QbD-based development of α-linolenic acid potentiated nanoemulsion for targeted delivery of doxorubicin in DMBA-induced mammary gland carcinoma: in vitro and in vivo evaluation

Article abstract of DOI:10.1007/s13346-018-0525-5

Breast cancer is the most common cancer of occurrence in women and has the highest mortality incidence rate therein. The present study envisaged to develop doxorubicin (Dox) loaded folate functionalized nanoemulsion (NE) for profound therapeutic activity

Full text of DOI:10.1007/s13346-018-0525-5

Drug Delivery and Translational Research
https://doi.org/10.1007/s13346-018-0525-5
ORIGINAL ARTICLE
QbD-based development of α-linolenic acid potentiated nanoemulsion
for targeted delivery of doxorubicin in DMBA-induced mammary gland
carcinoma: in vitro and in vivo evaluation
Chandra Bhushan Tripathi1 & Poonam Parashar1 & Malti Arya1 & Mahendra Singh1 & Jovita Kanoujia1
Gaurav Kaithwas1 & Shubhini A. Saraf1
&
#
Controlled Release Society 2018
Abstract
Breast cancer is the most common cancer of occurrence in women and has the highest mortality incidence rate therein. The
present study envisaged to develop doxorubicin (Dox) loaded folate functionalized nanoemulsion (NE) for profound therapeutic
activity against mammary gland cancer. NE was prepared using pseudo-ternary phase diagrams utilizing α-linolenic acid (ALA)
as lipid phase, to further enhance the anticancer potential of Dox. Box-Behnken design was employed to systematically develop
the NE. Optimized NE (f-Dox-NE) was evaluated for in vitro and in vivo performance. f-Dox-NE, with globule size 55.2
3.3 nm, zeta potential − 31 2 mV, entrapment 92.51 3.62%, drug loading 0.42 0.08% and percent drug release 94.86
1.87% in 72 h, was capable of reducing cell viability in MCF-7 cell lines vis-à-vis pure and marketed drug. Further, mechanistic
studies in MCF-7 cell lines demonstrated that f-Dox-NE induces cellular apoptosis by reactive oxygen species generated and
mitochondrial membrane mediated apoptosis. The antitumor effect was evaluated in 7,12-dimethylbenz[a]anthracene (DMBA)
induced mammary gland tumor in female Albino Wistar rats. f-Dox-NE exhibited enhanced antitumor targeting potential,
therapeutic safety and efficacy vis-à-vis pure and marketed drug, as revealed by tumor volume, animal survival, weight variation,
cardiotoxicity and biodistribution studies. f-Dox-NE restored the biochemical parameters viz., SOD, catalase, TBARS and
protein carbonyl, towards normal levels in comparison to DMBA induced animal group. f-Dox-NE displayed downregulation
of anti-apoptotic (Bcl-2 and MMP-9) proteins and upregulation of pro-apoptotic proteins (caspase-9 and BAX). The experimen-
tal results suggest that ALA augmented folate functionalized NE are able to overcome the challenges of developing safe and
effective delivery system with enhanced potential for mammary gland carcinoma therapy.
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Keywords Doxorubicin α-linolenic acid Nanoemulsion MCF-7 cell lines Western blot Anticancer
Introduction
pollution, presence of carcinogen in environment, radiation,
obesity, hormonal imbalance, development of drug resistance,
lack of specific targeted therapies etc. among other factors.
Lifestyle habits such as physical inactivity, smoking, poor
diet, and reproductive changes have further enhanced the tu-
mor burden [1, 2]. Lung cancer has surpassed the incidences
of breast cancer; however, mammary gland carcinoma re-
mains the leading factor for cancer deaths in females [1–4].
Doxorubicin (Dox) is an anthracycline anticancer drug ob-
tained from the fungus Streptocococcus peuceticus var.
caesius. It is one of the most potent chemotherapeutic agent
among modern day breast anticancer therapies. Anticancer
activity of Dox is due to its activity against topoisomerase II
which plays a vital role in the negative supercoiling of DNA.
Dox intercalates into the DNA causing the alteration in ste-
reochemical conformation of the double-helical structure.
Nowadays, tumor is one of the most prominent causes of
death in developing as well as developed countries. The bur-
den is expected to amplify owing to the environmental
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s13346-018-0525-5) contains supplementary
material, which is available to authorized users.
* Shubhini A. Saraf
shubhini.saraf@gmail.com
1
Department of Pharmaceutical Sciences, School of Biosciences &
Biotechnology, Babasaheb Bhimrao Ambedkar University (A
Central University), Vidya Vihar, Rae Bareli Road,
Lucknow 226025, India

