Engineered nanoparticles of Efavirenz using methacrylate co-polymer (Eudragit-E100) and its biological effects in-vivo

    Engineered nanoparticles of efavirenz using methacrylate Co-polymer (eudragit-E100) and its biological effects in-vivo B.N. Vedha Hari, N. Narayanan, K. Dhevendaran, D. Ramyadevi PII: DOI: Reference:

S0928-4931(16)30493-3 doi: 10.1016/j.msec.2016.05.064 MSC 6554

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

1 March 2016 21 April 2016 15 May 2016

Please cite this article as: B.N. Vedha Hari, N. Narayanan, K. Dhevendaran, D. Ramyadevi, Engineered nanoparticles of efavirenz using methacrylate Co-polymer (eudragit-E100) and its biological effects in-vivo, Materials Science & Engineering C (2016), doi: 10.1016/j.msec.2016.05.064

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ACCEPTED MANUSCRIPT Engineered Nanoparticles of Efavirenz using Methacrylate Co-Polymer (Eudragit-E100) and its Biological Effects in-vivo Vedha Hari BN*,bNarayanan N, aDhevendaran K, aRamyadevi D

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Department of Pharmaceutical Technology, School of Chemical & Biotechnology, SASTRA University,

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Thanjavur - 613401. Tamil Nadu, India

Department of Pharmaceutics, Jaya College of Pharmacy, Thiruninravur, Chennai, Tamil Nadu, India

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Dr. B.N.Vedha Hari, Asst. Professor,

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*Address for correspondence:

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School of Chemical &Biotechnology, SASTRA University, Thanjvaur-613401.

Phone: +914362264101; Moble: +919944185974 Email: [email protected]

Running title: In-vivo bio-distribution of Efavirenz-Eudragit E100 nanoparticles

ACCEPTED MANUSCRIPT Abstract Nanotechnology in drug delivery is explored widely to improve therapeutic efficacy and

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minimize undesirable effects of several anti-HIV drugs. Efavirenz is a non-nucleoside reverse

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transcriptase inhibitor, prescribed as first-line drug of choice for treatment of AIDS. It is poorly soluble and exhibits variable bioavailability hence, a high oral dose is recommended for therapy.

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The present work focuses on improving the dissolution and bioavailability of Efavirenz through

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nano drug delivery approach. Polymeric nanoparticles were developed using Eudragit E100 and characterized for size, stability, morphology, cytotoxicity (MTT assay in T-lymphatic (C8166)

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cell lines) and in-vivo biodistribution in mice models. The optimized nanoparticles exhibited average particle size of 110 nm, zeta potential of −33 mV and entrapment efficiency 99%. The

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SEM images displayed the formation of nano-size particles. The cell viability was significantly improved in the nanoparticles (99%) compared to pure drug (15%) at the concentration of 8

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µg/mL. The in-vivo biodistribution profile of the nanoparticles showed considerably higher drug concentration in serum and major organs, especially in the brain compared to the free drug. The optimized Efavirenz loaded nanoparticles clearly demonstrated an increase in dissolution, drug distribution, and bioavailability, which implies better control over the therapeutic dosing. Key Words

HIV/AIDS, Eudragit E100, MTT assay, biodistribution, Nanoparticle

ACCEPTED MANUSCRIPT ABBREVIATIONS

HPLC LC-MS-MS IS ANOVAPDI CD4 CmaxAUC

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Human immunodeficiency virus Highly active anti-retroviral therapy Acquired immunodeficiency virus syndrome Lamivudine Emtricitabine Biopharmaceutical Classification System Central nervous system Blood Brain Barrier Antiretroviral therapy Nucleoside Reverse Transcriptase Inhibitors Protease inhibitors Rate-determining step Reticuloendothelial Systems Food and drug administration Fetal bovine serum Central Animal Facility Animal ethical committee number Field emission scanning electron microscopy (3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) Roswell Park Memorial Institute Sodium dodecyl sulfate Enzyme linked immunosorbent assay Cytotoxicity concentration Committee for the Purpose of Control and Supervision of Experiments on Animals High performance liquid chromatography Liquid chromatography-Mass spectrometry- Mass spectrometry Internal standard Analysis of variance Polydispersity Index Cluster of differentiation Maximum plasma concentration Area under the curve

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HIV HAART AIDS 3Tc FTC BCS CNS BBB ART NRTIs PIs RDS RES FDA FBS CAF AEC No. FE-SEM MTT RPMISDSELISA CC50 CPCSEA

ACCEPTED MANUSCRIPT 1 Introduction An important global health threat termed as acquired immune deficiency syndrome caused by

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HIV infections is one among the lethal diseases. The world is still amidst the pandemic, with

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more than 37 million patients infected, especially 2.5 million children population. Highly active antiretroviral therapy (HAART) is considered as a boon for the reduction of mortality and

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morbidity among AIDS patients. The major challenge with this therapy is the requirement of

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high degree monitoring to administered dose levels, which otherwise leads to virus persistence (1). The HAART gives a ray of hope by a sequential decline in the viral load of HIV- infected

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patients but it is associated with the challenge of increased levels of virus resistance and its subsequent transmission. Plausible causes may be the choice of drugs, multifunctional charges

