http://informahealthcare.com/drdISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, Early Online: 1–10! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.920430

RESEARCH ARTICLE

Perspectives of nanoemulsion assisted oral delivery of docetaxel forimproved chemotherapy of cancer

Prerna Verma1,2, Jaya Gopal Meher1, Shalini Asthana1, Vivek K. Pawar1, Mohini Chaurasia2, and Manish K. Chourasia1

1Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, India and 2Amity Institute of Pharmacy, Amity University, Lucknow, India

Abstract

Context: Nanoemulsions (NE) are one of the robust delivery tools for drugs due to their higherstability and efficacy.Objectives: The purpose of present investigation is to develop stable, effective and safe NE ofdocetaxel (DTX).Methods: Soybean oil, lecithin, Pluronic F68, PEG 4000 and ethanol were employed as excipientsand NEs were prepared by hot homogenization followed by ultra-sonication. NEs wereoptimized and investigated for different in vitro and in vivo parameters viz. droplet size, polydispersity index, charge; zeta potential, drug content and in vitro drug release, in vitrocytotoxicity, in vitro cell uptake and acute toxicity. Transmission electron microscopy wasperformed to study morphology and structure of NEs. Stability studies of the optimizedformulation were performed.Results: Droplet size, poly dispersity index, zeta potential, drug content and in vitro drugrelease were found to be 233.23 ± 4.3 nm, 0.24 ± 0.010, �43.66 ± 1.9 mV, 96.76 ± 1.5%,96.25 ± 2.1%, respectively. NE F11 exhibited higher cell uptake (2.83 times than control) andstrong cytotoxic activity against MCF-7 cancer cells (IC50; 13.55 ± 0.21mg/mL at 72 h) whereas notoxicity or necrosis was observed with liver and kidney tissues of mice at a dose of 20 mg/kg.Transmission electron microscopy ensured formation of poly-dispersed and spherical dropletsin nanometer range. NE F11 (values indicated above) was selected as the optimized formulationbased on the aforesaid parameters.Conclusion: Conclusively, stable, effective and safe NE was developed which might be used asan alternative DTX therapy.

Keywords

Acute toxicity, droplet size, MTT assay,soybean oil, zeta potential

History

Received 22 March 2014Revised 29 April 2014Accepted 29 April 2014

Introduction

Most of the anticancer/cytotoxic drugs are known to be

notorious as far as their pharmacokinetics (ADME) and

adverse drug effects are concerned (Undevia et al., 2005).

Taxoids (paclitaxel, docetaxel (DTX) etc.) are one of the

representative classes of cytotoxic drugs, which show various

undesirable side effects as well as low oral bioavailability

(510%) (Ganesh, 2007). The reasons for diminished bioavail-

ability is attributed to their low water solubility and high-

affinity for the P-glycoprotein (P-gp), which limit absorption

of the orally administered taxoids and mediates their direct

excretion into the gut lumen (Eytan & Kuchel, 1999; El-Readi

et al., 2010). Moreover cytochrome P450 (CYP 450) isoen-

zymes, abundantly found in the liver and gut wall also

contribute to high first-pass effect leading to poor

bioavailability (Spratlin & Sawyer, 2007). At such situation

the major challenges for scientists are to overwhelm interfer-

ence of biological barriers that confine the access of drugs to

target sites and also to develop drug delivery systems/drug

products with utmost therapeutic value. In this quest, one of

the simplest ways to resolve first-pass effect and bioavail-

ability issues may be parenteral route of administration, but it

results into patient incompliance, therapeutic complications,

high expenses, and poor quality of life. Alternatively, P-gp or

CYP 450 inhibitors such as cyclosporine may be employed to

minimize the metabolism/elimination of drug, but again these

are found to suppress body’s immune system and thus cause

severe health complications (Sweetman, 2007).

Aforementioned problems associated with taxoids create a

platform for the development of a novel drug delivery

system(s)/product(s), which may resolve the undesired effects

of taxoids. DTX is one of the effective anticancer drug

(preferably used for locally advanced or metastatic breast

cancer), commercially available as Taxotere� and Duopafei�.

These products contain high concentration of Tween-80 and

reported to show fluid retention, hypersensitivity, musculo-

skeletal toxicity, neurotoxicity as well as neutropenia

(Manjappa et al., 2013). Several research groups have

Address for correspondence: Manish K. Chourasia, Sr. Scientist,Pharmaceutics Division, CSIR-Central Drug Research Institute,Lucknow 226031, India. Tel:+91 522 2612411-18. Fax: +91 5222623405. Email: manish_chourasia@cdri.res.inMohini Chaurasia, Amity Institute of Pharmacy, Amity University,Lucknow 226010, India. Tel: +91 522 2399418. Email:mohinichaurasia@gmail.com

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reported different delivery systems of DTX ensuring better

therapeutic efficacy which include nanoparticles (Cho et al.,

2011), hydrogels (Bae et al., 2013), liposomes (Jinturkar

et al., 2012; Mishra et al., 2012), polymer-DTX conjugates

(Lee et al., 2009), micelles (Liu et al., 2008), solid oral

dispersions (Moes et al., 2011), micro needles and elastic

liposomes (Qiu et al., 2008) as well as solid self-nano

emulsifying delivery systems (Seo et al., 2013). Despite such

advancement in drug delivery, commercial/industrial viability

of these models are questionable, which might be accredited

to many issues especially stability and scale up of products.