Drug Deliv. and Transl. Res.
Therefore, Dox inhibits DNA and RNA polymerases and ob-
structs the DNA replication and transcription [5]. There are
evidences that the treatment with Dox initiates cell signaling
pathways that lead to programmed cell death known as apo-
ptosis. The cellular proteins like Bcl-2, BAX, BAD, MMP-9,
caspase-9 etc., play an important role in the regulation of
apoptosis [6–9]. Over-expression of anti-apoptotic proteins
Bcl-2 [6] and MMP-9 [8] result in blockage of apoptosis; in
contrast, BAX [7] and caspase-9 [9] serve as pro-apoptotic
proteins that block the anti-apoptotic proteins, thereby, induc-
ing apoptosis. In addition, it has been demonstrated that the
high levels of Bcl-2 and MMP-9 induce cancer cell migration,
invasion, and cancer metastasis in mammary gland carcinoma
[6–8]. Dox is one of the most effective anticancer agents but
its clinical use is limited due to its severe cardiotoxicity owing
to oxidative stress, mitochondrial impairment, cell necrosis
and induction of apoptosis through the activation of both in-
trinsic and extrinsic pathways [5].
α-linolenic acid (ALA) is an ω-3 fatty acid which is an
essential polyunsaturated fatty acid (PUFA). It cannot be pro-
duced by the body and must be supplemented through dietary
resources. ALA is finally metabolized to eicosapentanoic acid
(EPA) and docosahexaenoic acid (DHA) through desaturation
and elongation reactions of its fatty acid chain [10]. EPA and
DHA are vital in modulating the cell membrane fluidity
functions of the cellular proteins, gene expressions and cell
signaling mechanisms. It has been established that ALA
reduces the progression of mammary gland carcinoma,
curtails histological changes and improves the survival rate
in animal models. ALA actively suppresses the over-
expression of human epidermal growth factor receptor
(HER2+) and stabilizes hypoxia-inducible factor-1 (HIF-
1 α) and downregulates fatty acid synthase (FASN) to pro-
mote mitochondrial apoptosis and inhibit metastasis in
mammary gland carcinomas [10–12].
Of late, lipidic drug delivery systems have extensively
proved their worth as one of the highly promising systems
for delivering the drug to the designated locations. Among
the lipidic systems, lipid-based nanoemulsions are effectively
utilized to improve efficacy of the anticancer drugs and sub-
sequently reduced side effects [13]. These nanoemulsions are
relatively newer delivery systems prepared by using synthetic
as well as natural lipids and have proved to be the promising
vehicles owing to the sustained release of drug from lipid
matrix. Through these lipidic drug delivery systems, one can
also achieve targeted drug delivery due to the inherent safety
and non-irritability. Oil matrix of the system provides a better
medium for the solubility of hydrophobic drug, stable micro-
environment and prolonged delivery of the drug moiety to the
specific site [14, 15]. Development in the targeted delivery
approaches has further improved the potency of such systems
to deliver the drug molecules specifically to the cancer cells
thereby avoiding dose-related toxicity. Among the various
targeting approaches like antibodies, proteins, peptides,
nucleic acid-based ligand and small molecules, folate is one
of the safest targeting ligand [16, 17]. Folate is required for the
normal cellular metabolism and cell survival particularly dur-
ing the period of rapid cell division which happen in cancer-
ous cells. Folate receptors (FR) are therefore upregulated in
different type of cancers such as lung, colon, mammary gland,
kidney and brain. Normal cells have relatively lower FR levels
on their cell surface, making them an attractive target to de-
velop cancer specific drug therapy. Folate has high affinity
(Kd~10−10 M) for FR. Therefore, the folate decorated lipid
nanoemulsion can prove to be a potential delivery system
for selectively enabling drug delivery to FR-positive cancers
cells/tissue [18–21].
In the present study, ALA-based nanoemulsion (NE) was
envisaged and systematically developed using Quality by
Design (QbD) concept for the targeted delivery of Dox to the
DMBA-induced mammary gland carcinoma. ALA has been
reported to improve the cellular uptake of Dox and also to
enhance its cytotoxicity [22]. ALA has been reported to be
highly efficacious in modulating the progression of cancerous
cells in DMBA induced mammary gland carcinoma [10].
Further, the drug-induced cardiotoxicity is a major limiting
feature for clinical use of Dox which may be attenuated by
the concomitant administration of ALA. Till date, no reports
are available regarding the scientific and systematic study of a
formulation containing ALA as a therapeutic agent as well as
excipient and Dox as a drug. In the present study, it has been
envisaged to use ALA as the lipid phase along with lecithin
and Tween 80 as emulgents and cholesterol as a co-emulgent
to develop a NE that is capable of co-delivery of ALA and Dox
to the cancer cells. The NE was prepared by emulsification
followed by sonication process. NE was surface decorated with
folate as a targeting ligand with help of N-hydroxysuccinimide
(NHS) and N,N′-dicyclohexylcarbodiimide (DCC). The sur-
face decorated NE shall be characterized for physicochemical
properties like globule size, zeta potential, drug entrapment,
drug loading, drug release profile and TEM. Subsequently,
in vitro evaluations like cellular cytotoxicity, cellular prolifer-
ation, reactive oxygen species (ROS) generation, mitochondri-
al membrane potential (MMP) changes and cell cycle analysis
would be performed. Thereafter, the in vivo efficacy of the
surface decorated NE would evaluated in DMBA-induced
mammary gland carcinoma in female rats by monitoring the
tumor progression through animal survival, animal weight var-
iations, biochemical parameters i.e., thiobarbituric acid reac-
tive substances (TBARS), protein carbonyl, superoxide dis-
mutase (SOD), catalase and glutathione GSH levels as well
as carmine staining of the mammary glands, upregulation/
downregulation of various apoptotic regulators like B cell lym-
phoma 2 (Bcl-2), Bcl-2-associated X (BAX), caspase-9 and
matrix metalloproteinase-9 (MMP-9), cardiotoxicity and
biodistribution of the drug to different body organs.

Drug Deliv. and Transl. Res.
Experimental
Materials
Experimental design
On the basis of above data, the most suitable range of
critical material attributes (CMAs), viz., lipid (200–
600 mg), emulgent (500–900 mg), cholesterol (100–
200 mg) and aqueous phase (upto 2000 mg) was selected;
and 3-factor 3-level Box–Behnken design (BBD) was
employed for systematic optimization of NE [23]. A total
of 15 experimental trials loaded with drug in each were
formulated which were characterized for critical quality
attributes (CQAs), viz., globule size, drug loading, entrap-
ment efficiency and drug release in 72 h.
Doxorubicin was provided ex-gratis by M/s Miracalus
Pharma, India. Oleic acid, Isopropyl myristate (IPM), Tween
80, Tween 20, lecithin and cholesterol were purchased from
M/s HiMedia Laboratories (Mumbai, India). Sesame oil,
soyabean oil and perilla oil were purchased from local vendors
(M/s Sugandhco Pvt. Ltd., India). N-hydroxysuccinimide
(NHS), N,N′-dicyclohexylcarbodiimide (DCC), poly(ethyl-
ene glycol) bis(amine) (PEG-bis-amine, MW 3.4 kDa), 2-
mercaptoethanol, Propidium Iodide, RNase A, Eagle’s
Minimum Essential Medium (EMEM), Fetal Bovine Serum
(previously heat-inactivated), Hank’s Balanced Salt Solution
(HBSS), MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide), DMBA were purchased from M/s
Sigma Aldrich, USA. α-Linolenic acid (ALA) was purchased
from Rolex Chemicals (Mumbai, India). DCFDA (2,7-
dichlorofluorescin diacetate), Alamar blue® reagent,
Rhodamine 123, Radio Immuno Precipitation Assay (RIPA)
lysis buffer were purchased from M/s Thermo Fisher
Scientific, USA. All the antibodies were purchased from
M/s Santa Cruz, USA. All solvents including water used for
extraction and in the mobile phase were of HPLC grade. All
other chemicals used in the study were of analytical grade.
Preparation of nanoemulsion
Dox loaded nanoemulsion (Dox-NE) was prepared by emul-
sification followed by ultrasonication technique. Briefly, Dox
(10 mg), Smix (lecithin, Tween 80 in ratio 1:1) and cholesterol
were added in ALA maintained at 60 °C and mixed with
continuous stirring at 1000 rpm. The aqueous phase was
added to the oil phase with continuous stirring to get a coarse
emulsion. Furthermore, the mixture was sonicated at 20 kHz,
750 W, 40% amplitude using probe sonicator, (M/s
Labsonic®M, Sartorius, Germany) for 10 min to obtain
nanoemulsion system.
Globule size determination
Methods
Aliquots (1 mL) of the NEs were diluted suitably using
distilled water and subjected to globule size analysis by
dynamic light scattering technique using NanoPlus
zeta/nano particle analyzer (M/s Micromeritics Instrument
Corporation, USA).
Equilibrium solubility studies
Solubility studies of doxorubicin were performed by addition
of an excess amount of drug with different solvents vis-à-vis
oils, emulgents, and co-emulgents using a water bath shaker at
37 2 °C for 48 h to reach equilibrium. The equilibrated sam-
ples were centrifuged at 10,000 rpm for 10 min and the
amount of solubilized drug was determined using UV–visible
spectrophotometer (M/s Labtronics-LT 2910) at 485 nm after
suitable dilutions [13].
Entrapment efficiency and drug loading
The drug loading and entrapment efficiency were determined
using reported procedure with some modifications [17].
Briefly, the entrapment was measured by keeping 1 mL of
drug loaded NEs in dialysis membrane (12–14 kDa
MWCO) for 2 h, and the dialyzed drug (free drug) was esti-
mated using UV-Vis spectrophotometer at 485 nm and
amount of Dox in the aqueous phase was calculated.
Further, drug loading in NEs was determined after dissolving
1 mL of NE (from donor compartment) in 10-mL chloroform/
methanol 1:1 mixture. After suitable dilution, absorbance was
taken using UV-Vis spectrophotometer at 485 nm, and percent
drug loaded was calculated.
Selection of excipients
The excipients were selected on the basis of solubility of drug,
stability and water absorption capacity for different combina-
tions of lipids, emulgents and co-emulgents. Various combi-
nations of lipid, emulgents with different co-emulgent were
titrated drop by drop with aqueous phase and observed visu-
ally for stability. The amount of aqueous phase at which the
system became turbid was considered as the end point [12].
Further, NE formulations of each of the combinations, as de-
scribed above, were kept at elevated temperature (40 2 °C)
for a month to access the stability of the system.
Total weight of DOX dialyzed
Entarpment Efficiency ¼
 100
ð1Þ
Total amount of DOX