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and the viral content (2). The truth is that the spread of this disease is observed to be faster in HIV-drug resistant patients than that of non-resistant patients, which contributes to the higher

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level of susceptibility to HIV infections. A wide variety of drugs namely 3TC (lamivudine), Abacavir, Delavirdine, Darunavir, FTC (Emtricitabine), Tipranavir, Ritonavir, Tenofovir and their modifications have been attempted for the treatment of HIV with an insight of targeting HIV virus receptors (3). Researchers are incorporating every tool at hands and suggest that the ultimate focus should be the development of suitable drugs and delivery systems that efficiently target the viral proteins and thus resulting in the termination of the replication of HIV virus cells. Efavirenz is a highly potent first-line anti-HIV drug that inhibits the reverse transcriptase enzyme present in HIV-1, but ineffective on HIV-2 (4). Efavirenz is chemically named as (S)-6-chloro-4(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one and it shows a half-life of 45–55 h with variable bioavailability (5). Recent research studies trust Efavirenz as a promising candidate for HAART in the treatment of HIV infection because of its higher

ACCEPTED MANUSCRIPT efficiency level. It is a yellowish/ white crystalline powder with solubility specifications as insoluble in water (0.00855 mg/mL) but soluble in organic solvents like methanol and

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dichloromethane. It belongs to Biopharmaceutical Classification System (BCS) class-II category

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which possesses a high degree of lipophilicity (log P=5.4), limited oral bioavailability (40–50%), and high inter-subject variability (6, 7). Debilitation occurs at a low level of therapeutic dosage

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and an adverse effect (CNS toxicity) is noted at a higher dose level (8). Efavirenz is

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commercially available as tablets, capsules (Sustiva/ Stocrin/ Atripla) and oral suspension. In spite of some potential side effects reported and the fact that the intake of the drug is seized by

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4% of patients (9), Efavirenz is still prescribed often as the first line drug in antiretroviral therapy (ART). It shows an effective reduction (>80%) of viral load in HIV patients and hence put

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forward as a better treatment option. This drug is also prescribed in combination with nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs) for the highly active

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antiretroviral therapy (HAART). It has been postulated that Efavirenz functions by the inducing cytochrome P450 enzyme but its relative dosage level is still not clear. It is expected that solubility enhancement can pave a way for an improved bioavailability because the dissolution step is considered to be the rate-determining step (RDS) in its absorption. In this view, nanoparticle designing, optimization, in-vitro characterization, cytotoxicity and in-vivo biodistribution have been addressed as the specific aims of this work (10). In general, polymeric nanoparticles are colloidal particulate systems of drug synthesized, either by encapsulation, adsorption or dispersion through a polymeric matrix with the size ranging from 10–100 nm. Nano-sized particles of drug and natural/synthetic polymers support in improving the solubility, permeability, distribution, sustained/controlled release, targeting the drug and henceforth, the stability. All these can be attained either with or without surface modification

ACCEPTED MANUSCRIPT (11). When the polymeric nanoparticles are administered in the human system, they may be captured by Reticulo-Endothelial Systems (RES). This improves the targeting efficacy of the

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drug along with subsided side effects (12). Various studies have pinpointed the induction of

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injuries, inflammation, and cytotoxicity by the materials used for the nanoparticle synthesis. But the toxicity of polymeric nanoparticles has not been reported extensively, owing to their

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biocompatible and degradable nature in addition to the usage of FDA approved excipients (13).

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A detailed survey of literature studies gives us a clear picture of the enhanced efficacy of drugs after being converted to the order of nano-size using polymeric materials when compared to that

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of bulk drugs. With the complete understanding of the knitted pros and cons, the current research was aimed to develop polymeric nanoparticles of Efavirenz using Eudragit-E100 as a polymer,

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and grab it for the in-vivo biodistribution study in non-infected (healthy) mice models. The mice model is considered as highly reliable and most commonly used for preclinical evaluations,

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possessing many advantages such as smaller size (body), availability of validated protocols for many disorders and similarity of their genome with the human genome etc. The incorporation of drugs into Eudragit-E 100 nanoparticles displays promising potential for the enhancement of parameters namely solubility, bioavailability, and performance, especially for the sparingly water-soluble drugs such as andrographolide, cyclosporine, genistein, meloxicam and quercetin (14, 15). Hence, we have preceded our experiment through tapping the potential of the EudragitE100 polymeric material.

ACCEPTED MANUSCRIPT 2 Materials and Methods 2.1 Materials

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Efavirenz was received as a gift sample from ISP Hongkong (P) Ltd., Hyderabad, India. Eudragit

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E100 was obtained as a gift sample from Gluchem Pharma, Hyderabad, India. Pluronic F68 was obtained from Himedia Lab (P) Ltd., Mumbai, India. Sodium alginate was purchased from S.D.

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Fine-Chem. Ltd., Mumbai, India. All the chemicals and reagents used in this study were of

(3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

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analytical grade. HEPES (N-2 (2-hydroxyothyl) piperazine-N'-(2-ethanesufonic acid), MTT tetrazolium

bromide),

DMF

(N,N’-Dimethyl

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formamide), penicillin, streptomycin sulfate and glutamine were purchased from Sigma, Hongkong, China; 2-ME (2-mercaptoethanol) was purchased from Bio-Rad, China. RPMI-1640 and

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fetal bovine serum (FBS) were purchased from Gibco, Auckland, New Zealand.