Realizing the need for development of DTX in a

comparatively safe, stable and effective drug delivery

system, our research group started investigating a suitable

alternative and hypothesized that NE may serve as the robust

delivery system. A nanometric amphotericin B-encapsulated

chitosan nanocapsules using polymer deposition technique

mediated by NE template fabrication, which was a cost-

effective immune-adjuvant chemotherapeutic delivery system

is previously reported from our laboratory (Asthana et al.,

2013). NE, because of its nano size is believed to escape from

the recognition of P-gp and thus enhances the oral bioavail-

ability of taxoid, at the same time all complications of

parenteral administration also circumvent by this novel

approach (Dasgupta et al., 2013). Into the bargain, NEs

could shield integrated drug molecules from the gastric

degradation, gut wall metabolism and also prevent the first-

pass metabolism. NEs are comparatively stable bi-/multi-

phasic systems than liposomes, ethosomes or microspheres

with additional advantage of improved solubility as well as

absorption of poor bioavailable drugs (Gupta et al., 2005;

Chaurasia et al., 2008, Chourasia et al., 2011).

Based on our hypothesis and the features associated with

NEs, a NE delivery system for DTX (model drug representing

taxoids) has been proposed in the present research work. DTX

(C43H53NO14) is a semi-synthetic analogue of paclitaxel

having very poor water solubility (4.93 mg/mL) (Gao et al.,

2008; Yin et al., 2009). Because of low water solubility

dissolution is the rate limiting step in the absorption and

bioavailability of DTX. For the development of DTX NE,

soybean oil was employed as a solubilizer of drug, Pluronic

F68 and lecithin as surfactant, whereas ethanol and PEG 4000

were used as co-surfactant and stabilizer respectively. The

developed NEs were characterized for different in vitro and

in vivo parameters as well as subjected to stability studies.

The objective of this research work was to develop stable,

effective and safe NEs containing DTX by utilizing drug

delivery approach with broad aim to improve the chemother-

apy of cancer.

Materials and methods

Materials

Chemicals and biochemicals

Docetaxel was generously provided by Intas Pharma,

Ahmedabad, India as a gift sample. Poloxamer (Pluronic

F68), soya lecithin, Polyethylene glycol (PEG) 4000, soybean

oil, bovine serum albumin (BSA), MTT (3-[4, 5-dimethylthia-

zol-2-yl]-2, 5-diphenyltetrazolium bromide), DMEM and all

other chemicals, bio-chemicals and solvents were purchased

from Sigma-Aldrich, (St. Louis, MO). Ultrapure water

obtained by Millipore Milli-Q system (Bedford, MA) was

utilized throughout the experiments. Analytical reagent grade

chemicals and ingredients were used in the present studies as

received from the distributor/supplier without any modifica-

tion/alteration.

Laboratory animals and cell lines

MCF-7 cell lines procured from Microbial Type Culture

Collection and Gene Bank, Chandigarh, India were used for

in vitro cytotoxicity studies. Cell lines were maintained in

RPMI-1640 medium supplemented with 10% heat inactivated

fetal bovine serum, 100mg/mL streptomycin and 100m/mL

penicillin at 37 ± 2 �C in humidified atmosphere (5% CO2/air

mixture). Female Swiss mice weighing 30–35 g were obtained

from institutional animal house (CSIR-CDRI, Lucknow,

India) for in vivo acute toxicity studies. In vivo studies were

carried out with prior approval of the Animal

Ethics Committee of Central Drug Research Institute,

Lucknow. All animal experimentations were conducted

according to the guidelines of the Council for the Purpose

of Control and Supervision of Experiments on Animals

(CPCSEA), Ministry of Social Justice and Empowerment,

Government of India.

Methods

Preparation of NEs

NEs were prepared by a prototype hot homogenization

followed by ultra-sonication method (Sakulku et al., 2009).

Briefly, DTX was dissolved in soybean oil by sonication

(Vibra-Cell, Sonics) with moderate heating to get a clear

solution. The aqueous phase was prepared by mixing ethanol

and water, in which PEG 4000 was dispersed. Pluronic F68

and/or lecithin were dispersed in aqueous phase, which was

added drop wise to the oil phase under stirring

(25000 ± 200 rpm, Ultra-Turax T25, IKA, Germany) for

30 min in order to get a coarse emulsion. Afterwards, the

coarse emulsion was homogenized (Vibra-Cell, Sonics, USA)

to get the NE. Placebo NEs were prepared by the same

procedure omitting drug. The developed NEs were stored at

room temperature (22 ± 1 �C) until used for further

characterizations.

