Preparation of liposomes encapsulated Epirubicin-gold nanoparticles

Biomedical Physics & Engineering Express

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Preparation of liposomes encapsulated Epirubicin-gold nanoparticles for Tumor specific delivery and release To cite this article before publication: Selvaraj Kunjiappan et al 2018 Biomed. Phys. Eng. Express in press https://doi.org/10.1088/20571976/aac9ec

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Preparation of liposomes encapsulated Epirubicin-gold nanoparticles for Tumor specific

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delivery and release

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Selvaraj Kunjiappan1*, Theivendran Panneerselvam2, Balasubramanian Somasundaram1,

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Sankarganesh Arunachalam1 Murugesan Sankaranarayanan3 and Pavadai Parasuraman4

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Krishnankoil-626126, India

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Sir CV Raman-KS Krishnan International Research Center, Kalasalingam University,

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Department of Pharmaceutical Chemistry, Karavali College of Pharmacy, Vamanjoor, Near

Reymond, Mangaluru-575028, India

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Department of Pharmacy, Birla Institute of Technology and Science, Pilani-333031, India

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Department of Pharmacy, M S Ramaiah University of Applied Sciences, Gnanagangothri

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Campus, Bengaluru-560054, India.

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*

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Dr. Selvaraj Kunjiappan,

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Research Faculty,

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Sir CV Raman-KS Krishnan International Research Center,

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Kalasalingam University,

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Krishnankoil-626126, India

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Email: [email protected]

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Ph: +919994972108

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AUTHOR SUBMITTED MANUSCRIPT - BPEX-101073.R1

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Abstract

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Liposome/metal nanoparticle conjugates are found to be a promising carrier tool for effectively

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delivering wide variety of drugs in a targeted site and without arguing toxic side effects of

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existing anticancer drugs. In this scenario, the present work was performed to choose Epirubicin-

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HCl (EPI) as model anticancer drug to formulate citrate stabilized EPI-gold nanoparticles (EPI-

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GNPs). Further, the biocompatible EPI-GNPs incorporated with lipid bilayer of liposomes

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formulated to exhibit effective drug delivery system. The biosynthesized EPI-GNPs and its

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incorporation in a lipid bilayer of liposomes was confirmed by UV-visible spectrophotometer,

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drug encapsulation, loading capacity, pH stability and drug releasing capacity were studied.

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Simultaneously, drug and carrier interaction, physical nature, stability, morphological and

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particle size characteristics were also studied by FTIR, XRD, SEM, TEM and Zeta size analyzer.

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The obtained characterization data confirmed the properties of EPI-GNPs encapsulated

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liposomes. The in vitro cytotoxic screening of EPI-GNPs encapsulated liposomes arrest the

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proliferation of MCF-7 breast cancer cell lines, at 0.1 µg/mL of liposomes displayed 52% of cell

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death at 24 h. The EPI-GNPs encapsulated liposomes induced time and dose dependent apoptosis

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compared with untreated control cancer cells.

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Keywords: Epirubicin, drug delivery, liposomes, gold nanoparticles, breast cancer, cytotoxicity

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1. Introduction

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Breast cancer is the most widely recognized malignancy in females and it is a leading cause for

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death rate in women (14% of all cancer deaths) worldwide [1]. However, cancer cells proliferate

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at much faster rate than the normal cells. Breast cancer tends to spread metastasis primarily to

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lymph nodes, liver, lungs, bones and brain [2]. Therefore, breast cancer has become major health

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concern and proper treatment of breast cancer has nowadays become a necessity. Recently, a

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number of recognized chemotherapeutic agents are in market for effective breast cancer therapy,

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but unfortunately, they have inability to selectively deliver the drugs to tumor targets, shorter life

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time and aggressively producing undesirable toxic side effects to normal tissues [3]. Therefore,

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there is a pressing need to develop alternative way of drug delivery system to deliver a sufficient

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dose of drug to the disease location with less invasive but more efficient cancer treatments by the

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currently available chemotherapeutic agents. The most promising drug delivery system would be

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to exploit nanotechnology based biodegradable carrier material to explore the treatment of cancer

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at its molecular level. Presently polymers, proteins, liposomes, solid lipid nanoparticles, and

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metal nanoparticle-conjugated biodegradable systems are helpful for therapeutic purposes [4].