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2.2 Animals

Eight weeks old Male Musmusculus (Swiss albino mice) were obtained from Central Animal Facility (CAF), SASTRA University, Thanjavur on prior approval from Institutional Animal Ethical Committee (AEC No. 81/SASTRA/IAEC/RPP). Animals were caged and maintained as per guidelines under dark and light conditions. Drinking water and conventional feed were provided ad libitum.

2.3 Preparation of nanoparticles Efavirenz loaded Eudragit E-100 nanoparticles (1:1 ratio of the drug and polymer, 100mg each) was synthesized by adopting an emulsion solvent evaporation method (16). Typically, an organic solution was prepared by mixing 100 mg of Eudragit E-100 and 100 mg of Efavirenz in 10 mL

ACCEPTED MANUSCRIPT methanol as a solvent. This was followed by the preparation of aqueous solution where a surfactant (50 mg of Pluronic F-68) and a stabilizing agent (25 mg of Sodium alginate) are

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dissolved together in distilled water. Finally, the organic solution was added slowly by injecting

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(2 mL/min) into the aqueous solution under continuous vigorous stirring using a magnetic stirrer at room temperature. The stirring was continued up to 4 h until the formation of nanosuspension

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and complete removal of the organic solvent. The organic solvent present in the formulated

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nanosuspension was removed by continuous stirring for 4 h followed by vacuum drying (Bio Nick, Mumbai) under reduced pressure at room temperature for 1 h to remove the trace amount

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of the solvent. The organic solvent would evaporate by two mechanisms: first, the solvent diffuses into the continuous phase and the second; the solvent evaporates at the continuous phase

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air interface. The freeze dried (17) nanoparticles were subjected for various characterization techniques such as, Particle size and zeta potential, % entrapment efficiency and morphological

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studies (18-19) (procedures are submitted as supporting information).

2.4 Characterization Techniques 2.4.1 Comparison of drug releases pattern In-vitro drug release from nanoparticles was studied using a well-established dialysis bag method in distilled water as release media. The nanoparticle dispersion (0.5 mL) containing drug concentration equivalent to 2 mg/mL of Efavirenz was placed in a dialysis bag and was immersed in a vial containing 10 mL of dissolution media. The set up was placed on a thermostatic magnetic stirrer and stirred at 100 rpm, maintained at 37°C. The dissolution media was completely withdrawn at predetermined time intervals and replaced with whole warm fresh media each time to maintain the sink condition, for the total period of 24 hours. The withdrawn

ACCEPTED MANUSCRIPT samples were then analyzed using a UV-Visible spectrophotometer (SL150, Elico Instrumentation Ltd, Mumbai, India) at 247 nm with the fresh media as the blank. The

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experiment was done in triplicate (n = 3) for each formulation and the percentage drug release at

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each time point was calculated using a standard calibration curve method (20). The mechanism of drug release from the nanoparticles was studied by fitting the release profile data obtained for

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different media for all the formulations to the various release kinetic modeling (21).

2.4.2 In-vitro Cytotoxicity assay

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C8166 cells were donated by Medical Research Council, AIDS Regent Project. The cells were maintained at 37 °C in 5% CO2 in RPMI-1640 medium supplemented with 10% heat-

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inactivating FBS (Gibco). The cellular toxicity of nanoparticles was assessed by the MTT assay

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using C8166 cell lines. Briefly, 100 μL of 4×105 cells were plated into 96-well plates, 100 μL of

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various concentrations of nanoparticle samples were added and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 72 h. The supernatant solution in the well plates was discarded; the MTT reagent was added and incubated for 4 h. Then, 100μL 50% DMF-20% SDS was added to completely dissolve the formed formazan crystals. The plates were read on a BioTek ELx 800 ELISA reader at 630 nm. The 50% cytotoxicity concentration (CC50) was calculated from the data (22, 23).

2.5 Bio-distribution Studies 2.5.1 Experimental design The study was performed in accordance with the strict guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of

ACCEPTED MANUSCRIPT Environment and Forest, Govt. of India. The experimental design was approved by the Institutional

Animal

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Committee

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(AEC

No.

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81/SASTRA/IAEC/RPP). Mice models were selected to study the biodistribution and

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pharmacokinetic parameters through an intra-peritoneal route of administration, for the optimized Efavirenz nanoparticles and compared with the pure drug. The experimental animals

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were divided into two groups each containing 30 animals, for treating with the pure drug

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suspension and nanoparticle formulation, respectively. Group-I was administered with the Efavirenz pure drug suspension prepared with 0.5% carboxy methyl cellulose, which was sub-

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divided into 5 sub-groups (n = 6 in each sub-group) for collecting blood and organs at different time points as 30 min, 1 h, 4 h, 8 h and 24 h. Similarly, group-II animals were sub-grouped for

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five-time points with n = 6 animals in each group and tested with optimized polymeric nanoparticle formulation prepared by the emulsion solvent evaporation method. A control group

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of animals (n = 6) was maintained without any treatment, for histopathology study comparison. The sample for intra-peritonial injection was prepared by centrifuging the nanosuspension (20000 rpm) followed by the dispersion of the pellet in distilled water using a vortex mixer (Remi CM101 DX, Mumbai, India). The suspension of pure drug and nanoparticles were clear and there was no sedimentation or agglomeration /aggregation.