Drug Deliv. and Transl. Res.
Drug Loading
Total weight of DOX in donar compartment
significant using the Student’s t test, were considered
in developing the polynomial equations (Eq. 3).
¼
Total amount of drug þ total amount of excipients
 100
Y ¼ β0 þ β1X1 þ β2X2 þ β3X3 þ β4X1X2 þ β5X2X3
2
2
2
þ β6X1X3 þ β7X1 þ β8X2 þ β9X3
þ β10X12X2 þ β11X22X3
ð2Þ
ð3Þ
where, Y represents the CQAs; β0 the intercept; β1 to β11
are the coefficients of various model terms. The response
surface analysis was carried out for understanding the
factor-response relationship and possible interaction(s)
among them. Search for the optimum formulations was
carried out by Btrading-off^ various CQAs by numerical
optimization using overlay plots with suitable application
of constraints on CQAs [13, 23].
Drug release study from NEs
The drug release was performed by loading NE formu-
lations into dialysis bag (3.5 kDa MWCO). The dialysis
bag was placed in 100 mL release media (PBS, pH 7.4)
maintained at 37 0.5 °C with stirring rate of 400 rpm.
Sample aliquots (1 mL) were withdrawn at different
time intervals up to 72 h and replaced with equal
amount of fresh media. The drug concentration in the
samples was analyzed using UV-visible spectrophotom-
eter at 485 nm after suitable dilutions.
Preparation of folate NHS ester
The active ester of folic acid was prepared using a previously
reported method [18, 24, 25]. Folic acid (300 mg, 0.68 mmol)
and triethylamine (0.15 mL, 1.0 mmol) were dissolved in
10 mL dry dimethyl sulfoxide (DMSO), to which DCC
(140 mg, 0.68 mmol) was added. The mixture was stirred
for 1 h at room temperature in the dark, and NHS (115 mg,
1.0 mmol) was added. The reaction was stirred overnight at
room temperature in the dark. The dicyclohexylurea precipi-
tate (a side product of the reaction) was filtered via glass wool
and folate-NHS ester was precipitated using diethylether. The
Mathematical model development and optimization of NE
The data obtained (Table 1) from the above studies was
applied to fit the second-order polynomial equations, the
multiple linear regression analysis (MLRA) with added
interaction terms to establish the correlation between the
CMAs with the CQAs through Design Expert 8.0 soft-
ware (Trial version, M/s Stat-Ease, Minneapolis, USA).
The coefficients which were found to be highly
Table 1 Physicochemical parameters for Dox loaded nanoemulsions prepared as per the BBD. [Data presented as mean SD, n = 3]
Code
Drug (mg/g of
formulation)
Lipid (mg)
(X1)
Smix (mg)
(X2)
Cholesterol
(mg) (X2)
Globule size
(nm) (Y1)
Drug release
(%) (Y2)
Drug loading
(%) (Y3)
Entrapment
efficiency
(%) (Y4)
NE1
NE2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
200
600
200
600
200
600
200
600
400
400
400
400
400
400
400
500
500
900
900
700
700
700
700
500
900
500
900
700
700
700
150
150
150
150
100
100
200
200
100
100
200
200
150
150
150
250.4 3.5
380.5 2.8
70.6 4.5
300.5 4.2
139.7 6.1
312.6 5.9
68.2 4.1
230.3 3.4
280.5 5.1
120.6 3.9
210.9 2.2
80.2 3.1
57.4 2.4
55.1 3.2
59.4 3.1
80.12 5.13
70.36 3.94
95.52 2.63
75.44 3.54
89.21 4.11
70.33 3.33
92.21 2.74
78.71 5.52
75.23 6.15
90.32 4.35
82.33 3.63
93.61 4.21
92.77 4.74
94.33 3.93
91.43 4.51
0.43 0.02
0.36 0.02
0.47 0.08
0.41 0.01
0.43 0.03
0.34 0.04
0.49 0.03
0.45 0.02
0.36 0.04
0.39 0.02
0.40 0.03
0.43 0.03
0.48 0.02
0.47 0.01
0.48 0.03
86.77 3.21
72.12 4.36
95.22 4.84
82.99 3.16
85.47 4.93
67.44 4.67
99.29 2.16
89.52 3.55
72.44 3.63
78.63 2.12
80.61 3.74
86.71 4.51
96.12 3.36
94.33 2.49
97.21 3.65
NE3
NE4
NE5
NE6
NE7
NE8
NE9
NE10
NE11
NE12
NE13
NE14
NE15

Drug Deliv. and Transl. Res.
yellow precipitate (active ester of folic acid) was filtered,
washed several times with dry tetrahydrofuran, dried under
vacuum, and stored as a yellow powder (Scheme 1).
FT-NMR) using DMSO-D6 as solvent. Chemical shifts (δ)
were expressed as parts per million (ppm) relative to the
NMR solvent signal (d6-DMSO) using tetramethylsilane (in-
ternal standard).
Activation of fatty acid
Estimation of folate content attached to the surface
The ALA (100 mg, 0.36 mmol) was dissolved in DMSO and
DCC (74.11 mg, 0.36 mmol) (ALA: DCC, 1:1 M ratio) added
to it with subsequent stirring in dark for 12 h to complete the
reaction. Thereafter, PEG-bis-amine (1257 mg, 0.36 mmol)
(ALA:DCC:PEG-bis-amine, 1:1:1 M ratio) was added to the
above mixture and the reaction was stirred for 3 h to obtain
PEG-amino derivative of fatty acid with primary amine at ter-
minals i.e., ALA-PEG-NH2 (Scheme 2). The dicyclohexylurea
precipitate, by product of reaction, was filtered via glass wool
and the filtrate was precipitated using diethylether and dried
under vacuum to collect ALA-PEG-NH2.
The amount of folate present on the surface of nanoemulsion
was determined by UV spectrophotometer. Analysis was car-
ried out in CH2Cl2/DMSO (1:4) solvent. The nanoparticles
were evaluated by measuring the absorbance of the sample at
358 nm (folic acid ε = 15,760 M−1 cm−1) [26].
Physicochemical characterization of folate modified
nanoemulsion
Particle size, drug loading, entrapment efficiency, zeta poten-
tial and drug release comparisons of folate modified
nanoemulsion were carried out as per methods previously
described.
Folate modified nanoemulsion
Folate-NHS ester (20 mg/mL) suspended in phosphate buffer
saline (PBS pH 7.4) was poured in equimolar ratio into NE
prepared with mixture of activated ALA (ALA-PEG-NH2)
and ALA (1:20 w/w; 19 mg, 381 mg, respectively) for surface
conjugation as per Scheme 3. This mixture was stirred gently
overnight at room temperature. To complete the reaction 2-
mercaptoethanol (10 mmol) was added in to the above mix-
ture. Subsequently, folate decorated doxorubicin loaded NE
(f-Dox-NE) was dialyzed (12–14 kDa MWCO) to remove
unreacted 2-mercaptoethanol and folate-NHS ester [26].
Robustness to dilution
Robustness to dilution for the optimized nanoemulsion was
determined by suitably diluting (100, 500, 1000 folds) the
optimized formulation with phosphate buffer pH 7.4 and ob-
served for precipitation and cloudiness, if any, after 24 h.
Thermodynamic stability studies of formulation
FTIR characterization Synthesis of folate NHS, ALA-PEG-
NH2 and f-Dox-NE was confirmed by FTIR spectra recorded
by adsorbing the samples on KBr disk using PerkinElmer FT-
IR Spectrometer. Conjugation of folate to fatty acid (f-PEG-
ALA) was characterized after freeze drying of f-Dox-NE by
FTIR analysis.
To assess the stability of the optimized nanoemulsion, the
formulation was subjected to various stress conditions as
discussed below.
Centrifugation study The optimized nanoemulsion (1 g) was
diluted to 100 mL and centrifuged at 5000 rpm for 15 min and
analyzed for any instability manifested through phase separa-
tion, creaming or cracking, if.
1H-NMR characterization
The conjugation of folate to ALA through PEG was charac-
terized through H-NMR spectroscopy (Bruker Avance 400,
Freeze thaw cycle Three freeze-thaw cycles were performed
for the optimized nanoemulsion maintaining the
1
Scheme 1 Synthesis of folate
NHS ester [NHS: N-
hydroxysuccinimide]