Optimization of NEs

The effect of formulation variables viz. type of surfactant, oil,

co-surfactant, and surfactant mixture (surfactant and

co-surfactant) ratio on NEs and their characteristics were

studied. Optimization of NEs was done with monitoring of

some selected parameters viz. droplet size, zeta potential and

drug content (Muller et al., 2012).

Characterization of NEs

NEs were characterized for various in vitro and in vivo

parameters viz. size, shape, surface morphology, charge (zeta

potential), drug content, in vitro drug release, in vitro cell

uptake, in vitro cytotoxic activity, in vivo acute toxicity and

stability studies.

2 P. Verma et al. Drug Deliv, Early Online: 1–10

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Droplet size and zeta potential

The mean droplet size and size distribution were measured by

photon correlation spectroscopy with a Zetasizer NanoZS

(Malvern, UK) at 22 ± 1 �C (Oh et al., 2011). DTX NE

samples were diluted to the appropriate concentration with

distilled water before measurement, filled in the cuvette and

measured for the mentioned parameters. The size distribution

of NEs droplets was represented by the polydispersity index

(PDI) values. Zeta potential was determined using electro-

phoretic mobility with laser-based multiple-angle particle

electrophoresis analyzer at 22 ± 1 �C (Zetasizer Nano-ZS,

Malvern, UK) (Oh et al., 2011).

Droplet shape and surface morphology

Transmission electron microscopy (TEM; Tecnai� G2 F20,

Eindhoven, Netharlands) was employed for the analysis of

shape and surface morphological characteristic of DTX NEs.

NE sample was prepared as a thin aqueous film held on a

300-mesh copper grid. A droplet of 2% (w/v) phosphotungstic

acid was used for negative staining and excess was drawn off.

Samples were dried and observed by TEM and images were

taken at a 200 kV acceleration voltage and recorded for

interpretation (He et al., 2013).

Drug content

High performance liquid chromatography (LC-10ATvp HPLC

instrument, Tokyo, Japan) with Lichrosphere reverse-phase

C18 column (250� 4 mm, 5 mm; Merck, Darmstadt,

Germany) was employed for the determination of quantity

of DTX in DTX NEs (Asthana et al., 2013). In brief,

accurately measured quantity of DTX NEs was dissolved in

0.5 mL dichloromethane (DCM) and mixed with 2 mL of

mobile phase (acetonitrile: deionized water; 65:35, v/v). The

above mixture was vortexed for 5 min, and DCM was

evaporated under a nitrogen stream until a clear solution

was obtained. The solution was analyzed by HPLC after

filtering through 0.45mm syringe filter. The flow rate of

mobile phase was set at 1.0 mL/min and column effluent was

detected with a UV detector at 230 nm. DTX was eluted

with mobile phase at 3.2 min. Linearity of response for

DTX quantization was verified in the concentration range

400–64 000 ng/mL with a correlation coefficient (R2) 0.998.

In vitro drug release

The in vitro drug release study was performed using dialysis

membrane diffusion method (Jiang et al., 2013). Accurately

the measured amount of DTX NE was filled in a dialysis bag

(cellulose membrane, MWCO: 12 KDa), sealed at both ends

and suspended in dissolution apparatus (DISSO 2000,

Labindia, India) containing 250 mL of phosphate buffer

saline (PBS; pH 7.4) with 0.1% w/v Tween 80 in order to

maintain sink conditions. Temperature of dissolution medium

was maintained at 37 ± 1 �C with moderate shaking at

100 ± 5 rpm. At predetermined time intervals, 1 mL of the

release medium was withdrawn and replaced with equal

volume of fresh pre-warmed medium. The withdrawn samples

were diluted and analyzed for the drug content by developed

HPLC method at 230 nm.

Stability studies

DTX NEs (F11) were stored in screw capped, amber colored

small glass bottles at 4 ± 1 and 22 ± 1�C temperature.

Samples were analyzed for droplet size and residual drug

content at time intervals of 10, 20, 30, 60 and 90 days (Kong

& Park, 2011).

In vitro cell uptake studies

A qualitative estimation was performed using fluorescence

activated cell sorting method (FACS) to depict the cell uptake

affinity of the developed NEs (Liu et al., 2011). MCF-7 cells

were seeded at a density of 2� 105 cells/well in a 6-well plate

and allowed to adhere over night with surface of the culture

plate. After 24 h, old media was exchanged with fresh media

containing FITC loaded NE and allowed incubation in a

controlled environment overnight. Subsequently, cells were

washed three times with PBS to remove any surface adhered

formulation. Washed cells were suspended in PBS and cell

assisted florescence was measured using flow cytometer (BD

Biosciences, FACS Aria, Germany) at an excitation wave-

length of 485 nm and an emission wavelength of 538 nm.

