoptosis

335

(both early as well as late apoptosis) was observed in rotenone treated cells as compared to

336

control group. However, when treated in combination with curcumin and piperine both in

337

native as well as in nanoformulation protected from apoptosis induced by rotenone (Figure

338

6E and 6F) suggesting the activation of cell survival pathway.

339

Enhanced Plasma Bioavailability and Brain Biodistribution of Dual Drug

340

Loaded NPs. Poor bioavailability of curcumin is a major impediment towards therapeutic

341

success of this novel molecule in preclinical settings. To this end Shoba et al. have explored

342

piperine as a bioavailability enhancer and documented superior enhancement in the

343

bioavailability of curcumin in preclinical studies and studies conducted on human volunteers.

344

14

345

bioavailability of curcumin.

346

curcumin in lipid based NPs to enhance the bioavailability of curcumin in plasma to achieve

347

a therapeutic dose in the brain to combat PD. Further, lipid based NPs may also aid towards

348

crossing BBB (owing to lipophilic nature and nanometer size) to deliver the payload in an

349

enhanced way at the site of action. Our in vivo result indicates a low bioavailability (in

350

plasma) (Figure 7A) and low biodistribution (in brain) (Figure 7B) of native curcumin

Further numerous researchers have exploited novel nano-delivery systems to increase 58

In the present investigation, we explored piperine along with


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351

administered orally, at all the time point studied. Further, the amount of curcumin decreased

352

in a time dependent manner. The lower bioavailability of native curcumin may have resulted

353

due to poor aqueous solubility, degradation under alkaline conditions, limited gastrointestinal

354

absorption and pre-systemic transformation to glucornides in the liver leading to faster

355

elimination.

356

mg/kg orally administered curcumin) was documented by Yang et al. thus, corroborating our

357

observation with native curcumin.

358

piperine (in native or in NPs) resulted in significant enhanced bioavailability and distribution

359

of curcumin in brain tissue compared to only curcumin treatment. The enhanced

360

bioavailability of curcumin might have resulted due to the inhibitory effect of piperine on

361

hepatic and intestinal glucoronidation process (that causes curcumin metabolic degradation).

362

14

363

curcumin in plasma as well as its distribution in brain tissue compared to respective native

364

counterpart. The improved efficacy in drug loaded NPs might have resulted due to better

365

solubility of curcumin in nanoformulation and BBB crossing ability of lipid based NPs. In

366

accordance with our observation, in a recent study Ramalingam et al. have shown the

367

improved oral bioavailability and brain biodistribution of curcumin loaded with N-trimethyl

368

chitosan coated solid lipid NPs compared to native drug.

369

loaded NPs elucidated superlative bioavailability and brain biodistribution of curcumin than

370

other treatment that could be due to combined approach of using piperine and lipid based

371

NPs. As our drug loaded NPs exhibited prolonged plasma retention and augmented

372

bioavailability and enhanced brain tissue distribution of curcumin, we anticipated a

373

substantial delivery of our dual drug loaded NPs to the substatia nigra region of brain. To

374

endorse the above view, uptake of NPs in substantia nigra of brain was evaluated by confocal

375

microscopy (Figure 7C). The confocal microscopy images of the mid brain section clearly

10

Low bioavailability of native curcumin in plasma (0.06 ± µg/ml) of rat (500

59

Importantly, co-administration of curcumin with

Further, encapsulation of drug in NPs significantly enhanced the bioavailability of

60

Note to mention, that dual drug



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

376

indicates the presence of CNPs or CPNPs in the substantia nigra region as evident from green

377

fluorescence of curcumin, thus narrating the putative role of our lipid based NPs in crossing

378

BBB to deliver the therapeutic payload efficiently. NPs have shown remarkable potentiality

379

as a brain targeting system compared to native drugs. In relation to this, in a recent study

380

Kakkar et al. have shown the brain targeting and BBB crossing ability of lipid based NPs to

381

deliver curcumin to the brain, thus substantiating with our observation of enhanced brain

382

targeting with lipid based NPs. 61

383

Restoration of Functional Deficits in a Rotenone Induced Mouse Model of PD.

384

Resting tremor, bradykinesia, muscular rigidity and postural instability are the cardinal

385

manifestations of PD. Therefore, therapeutic molecule that imparts a symptomatic relief in

386

PD may consider beneficial. In the present study, curcumin and piperine has shown

387

substantial therapeutic benefits in preclinical testing by modulating various molecular and

388

biochemical aspects of pathogenesis of PD therefore, we next focused to explore the utility of

389

curcumin and/or piperine loaded NPs in providing functional relief in a rotenone induced

390

mouse model of PD. 62 This model exhibits key symptomatic features of PD (impaired motor

391

balance and coordination) as evident from rotarod motor performance study in which mice

392

treated with rotenone spent less time in the rod compared to untreated control (Figure 8A).