Drug Deliv. and Transl. Res.
Scheme 2 Synthesis of PEG-
NH2 conjugated α-linolenic acid
(ALA-PEG-NH2)
temperatures between − 21 and + 25 °C, and was observed
for instability, if any.
CPCSEA guidelines for laboratory animals and ethics,
Department of Animal Welfare, Government of India, after
getting approval from Institutional Animal Ethical
Committee (SDCOPVS/AH/CPCSEA/01/0078).
Transmission electron microscopy (TEM) studies
The TEM analysis (TEM Fei Tecnai 200 Kv, Electron Optics)
of f-Dox-NE was performed for morphological characteriza-
tion of emulsion droplets. The sample was diluted with deion-
ized water (1:25). A drop of above solution was placed on
copper grids, stained with phosphotungstic acid (1%) for
30 s, and finally observed under electron microscope.
Stability studies
The stability studies on the f-dox-NE formulations were per-
formed by filling the same in amber colored glass vials and
storing them at 5 3 °C (Refrigerator), 25 2 °C/60 5%
RH, 40 2 °C/75% 5% RH, as per the ICH Q1A(R2)
guidelines(stability chamber -M/s Thermolab, India). The for-
mulations, kept in air-tight amber colored glass vials, were
assayed periodically at 0, 1, 3 and 6 months, respectively,
for globule size, similarity factor (F2 values, for dissolution
comparison) and drug content [13].
Stability of formulations in plasma and intravenous infusion
solutions
Stability of NEs was evaluated in various media viz., 90%
fresh rat plasma, phosphate buffer pH 7.4, normal saline
(0.9% w/w NaCl), respectively. Each NE was diluted (1:10
w/w) with respective media and incubated in amber color glass
vials for 24 h at 37 2 °C. Aliquots of 10 ml of each sample
were withdrawn for analysis at 1, 2, 4, 6, 8 and 24 h, respec-
tively, and observed visually for globule aggregation. Further,
the aliquots were diluted 1000-fold with distilled water and
evaluated for any significant change in globule size using
nanoplus particle size analyzer. No particle aggregation and
change in globule size distribution towards higher size range
was observed, which was taken as an indicator for stability on
storage. The experiments were performed according to the
In vitro studies
Cell culture conditions MCF-7 cells were cultured in EMEM
(Eagle’s Minimum Essential Medium) with 10% v/v fetal bo-
vine serum (FBS) and 1% v/v antibiotic drug (Penicillin/strep-
tomycin) and maintained in a humidified 95% O2/5% CO2
atmosphere at 37 C. Cells were grown until 60–70% confluent
in the flasks and trypsinized with a solution 0.25% v/v Trypsin
with 2.25 mmol EDTA in HBSS. Trypan blue exclusion meth-
od was employed to determine the viable cells.
Scheme 3 Synthesis of folate
attached α-linolenic acid (f-PEG-
ALA)

Drug Deliv. and Transl. Res.
Cell cytotoxicity study (MTT assay) Cell cytotoxicity of Dox,
Dox-NE, f-Dox-NE and ALA-NEs was performed using the
MTT assay. In this assay, the conversion of this salt by mito-
chondrial enzymes reflects the number of viable cells. The
MCF-7 cells were seeded into 96-well plates (density 1.0 ×
103 cells per well) and incubated for 24 h maintained 5% CO2
humidified atmosphere conditions at 37 °C to allow adhesion
of cell to surface. Freshly prepared samples of formulations
were used to treat cells adhered to the wells at the concen-
tration ranging from 0 to 50 μg/mL equivalent Dox (n = 3).
Plain PBS was taken as an untreated control. The culture
cells were further incubated for 48 h. 20 μL of MTT solu-
tion (5 mg/mL MTT in PBS) was added to each well and
cells were further incubated for 4 h, at 37 °C, leading to
formation of blue MTT-formazan crystals. Following incu-
bation, the MTT reagent was dissolved in 200 μL of
DMSO. The optical density was assessed at 540 nm by
ELISA plate reader (M/s Biotek, USA) [12, 27].
Reactive oxygen species estimation MCF-7 cells were plated
in 96 well culture plates (approximately 3000 cell/well) and
incubated at 37 °C with 5% CO2 for about 24 h. The cells were
treated at different concentration (0–200 μg/mL) of samples
for 24 h. After incubation, DCFDA was added (final concen-
tration 10 μmol) and the plate was incubated for 30 min in
dark. DCFDAwas removed and wells were washed once with
PBS and finally cells were suspended in 100 μl of PBS. The
plate was read at 485/528 nm (Excitation/Emission) at 100%
sensitivity in a multimode reader (Synergy H1, M/s Biotech
Inc., USA) and readings were presented as relative fluores-
cence unit for ROS generation. The cells without treatment
were considered as control [30].
Mitochondrial membrane potential (MMP) The MCF-7 cells
were plated in 96 well culture plate (approximately 3000
cell/well) and incubated at 37 °C in EMEM (Eagle’s
Minimum Essential Medium with 10% FBS and 1% antibiot-
ic) with 5% CO2 for about 24 h. The cells were treated at
different concentration (0–200 μg/mL) of samples for 24 h.
After incubation, Rhodamine 123 (Rh 123) was added (final
concentration 10 μM) and the plate was incubated for 30 min
in dark. Subsequently, the medium was removed and wells
were washed once with PBS and finally 100 μL of PBS was
added to each well. The plate was read at 507/529 nm
(Excitation/Emission) at 100% sensitivity in multimode read-
er (Synergy H1, Biotech Inc., USA) and readings were pre-
sented as relative fluorescence unit for mitochondrial mem-
brane potential (MMP). The cells without treatment were con-
sidered as control [31].
Cell cycle analysis through fluorescence-activated cell sorting
(FACS) analysis MCF-7 cells (1 × 106 cells mL−1) were
suspended in 1 mL of PBS in centrifuge tube. The cells were
centrifuged at 200g for 5 min at room temperature. PBS was
removed and cell pellet was resuspended in 500 μL of PBS.
Cells were fixed by adding 4 mL of 70% (v/v) cold ethanol to
the cell suspension and kept overnight at 4 °C. Cell suspension
was centrifuged at 400g for 5 min and the supernatant (ethanol
solution) was removed. Again the cells were washed in 5 mL
of PBS and centrifuged at 400g for 5 min. Supernatant was
removed and cells were resuspended in 1 mL of DNA staining
solution (500 μL/106 cells; 200 mg of PI in 10 mL of PBS +
2 mg of DNase free RNase A) and incubated for at least
30 min at room temperature in the dark. The samples were
analyzed by flow cytometer (488-nm laser line for excitation,
FACS Calibur, M/s BD Biosciences, Franklin Lakes, NJ) in
PI/RNase A solution without washing the cells at a low flow
rate under 500 events/s. The percentage of cells in different
phases of cell cycle was analyzed by ModFit LT 3.0 software
(Verity Software House). The cells without treatment were
considered as control [28].
In vivo study Female albino wistar rats, weighing 120–150 g,
were used for the experiment. Animals were housed in stan-
dard condition (25 °C, 12 h light/dark cycle) in polypropylene
cages. The animals were fed with standard pellet diet and
water ad libitum. The experiment was performed according
to the CPCSEA guidelines for laboratory animals and ethics,
Department of animal welfare, Government of India
(SDCOPVS/AH/CPCSEA/01/0078). Animals were random-
ized and divided into seven groups of 10 animals each [Group
I (control, received normal saline only); Group II (toxic con-
trol, received normal saline); Group III (ALA NE, NE with
ALA), Group IV (standard drug/pure drug solution); Group V
(Marketed formulation); Group VI (Dox-NE); and group VII
(f-Dox-NE)]. Mammary gland carcinoma in each group (ex-
cept Group I) was induced by single tail vein injection of
DMBA (8 mg/kg) on day 1. The drug/formulations were ad-
ministered (10 mg/kg e.q. Dox) thrice/weeks in last 6 weeks of
total 16 weeks of in vivo studies. A gap of 10 weeks was
provided to develop the cancer in animals. Tumor incidence
and size were monitored by measuring the diameter of mam-
mary glands of rats by using Vernier caliper. Tumor progres-
sion was further monitored by weight and survival of animals
Cell proliferation studies The cell proliferation assay was per-
formed by Alamar blue® method. The MCF-7 cells were
plated in 96 well culture plate (approximately 3000 cell/well)
and incubated at 37 °C in EMEM with 10% FBS and 1%
antibiotic with 5% CO2 for 24 h. The cells were treated with
different samples at concentration 3.31 μg/mL equivalent of
Dox for 24 h. After incubation, Alamar blue® was added at a
final concentration of 5% v/v and plates were allowed to stand
at 37 °C for 4 h. The plated were read at 570/600 nm in a plate
reader (M/s Biotek, USA) and cell proliferation was calculated
using colorimetric measurements. The cells without treatment
were considered as control [29].