2.5.2 Reagents and standards All chemicals used were of analytical grade and of high purity. HPLC grade acetonitrile and formic acid were used for the LC analysis. Formic acid buffer: 0.1% formic acid in water obtained using a reverse osmosis system, Milli-Q/HPLC grade millipore corporation (Fisher scientific Mumbai, India).

ACCEPTED MANUSCRIPT 2.5.3 Sample preparation Serum samples (n = 6) were isolated by clotting the collected blood through the centrifugation

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process. The organs such as heart, liver, kidney, lung, spleen, testis and brain were harvested,

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and stored at −80oC until further process. The collected tissues (n = 6) were individually weighed and then 5 mL of acetonitrile was added to the tissue and serum samples. Each tissue was cut

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into small pieces and homogenized separately using a pellet homogenizer, equilibrated at 4oC for

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30 min and then centrifuged at 10000 rpm for 15 min at 4oC. The aliquot of the supernatant solution containing the extracted drug from tissue samples was injected into auto sampler vials

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and analyzed using LC-MS (24).

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2.5.4 Equipment condition (LC/MS-MS)

Efavirenz sample analysis using liquid chromatography coupled with a mass spectra (LC-MS)

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technique was validated and the specifications are detailed as follows: the mobile phase was buffer (0.1% formic acid in water), acetonitrile 30:70 (v/v) with a binary pump at a flow rate of 0.6 mL/min with a splitter, column oven temperature of 40oC±2oC and auto sampling temperature of 10oC ± 4oC. The volume of injection was maintained to be 10 µL with a run time of 2.0 min. The column used was BDS Hypersil C18, 50x4.6mm (5 µm particle size) column (Thermo, USA). The analyte and internal standard (IS) detection were performed with a turbo ion spray ionization source, operating in the positive ion mode on a triple quadrupole mass spectrometer (Micromass, Quattro, API tandem quadrupole system). Quantification was performed using multiple reactions monitoring (MRM) modes based on the precursor m/z and its fragment m/z (MRM transition) for each analyte. Source dependent parameters such as source temperature of 120oC, desolvation gas

ACCEPTED MANUSCRIPT flow rate as 900 L/h, desolvation temperature of 425oC and capillary maintained at 3 V were optimized. Compound dependent parameters were cone gas flow 50 L/h, IS cone gas flow

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volume 15 V, analyte cone gas flow 23 V and collision for the analyte (Efavirenz) 14 V and for

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above method was validated for various parameters (26,27).

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IS (Valsartan) 14V with nitrogen as a collision activating agent maintaining at 6psi (25). The

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2.5.5 Estimation of Bioavailability

The concentration of the drug present in plasma collected at various time points was calculated

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for both the pure drug and nanoformulation treated animal groups. The plasma concentration Vs. time plot curve was constructed, from which the maximum concentration reached (Cmax) and

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time to reach the peak plasma concentration (Tmax) was estimated. The area under the curve (AUC) was determined using Graphpad Prism software from which the bioavailability was

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determined by comparing the data of nanoformulation with respect to the pure drug.

2.5.6 Histopathological studies

After 24 h exposure of the free drug sample and the nanosuspension, the tissues such as lung, kidney, liver, heart, spleen, brain and testis were collected from the mice models (n = 6), stored in formalin solution and subjected to the histopathological study.

Each tissue was sliced

individually with a thickness of 2mm using a microtome, after paraffin embedding. The sections obtained were stained with haemotoxylin and eosin using an automatic staining technique and viewed under a microscope (28).

ACCEPTED MANUSCRIPT 2.6 Statistical analysis: The experiments were carried out with n = 6 animals in each group and the drug deposition in

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various organs and blood were reported as mean ± SD. The data were subjected to statistical

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analysis using two-way analysis of variance (ANOVA) at a significance level of 0.05 using

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graph pad prism-5 software.

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3. Results

3.1 Particle size, charge distribution and entrapment efficiency

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The particle size of the optimized nanoparticles was analyzed using a low angle light scattering technique. The average particle size was found to be 110 ± 5 nm with a Poly Dispersity Index

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(PDI) of 0.201 ± 0.05 for the optimized formulation (Fig. 1A). The surface charge of the

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Efavirenz –Eudragit E100 nanoparticles was found to be −28.3±2 mV (Fig. 1B), whereas the

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zeta potential value of −38.8±3 mV was observed for the blank Eudragit E100 nanoparticles prepared without the drug. The formulated nanoparticles showed an entrapment efficiency of 95±2% which confirmed the effective drug loading in the polymer.