393

Importantly, rotenone induced mice co-administered with curcumin and piperine in

394

combination (native or NPs) showed significant extension of time spent on the rod, with dual

395

drug loaded NPs exhibiting significant effect than native counterpart. These results suggest

396

the potent role of drug combination, specifically dual drug loaded NPs in ameliorating motor

397

dysfunction in rotenone induced PD model. The motor coordination restoration observed with

398

dual drug combination in an enhanced way, can be explained by considering the putative role

399

of both curcumin and piperine in modulating various molecular and biochemical aspects of

400

pathogenesis of PD in vitro observed in the present investigation. Further, the superlative 16 ACS Paragon Plus Environment

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401

efficacy of dual drug loaded NPs over native drug combination may have resulted following

402

enhanced plasma bioavailability, superior brain targeting by crossing BBB, increased

403

delivery to substantia nigra of brain and augmented cellular internalization. In a recent study,

404

da Rocha Lindner et al. have demonstrated the superlative protective efficacy of resveratrol

405

(RVT)-loaded polysorbate 80 (PS80)-coated poly(lactide) nanoparticles compared to native

406

resveratrol in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induces PD model, thus

407

suggesting the advantage of NPs over native drug in neuroprotection.

408

dopaminergic neurons is another cardinal sign of pathogenesis of PD. We therefore further

409

tried to evaluate the effect of curcumin and piperine in protecting the degeneration of

410

dopaminergic neurons in rotenone induced PD mice. As the gold standard marker in the

411

identification of dopaminergic neurons is tyrosine hydroxylase (TH), the rate limiting enzyme

412

in dopamine synthesis, we studied the presence of TH positive neurons to mark the presence

413

of dopaminergic neuronal cells. 16 Immunohistochemistry study of the substantia nigra region

414

clearly indicates low density of TH positive neurons in rotenone treated mice compared to

415

untreated control (Figure 8B, C). Noteworthy, co-administration of curcumin and piperine (in

416

native or NPs) resulted in higher density of TH positive neurons, with dual drug loaded NPs

417

exhibiting more intense effect in protecting against rotenone induced degeneration of

418

dopaminergic neurons.

419

CONCLUSIONS

420

In the present study, we have formulated a dual drug (curcumin and piperine) loaded lipid

421

based nanoformulation and studied its anti parkinsonism effect through various in vitro and in

422

vivo studies. Our present data demonstrate the neuroprotective effect of our dual drug loaded

423

NPs by inhibiting the aggregation of αS protein, reducing the cytotoxicity and oxidative

424

stress induced by rotenone, activation of autophagy mediated protein degradation and

425

induction of anti-apoptotic events. However, a detail investigation on primary target of these 17 ACS Paragon Plus Environment

63

Degeneration of


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Page 18 of 41

426

drugs to ameliorate PD pathogenesis is warranted in near future. Further, the drug loaded NPs

427

also significantly reversed the neurobehavioral abnormalities and neuronal degeneration in

428

the substantia nigra in a PD mice model. The better therapeutic effect of curcumin and

429

piperine in dual drug loaded NPs may be due to improved bioavailability of curcumin, ability

430

to cross BBB and synergistic effect exhibited by both the drugs. Thus, the present study

431

suggest towards the potentiality of our dual drug loaded NPs in ameliorating Parkinson’s

432

pathogenesis in clinical settings.

433

434

METHODS

435

Materials. CUR-500, containing curcumin (> 95 %), was purchased from UNICO

436

Pharmaceuticals (Ludhiana, India). GMO was purchased from Eastman (Tennessee, USA).

437

Sodium chloride and piperine were obtained from MP Biomedicals (Illkirch, France).

438

Acetonitrile was purchased from Spectrochem, India. Dimethyl sulphoxide (DMSO),

439

Methanol, Ethanol and Acetic acid were procured from E-merk (Mumbai, India).

440

Haematoxylin was obtained from Thermo Fisher Scientific, Mumbai, India. Lipofectamine®

441

2000 transfection reagent, Nerve Growth Factor (NGF Mouse protein, Native, 7S subunit)

442

was purchased from Invitrogen Corp. (CA, USA). Skimmed milk powder was procured from

443

Himedia Laboratories Pvt. Ltd., Mumbai, India. mRFP-LC3 (plasmid # 21075) was obtained

444

from Addgene Inc. (MA, USA). Sodium deoxycholate, Ethylene glycol-bis (2 amino ethyl

445

ether)-N, N, N, N-tetraacetic acid (EGTA), Ethylene diamine tetra acetic acid (EDTA), 5,5' -

446

dithiobis-2-nitrobenzoic

447

polyethylene glycol 1000 succinate (vitamin E-TPGS), 3-(4,5-dimethylthiazol-2-yl)-2,5-