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Among the above carrier systems, liposomes with metal nanoparticle conjugate have many

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advantages in the field of drug delivery systems because of their nanosize, high surface to

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volume ratio, less severe adverse reaction with enhanced cancer treatment [5]. Liposomes are the

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simplest artificial biological cell, and are well known to reduce toxicity related with many

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compounds including antitumor drugs, antiviral drugs and antibiotics [6]. Further, it is currently

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being introduced into clinical use and several more are under clinical trials or pre-clinical

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development [7]. Liposomes are submicronic spherical vesicular structure consisting of mono or

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bilayer of phospholipids/cholesterol having a hydrophobic tail and a hydrophilic head, which can

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be encapsulated with both hydrophilic and hydrophobic drugs depending on the preparation

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method [8].

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In the recent decade, gold nanoparticles (GNPs) have been remarkably being used in a wide

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range of biomedical applications in view of their stability, nanosize, bio compatibility and have

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displayed unique and tunable surface plasmon resonance (SPR) [9]. These points of interest

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empower the GNPs to target tumor all the more effectively without severe accumulation in

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reticuloendothelial system, making them exceptionally encouraging for cancer diagnosis and

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treatment [10, 11]. Further, GNPs have demonstrated high affinities with highly selective

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targeting properties to either tissues or cells and passively aggregate at tumor sites because of

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upgraded permeability and retention (EPR) effects [12]. It is also flexible to surface functionalize

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with various biomolecules such as protein, DNA, algae, enzymes, and plant-derived bioactive

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compounds [13, 14]. Therefore, GNPs are currently the subject of exceptional research for

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potential clinical applications.

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The hybrid GNPs/liposomal conjugate can be prepared by GNPs encapsulation or surface

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coating with lipids, which is a useful non-covalent approach to stabilize surface chemistry for

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prolonged plasma circulation improving pharmacokinetic profile and reducing clearance by

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circumventing reticuloendothelial system (RES) uptake [15]. Epirubicin-HCl (EPI) is an

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anthracycline antineoplastic agent that acts by DNA intercalation and inhibiting topoisomerase

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II, which further interfere with DNA, RNA and protein synthesis [16]. EPI is a 4’-epimer of

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Doxorubicin (DOX) and differs in the orientation of the C-4 hydroxyl group on the sugar that has

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several beneficial effects over DOX including improved efficacy, lower toxicity, rapid clearance

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from plasma resulting in less cumulative dose, and thus can successfully replace DOX in breast

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cancer treatment [17, 18]. But the use of free EPI is rather limited due to the severe side effects

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and multidrug resistance [19]. Hence, the investigation reported here is aimed to enhance the

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therapeutic effects of EPI and reduce its toxic side effects by synthesizing GNPs and

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encapsulating them in a lipid bilayer on nanoparticles. In the present study, a novel strategy was

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employed to exploit the use of sodium citrate dihyrate as a reducing and stabilizing agent for the

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biosynthesis of GNPs and subsequently attached with EPI. The stabilized EPI-GNPs were

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encapsulated in liposomes by conventional thin film hydration method. Then, the formulated

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GNPs/liposomal conjugate were characterized by analytical techniques to evaluate their size,

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size-distribution, morphology, stability, encapsulation efficiency, and drug releasing capacity.

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Further, in vitro anticancer activity was evaluated against breast cancer cell lines (MCF-7).

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2. Materials and Methods

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2.1. Materials

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The chemicals, Hydrogen tetracholoroaureate (III) hydrates (HAuCl4.3H2O (99.9%)), Sodium

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citrate dihydrate (99%), 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT),

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fetal bovine serum (FBS), Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Dulbecco’s

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Modified Eagles Medium (DMEM) and the solvents, analytical grade ethanol (95%), chloroform

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(98%) and toluene (99.7%) were procured from Himedia Laboratories Pvt., Ltd., Mumbai, India.