3.2 Morphological characteristics of Efavirenz and its nanoparticles Scanning electron microscopy images of the pure drug and the freeze-dried drug loaded nanoparticles are shown in Fig. 2 A and Fig. 2 B, respectively. The images clearly depict the formation of nanoparticles and solid state transition of the drug from a long, smooth, blade structure converted to small, rough and irregular spherical forms of the nanosize range. Transmission electron microscopy images of the drug loaded nanoparticles and unloaded form of

ACCEPTED MANUSCRIPT Eudragit E100 nanoparticles (Fig 2C and Fig 2D) also clearly display the spherical morphology

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of the nanoparticles.

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3.3 Enhancement of dissolution

The comparative in-vitro drug release profile of pure Efavirenz and the nanoparticle formulation

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in phosphate buffer (pH 7.4) and distilled water as media are shown in Fig. 3 A and Fig. 3 B,

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respectively. Pure drug and nanoparticles formulations showed approximately similar trend of drug release in phosphate buffer media, reaching the maximum of 90% and 100% drug

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concentrations at the end of 16–18 h, respectively. However, in the case of distilled water as media, the pure drug exhibited 40% dissolution in 4 h followed by very slow increase in the drug

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release reaching maximum of 50% at the end of 24 h. Whereas the polymeric nanoparticles showed 40% release of Efavirenz within 2 h and gradually increased to 70% at 4 h. The further

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release was more linear reaching the maximum drug release (100%) at the end of 18 h. The kinetics data of drug release for the nanoparticles as studied by fitting the release data with respect to different models such as zero order, first order, Higuchi, Korsemeyer-Peppas and Hixson Crowell is shown in table 1. The drug release from the nanoparticle formulations was based on the diffusion mechanism as indicated by the high R2 value (0.98 – 0.99) in KorsmeyerPeppas kinetics model. Also the n-value <0.5 revealed that the drug release followed the Fickian diffusion transport from the polymeric matrix system.

3.4 Cytotoxicity of the formulated nanoparticles Cytotoxicity of Efavirenz polymeric nanoparticles and the free drug was evaluated by the MTT assay using C8166 cell lines. This study was performed for 72 hours, and the cell viability was

ACCEPTED MANUSCRIPT found to be 100% in all formulations for the 1.6 µg/mL concentration (Fig. 4). As the concentration was increased to 8 µg/mL, the cell viability was declined by 15% in the free drug,

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whereas EVN-2 exhibited 100% cell viability even at the 40 µg/mL concentration. The cell

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viability for the formulation became less than 10% with an increase in the concentration as 200

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3.5 Validation of the LC-MS method of analysis

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µg/mL.

The calibration curve with six different concentrations was plotted and found to be linear with a

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correlation coefficient of 0.9992 with RSD of 1.4. The precision of the method was assessed by inter- and intra-day assay analyses, where the assay was proved to be in the range of 95.09 to

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103.76 % with RSD of 1.69 and 1.352, respectively. The extraction efficiency was found to be

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93–95% through recovery studies and the limit of detection was identified to be 0.0012 µg/mL.

3.6 Effect of nanoparticles on in-vivo Biodistribution The biodistribution of the drug by nanoparticles delivery and its retention in the biological system were evaluated by analyzing the concentrations of the free drug in serum and different organs, at various time points, for a period of 24 h. Both the free Efavirenz and Eudragit-E100 nanoparticles of Efavirenz were administered through an intraperitoneal route. The Efavirenz concentration in the collected tissue samples and plasma was measured for both the free drug sample and nanoparticles and is shown in Fig. 5 A to Fig. 5 H. The free drug showed a higher level of deposition in the testes followed by brain, liver, spleen and kidney at the 8th h and the concentration of the drug surged in all organs after the 24th h.

ACCEPTED MANUSCRIPT The distribution of nanoparticles and the free drug in various organs like liver, kidney, spleen and lung showed a gradual increase with an increase in time. The drug deposition in the heart

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showed maximum accumulation within 30 min, followed by a sudden drop in the concentration

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of the pure drug as well as nanoparticles.

In the Efavirenz-Eudragit-E100 nanoparticle injected a group of mice models, the distribution of

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nanoparticles to specific organs showed about two to three-fold increase in deposition compared

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to the free drug treated animals. The maximum deposition of drug from the nanoparticles occurred in the testis (280 ng/g) (p<0.05 statistically significant) and this elevation was notable

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up to 8 h but declined after 24 h. The brain tissue concentration was found to be 66 ng/g for the free drug sample and 170 ng/g (p<0.05) for nanoparticles at the end of 8 h. With respect to the

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nanoparticles in the highly perfused tissues, a higher level of drug deposition was observed in the brain followed by the liver at 4 h, and the levels of deposition increased until 24 h. The liver

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showed the maximum drug accumulation of 100 ng/g of the tissue after 24 h for the nanoparticles, whereas pure drug reached only 40% at 8 h and decreased further.

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nanoparticles achieved the drug concentration of 70–80 ng/g of tissue in the spleen, kidney, and lungs at 24 h time point.

3.7 Pharmacokinetics of Efavirenz nanoparticles The pharmacokinetic parameters were determined from the plasma concentration of the drug vs. the time profile. The peak plasma concentration (Cmax) of 25 ng/mL was achieved by the nanoparticles at 24 h through a gradual increase in the drug level from the time of administration. Whereas the pure drug showed Cmax of 23 ng/mL within one hour and suddenly decreased in the concentration up to the level of 3 ng/mL at 24 h. The AUC for the formulated Efavirenz

ACCEPTED MANUSCRIPT nanoparticles was found to be 1.5 folds higher than the pure drug administered through the intra-

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peritoneal route.