448

diphenyltetrazolium bromide (MTT), Pluronic F-68, Polyethylene glycol (PEG)-10,000,

449

Phenylmethylsulfonyl

acid

fluoride

(DTNB),

Poly-L-lysine,

(PMSF),

Sodium

Vitamin

E

orthovanadate


D-α-Tocopherol

(NaVO4),

β-

Page 19 of 41

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450

glycerophosphate, Protease inhibitor cocktail, Thiobarbituric acid (TBA), Butylated

451

hydroxytoluene (BHT), L-Glutathione reduced, Thioflavin T, Sodium dodecyl sulphate

452

(SDS), 4',6-diamidino-2-phenylindole (DAPI) were obtained from Sigma Aldrich (St. Louis,

453

MO, USA). All other chemicals used were purchased from Sigma Aldrich (St. Louis, MO,

454

USA) without further purification.

455

Preparation of Drug Loaded Lipid Based NPs. Dual drug loaded NPs was 25

456

formulated by following our previous published protocol with little modifications.

457

50 mg of curcumin and 50 mg of piperine was dispersed in fluid phase of GMO (500 µl at 40

458

°C) and vortexed. The above mixture was subjected to emulsification with 10 ml of Pluronic

459

F- 68 solution (5 % w/v) by sonication using a microtip probe sonicator (VC 505, Vibracell

460

Sonics, MA, USA) set at an amplitude of 30 % for 2 mins in an ice bath. The resultant

461

solution was further emulsified with 10 ml of vitamin E-TPGS (5 % w/v) as mentioned

462

above. The above emulsion obtained was centrifuged at 1,000 rpm for 1 min to remove the

463

unentrapped curcumin/piperine, followed by addition of PEG-10,000 (20 mg/ml) as a

464

lyoprotectant with constant vortexing for 5 mins.

465

six days (-50 °C and < 0.05 mBar, Labconco Free Zone 12, Labconco Corporation, Kansas,

466

USA) to obtain the lyophilized powder for further use. Single drug loaded NPs

467

(curcumin/piperine) was also formulated following the above protocol.

64

Briefly,

Finally the emulsion was lyophilized for

468

Physico-Chemical Characterization of Dual Drug Loaded NPs. The particle size

469

and zeta potential of the NPs was measured by Zetasizer (Nano ZS, Malvern Instruments,

470

Malvern, UK) using our previously published protocol.

471

NPs was further assessed by TEM and AFM respectively following our previously published

472

protocol.

473

estimated by reverse phase isocratic mode of RP-HPLC (Waters

474

USA). Briefly, ~ 1 mg/ml of curcumin NPs (CNPs) / piperine NPs (PNPs) or (curcumin and

34

65

Size and surface topology of the

Entrapment efficiency of curcumin and piperine in drug loaded NPs was


TM

600, Waters Co., MA,


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

475

piperine) loaded NPs (CPNPs) was dissolved in ACN, sonicated for 2 min at an amplitude of

476

30 % in an ice bath and the supernatant was collected following centrifugation. Curcumin

477

concentration was measured using the mobile phase ACN: sodium acetate buffer (20 mM, pH

478

3.0): methanol in a ratio of 6:1:3 at a wavelength of 420 nm and piperine was measured using

479

the mobile phase ACN: potassium dihydrogen phosphate (25 mM, pH 4.5) in a ratio of

480

6.5:3.5 at a wavelength of 345 nm.

481

the peak area correlated to a standard curve prepared in an identical condition. In vitro release

482

kinetics of (curcumin and piperine) loaded NPs were performed in PBS as per previously

483

published protocol. 25 All analysis was performed in triplicates.

484

66, 67

The amount of drug in the NPs was obtained from

α-Synuclein Aggregation Assay. α-synuclein aggregation analysis was performed 68

485

following the protocol of Danzer et al.

In brief, stock solution of purified αS (Sigma-

486

Aldrich, St. Louis, MO, USA) was prepared in distilled water and subsequent dilutions were

487

made in 50 mM sodium phosphate buffer (pH 7). Oligomeric forms of αS were generated by

488

dissolving 10 µM of the protein in reaction buffer (50 mM sodium phosphate buffer,

489

containing 20 % ethanol and 10 µM of FeCl3) at room temperature under continuous shaking

490

with overnight incubation while predominantly fibrillar forms of αS were generated by

491

following the same condition with the incubation time increased up to 6 days. Further, to

492

evaluate the inhibitory effect of curumin and piperine on αS aggregation, 10 µM of the

493

protein dissolved in reaction buffer was co-incubated with 7.5 µg/ml of curcumin or piperine

494

(in single or in combination) both in native as well as in nanoformulations and kept in

495

shaking condition overnight (oligomer study) or 6 days (fibrillar study). Following incubation

496

period, the morphology of αS with different treatments was visualized by AFM analysis. For

497

this, ~ 10 µl aliquot of each sample was applied onto freshly cleaved mica surface and left to

498

dry at room temperature for 10 min. Samples were then rinsed with Milli Q water and dried

499

under nitrogen flow. Samples were imaged in contact mode set at a frequency of 13 kHz and


Page 20 of 41

Page 21 of 41

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500

scanned at a speed of 1 Hz. Topographic images were analyzed and height distribution plot

501

was generated using JPK data processing software. Experiment was performed in triplicates

502

and representative images have been provided.