Drug Deliv. and Transl. Res.
during the study. The blood samples were collected under
mild chloroform anesthesia through retro orbital puncture for
further analysis. The blood samples were incubated at 37 °C
for 1 h and centrifuged at 10,000 rpm for 20 min to collect
serum and the serum samples were stored at − 20 °C till fur-
ther use. Animals were sacrificed on the 112th day after treat-
ment and mammary tissues were removed and stored at −
80 °C till further evaluation [10].
route for 5 weeks. Animals were sacrificed after 1 week of
receiving the last injection of Dox and heart tissues were re-
moved. Biochemical estimation for lipid peroxidation (MDA
levels) and GSH content was measured from tissue
homogenates.
Biodistribution of doxorubicin Biodistribution of Dox was
examined by injecting f-Dox-NE, Dox-NE, standard drug so-
lution and marketed formulation at 10 mg/kg Dox equivalent
dose through tail vein of cancer induced rats. Different organs,
viz., mammary glands, liver, heart, kidney, spleen were re-
moved post injection (at time intervals 4, 12, 24 and 48 h)
and washed to remove the blood, weighed and homogenized
and the Dox concentration was measured through the validat-
ed HPLC method (M/s Waters 2489, Milford, Massachusetts,
USA). The system was equipped with Spherowsorb C18,
3.5 μm, 4.6 × 250 mm column, having flow rate 1.0 mL/min
and ambient temperature. The mobile phase consisted of water
at pH 3 adjusted with 85% w/v phosphoric acid and acetoni-
trile (30:70) and the total run time was 10 min. The HPLC was
run through Empower software ver.2 and absorbance was
measured at 233 nm wavelength using Waters 2489 UV-Vis
detector [27, 33].
Western blotting The mammary gland tissues were lysed in
RIPA lysis buffer and total protein lysates were obtained and
the protein content was quantified with help of the Bradford
reagent. Equal amount of protein was separated by SDS-
PAGE from each sample and transferred to nitrocellulose
membranes. Subsequently, the membranes were blocked
and immunoblotting was performed with appropriate anti-
bodies viz., Bcl-2 (mouse monoclonal, SC-7382), BAX
(mouse monoclonal, SC-23959), Caspase-9 (mouse
monoclonal, SC-56073) and MMP-9 (goat polyclonal, sc-
6840). β-actin (MA5-15739-HRP) was used as a standard
reference in this study. Nitrocellulose membranes were in-
cubated with an appropriate secondary antibody conjugat-
ed to horseradish peroxidise (HRP) followed by detection
of protein using enhanced chemiluminescence (Western
Bright ECL HRP substrate, Advansta, US). Quantitative
analysis of protein expression was performed by scanning
densitometry (ImageJ, NIH) [10, 32].
Statistical analysis
One-way ANOVA with Bonferroni multiple comparison tests
were used to find out significant statistical differences be-
tween the groups. Values of *p < 0.05, **p < 0.01,
***p < 0.001 were considered as statistically significant.
Statistical analysis was performed using Graph Pad Prism
software (ver. 5.02).
Biochemical estimation The mammary gland tissue (10% w/v)
was homogenized in 0.15 mol. KCl and centrifuged at
10,000 rpm. The supernatants were scrutinized for biochemi-
cal parameters including TBARS, superoxide dismutase
SOD, catalase, GSH and protein carbonyl. All the experiments
were performed in triplicate and were subjected to statistical
analysis using Graph Pad Prism (ver. 5.01, San Diego,
California).
Results
Equilibrium solubility studies
Microscopy of whole mounts of mammary glands through
carmine staining The mammary glands obtained from rats
were stained with carmine according to reported procedure.
The whole mounts were examined under the 4X microscope
and evaluated for the number of alveolar buds/terminal end
buds (AB/TEB) [10].
The drug solubility plays a vital role for the development of
effective nanoemulsion formulations using various lipidic
components. Dox displayed maximum solubility in isopropyl
myristate (30.55 3.25 mg/mL) followed by the ALA (25.33
3.78 mg/mL) among various lipids that were taken. It also
displayed maximum solubility in lecithin (55.49 4.76 mg/
mL) among various emulgents and co-emulgents (Fig. 1).
Cholesterol was used as co-emulgent as well stabilizer for
the nanoemulsion globules.
Cardiotoxicity Dox has been reported to induce cardiotoxicity
at cumulative dose of 15 mg/kg over a period of 6 weeks in
rats. The study was designed according to the previously pub-
lished report [5]. Briefly, the animals were divided into four
groups of 6 animals each. Group I served as control and re-
ceived normal saline, Group II received Dox 4 mg/kg solu-
tion, Group III received ALA-NE as vehicle control, and
Group IV received f-Dox-NE (4 mg/kg eq. Dox). All treat-
ments were given once a week via intraperitonial injection
Selection of excipients
The lipid, emulgents and co-emulgents were selected on the
basis of solubility, stability and water absorption capacity. The
ALA was selected as lipid phase, Lecithin and Tween 80 as