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Distearoyl phosphatidylcholine (DSPC) with >99% purity was obtained from Lipoid (Germany)

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and used without further purification. Cholesterol was purchased from Sigma Aldrich Company

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(St. Louis, USA). All other chemicals and solvents used in this study were of analytical grade

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and obtained from Merck, Mumbai, India. Epirubicin-HCl (4′-epidoxorubicin hydrochloride;

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(EPI)) was received as kind gift from Alkem Laboratories Ltd., Mumbai, India. Glassware were

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washed with dilute nitric acid and thoroughly rinsed with double distilled water and dried in hot

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air oven with before use. MCF-7 cell line was obtained from National Centre for Cell Science

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(NCCS, Pune, India).

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2.2. Synthesis of GNPs

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Citrate-stabilized GNPs were synthesized by a modified procedure similar to that of Frens, 1973

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[20]. In short, 10 mL of sodium citrate di hydrate (0.01 M) was added to 90 mL of aqueous

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solution of 1 × 10−4 M HAuCl4∙3H2O and the solution was kept under magnetic stirring at 60°C

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for 30 min. This procedure results in a ruby red solution containing monodisperse GNPs. The

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formation of GNPs was monitored with UV-visible spectrophotometer. GNPs were obtained by

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centrifugation at 12000 × g for 30 min and washed several times with de-ionized water by

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centrifugation and re-dispersion in water to remove excess gold.

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2.3. Preparation of EPI-GNPs

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2 mg of EPI was added to GNPs dispersion, obtained as described above, resulting in a final EPI

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concentration of 10−4 M in solution. The mixture of EPI and GNPs dispersion was incubated for

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24 h at room temperature and then centrifuged at 10000 × g for 15 min. The obtained pellet after

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centrifugation was separated from the supernatant and re-dispersed in de-ionized water prior to

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further characterization.

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2.4. Preparation of Liposome encapsulated EPI-GNPs

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Liposomes encapsulating EPI-GNPs were formulated by the method of Rengan et al. 2014 [21].

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Briefly, a mixture of lipids DSPC and cholesterol were dissolved in a mixture of chloroform:

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methanol (2:1), followed by removal of chloroform and methanol using a rotary evaporator

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(Buchi rotavap). The EPI-GNPs solution (1.0 mL) was then added to the dried cationic lipid film

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and the mixture was incubated at room temperature for 4 h with intermittent mixing, resulting in

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a final lipid concentration of 10 mM. To achieve a homogeneous mixture of liposomes, the

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suspension was sonicated for 5 min. Liposomes were annealed for 1 h at 60 °C in a water bath

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and cooled to room temperature.

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2.5. Determination of drug encapsulation efficiency and loading capacity

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The EPI encapsulation and loading capacity of the formulated liposomes was measured

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according to the method described by David et al. 2015 [22]. In short, EPI concentration in the

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supernatant after the centrifugation of the formulated liposomes solution was detected using the

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UV-visible spectrophotometer. The resultant supernatant was examined under UV-Visible

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spectrophotometer which yielded an absorbance at 490 nm for EPI. Calibrated standard plots and

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encapsulation efficiency and drug loading capacity were estimated using the absorbance values

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and converted to quantity of EPI using the equation as given below:

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Encapsulation efficiency (%)= (Total drug - Free drug)/Total drug × 100

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Drug Loading capacity (%)= (Total drug - Free drug)/Nanoparticle weight × 100

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2.6. In vitro drug release study

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In vitro drug release characteristics of liposomes was studied by dialysis method [23]. In short,

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free drug (EPI) was separated from liposomes (50 mg) by passing through a sephadex column of

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dialysis bag and the dialysis bag was then immersed in beaker containing 10 mL of PBS (0.01

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M, pH 7.4) or 10 mL of PBS (0.01 M, pH 5.0) and was subjected to separation after

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centrifugation. After completion of experiment (as the experiment was performed in duplicate),

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the liposomal suspension was equally divided into 10 mL and transferred to two tubes kept in a

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shaker at 150 rpm and temperature 37 °C. Samples were withdrawn at scheduled time points,

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the released medium was completely removed and replaced with an equal volume of fresh

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medium. DMSO was used to dissolve the collected resultant supernatant, further the

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centrifugation of this solution at 14000 × g at 25 °C ensured collection of the drugs which was

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used for UV-visible spectrophotometric analysis.