In vitro cytotoxicity studies

Cancer cell viability affected by DTX NEs and Taxotere

(marketed formulation) was evaluated using the 3-[4, 5-

dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide

(MTT) assay (He et al., 2013). Briefly, 104 MCF-7 cells/

well were seeded in 100 mL DMEM, supplemented with 10%

FBS in each well of 96-well micro culture plates and

incubated for 24 h at 37 ± 1 �C in a CO2 incubator till 70%

confluence was reached. Cells were exposed to various

concentrations of DTX NEs, Taxotere and free drug (DTX) at

5, 10, 15, 20, 25, 30 mg/mL equivalent drug concentrations as

well as placebo NE for 24 and 72 h. At predetermined time,

the formulations were replaced with DMEM containing MTT

(500 mg/mL) and cells were incubated for additional 4 h. MTT

was aspirated off and dimethylsulfoxide (200mL) was added

to dissolve the formed crystals. Absorbance was measured at

570 nm by multi-well microplate reader (BIO-TEK, Model-

Power wave XS, Crailsheim, Germany). Cell death (%) was

expressed as the percentage of absorbance of transformed

MTT in cells incubated with formulations relative to that of

the untreated cells as control. The IC50 values were calculated

graphically (logarithmic scale) by plotting percent cell death

versus concentration.

In vivo acute toxicity studies

Female Swiss mice weighing 30–35 g were randomly grouped

(6 mice/group) into (1) DTX NEs treated, (2) free drug (DTX)

treated, (3) placebo NEs treated and (4) untreated control

group. Before administration, free drug (DTX), DTX NEs and

placebo NEs were re-suspended separately in purified water

to reach the desired concentration (20 mg/kg). Mice in all the

4 groups were orally administered with respective formula-

tions. Body weight and behavior of the animals were observed

and recorded throughout the acute toxicity studies. After

14 days animals were sacrificed and kidney and liver tissues

were isolated, fixed in 10% formalin. The tissues were

DOI: 10.3109/10717544.2014.920430 Nanoemulsion assisted oral delivery of docetaxel 3

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embedded in paraffin and sectioned as well as stained with

haematoxylin and eosin for histo-pathological analysis. The

sections were observed under an optical microscope (Eclipse

50i, Nikon, kawasaki, Kanagawa, Japan) and photomicro-

graphs of the sections, were recorded for comparison and

interpretation of acute toxicity (Tang et al., 2012).

Statistical analysis

Values of experimental results are expressed as mean ± SEM

(n¼ 3). One way ANOVA was employed to investigate any

significant difference in evaluation parameters (particle size,

PDI, zeta potential, in vitro drug release, in vitro cell uptake

and in vitro cytotoxicity evaluation) and student’s t test was

used to analyze any statistical difference in evaluation

parameters (particle size and drug content) during stability

studies, using GraphPad Prism 5 (Version 5.01) statistical

software.

Results and discussion

The objective of the present investigation was to develop NEs

of DTX which could ensure better therapeutic efficacy. NEs

were successfully developed and characterized for different

in vitro, in vivo parameters as well as subjected to stability

analysis. Developed NEs were optimized on the basis of

droplet size, zeta potential and drug content which were

influenced by the surfactant, co-surfactant and oil used in the

formulation development. The optimized NEs were evaluated

for cytotoxic potential using MCF-7 cancer cell lines and the

in vitro cell uptake as well as in vivo toxicity aspects were

also experimentally scrutinized.

Formulation and optimization of NEs

Pharmaceutical ingredients and their ratio used in the

formulation of NEs are depicted in Table 1. Five different

series of NEs (F1-F3, F4-F5, F6-F7, F8-F9 and F10-F11)

were formulated by varying the surfactants/co-surfactant/oil/

drug concentration. In the first two series the concentration of

soybean oil and ethanol was fixed whereas lecithin and

Pluronic F68 were varied. In the third and fourth series,

concentration of soybean oil and ethanol were altered but

lecithin and Pluronic F68 were kept constant. In the last

i.e. fifth series of NEs, the concentration of DTX was varied

keeping other ingredients unchanged as F1. The changes in

the ingredients/drug were not random, but guided by the

characterization parameters viz. droplet size, charge as well as

drug content. In the subsequent section (3.2 characterizations

of NEs) the optimization of NEs as directed by the aforesaid

parameters is discussed ins and outs.

Characterization of NEs

Parameters influencing thermodynamic activity viz. droplet

size, PDI and zeta potential are very much crucial in the

formulation of NEs (Muller et al., 2012). The results of these

characterization parameters are shown in Table 2. Among the

above said parameters, droplet size of the NEs is central

protagonist, as it regulates the rate and extent of drug release

as well as absorption. Drug can diffuse faster from smaller

droplets into the aqueous phase, thereby increasing the drug

dissolution. Moreover, smaller droplet provides larger surface

area for drug absorption. Reduction in droplet size and PDI

improves bio-availability of emulsion as compared to a coarse

emulsion (Asthana et al., 2013).