503

Cell Culture. Rat PC12 cell line was obtained from National Centre for Cell Sciences

504

Cell Repository, Pune, India. Cells were cultured in RPMI media supplemented with 10 %

505

heat inactivated horse serum, 5 % fetal bovine serum (FBS), 1 % L-glutamine and 1 %

506

penicillin-streptomycin (Himedia Laboratories Pvt. Ltd., Mumbai, India) and maintained at

507

37 °C in a 5 % CO2 atmosphere incubator (Hera Cell, Thermo scientific, Waltham, USA). All

508

chemicals for cell culture were purchased from PAN Biotech (GmbH, Germany) unless

509

mentioned. All the cellular experiments were performed in differentiated PC12 cells,

510

obtained by culturing the cells in RPMI media containing NGF (50 ng/ml) and 1 % FBS for 3

511

days and used subsequently for further experiments.

512

Cellular Uptake Study. Quantitative and qualitative cellular uptake of native

513

curcumin and curcumin loaded NPs in PC12 cells were evaluated by fluorescence

514

spectrophotometer and confocal microscopy respectively following our previously published

515

protocol. 25 For quantitative cellular uptake study, 1 × 105 PC12 cells seeded in poly-L-lysine

516

coated twelve well plates (Corning Inc., NY, USA) were treated with 2 µg/ml of native

517

curcumin or equivalent concentration of curcumin loaded NPs for different time points and

518

intracellular concentration of curcumin was quantified using fluorescence spectrophotometer

519

(Ex: 420 nm, Em: 525 nm). Experiments were performed in triplicates. For qualitative

520

cellular uptake study, cells were exposed to above drug treatments for 2 hrs and

521

counterstained with propidium iodide for nuclear staining. Images were visualized in

522

confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems GmbH, Germany)

523

equipped with an argon laser with an FITC filter (Ex: 488 nm, Em: 525 nm) and PI filter (Ex:



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Page 22 of 41

524

535 nm, Em: 617 nm). The images were processed using Leica Application Suite software.

525

Experiment was performed in triplicates and representative image has been provided.

526

In Vitro Cell Viability Assay. Cell viability was analyzed using MTT based 69

527

colorimetric assay as described in our previously published protocol.

528

seeded at a density of 5 × 103 cells per well in a poly-L-lysine coated 96 well plate (Corning

529

Inc., NY, USA) was treated with different concentration of rotenone (2 µg/ml) or co-treated

530

with rotenone (2 µg/ml) along with different concentration of curcumin or piperine (single or

531

in combination) in native as well as in NPs for 48 hrs. Cells treated with only media was used

532

as control for the experiment. At the end of the incubation period cell viability was assessed

533

by MTT assay. Data represented as mean ± S.E.M (n = 4).

534

Briefly, PC12 cells

Western Blot Analysis. Western blot analysis of different proteins in PC12 cells 34

535

following various treatments was carried out following previously published protocol.

536

Briefly, PC12 cells (1 × 106 cells) seeded in poly-L-lysine coated T-25 flask was exposed to

537

rotenone (2 µg/ml) or co-administered with rotenone (2 µg/ml) along with curcumin or

538

piperine (2 µg/ml, in single or in combination) both in native as well as in nanoformulations.

539

After 48 hrs of treatment, the cells were harvested, washed with PBS and whole cell lysate

540

was prepared with RIPA buffer. Western blot analysis of various proteins were performed

541

using specific primary antibody recognizing LC3 (Novus Biologicals, Colorado, USA), α-

542

synuclein, Lamp2, β-actin (Santa Cruz Biotechnology, Inc., CA, USA), BAX, Bcl-2, PARP,

543

Caspase 3 (Cell Signaling Technology, Inc., MA, USA) and their respective secondary

544

antibody. The band intensity was measured by ImageJ software.

545

Fluorescence Imaging of Red Fluorescent Protein (RFP) LC3 for Autophagy

546

Study. The RFP-LC3 transfected PC12 cells were generated by transfecting pRFP-LC3

547

plasmid into subconfluent PC12 cells using Lipofectamine® 2000 transfection kit.