Drug Deliv. and Transl. Res.
using modified quadratic equations as revealed by nonsignif-
icant lack of fit (p > 0.05) form the ANOVA data of each
CQAs (Supplementary Table 1–4).
Optimization of formulation and validation
The optimized formulation was chosen by graphical opti-
mization methodology using overlay plot (Fig. 3). The
constraints applied for the graphical optimization of the
nanoemulsion were globule size < 100 nm, drug release
> 80%, entrapment efficiency > 90% and drug loading >
0.4% fixed for the purpose. The optimized formulation was
selected based upon the afore-listed criteria. The optimized
nanoemulsion contained ALA (400 mg), Lecithin
(350 mg), Tween 80 (350 mg) and cholesterol (150 mg),
exhibiting observed values of the CQAs as globule size of
52.7 nm, drug release of 94.86 1.87% in 72 h, entrapment
efficiency 92.51 3.62% and drug loading 0.42 .08%
(Fig. 2).
Fig. 1 Plot representing solubility of Dox in various excipients of the
nanoemulsion. [Data presented as mean SD, n = 3]
emulgents while cholesterol as co-emulgent. The lipid (ALA),
emulgents (Lecithin and Tween 80) and co-emulgent
(cholesterol) were considered as critical material attributes
(CMAs) for nanoemulsion.
Validation of the optimization methodology revealed the
percent prediction error ranging between − 0.57 and 7.46%
for the optimized nanoemulsion, thus demonstrating high de-
gree of the predictive ability of the design methodology.
Preparation of nanoemulsion and data analysis
Nanoemulsion was prepared as per DoE and characterized
for various CQAs. Mathematical data analysis was carried
out by employing second order quadratic model by multi-
ple linear regression analysis to fit the data (Table 1).
Excellent goodness of fit of the experimental data was ob-
served for the generated coefficients for each of CQAs of
the product. [13, 23].
Folate-conjugated nanoemulsion
FTIR characterization
The conjugation of folic acid was characterized through IR
spectroscopy. Important peaks obtained for the folic acid were
at 897.2 cm−1 (aromatic C–H bending and benzene 1,4-
disubstitution), 1478.9 cm−1 (CH–NH–CO amides bending),
1702.5 cm−1 (aromatic C-C bending and stretching),
2996.4 cm−1 (alkyl C–H and O-H stretch),3439.3 cm−1 (N–
H stretch of primary amine and amide, C-H stretch) and
1310.11 cm−1 (C-O stretch ester) (Supplementary Fig. 2A).
For ALA characteristic peaks were observed at 2923.7 cm−1
(carboxylic acid COOH and O–H unconjugated stretching)
1708.8 cm−1 (C=C Alkenyl stretch), 2854.3 cm−1 (alkyl C-H
stretching), 1463.8 cm−1 (CH2, CH3 deformation)
(Supplementary Fig. 2B). For the Folate-NHS ester, the
peaks were observed at 3491.8 cm−1 (amide N–H and
C=O stretching), 1551.37 cm−1 (CH–NH–CO amides
bending), 1663.8 cm−1 (ketone C=O stretch), 2913.7 and
2996.1 cm−1 (carboxylic acid COOH and O–H unconjugat-
ed stretching), 697.4, 896.9, 931.29 cm−1 (aromatic C-H
bending and stretching) confirmed the formation of folic
acid NHS ester. (Supplementary Fig. 2C). For the Folate-
PEG-ALA, the peaks were observed at 3441.5 cm−1 (am-
ide N–H and C=O stretching), 2913.8 and 2997.08 cm−1
(carboxylic acid COO-H, alkane C-H stretching and O–H
unconjugated stretching), 1436.9 cm−1 (CH–NH–CO am-
ides bending), 1712.5 cm−1 (aromatic C-C bending and
The response surface plots (Supplementary Fig. 1 A-D)
explicitly describe the effects of lipid, emulgent and co-
emulgent on various CQAs of nanoemulsion. Briefly, the lip-
id, emulgent and co-emulgent conferred significant
(p < 0.001) effect on globule size of the nanoemulsion
(Supplementary Fig. 1A). The lowest value for globule size
was observed at middle levels of the excipients. Further, sig-
nificant interaction among the excipients (except emulgent
and cholesterol) was observed for selected modified quadratic
model. At middle level of excipients inverse effect
(Supplementary Fig. 1B) to the globule size was observed
with drug release data when analyzed through quadratic mod-
el, indicating significant effect (p < 0.05) of the selected ex-
cipients on drug release. Here, ALA and cholesterol played a
vital role in controlling the drug release. In case of drug load-
ing (Supplementary Fig. 1C) ALA and cholesterol showed
significant effect (p < 0.05) while Smix showed no significant
effect on loading efficiency of drug in the nanoemulsion
(p > 0.05). ALA, Smix and cholesterol each demonstrated sig-
nificant role in the entrapment of Dox in the NE (p < 0.01)
with significant interaction between lipid and emulgent (p <
0.05) (Supplementary Fig. 1D). Data was appropriately fitted

Drug Deliv. and Transl. Res.
Fig. 2 Overlay plot depicting the
desirable region (design space) as
highlighted area for the
nanoemulsion. Inset flag presents
the composition of optimized
nanoemulsion (X1–400 mg, X2
700 mg, X3–150 mg) and
predicted values of CQAs
stretching), 698.2, 897.8, 932.2 cm−1 (aromatic C-H bend-
ing and stretching) and 1649.7.8 cm−1 (ALA C=C Alkene
stretch), 1212.2 cm−1 (PEG C-O-C stretching) confirmed
the attachment of the folic acid to ALA through PEG
(Supplementary Fig. 2D).
Estimation of folate content attached to the surface
The content of folate attached to the nanoemulsion surface
was observed to be 10.33 μmol g−1 of nanoemulsion
formulation.
1H-NMR characterization
Physicochemical characterization
1H-NMR of folate attached nanoemulsion was performed
and characteristic peaks were identified and appropriately
marked that confirm the conjugation of folate to ALA.
Important peaks of ALA-PEG-NH2 were 1 (3.66, 2H, d,
broad peak of protonated NH2 protons), 2 (3.31–3.46, 2H,
dd, two adjacent peaks for PEG); 3 (7.85, 1H, m, CONH),
4 (2.14, 2H, t, methylene), 5 (1.18–1.46, 12H, m, methy-
lene); 6 (5.28, 6H, m, ethene); 7 (2.66, 4H, s, methylene); 8
(2.13–2.19, 2H, m, methylene); 9 (0.78–0.80, 3H, t, meth-
yl) (Fig. 3a). Important peaks of folate decorated ALA
(ALA-PEG-folate) were 1 (8.35, 1H, s, dihydropteridin
NH); 2 (3.76, 2H, d, broad peak of protonated NH2 pro-
tons); 3 (8.58, 1H, s, dihydropteridine CH); 4 (2.42, 2H, s,
methylene); 5 (3.44, 3H, m, aliphatic NH); 6 (6.58 & 7.54,
4H, dd, aromatic CH); 7,11,13 (6.83, 3H, m, CONH); 8
(1.18, 1H, s, aliphatic CH); 9 (2.27, 4H, s, methylene); 10,
14 (2.14, 4H, t, methylene); 12 (3.35–3.46, 2H, dd, two
adjacent peaks for PEG); 15 (1.18–1.53, 12H, m, methy-
lene); 16 (5.27, 6H, m, ethene); 17 (2.66, 4H, s, methy-
lene); 18 (2.10–2.18, 2H, m, methylene); 19 (0.70–0.80,
3H, t, methyl) (Fig. 3b).
Folic acid was coupled with the Dox nanoemulsion with the
help of the NHS and DCC. Attachment of the folate over the
emulsion droplets increased the size of Dox-LNE up to 55.2
3.3 nm from 52.7 2.8 nm; however, the surface charge did
not change (zeta potential − 31 2 mV). The drug entrapment
efficiency and the drug loading in the f-Dox-NE were ob-
served to be 92.51 3.62% and be 0.47 .03%, respectively.
In vitro drug release comparison
Significant improvement was observed in the drug release
profiles of the optimized nanoemulsion in comparison to the
standard drug and the marketed formulation. The cumulative
drug release of the pure drug (standard) and the marketed
formulation was 82. 2.30% and 101.71 3.10%, respective-
ly, in 2 h; however, the f-Dox-NE released about 20% in the
same time period. The optimized nanoemulsion (f-Dox-NE)
showed cumulative drug release of 55.47 3.40%, 70.22
5.02, 86.97 3.50%, and 94.86 1.87% in 12, 24, 36, and
72 h, respectively (Fig. 4). No significant variation in the drug
release profiles was observed before (Dox-NE) and after (f-