For the qualitative selection of surfactant and

co-surfactant, NEs were prepared, in which one of the

ingredients was omitted and other ingredients were kept

constant. As discussed in the formulation and optimization

(section 3.1), in formulation F1, the surfactants lecithin:

Pluronic F68 (1:1) and co-surfactant – PEG 4000 were taken

in equal amount. In F2, Pluronic F68 was excluded and other

contents were kept constant as in F1 whereas in F3, PEG-400

has been skipped keeping rest of the excipients unaltered as in

F1. Between these three NEs only F1 (lecithin: Pluronic F68:

PEG in 1:1:1; Table 1) passed the stability test and was of

smallest size (252.93 ± 7.4 nm; Table 2). There was signifi-

cant difference (p50.05) in the particle size, PDI, and zeta

potential of these formulations. Based on the outcomes,

lecithin, Pluronic F68 and PEG 4000 were included for

further optimization study in next series of NEs. NEs without

Pluronic (F2) was degraded few minutes after preparation,

Table 1. Composition of the NE formulations.

Formulationcodea

Soya beanoil (mL)

Egg lecithin(mg)

PluronicF68 (mg)

PEG 4000(mg)

Ethanol(mL)

Docetaxel(mg)

Water(mL)

First series Quantity sufficient (up to 10 mL)F1 2 100 100 100 3 0.5F2 2 100 – 100 3 0.5F3 2 100 100 – 3 0.5

Second seriesF4 2 50 50 100 3 0.5F5 2 100 100 50 3 0.5

Third seriesF6 1 100 100 50 3 0.5F7 3 100 100 50 3 0.5

Fourth seriesF8 2 100 100 50 2 0.5F9 2 100 100 50 4 0.5

Fifth seriesF10 2 100 100 100 3 1F11 2 100 100 100 3 10

aplacebo NE for each series were prepared.

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which might be attributed to the inadequate balance of

surfactant-PEG combination in NE. On the contrary, F3 NE,

which was devoid of PEG was prepared, however the

formulation was appeared to be unstable upon storage. Any

bi/multi-phasic system requires a specific hydrophilic lipo-

philic balance (HLB) in order to attain stability for a definite

period of time and a combination of surfactants in specific

ratio could achieve stability by reducing the interfacial

tension of immiscible phages (Meher et al., 2013; Yadav

et al., 2013). In the present formulation, a combination of

lecithin, Pluronic and PEG in the same ratio was indeed

required to develop a stable NE containing soybean oil and

DTX. Hosny et al. and Brusewitz et al. have employed PEG

and Poloxamer in the development of nanoemulsion and

reported these to be effective in maintaining the stability of

the nanoemulsions (Brusewitz et al., 2007; Hosny & Banjar,

2013).

Furthermore, for the quantitative optimization of the

surfactant and co-surfactant ratio, two NE formulations

(F4 and F5) were prepared, where F5 (droplet size;

264.50 ± 4.3 nm, PDI; 0.25 ± 0.005; Table 2) was found to

be superlative between these formulations. Observation of

particle size and PDI indicated that higher concentration of

PEG was not in favor of stable NE, rather less i.e. half

concentration of PEG and equal ratio of surfactant (lecithin

and Pluronic, Table 1) were suitable for the development of a

stable NE. This behavior of NEs was attributed to the

thermodynamic commotion of bi/multi-phasic system and

surfactant-co-surfactant ratio (Dasgupta et al., 2013). PEG

4000 is one of the commonly used excipients of generally

recognized as safe (GRAS) category which has got wide

application as co-surfactant, solubilizer, co-solvent as well as

drug-targeting agent like chitosan, PLGA and Azo polymers

etc. (Jain et al., 2005; Cheng et al., 2007). In the current

formulation PEG exhibited promising activity in stabilization

of emulsion.

In next series (3rd series) of formulations, concentration of

soybean oil and stability of NEs were examined. Soybean oil

is an edible oil and has been used in the preparation of

pharmaceutical formulations as solubilizer of drug molecules

(Bilbao-Sainz et al., 2010; He et al., 2011). In the present

study it was employed as the solubilizer of DTX.

In formulation F6 and F7, the concentration of oil was

1 and 3 mL, respectively (Table 1). Apart from the stability,

drug content was another parameter which was optimized

for these formulations. Results of these parameters are

exhibited in Table 2. Between F5, F6 and F7 NEs, F5 was

found to display higher content of the drug (82.30 ± 1.2%).

NE F6 showed lower drug content (75.38 ± 1.8%) as

compared to F5 but it could pass the stability evaluation.

On the other hand F7 was degraded out after few hours of

preparation, which was assumed due to higher concentration

of soybean oil (3 mL) leading to imbalance in HLB require-

ment of NE. Based on these results, NE F5 was further

continued in the optimization process in order to get the best

formulation.