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%DRC = (A0 - A1) × 100/A0

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Where, A0 = absorbance of the control; A1 = absorbance of the sample.

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2.7. Stability studies

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In vitro stability of EPI-GNPs encapsulated liposomes were evaluated in various physiological

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medium (10% NaCl, 0.5% BSA) and in phosphate buffer solutions at room temperature with

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different pH values of 1.5, 3, 5, 6, 7.4 and 9 in order to investigate pH responsive behavior of

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liposomes. Briefly, 1 mL of EPI-GNPs encapsulated liposomes suspension was mixed with 1 mL

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each of the physiological medium and buffer solutions and its maximum absorbance was

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recorded after 24 h using the UV-Visible spectrophotometer.

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2.8. Physicochemical properties of EPI-GNPs encapsulated liposomes

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2.8.1. Fourier transform infrared spectroscopy (FTIR)

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Infrared spectra of free EPI, EPI-GNPs and EPI-GNPs encapsulated liposomes were recorded on

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Shimadzu IR Tracer-100, FTIR spectrophotometer. A small amount (≈1) of free EPI, EPI-GNPs

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and EPI-GNPs encapsulated liposomes (lyophilized powder) were mixed with IR grade

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Potassium Bromide (KBr) to prepare a round disc using a small hydraulic press to study FTIR

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characteristic spectrum in the spectral range of 4000-400 cm-1 with resolution of 4 cm-1.

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2.8.2. Powder X-ray Diffraction analysis

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The purity and crystalline properties of plain EPI, EPI-GNPs and EPI-GNPs encapsulated

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liposomes were studied by Powder XRD. The XRD scanning was done at a voltage of 20 keV

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and a current of 30 mA with Cu Kα 1 radiation (l = 0.1542) in a two-theta (degree) configuration

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by using A BRUKER D 8 Advance ECO XRD system with SSD160 1 D Detector.

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2.8.3. Particle characterization

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The surface charge and sizes of the freshly prepared EPI-GNPs encapsulated liposomes were

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measured by Zetasizer Nano ZS ver.7.03 (Malvern Instrument, Malvern, Worcestershire, United

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Kingdom).

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2.8.4. SEM analysis

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Scanning Electron microscopic study was carried out to study and compare the morphological

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characteristics of EPI-GNPs and EPI-GNPs encapsulated liposomes. A sample of EPI-GNPs and

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EPI-GNPs encapsulated liposomes were placed in a 200 mesh copper grid and observed it for

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particles shape and size using SEM detector attached to scanning electron microscopy (Carl

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Zeiss EVO 18).

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2.8.5. TEM analysis

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The morphological shape and size of EPI-GNPs and EPI-GNPs encapsulated liposomes were

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studied using transmission electron microscope (TEM-JEOL model 2100) operated at an

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acceleration voltage of 200 KV. For this purpose, EPI-GNPs encapsulated liposomes was re-

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dispersed with 1 mL distilled water. Few drops of dispersed nanoparticles in suspension was

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placed on the carbon coated copper grid, which was air-dried at 60 °C for 5 min.