Drug Deliv. and Transl. Res.
Fig. 3 1H-NMR spectrum of (a) PEG-NH2 attached ALA (ALA-PEG-NH2) (b) folate modified nanoemulsion (f-Dox-NE)
Dox-NE) the surface modification of the nanoemulsion with
there was no evident sign of precipitation and cloudiness
folate (p > 0.05).
even after 24 h.
Robustness to dilution
Thermodynamic studies of formulation
The optimized nanoemulsion exhibited robustness on dilu-
tion of the nanoemulsion with the PBS pH 7.4. Further,
To ensure the stress stability of the formulation, one must pass
through the thermodynamic stability studies. The optimum

Drug Deliv. and Transl. Res.
Fig. 6 Cell viability through MTT assay of the differently treated cell
groups, Standard, marketed formulation, Dox-NE and f-Dox-NE. All
the experiments were performed in triplicate
Fig. 4 Drug release comparisons of standard drug, marketed formulation
with Dox-NE and f-Dox-NE. [Data is presented as mean SD (n = 3)]
size was observed demonstrating that the formulation was
non-aggregated and showed no sign of disruption even in
presence of plasma proteins and electrolytes.
formulation was found to be stable as there was no drug precipi-
tation, creaming or cracking after centrifugation and freeze thaw
cycles which indicate stability of the nanoemulsion system.
Stability studies
Transmission electron microscopy (TEM) studies
The stability studies of the formulation confirmed the robust
nature of the system under the specified stability conditions
without any significant change in globule size, F2 values and
drug content (Supplementary Table 5).
All the globules of the formulation (f-Dox-NE) displayed
smooth surface and spherical shape within the size range of
20.80–65.99 nm as observed from TEM images (Fig. 5).
Stability of formulations in plasma and intravenous infusion
solutions
In vitro studies
Cell cytotoxicity study (MTT assay)
The stability of the f-Dox-NE was accessed visually for glob-
ule aggregation and by diluting sample in plasma and infusion
solutions and observed for changes in globule size, if any, over
24 h at 37 °C. No particle aggression and change in globule
The changes in percent cell viability against the drug treat-
ment of Dox (positive control), Dox-NE, f-Dox-NE and
ALA-NE are presented in Fig. 6. IC50 values of the Dox-
NE, f-Dox-NE, Dox and marketed formulation were re-
corded as 1.92 0.21, 1.02 0.16, 3.31 0.20 and 2.83
0.18 μg mL−1, respectively. For nanoemulsion with ALA
only (ALA-NE), the IC50 value was 4865.17
448.12 μg mL−1 (data not shown).
Cell cycle analysis through FACS analysis
The distribution of cells in different stages of the cell cycle
evaluated through FACS analysis is presented in Fig. 7. The
cell distribution in different phases for the control cells was
67.52% in S phase, 7.70% in G1 phase and 24.78% in G2/M
phase (Fig. 7a). While, the cell distribution in the f-Dox-NE
Fig. 7 Flow cytometric analysis of the cell cycle phase distribution for the
„
differently treated cells. (a) Control (no treatment), (b) Standard (Pure
Dox), (c) ALA-NE (blank NE formulation with ALA) (d) f-Dox-NE;
(e) Graph depicting the comparative account of the cells present in the
different phases for treatment groups (a–d)
Fig. 5 TEM image of final formulation of f-Dox-NE depicting globules
in size range of 20.80–65.99 nm. Mean size was in range of 50–55 nm

Drug Deliv. and Transl. Res.

Drug Deliv. and Transl. Res.
treated cell was 38.29% in G1 phase, 19.82% in S phase and
41.90% in G2/M phase. The standard formulation (Fig. 7b)
showed highest percentage of cells in the G1 phase
(53.34%) followed by S phase (27.24%) and G2/M phase
(25.00%), while the ALA-NE showed highest percentage
of cells in G2/M phase (88.38%) of the cell cycle (Fig. 7c).
Apoptotic cell population as visualized in ALA-NE and f-
Dox-NE treated cell groups suggested that these formula-
tions forced the cells to enter in the process of programmed
cell death (Fig. 7c, d).
3.77%) and the marketed (53.34 4.02%) formulations
(p < 0.05) as well. The formulation having ALA only (ALA-
NE) showed 21.89 4.56% of the cell growth inhibition as
compared with the control. Similar results could be inferred
from the data of Alamar Blue® reduction (Fig. 8b) The f-Dox-
NE group showed least dye reduction indicating least cell
viability and therefore caused the highest cell death of the
MCF-7 cell lines [29].
Evaluation of reactive oxygen species
The results of DCFDA fluorescence showed that the f-Dox-
NE and Dox-NE caused significant (p < 0.01) cellular mortal-
ity in comparison with standard and the marketed formulation
(Fig. 8c) [30].
Cell proliferation studies
The percent inhibition in the cell proliferation data revealed
highest percent inhibition of the cellular proliferation for the f-
Dox-NE (89.12 2.55%) and Dox-NE (71.40 2.43%) of the
MCF-7 cells compared to the control cells (p < 0.001) mea-
sured by the amount of reduced form of the Alamar Blue®
(Fig. 8a). The f-Dox-NE and Dox-NE showed significant cell
proliferation inhibition as compared to the standard (48.98
Mitochondrial membrane potential (MMP)
MMP-dependent Rh-123 uptake and preferential accumula-
tion in the mitochondria of the living cells accompanied by
Fig. 8 In vitro studies on MCF-7 cell line of differently treated cells
groups, i.e., control (no treatment), ALA-NE (blank NE with ALA only),
Standard (Dox solution), f-Dox-NE (folate decorated Dox loaded NE).
(a) % reduction in cell proliferation, (b) %Alamar Blue® reduction as
compared to the control (no treatment), (c) DCFDA fluorescence units
presenting the ROS levels, and (d) Rh-123 fluorescence unit depicting
variation in the MMP. [Data presented as the Mean SD, n = 3.
***p < 0.001, **p < 0.01, *p < 0.05 with respect to control, **a
p < 0.01 with respect to the marketed and the standard groups]