The next excipient to be optimized was ethanol, which was

playing the role of a co-surfactant. In the 4th series of NE

formulation, F8 and F9 were prepared containing 2 and 4 mL

of ethanol, respectively. Both of these NEs were degraded in

the stability evaluation. The role of co-surfactant is to impart

either rigidity or flexibility to the tight layer of surfactant-

co-surfactant mixture surrounding the oil droplets in the NEs

(Peng et al., 2010). As the HLB requirement of a particular oil

(mixture of oils) is very much specific, any fluctuation in the

same may disrupt the thermodynamic stability causing

degradation of NEs (Hosny & Banjar, 2013). Here, in this

case the change in concentration of ethanol caused a change

in the required HLB by increasing the hydrophilicity and led

to destabilization of F8 and F9.

So far, the NEs F1, F4, F5 and F6 could clear the stability

tests showing thermodynamic steadiness. There was signifi-

cant difference (P50.05) in thermodynamic parameters

(particle size, PDI and zeta potential) and in vitro drug

release of the above NEs. Among these NEs, F5 and F6

demonstrated excellent results as far as thermodynamic

parameters were concerned. But as and when, the other

parameters viz. drug content and in vitro drug release were

taken into consideration, F1 dominated both F5 and F6

(Table 2). Based on the above discussion, F1 was selected as

the optimized NE and further, concentration of drug was

attempted to be optimized.

Table 2. Physicochemical parameters of the DTX NEs.

Formulation code Particle size (nm) Zeta potential (mV) PDI Stability status Drug Content (%) Drug Releasea (%)

First seriesF1 252.93 ± 7.4 �46.16 ± 2.2 0.34 ± 0.019 Pass 97.53 ± 0.9 94.64 ± 1.3F2 ND ND ND Fail ND NDF3 277.30 ± 5.5 �44.86 ± 2.0 0.34 ± 0.009 Fail ND ND

Second seriesF4 286.66 ± 3.8 �35.90 ± 2.5 0.36 ± 0.008 Pass 80.40 ± 1.3 71.73 ± 3.0F5 264.50 ± 4.3 �35.13 ± 1.8 0.25 ± 0.005 Pass 82.30 ± 1.2 81.81 ± 1.5

Third seriesF6 228.56 ± 4.2 �41.11 ± 0.9 0.16 ± 0.008 Pass 75.38 ± 1.8 59.68 ± 5.2F7 229.03 ± 5.6 �45.13 ± 1.6 0.20 ± 0.009 Fail 71.96 ± 1.1 ND

Fourth seriesF8 275.40 ± 5.0 �54.83 ± 1.8 0.15 ± 0.009 Fail 41.46 ± 2.4 NDF9 385.90 ± 8.1 �53.73 ± 1.3 0.40 ± 0.006 Fail 45.51 ± 1.7 ND

Fifth seriesF10 241.50 ± 3.9 �45.00 ± 1.5 0.28 ± 0.006 Pass 96.57 ± 0.9 94.46 ± 2.4F11 233.23 ± 4.3 �43.66 ± 1.9 0.24 ± 0.010 Pass 96.76 ± 1.5 96.25 ± 2.1

Mean ± SEM (n¼ 3), PDI; Poly dispersity index; ain vitro drug release at 12 h, ND; not determined.

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In the 5th series of NE formulations, F10 and F11 were

prepared where concentration of drug was 1.0 and 10.0 mg/

mL respectively. F11 exhibited smaller droplet size

(233.23 ± 4.3 nm), lower PDI (0.24 ± 0.010) and higher zeta

potential (�43.66 ± 1.9 mV) in comparison to F10 (Table 2).

Although there were no significant difference in the thermo-

dynamic profile of F11 and F10, the higher accommodation

of DTX suggested F11 NEs to be the optimized formulation.

The percent drug release from F11 formulation was found to

be 96.25 ± 2.1% and drug content was determined to be

96.76 ± 1.5%. The drug release mechanism was found to be

zero order and non-fickian diffusion controlled. The drug

release kinetics data of F1, F4, F5, F6, F10 and F11 NEs

[those passed stability test (Table 2)] is presented in Table 3.

Finally, optimized NE F11 was characterized for shape and

surface morphology, in vitro cytotoxic potential, cell uptake

in animal cell lines and in vivo acute toxicity studies in mice.

The NE (F11) appeared bright and the surrounding was dark

in TEM as it was clear from the image (Figure 1).

The droplets were clearly visible as a poly-dispersed system

of emulsified soybean oil (Figure 1A). Some equally

distributed droplet sizes were measured using TEM, as it

was capable of point to point resolution. Figure 1(B) exhibits

the magnified view with droplet size. The droplet size is in

agreement with the results obtained from droplet size analysis

(Table 2). TEM analysis ensured the development of a

polydisperse NE.