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3.8 Histopathology:

No significant difference in the pathological conditions of the tissues was noted on the

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administration of the free drug and Eudragit-E100 nanoparticles after 24 h exposure, when

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compared with the untreated control group (n = 6) mice tissues, as shown in Fig. 6.

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3.9 Statistical analysis:

Statistically significant difference (P<0.05) was observed in the drug concentration in serum and

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organs collected at different time points, for the free drug and nanoparticles treated animal

4 Discussion

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groups (n = 6), as established by two-way ANOVA using graph pad prism 5 software.

The average particle size measuring by dynamic light scattering and the PDI value of the optimized nanoparticles indicate narrow size distribution. In general, PDI is the measure of the width of a particle distribution where 0.0 and 0.5 represent the narrow and very broad distributions, respectively. Park et al. 2013 (14) reported the efficiency of nanoparticle formation of Efavirenz with a narrow size distribution in addition to the clear insight of critical formulation and process parameters involved. The narrow size distribution and uniform spherical nanoparticles formation could also be confirmed by the SEM analysis. The morphology clearly demonstrated the size reduction of pure drug flakes into the order of nanoscale during the formulation processing (29).

ACCEPTED MANUSCRIPT Zeta potential value refers to the measure of the charge at the surface of the particles and thus an indicator of the physical stability of colloidal systems. The charge measurement of the drug

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loaded nanoparticles, as well as the blank nanoparticles revealed their inherent stability.

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Although the polymer is cationic in nature, the negative charge is substantiated the presence of stabilizing agent (25 mg of sodium alginate) and surfactant (50 mg of Pluronic F-68) (n = 3),

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which are the contributing factors.

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An important parameter contributing to the effective dose fixing and in-vivo administration is the entrapment efficiency of nanoparticle formulation. The higher entrapment level of drug in the

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developed nanoparticles offers the specific advantage for the dose calculation for further studies. The increased level of entrapment efficiency may be ascribed to the good interaction between the

interactions (14, 30).

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cationic polymer and the anionic drug with respect to the drug–drug and polymer–drug

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The comparison of in-vitro drug release study is one the important tool to assess the improvement in solubility, dissolution and indirectly bioavailability. In the present study, even though the release profile of the pure drug and formulated nanoparticles was similar in phosphate buffer media, a significant variation was observed in the distilled water media. The pure drug could reach the level of only 40-50% release due to non-uniform particle size, crystalline nature and low saturation (aqueous) solubility of the pure drug (31). In case of polymeric nanoparticles maximum drug release was achieved by significant features like reduction in crystallinity, effective size reduction (particle size ≈ 100 nm) of drug that ultimately improves the surface contact area and improved solubility in aqueous media (32). The release kinetics clearly indicated that diffusion as the basic mechanism for the drug to release into the media. Further, the n-value less than 0.5 indicated the mode of diffusion to be

ACCEPTED MANUSCRIPT Fickian transport. Additionally, the high R2 value (0.99) in phosphate buffer with respect to Higuchi model for the nanoparticles supported the type of drug release from the smaller

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polymeric matrix particles (33, 34).

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MTT assay was adopted as a suitable technique to assess the cytotoxicity effect of the formulated nanoparticles. This technique yields the quantitative results of cytotoxicity based on the level of

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metabolism of water-soluble dye MTT into water insoluble purple colored crystalline formazan.

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The C8166 cells lines served as an onset indicator of toxicity as many researchers have proved the potentiality of these cell lines. The viability of cells was measured as a function of the

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concentration of the drug added (35). The maintenance of 100% viability of the cells even up to 40 µg/mL drug equivalent concentration of the nanoparticles provides an evidence for lesser

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toxicity of the developed formulation. Especially, when there was a significant reduction in cell viability in the pure drug treated wells even at the lower concentration. Yet, reduced live cells

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were observed with further increase in the concentration of the drug (36). The CC50 results revealed that the nanoparticles were less toxic than the free drug. The same was in agreement with the previously published data (37-39). In-vitro cytotoxicity studies have provided rapid, cost-effective and reliable results.

The intra-peritoneal route of administration was selected for the in-vivo biodistribution study of Efavirenz nanoparticles as it was reported to be the most suitable route since it extends the residence time of the drug in the peritoneal cavity thereby improving the systemic absorption, as well as enhancing the drug entry through lymphatic circulation and portal veins (40,41). It is suitable to target the CD4 cells through peritoneal cavity which reaches the lymphatic system easily. It has provided effective treatment for HIV infections, especially reduction in the HIV viral load, has been observed at the later stage of the treatment (42). Even though the intra-

ACCEPTED MANUSCRIPT peritoneal route of drug delivery is considered as a parenteral route of administration, the pharmacokinetics of substances administered through the intra-peritoneal route are more similar

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to those observed in oral administration, because the primary route of absorption is through the

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mesenteric vessels, which drain into the portal veins and pass through the liver. Hence, this route could also be used to predict the oral bioavailability indirectly (43).