548

PC12 cells at density of 1 × 106 were seeded in 60 x 15 mm petridish (Corning, NY, USA)


70

Briefly,

Page 23 of 41

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549

for overnight attachment. Next day, 8 µg of plasmid DNA was mixed with 20 µl of

550

Lipofectamine® 2000 reagent in serum free RPMI media, incubated for 20 mins at room

551

temperature and then added to the cells. After 8 hrs of incubation, the transfection media was

552

replaced with fresh culture medium. Next day, RFP-LC3 transfected PC12 cells were seeded

553

at a density of 1 × 105 cells in poly-L-lysine coated coverslips. The cells were then

554

differentiated with 50 ng/ml NGF media for 3 days. The cells were then treated for 48 hrs

555

with rotenone (2 µg/ml) or co-administered with rotenone (2 µg/ml) along with curcumin or

556

piperine (2 µg/ml, in single or in combination) both in native as well as in nanoformulations.

557

After 48 hrs, the cells were washed with PBS and fixed with 4 % paraformaldehyde. Cells

558

were again washed with PBS incubated with DAPI to stain the nucleus and finally mounted

559

with aqueous mounting media (Vector Laboratories, California, USA) and observed under

560

confocal microscopy (Leica TCS SP5, Leica Microsystems, Germany) for the formation of

561

RFP-LC3 puncta, a primary marker of autophagosomes formation. Further, the effect of

562

impaired autophagosome pathway on cell cytotoxicity, cellular viability was assessed in

563

PC12 cells treated with 3MA (autophagosome inhibitor). In brief, 5 × 103 PC12 cells seeded

564

in 96 well plates were treated with 3MA (10 mM), rotenone (2 µg/ml) and different

565

concentration of curcumin and piperine in combination (in native or in NPs) for 48 hrs and

566

cell viability was assessed by MTT assay as mentioned before.

567

Apoptosis Study. Apoptotic cell death in PC12 cells following different treatments 34

Briefly, 3 x 105

568

was studied by flow cytometry following previously published protocol.

569

PC12 cells seeded in six well plates were treated with rotenone (2 µg/ml) or co-administered

570

with rotenone (2 µg/ml) along with curcumin and piperine (2 µg/ml in combination) in native

571

as well as in nanoformulations for 48 hrs. Cells treated with only media served as control for

572

the experiment. After the incubation period, cells were washed thrice with PBS and processed

573

for apoptosis analysis using Annexin V-PE and 7-aminoactinomycin D (7-AAD) (BD



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

574

FACSCalibur Flow Cytometer, BD Biosciences, CA, USA) in FL2-H and FL3-H channel

575

respectively using FlowJo software.

576

In vivo Study. Male Balb/c mice and male C57BL/6 mice were used for different in

577

vivo experiments with the approval of the Institutional Animal Ethics committee of the

578

Institute of Life Sciences, Bhubaneswar. The animals were housed at a constant temperature

579

and relative humidity with alternating 12-hr cycles of light and dark. Mouse was housed in

580

standard laboratory cages and had free access to food and water throughout the study period.

581

For the pharmacokinetics study animals were fasted overnight before dosing.

582

In vivo Bioavailability Study. Male Balb/c mice (4–6 weeks old), weighing 22 ± 10

583

g, were divided into five groups (n = 3). Group 1: control, administered with 0.5 %

584

carboxymethyl cellulose sodium salt (CMC). Group 2: administered with native curcumin

585

dispersed in 0.5 % CMC at a dose of 100 mg/kg body weight, Group 3: administered with

586

CNPs dispersed in distilled water at an equivalent dose of native curcumin (100 mg/kg body

587

weight). Group 4: administered with native curcumin and piperine (1:1 ratio) dispersed in 0.5

588

% CMC at a dose of 100 mg/kg body weight. Group 5: administered with CPNPs dispersed

589

in distilled water at an equivalent dose of (curcumin and piperine) native of 100 mg/kg body

590

weight. After oral administration by gavage with a catheter at different time points (0.5, 2, 6,

591

48 hrs) blood samples were collected from retro-orbital plexus into pre-coated heparin tubes.

592

Curcumin concentration in plasma was estimated by HPLC as mentioned before using the

593

protocol of Shaikh et al.

594

biodistribution analysis was carried out at the above time points for all the five groups.