Stability of NEs

Stability of a product during its self-life is an important aspect

in the successful development of dosage form (Singh et al.,

2013). Hence, a well-designed stability-testing plan is essen-

tial for the development of NE. NEs were stored at two

different storage conditions i.e. 4 ± 1 and 22 ± 1 �C and were

subjected to stability studies. Droplet size and residual drug

content upon aging were selected as the evaluation criteria for

the stability analysis. Mean droplet size of NEs was found to

increase on storage, which could be due to coalescence of oil

droplets over time. Figure 2(A and B) demonstrate the pattern

of increasing NE droplet size upon storage at 4 ± 1 �C and

Figure 2. Plots showing effect of storage at (A) 4 ± 1 �C and (B) 22 ± 1 �C on particle size and drug content of F11 NE.

Figure 1. TEM photomicrograph of DXT NEs (A) broad view (B) magnified view.

Table 3. Tumor cells inhibition activity of NE and Taxotere after 24 and72 h incubation with MCF cells in 96-well plate.

IC50 (mg/mL)a DTX NE (F11) Taxotere Free Drug (DTX)

After 24 h incubation 23.19 ± 0.12 17.38 ± 0.65 18.88 ± 0.38After 72 h incubationb 13.55 ± 0.21 16.56 ± 0.34 17.11 ± 0.16

Results represented in terms of IC50; calculated from percent cell deathagainst various equivalent concentration of DTX represented viadeveloped NE and marketed formulation.

aMean ± S.E.M. (n¼ 3).bSignificant difference (p50.05) between DTX NE versus Taxotere and

DTX NE versus free drug (DTX).

6 P. Verma et al. Drug Deliv, Early Online: 1–10

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22 ± 1 �C, respectively. There was a significant difference

(p50.05) in the mean droplet size of NEs (F11) at these two

different storage conditions. Such behavior leading to

thermodynamic instability was observed to be least with

NEs stored at 22 ± 1 �C, which indicated that coalescence

could be controlled by storage of formulation at this

temperature. The NEs stored at 4 ± 1 �C exhibited higher

increase in droplet size (coalescence) which might be

attributed to the fact that at low temperature, NE droplets

tend to aggregate/coalesce (Rahn-Chique et al., 2012). Jiang

et al., have reported an increase in particle size up to 100 nm

at room temperature in a three months stability study of

nanoemulsion but they did not find any variation in the

entrapment efficiency (Jiang et al., 2013). Percent of residual

drug remaining in NEs over storage was investigated by

determining the drug content at different time intervals as

described in the section 2.2.3.5. In both the storage conditions

a decrease in drug content was detected, however no

significant difference (p50.05) was found in the residual

drug content on aging at these two different storage condi-

tions. Figures 2(A) and (B) exhibit the residual drug content

at 4 ± 1 �C and 22 ± 1 �C respectively. It was comprehended

that after 90 days of storage, NEs stored at 4 ± 1 �C illustrated

86.93 ± 2.13% drug whereas 91.1 ± 0.96% was found with

NEs stored at 22 ± 1 �C. Based on the results obtained from

stability analysis, NE F11 stored at 22 ± 1 �C was found to be

comparatively stable and further subjected to in vitro cell

uptake, cytotoxicity and acute toxicity studies.

Cell uptake and cytotoxicity

The qualitative NE ingestion capability via MCF-7 cells was

evaluated using FACS. Results demonstrated a 2.83 times

higher mean florescence intensity of FITC loaded NE in

comparison to control cells. FACS histogram and mean

florescence intensity of control NE are represented in

Figure 3. Higher mean florescence intensity signifies higher

internalization of the fluorescence tagged moieties. The

internalization of the NEs would be processed through core

triglyceride (soybean oil) which undergoes a partially solu-

bilization upon contact with cellular phospholipid layer. Acyl

chains of triglyceride cause a partial penetration into the

hydrophobic domain of the surface phospholipid monolayers

resulting in separation of surface head group as to engage

Figure 3. Cell uptake by flow cytometer (A) FACS histogram showing uptake of NE in MCF-7 cells. (B) Mean florescence intensity of control andNE; *a significant difference was noticed at level p50.01.

Figure 4. Cytotoxic effect of developed and marketed formulation in terms of percent cell death. Cytotoxic potential after (A) 24 h and (B) 72 hincubation of DTX NE, Taxotere, free drug (DTX) and placebo NE with MCF cells.

DOI: 10.3109/10717544.2014.920430 Nanoemulsion assisted oral delivery of docetaxel 7

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Figure 5. Acute toxicity in animals. (A) Percent body weight change in mice after treatment with DTX NEs, placebo NE, Taxotere and free drug(DTX). (B) Representative photographs showing histopathology of liver and kidney tissues of mice after 14 days of treatment (dose 20 mg/kg) DXTNEs treated (a) liver, (b) kidney, free drug (DXT) treated (c) liver, (d) kidney, placebo NE treated (e) liver, (f) kidney.

8 P. Verma et al. Drug Deliv, Early Online: 1–10

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space for apoprotein binding (Saito et al., 2001; Tanaka et al.,

2003).