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The higher amount of Efavirenz distribution was observed in the testis which could be due to the

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increased affinity of drug towards the genital organs as reported by earlier studies (44, 45), where the human and animal studies showed the similar kind of drug deposition. The drug

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concentration of 100 ng/g of tissues observed in the liver tissue after 24 h for the nanoparticles was in accordance with the previously published data (46).

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In order to attain high therapeutic activity in the HIV treatment, the drug should possess the ability to cross the blood-brain barrier (47). Henceforth, two-fold increase in drug concentration

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in the brain in the case of nanoparticles compared to the free drug could be more beneficial and this could be attributed to the high permeability of nanoparticles (<100 nm) to brain through the blood brain barrier. Targeting and enhancing the drug deposition in the brain are important for controlling the severity of HIV infections because, in the HIV infected patients, the brain involvement (CNS) is passive; it serves as a shelter for HIV virus and provides the energy to reactivate the infections (48). A size dependent distribution of nanoparticles to the specific organs has been proved by prior reports where high permeation and distribution were observed with particles having a size of 100 nm compared to the particles of either smaller or bigger size than the 100 nm [1]. Another reason for high blood brain barrier permeation is the intraperitoneal route of administration (49). The Brain is one of the reservoir sites for the HIV virus along with reticuloendothelial systems. The complete eradication of the viral load with the

ACCEPTED MANUSCRIPT current treatment mode and dosage forms is limited, due to its inability in permeation of drugs through the blood-brain barrier. However, nanotechnology shows a promising trend in targeting

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the brain by crossing BBB with less complication. Many studies emphasize in the targeting

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reservoir site of HIV virus to reduce the viral load and, therefore, techniques to enhance the drug level and distribution in the brain is need of the hour (50, 51). Moreover, studies have confirmed

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that the 1000–4000 µg/L therapeutic plasma concentration of Efavirenz showed lesser CNS side

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effects in HIV-infected patients compared to higher levels than 4000 µg/L (52). Hence, for the achieved concentration of 4200 ng/mL of the drug for the nanoparticle formulation, the brain

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distribution was 170ng/g of the tissue, which could not cause neurotoxicity. In the lines of distribution of the drug to several organs, the deposition of nanoparticles was found to be more

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than the free drug. Finally, the nanoparticle in the serum was analyzed where only a low concentration was observed. This could be ascribed to the low solubility of the polymer in

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neutral (near alkaline) media, and this was supported by the presence of the low concentration of Eudragit nanoparticles when compared to that of the free drug sample (53). As the accepted range of therapeutic concentration limit of Efavirenz is 1000–4000 ng/mL in humans (54), a higher level of drug concentration is expected to produce the unwanted side effects.

The therapeutic range was achieved and maintained for the formulated Efavirenz

Eudragit E100 nanosuspension (4200 ng/mL), whereas in the case of the pure drug, the level of the drug concentration (7000ng/mL) has exceeded the limit. 5 Conclusions Efficacy of Efavirenz was improved through the nano-drug delivery using polymeric nanoparticles prepared by an emulsion solvent evaporation method with Eudragit-E100 as the polymer.

In-vitro characterization confirmed the formation of stable nanoparticles and the

ACCEPTED MANUSCRIPT cytotoxicity assay using NIH3T3 cell lines confirmed their high permeability than the free drug. Biodistribution studies proved the penetration ability of nanoparticles through blood-brain barrier

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and other organs. Thus, a potential nano-drug delivery of the active pharmaceutical ingredient is

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established by demonstrating its in-vivo parameters. The efficacy of polymeric nanoparticles of Efavirenz in an HIV induced animal (mice) model may be needed for better understanding and

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this could open up the windows for the reliable treatment of HIV with significant benefits.

6 Acknowledgements

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The authors are thankful to the SASTRA University, Thanjavur for the financial support through Prof. T.R.R. research scheme and also thankful to the Central Animal Facilities (CAF)

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established at SASTRA University. Yong-Tang Zheng, Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Science & Yunnan province, Kunming

studies.

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Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China for cytotoxicity

7 Conflicts of Interest

The authors have No conflict of interest

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54. A.F. Mello, T. Buclin, L.A. Decosterd, C. Delhumeau, J. Iulio, A. Fleurent, M. Schneider, M. Cavassini, A. Telenti, B. Hirschel, A. Calmy, Successful efavirenz dose

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Figure Captions

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reduction guided by therapeutic drug monitoring, Antivir. ther. 16(2) (2011) 189-197.