595

Briefly, the brain was dissected out at different time points, homogenized with PBS (BD-144

596

Tissue Homogeniser, BD Bioscience, Haryana, India) and lyophilized. The lyophilized

597

samples were processed for estimation of curcumin present in brain tissue by HPLC using

598

previously published protocol. 67

67

To study the amount of curcumin present in the brain tissue,


Page 24 of 41

Page 25 of 41

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599

Immunofluorescence. Male Balb/c mice (4–6 weeks old), weighing 22 ± 10 g were

600

divided into five group (n = 3) as mentioned above. The mice were administered with the

601

above treatments for 2 hrs. Following treatment, transcardial perfusion was performed with 4

602

% paraformaldehyde (pH 7.4) under deep anesthesia with xylazine and ketamine. After

603

perfusion, the brain was quickly removed and post fixed in 4 % paraformaldehyde solution at

604

4 °C overnight. Post fixed mid brain region was trimmed out and were embedded in paraffin,

605

followed by preparation of multiple coronal sections (5 µm) using a microtome. Slides

606

containing paraffin embedded brain sections were deparaffinized with xylene and rehydrated

607

by ethanol with concentration gradient of 100-70 % followed by washing with water. The

608

slides were then boiled in antigen retrieval solution for 20 min and were allowed to cool

609

down at room temperature. The sections were then washed with PBS containing 0.1 % Tween

610

20 (PBST), and then blocked with 2.5 % horse serum for 1 hr at 37 °C. After blocking,

611

sections were incubated with a rabbit polyclonal anti-TH antibody (1: 200 dilutions)

612

overnight at 4 °C. After a 10 min rinse in PBST, the sections were incubated with anti-rabbit

613

IgG secondary antibody Alexa Fluor® 594 conjugate (Invitrogen Corp., CA, USA) for 45

614

min. Finally the sections were washed with PBST and mounted with aqueous mounting

615

media and fluorescence (green for curcumin and red for TH positive cells) was observed

616

under confocal microscope (Leica TCS SP5, Leica Microsystems, GmbH, Germany).

617

Animal Model. Male, 8-10 weeks old C57BL/6 mice (25-30g) were randomly

618

assigned to 5 groups (n = 4). Group 1: control, administered orally with 0.5 % CMC as a

619

vehicle once daily for 28 days; Group 2: administered orally with rotenone suspended in 0.5

620

% CMC, once daily at a dose of 30 mg/kg body weight for 28 days; Group 3: administered

621

orally with native curcumin and native piperine (1:1 ratio) at a dose of 200 mg/kg body

622

weight, every alternate day, 30 min before administration of rotenone. Group 4: administered

623

orally with CPNPs at an equivalent dose of 200 mg/ kg body weight, every alternate day, 30



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

624

min before administration of rotenone. Group 5: administered orally with void NPs at an

625

equivalent dose of 200 mg/kg body weight every alternate day 30 min before administration

626

of rotenone. At the end of the experiment, on 29th day, the mice were preceded to rotarod task

627

for monitoring the behavioral pattern of mice and further studied the presence of

628

dopaminergic neurons (TH positive) in brain tissue through immunohistochemistry.

629

Motor Performance Study. The behavior of each mouse was assessed by the rotarod 62

630

test, as described by Inden et al.

by using rotarod treadmill (Orchid Scientific and

631

Innovative India Pvt Ltd, Nashik, India). In the present study, mice (from all groups of

632

treatment along with control) were placed on the rod rotating at 20 rpm, and the falling

633

latencies were recorded for up to 250 sec. All mice were tested on 29th day after the initial

634

rotenone administration.

635

Immunohistochemistry. In brief, transcardial perfusion was performed with 4 %

636

paraformaldehyde (pH 7.4) under deep anesthesia with xylazine and ketamine. The brain was

637

collected and multiple coronal sections (5 µm) were obtained using a microtome. Slides

638

containing paraffin embedded brain sections were deparaffinized with xylene and rehydrated

639

by ethanol with concentration gradient of 100-70 % followed by washing with water. The

640

slides were then boiled in antigen retrieval solution for 20 mins and were allowed to cool

641

down at room temperature followed by washing with PBST. Sections were then incubated

642

with 3 % hydrogen peroxide for 15 min at room temperature to remove the endogenous

643

peroxidase activity and then blocked with 2.5 % horse serum for 1 hr at 37 °C. After blocking

644

for 1 hr, sections were incubated with a rabbit polyclonal anti-TH antibody (1:200 dilutions,

645

Millipore, Darmstadt, Germany) overnight at 4 °C. After a 10 min rinse in PBST, the sections

646

were incubated with biotinylated secondary universal horse anti-rabbit/mouse IgG

647

(Vectastain Kit; Vector Laboratories, California, USA) for 45 mins, followed by incubation

648

with avidin-biotin peroxidase complex for 30 mins at room temperature. Sections were


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649

washed with PBST and exposed to diaminobenzidine (DAB) and the photographs of the brain

650

sections were taken using phase contrast microscope (Leica EZ, UK) with 40 X objective and

651

then TH positive neurons were counted. A certified human pathologist evaluated all the

652

stained slides.

653

Statistics: Results are expressed as mean ± S.E.M. or mean ± SD. Statistical analysis

654

of the data was performed by applying student’s t-test, one way and two way ANOVA using

655

GraphPad Prism Software and values of p < 0.05 were indicative of significant differences.