In order to confirm the cytotoxic potential of the NEs MTT

assay was performed employing MCF-7 cancer cell line.

A comparative cytotoxic effects of DTX NEs (F11), DTX and

Taxotere at 5, 10, 15, 20, 25, 30 mg/mL equivalent drug

concentrations as well as placebo NE were evaluated.

Figure 4(A and B) illustrate the percent cell death of MCF-

7 cells following incubation for 24 and 72 h, respectively.

There was significant difference (p50.05) in the percent cell

death of placebo NE, DTX NE (F11), Taxotere and free drug

(DTX). Experimental findings clearly suggested an aug-

mented reduction in viability of MCF-7 cells with increase in

drug concentration. Cytotoxicity of DTX NE was found to be

increased with rise in drug concentration but after the initial

incubation period (24 h) it was found to be less than both free

DTX and Taxotere. Similarly after 72 h of incubation

cytotoxicity of DTX NE was observed to be increasing but

higher cytotoxicity was noticed with DTX NE in comparison

to the free DTX and Taxotere. Such behavior of DTX NE

might be attributed to the sustained release of DTX from NE,

which is in agreement with the drug release kinetics of DTX

NEs (Table 3). The IC50 values further support the results that

DTX NE exhibited IC50 13.55 ± 0.21mg/mL, whereas

Taxotere and free drug (DTX) showed IC50 16.56 ± 0.34

and 17.11 ± 0.16mg/mL respectively. These results suggested

stronger cytotoxic activity of DTX NE in comparison to both

marketed formulation Taxotere and free drug (DTX), which in

turn indicate less drug is required for cytotoxic action in

NE formulations. Current findings are in agreement with

previously reported NEs, liposomes and nanoparticles,

advocating higher cytotoxic effect of these delivery systems

than the conventional systems (Dıaz et al., 2006; Choudhury

et al., 2014).

Acute toxicity

Many authors have demonstrated acute toxicity of nano-

particulate formulations. Such toxicity is due to the accumu-

lation of nanoparticles in some specific tissues (Beer et al.,

2012; Hu et al., 2012). In order to verify the toxicity potential

of DTX NE (F11), it was exposed to in vivo environment in

mice. The behavior of all the animals was found normal

throughout the study however, a significant variation

(p50.05) was observed in the body weights of the animals

treated with free drug (DTX) and DTX NE. Figure 5 displays

the percent body weight change in mice after treatment with

DTX NEs, placebo NE, Taxotere and free drug (DTX). Based

on the findings of histo-pathological studies, no particular

toxicity and/or necrosis were observed in liver and kidney

tissues with single dose of DTX NEs (Figure 5a and b), which

makes ground to claim that developed NE can be safely used

in vivo at concentration up to 20 mg/kg body weight. On the

contrary examination of liver tissues in free drug (DTX)

treated group revealed focal lesions surrounding a cluster of

necrotic hepatocytes, isolated foci of necrotic hepatocytes and

hepatocytes in the centrilobular zone showing degenerative

features (Figure 5c). Moreover, histo-pathological analysis of

kidney tissue treated with free drug (DTX) showed patchy

tubular epithelial necrosis that varied in extent from focal to

extensive (Figure 5d). The untreated group exhibited no sign

of any of the above mentioned abnormalities either in liver

(Figure 5e) or kidney (Figure 5f). The excipients used in NE

formulations are of GRAS category and hence the toxicity

seen in the free drug treated group is due to the DTX.

Comparatively lower toxicity of NEs is also reported by

several authors which is attributed to controlled exposition of

drug to tissues (Borhade et al., 2012; Asthana et al., 2013).

Conclusion

Stable (up to three months) NEs containing DTX (10 mg) was

successfully formulated with lecithin, Pluronic and PEG in

equal ratio (1:1:1) in combination with soybean oil (2 mL) and

ethanol (3 mL). In vitro cytotoxicity evaluation in MCF-7

cancer cell lines ensured the selective uptake of DTX NEs

to cells and exhibited promising cytotoxicity (IC50

13.55 ± 0.21 mg/mL) corroborating it to be effective. In vivo

acute tissue toxicity in mice demonstrated not any specific

toxicity and/or necrosis in the liver and kidney tissues with

single dose of DTX NEs (520 mg/kg), supporting it to be safe

for use.

In conclusion a stable, effective and safe NE was

formulated as well as characterized, however the clinical

investigations and scale up issues remain as the future scope

of present investigation.

Acknowledgements

Authors are thankful to SAIF, CSIR-Central Drug Research

Institute, Lucknow for providing facilities to perform cell

uptake using FACS. Authors extend their sincere thanks to

CSIR for granting financial assistance (network project

CSC0302) to the present investigation. Intas Pharma,

Ahmedabad, India is duly acknowledged for providing

docetaxel drug as gift sample.

Declaration of interest

All authors involved in the present research work declare no

conflict of interest. CSIR-CDRI Communication No.: 8677.

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