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2.9. Cytotoxicity studies

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The potential cytotoxicity of EPI-GNPs encapsulated liposomes was assessed by MTT assay

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[24]. Shortly, breast cancer cell lines (MCF-7 cell) were seeded in to 96-multiwell plate at

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approximately 1 × 105 cells/well at 37 °C in a 5% CO2 atmosphere for 24 h before treatment,

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allowing for cell attachment to the wall of the plate. Cells were treated with different

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concentrations of EPI-GNPs encapsulated liposomes (0.003, 0.006, 0.01, 0.05, 0.1, 0.2 and 0.3

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μg/mL) for 24 h incubation. The respective amounts of DMSO in PBS buffer instead of EPI-

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GNPs encapsulated liposomes was used as control. The medium was removed after 24 h of

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treatment and cells were washed with phosphate-buffered saline (PBS, 0.01 M, pH 7.4). This

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was followed by addition of 200 μL (5 mg/mL) of 0.5% 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-

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diphenyl-tetrazolium bromide (MTT) prepared in serum free medium solution to each well and

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incubated for 4 h at 37 °C in a 5% CO2. Thus, the obtained cells were fixed, washed and stained

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with MTT. Acetic acid was used to remove excess stain while Tris-EDTA buffer was used to

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remove any attached cells. The color intensity was measured in a microplate reader at 564 nm.

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The ratio of the absorption of treated cells to absorption of non-treated cells expressed in

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percentage resulted in percentage of death cells. Thus, every treatment condition was repeated in

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triplicate and IC50 of EPI-GNPs encapsulated liposomes obtained were used for treatment for all

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further studies.

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2.10. Analysis of apoptotic cells by Acridine Orange/Ethidium bromide (AO/EB) double

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Apoptosis of the cancer cells were investigated by fluorescence microscopy after staining with

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AO/EB [25]. Briefly, MCF-7 cancer cells were seeded on a cover slip in a 24-well plate (1×105

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cells/well), and addition of appropriate amount of EPI-GNPs encapsulated liposomes of IC50

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value and incubated. After being cultured for 72 h, 20 μL of trypsin was added to each well.

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Cover slip removed from 24-well plate and washed with 1×PBS buffer and treated with dual

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fluorescent stain 10 µL/mL containing acridine orange (10 mg/mL) and ethidium bromide (10

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mg/mL) and incubated again 30 min. After incubation, unbound dyes were washed with 1 × PBS

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buffer. The morphology of apoptotic cells was examined under fluorescence microscope and

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representative fields were captured at 40× magnification. Dual AO/EB staining method was

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repeated 3 times at least.

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2.11. Apoptotic analysis by Annexin V-FITC and Propidium Iodide

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Cell apoptotic activity of EPI-GNPs encapsulated liposomes was assessed by Nouri and

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Yazdanparast (2011) method with slight modification [26]. MCF-7 cancer cells were treated with

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EPI-GNPs encapsulated liposomes (IC50 value) for 24 h and 48 h. After stipulated time of

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treatment, the cells were harvested and washed twice with 1 × PBS and re-suspended in 100 µL

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binding buffer (10 mM HEPES/NaOH of pH 7.4, 140 mM NaCl and 2.5 mM CaCl2). Next,

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Annexin V-FITC (10 µL) was added to the cells followed by the addition of 10 µL of PI (50

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µg/mL in PBS). The samples were incubated for 10 min in the dark at 4 °C and then analyzed by

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flow cytometry.

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2.12. ROS generation assay

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Intracellular ROS generation in control and treated MCF-7 cancer cells were monitored by

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measuring changes in fluorescence resulting from intracellular probe oxidation [27]. The cancer

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cells seeded in 6-well culture plate (1×105 cells/well) and incubated for 24h. After incubation,

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the medium was replaced with 10% FBS supplemented with IC50 value of EPI-GNPs

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encapsulated liposomes, and again incubated for 24 and 48 h. The cells were washed twice with

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1× PBS and 20 μM DCFH-DA was added and incubated for another 10 min. The DCFH-DA was

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removed, and then 100 μM H2O2 was added into the cells and incubated for 45 min. The change

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in the fluorescence was monitored by fluorescence spectrophotometer at λex=475 nm, λem=525

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nm.

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2.13. Statistical analysis

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Experiments for each sample were performed three times, and obtained values were presented as

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mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA)

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and Student-t test. A value of p