656

Supporting Information: Histogram analysis of oligomers and fibrils obtained from

657

AFM study; Dose dependent cytotoxicity of rotenone in PC12 cells; Cellular GSH and lipid

658

peroxidation assay.

659

AUTHOR INFORMATION

660

Corresponding Author

661

*Sanjeeb K Sahoo, Ph.D

662

Laboratory for Nanomedicine,

663

Institute of Life Sciences,

664

Nalco Square, Chandrasekharpur,

665

Bhubaneswar, Orissa, INDIA

666

Phone- 91-674-2302094

667

Fax- 91-674-2300728

668

E-mail- [email protected]

669

Author Contributions

670

Conceived and designed the project P.K and S.K.S. Performed the experiments P.K and M.D.

671

Analyzed the data P.K, M.D, K.T and S.K.S. Contributed to the preparation of manuscript

672

P.K, M.D and S.K.S

673 27 ACS Paragon Plus Environment


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674

Notes

675

The authors declare no competing financial interest.

676

ACKNOWLEDGEMENTS

677

P.K. acknowledges University Grants Commission (UGC), New Delhi, India for providing

678

the award of Junior Research Fellowship (JRF). S.K.S and P.K. acknowledge Dr. Rupesh

679

Dash, Scientist at Institute of Life Sciences and lab members for experimental help in

680

autophagy study. S.K.S and P.K are also thankful to Dr. Shantibhusan Senapati, Scientist at

681

Institute of Life Sciences and lab members for their help in in vivo studies. Technical help of

682

Mr. Priyadarshi Ray in AFM and Mr. Madan Mallick for histological sectioning is

683

acknowledged.

684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 28 ACS Paragon Plus Environment

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699 700 701 702 703 704

705 706 707 708 709 710 711

Figure 1. Physicochemical characterization of dual drug loaded NPs. (A) Size of (curcumin and piperine)

712

loaded NPs (CPNPs) as measured by dynamic laser light scattering. (B) Transmission electron micrograph of

713

CPNPs, depicting that the formulated particles are of nanometer size range. Inset shows a higher magnification

714

of the particle. (C) AFM image of CPNPs, depicting their smooth and spherical topology. All experiments were

715

performed in triplicates and representative image has been provided. (D) In vitro release kinetics of curcumin

716

and piperine from dual drug loaded NPs as percent of drug release. Data represented as mean ± S.E.M (n = 3).

717 718 719 720 721 722 723 724 725 726



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

727

A

Oligomer

VNPs

CN

PN

PNPs

CPN

Fibrils

VNPs

CN

PN

PNPs

CPN

Page 30 of 41

CNPs

728 729 730 731 CPNPs

732 733 734 735

B CNPs

736 737 738 739 CPNPs

740 741 742 743 744 745

Figure 2. Atomic force microscopy analysis to study the aggregation of α-synuclein (αS) protein.

746

Representative AFM images of αS, showing inhibitory effect of different treatments on the formation of (A)

747

oligomers and (B) fibrils. The reaction mixture containing 10 µM αS, 50 mM sodium phosphate buffer pH 7, 20

748

% ethanol and 10 µM FeCl3 were treated with 7.5 µg/ml of native curcumin (CN), native piperine (PN) and

749

combination of native curcumin and native piperine (CPN) or equivalent concentration of curcumin NPs

750

(CNPs), piperine NPs (PNPs) and (curcumin and piperine) NPs (CPNPs), or equivalent amount of void NPs

751

(VNPs) incubated at room temperature under continuous shaking overnight (for oligomer formation) and 6 days

752

(for fibril formation). Experiment has been performed in triplicates and representative image has been provided.

753 754 755 756 30 ACS Paragon Plus Environment

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757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772

Figure 3. In vitro cellular uptake study. (A) Quantitative cellular uptake study of CN and CNPs in PC12 cells

773

for different time period by fluorescence spectrophotometer (Ex: 420 nm, Em: 525 nm). Data are presented as

774

mean ± SEM (n = 3). ***p < 0.001 CNPs in comparison to CN. (B) Qualitative cellular uptake analysis of CN

775

and CNPs in PC12 cells at 2 hr time point by confocal microscope equipped with FITC filter (Ex: 488 nm, Em:

776

525 nm) and with PI filter (Ex: 535 nm, Em: 617 nm). Experiment has been performed in triplicates and

777

representative image has been provided.

778 779 780 781 782 783 784 785 786 787



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

788 789 790 791 792 793 794 795 796 797 798 799 800 801 802

Figure 4. Cytotoxicity study to assess the protective effect of different treatments on rotenone induced

803

cytotoxicity in PC12 cells for 48 hrs by MTT assay. Cells were co-incubated with various concentrations of the

804

drug with 2 µg/ml rotenone (R) and cellular viability was determined. Results are presented as mean ± S.E.M (n

805

= 4). p< 0.05 is considered significant. ###p corresponds to rotenone vs control and *p **p or ***p corresponds

806

to different treatments vs rotenone. 1. CN, 2. CNPs, 3.PN, 4.PNPs, 5.CPN, 6.CPNPs, 7.VNPs.