Fig. 1 (A) Particle size distribution and (B) Zeta potential of nanoparticle formulation Fig. 2 Scanning electron microscopic image of A) Pure Efavirenz powder B) Efavirenz-Eudragit E100 nanoparticles; Transmission electron Microscopic images of C) Drug loaded nanoparticles and D) Eudragit E100 nanoparticles without drug. Fig. 3 Efavirenz release profile from Pure drug solution and Nanoparticulate formulation in A) Phosphate buffer (pH 7.4) as a medium (n = 3) B) Distilled water as a medium (n = 3) Fig. 4 Cytotoxicity study of pure Efavirenz and Efavirenz nanoparticle in T-lymphatic (C8166) cell lines

ACCEPTED MANUSCRIPT Fig. 5 Biodistribution profile of the Efavirenz pure drug and nanoparticulate formulation in arious organs and blood

(A) Liver, (B) Spleen, (C) Heart, (D) Lungs, (E) Testes, (F) Kidney,

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(G) Brain and (H) Blood. Data represented as mean ± SD (n=6)

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Fig. 6 Histophathalogy images of tissues treated with the free drug and nanoparticle compared

Table legends

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with the control (n = 6)

Table 1 Drug release kinetics mechanism of the Efavirenz nanoparticles in different media

ACCEPTED MANUSCRIPT

First

Order

order

Higuchi

Hixson

Korsemeyer

Crowell

- Peppas

n-value

Phosphate Buffer (pH 7.4)

0.1365

0.8887

0.8895

0.8047

0.9889

0.331

0.6550

0.9658

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Distilled Water

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R2 values

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Media

Zero

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Table 1: Drug release kinetics of the Efavirenz nanoparticles

0.9951

0.9384

0.9983

0.462

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2 n – release exponent; R – correlation co-efficient

ACCEPTED MANUSCRIPT

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(A)

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(B)

Fig.1. (A) Particle size distribution and (B) Zeta potential of nanoparticle formulation at pH 7

ACCEPTED MANUSCRIPT (B)

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(A)

(D)

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(C)

Fig.2. Scanning electron microscopic image of A) Pure Efavirenz powder B) Efavirenz- Eudragit E100 nanoparticles; Transmission electron Microscopic images of C) Drug loaded nanoparticles and D) Eudragit E100 nanoparticles without drug.

ACCEPTED MANUSCRIPT 110 100 90

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NF (1:1) Pure

70

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60 50 40

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% Drug Release

80

30

10 0 2

4

6

8

10

12

14

16

18

20

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24

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0

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20

Time (h)

Fig. 3.A) Efavirenz release profile from Pure drug solution and Nanoparticulate formulation in

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phosphate buffer (pH 7.4) as medium (n=3)

100 90 80

Drug release (%)

Free drug

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110

70 60 50

NF (1:1)

40 30 20 10 0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

Time (h)

Fig. 3.B) Efavirenz release profile from Pure drug solution and Nanoparticulate formulation in distilled water as medium (n=3)

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Nanoparticle Pure

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80 60

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% Cell vaibility

100

40

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20 0 0.32

1.6

8

40

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0.064

200

Conc. (µg/mL)

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Fig. 4. Cytotoxicity study of Efavirenz pure drug and Efavirenz nanoparticle in T-lymphatic

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(C8166) cell lines

Liver

Spleen

NF Free drug

AC CE P

120

80 60 40 20 0 0

0.5

1

60 50 40 30 20 10 0

4

8

0

24

0.5

1

4

8

24

Time (h)

Time (h)

(C)

(D) Lungs

NF Free drug

Heart

8000

NF Free drug

120 100

7000 6000

ng/g tissue

ng/g tissue

NF Free drug

70

ng/g tissue

100

ng/ g tissue

(B)

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5000 4000 3000 2000

80 60 40 20

1000

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Time (h)

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24

0

0.5

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Time (h)

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(F) Kidney

ng/g tissue

ng/ g tissue

300 250 200 150 100 50 0 0

0.5

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NF Free drug

Brain

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ng/ mL

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ng/g tissue

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1

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NF Free drug

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90 80 70 60 50 40 30 20 10 0

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NF Free drug

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Testes

1

4

8

24

Time (h)

(H) NF Free drug

Blood

30 25 20 15 10 5 0

8

24

Time (h)

0

0.5

1 4 Time (h)

8

24

Fig.5. Biodistribution profile of Efavirenz pure drug and Nanoparticulate formulation in various organs and blood. (A) Liver, (B) Spleen, (C) Heart, (D) Lungs, (E) Testes, (F) Kidney, (G) Brain and (H) Blood. Data represented as mean ± SD (n=6)

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Control

Free drug

Nanoparticle

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Brain

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Heart

Lungs

Spleen

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Liver

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Kidney

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Testes

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Fig.6. Histophathalogy images of different organs treated with free drug and nanoparticles

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compared with control (n=6)

ACCEPTED MANUSCRIPT Table I: Drug release kinetics of the Efavirenz nanoparticles

First

Order

order

Higuchi

Hixson

Korsemeyer

Crowell

- Peppas

n-value

0.9889

0.331

0.9983

0.462

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Media

Zero

(pH 7.4)

0.8887

0.8895

0.6550

0.9658

0.9951

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2 n – release exponent; R – correlation co-efficient

0.8047

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Phosphate Buffer

0.1365

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Distilled Water

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R2 values

0.9384

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Graphical abstract

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The highlights of the manuscript are: 1. Efavirenz loaded polymeric nanoparticle developed with small size and high drug loading 2. Cytotoxicity Nanoparticle was evaluated using Human Lymphatic cell line (C8166) 3. Nanoparticle enhanced the dissolution in-vitro 4. Nanoparticle accumulated more in testes and brain after Intraperitoneal administration