807 808 809 810 811 812 813 814 815 816


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817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839

Figure 5. (A) Western blot analysis was performed to investigate the expression of LC3 II, Lamp 2, α-synuclein

840

proteins following different treatments in PC12 cells. Cells were co-treated with R (2 µg/ml) along with 2 µg/ml

841

of CN, PN, CPN and CNPs, PNPs and CPNPs for 48 hrs. 1. Control, 2. R, 3.(R + CN), 4.(R + CNPs), 5. (R +

842

PN), 6.(R + PNPs), 7.(R + CPN), 8.(R + CPNPs). (B) Study of autophagy in PC12 cells through confocal

843

microscope. In brief, cells were transfected with RFP-LC3 plasmid and exposed to above treatments for 48 hrs.

844

Cells exhibiting RFP-LC3 puncta (indicator of autophagosome formation) were observed under confocal

845

microscope. Experiment has been performed in triplicates and representative image has been provided. (C)

846

Investigating the effect of autophagy inhibition towards protective effect of different treatments on rotenone

847

induced cytotoxicity in PC12 cells. In brief, cells were exposed to R (2 µg/ml) or 10 mM of 3MA and R (2

848

µg/ml) or co-administered with 10 mM 3MA, R (2 µg/ml) and different concentration of (curcumin and

849

piperine) in native or in NPs for 48 hrs and cell viability was assessed by MTT assay. Data presented as mean ±

850

S.E.M (n = 4). p< 0.05 is considered significant. **p or ***p corresponds to treatment with inhibitor and drug

851

treatment vs with only drug treatment.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878

Figure 6. Study of apoptosis. (A) Representative immunoblots of Bcl2 and BAX expression and (B)

879

quantification of Bcl2/ BAX. (C) Representative immunoblot of apoptotic protein Caspase 3. (D) Representative

880

immunoblot of apoptotic protein PARP. 1. Control, 2. R, 3.(R + CPN), 4.(R + CPNPs). (E) Analysis of

881

apoptosis by flow cytometry to study the protective effect of CPN or CPNPs against rotenone induced apoptosis

882

in PC12 cells for 48 hrs and apoptosis percentage was analyzed by annexin V-PE and 7-AAD staining.

883

Experiment has been performed in triplicates and representative image has been provided. (F) Bar diagram

884

depicting total percentage of apoptotic cells. p< 0.05 is considered significant. ###p corresponds to rotenone vs

885

control and ***p corresponds to different treatment vs rotenone.

886 887 888


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889 890

B

A

891 892 893 894 895 C

896

CN

CPN

CNPs

CPNPs

897 898 899 900 901 902 903 904 905 906 907 908 909

Figure 7. Pharmacokinetics of curcumin in mice (A) plasma (B) brain tissue after single oral administration of

910

CN, CNPs, CPN and CPNPs at a dose of 100 mg/kg body weight. Values are presented as mean ± S.E.M (n =

911

3). *p < 0.05, **p < 0.01, or ***p < 0.001 for CN vs CPNPs; #p or ##p or ###p corresponds to CPN vs CPNPs.

912

(C) Histological sections of the substantia nigra region of brain tissues (marked by immunoexpression for TH,

913

red) showing efficient accumulation of curcumin (green) after 2 hrs oral treatment and representative image has

914

been provided (n = 3). Insets are the higher magnification of area showing the presence of CNPs and CPNPs

915

(white arrow).

916 917 918 919 920 921 922 923 924 925 35 ACS Paragon Plus Environment


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926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957

Figure 8. Effect of different drug treatments on rotenone induced (A) behavioral deficit in mice as assessed by

958

rotarod and (B) representative image of TH positive neurons in the substantia nigra region of the brain following

959

different treatments has been provided (black arrow). (C) TH positive neurons count per field to study the

960

dopaminergic neuronal cell death in the substantia nigra. Data are presented as mean ± SEM (n = 4). p< 0.05 is

961

considered significant. ###p corresponds to rotenone vs control and *p or ***p corresponds to different

962

treatment vs rotenone and •••p corresponds to CPN vs CPNPs. Experiment has been performed with n = 4

963

animals.

964 965 966

REFERENCES


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Delivery of dual drug loaded lipid based nanoparticles across blood brain barrier impart enhanced neuroprotection in a rotenone induced mouse model of Parkinson’s disease Paromita Kundu¶, Manasi Das¶, Kalpalata Tripathyǂ, Sanjeeb K Sahoo*, ¶

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Delivery of Dual Drug Loaded Lipid Based Nanoparticles across the

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