Drug Deliv. and Transl. Res.
the red spectral shifts and the quenching of fluorescence
makes it a suitable agent to monitor the MMP of the cells
undergoing apoptosis. The effect of the formulations on the
MMP was elucidated using Rh-123 efflux, because the cell
apoptosis triggers a collapse of the MMP resulting in the lower
uptake and retention of the Rh-123 in the mitochondria [31].
The data of the fluorescence studies revealed that after the
treatment with the various formulations, the f-Dox-NE and
the Dox-NE induced significant disruption of the MMP com-
pared to the standard and the marketed formulation; indicating
significant (p < 0.01) mitochondrial dysfunction in the MCF-7
cell lines by the former groups (Fig. 8d).
which was in the corroboration with the previous study [10].
The marginal advantage for the f-Dox-NE over Dox-NE is
clearly evident in the data suggesting targeted delivery to the
diseased site owing to folate guided delivery to FR
overexpressed tumor cells.
Biochemical estimation of antioxidant marker
As illustrated in Table 3, the mean levels for TBARs, protein
carbonyl, glutathione GSH, SOD, and catalase were signifi-
cantly disturbed in the DMBA treated groups as compared to
the control group. f-Dox-NE and the standard drug treatment
successfully restored the level of TBARs, protein carbonyl,
GSH, SOD and catalase as compared to the toxic control.
The notable restoration of the antioxidant defense system
was observed in the ALA-NE treated group.
In vivo studies
The results of the in vitro cell lines studies encouraged the
authors to further validate the efficacy of the folate decorated
nanoemulsion through the in vivo experiments against
DMBA induced mammary gland carcinoma in rats.
Western blotting
To quantify the changes in the protein expression, the mam-
mary gland samples of different groups were investigated by
western blot analysis [5–10, 32]. The expression of anti-
apoptotic and pro-migratory proteins (Bcl-2, MMP-9) was
upregulated, while pro-apoptotic and anti-migratory markers
like BAX and caspase-9 were downregulated after DMBA
administration to rats. The treatment with different formula-
tions offered the reinstatement of the anti-apoptotic and pro-
apoptotic markers up to the extent of the efficacy of different
formulations (Fig. 10). The densitometric data suggested that
the treated groups restored the markers as compared to the
toxic group favorably signifying apoptosis (Fig. 10).
Tumor progression monitoring
Tumor incidence, total tumor burden, weight variation and
animal survival studies are well established methods to mon-
itor the tumor progression in the cancer induced animal
models. The tumor incidence and total tumor burden data
depicted toxic control showed highest tumor progression
while f-Dox-NE showed least progression in tumor
(Table 2). Weight variation plots for the test groups
(group IV-VII) during study duration showed significantly
less weight reduction in animals as compared to the DMBA
induced group (Group II, toxic control) (p < 0.05) (Fig. 9a).
The survival plot illustrated lowest animal deaths and
highest percent animal survival for f-Dox-NE at the end
of the study (Fig. 9b).
Microscopy of whole mounts of mammary glands
through carmine staining
The f-Dox-NE revealed marked reduction in animal mor-
tality when compared with standard and marketed (p < 0.01)
formulation. The effect of ALA-NE (Fig. 9b) as compared to
the toxic group is correlated to the anticancer potential of ALA
The carmine staining represented that DMBA administration
resulted in marked increase in the number of AB/TEB, dem-
onstrating the cellular proliferation in the mammary gland.
The treated groups afforded the significant defense against
cellular proliferation. As evident in the histogram, the effect
of Dox-NE and f-Dox-NE was more efficient in diminishing
the count of AB/TEBs as compared to the toxic (p < 0.001),
the standard and the marketed groups (p < 0.05) (Fig. 11).
Table 2 Tumor incidence and tumor burden of mammary gland
carcinoma in DMBA induced female albino wistar rats
Treatment groups
No. of rats with
tumors/total rats
Total tumor burden
(cm3)
Cardiotoxicity studies
Control
0/10
8/10
7/10
5/10
5/10
4/10
3/10
–
Toxic
412.67 43.22
250.39 20.72
185.48 21.31
180.89 18.15
127.81 12.44
95.61 11.23
Cardiotoxic effect of Dox was evident with the profound ele-
vation of MDA levels and the lowering of GSH levels in the
heart tissue as compared to the control. However, significant
favorable improvement in the MDA and GSH levels were
evident with the ALA-NE, f-Dox-NE treatment in respective
groups (Fig. 12a, b).
ALA-NE
Standard drug
Marketed formulation
Dox-NE
f-Dox-NE

Drug Deliv. and Transl. Res.
Fig. 9 (a) Weight variation in
animals. (b) Survival graph with
percent animal survived treated
with Control (normal saline),
toxic control, ALA-NE, standard
drug solution, Marketed
formulation, Dox-NE, f-Dox-NE.
[Data is presented as mean of
group, n = 10]
Biodistribution of doxorubicin
conjugates etc. have attracted much interest of the formulation
scientist. Targeted nanomedicine can also be best designated
as a ‘molecularly designed’ delivery system capable of carry-
ing multiple drugs, track and accumulate to the specific dis-
ease site through relevant receptors overexpressed on the tar-
get cell.
Higher targeting efficiency of the delivery system is often
anticipated for the successful outcome of anticancer therapy.
The Dox levels mammary carcinoma in case of standard and
the marketed formulation were significantly lower in compar-
ison to f-Dox-NE (p < 0.001) treated group (Fig. 13). The Dox
level was significantly higher at 4 h post injection and was
barely detectable after 48 h (except f-Dox-NE). Further, sig-
nificant Dox levels were maintained in the mammary cancer
cells for 48 h (25.78 1.42 μg/g organ) in the f-Dox-NE
group and 12 h (25.55 2.22 μg/g organ) in the Dox-NE
treated groups vis-à-vis the pure Dox and the marketed formu-
lation treated groups. Similar inference can be drawn for other
time points as well. Among different organs, highest Dox
concentration was observed in the liver followed by the
spleen, the heart and the kidney. The Dox level in the heart
tissue in f-Dox-NE treated group was lesser when compared to
the other groups (p < 0.05) (Fig. 13a–d). Overall, the Dox
levels in different organs of the folate decorated NE were
relatively lower than that of other animal groups, which was
in accordance with published literature [18, 26, 27].
For an ideal nanoemulsion system, the drug should remain
solubilized in the dosage form to demonstrate better therapeu-
tic efficacy. Lower drug solubility in the lipid may lead to
supersaturation resulting into drug precipitation [13]. The sys-
tematic and scientific tools like QbD and DoE are often ap-
preciated in the development and optimization of the drug
delivery systems. Excipients (CMAs) were selected consider-
ing the delivery system, route of administration, safety, nature
of excipients and solubility of the drug. The selected CMAs
significantly influenced the performance of the final product.
Cholesterol, selected as co-emulgent, has been shown to im-
prove stability of lipid nanocarriers and to control the drug
release from the delivery system [18]. The cholesterol and
lecithin are nontoxic, biocompatible and natural components
which can safely be used in the systemic delivery system.
Non-ionic emulgents like Tween 80 are considered safer than
their ionic counterparts and are frequently accepted for sys-
temic administration [34–37]. The globule size of a
nanoemulsion is a vital factor for the in vitro and in vivo
performance because it modulates the rate and extent of the
drug release, solubility of the drug into the system and passage
of oil globules through the biological membranes and blood
Discussion
In the era of nanomedicine therapies, advancement in the
targeted therapies, antibody therapies, small molecules drug
Table 3 Effect of treatment groups on oxidative stress markers in DMBA induced rat mammary gland carcinoma
Groups
TBARs
Protein carbonyl
GSH
SOD
Catalase
(nm of MDA/μg
of protein
nmol/mg of protein
(Mg %)
(units of SOD/mg
of protein)
(nmol of H2O2/min/mg
of protein)
Control
0.20 0.02
0.49 0.14
0.39 0.12
0.30 0.13
0.31 0.15
0.25 0.06
0.22 0.06
40.35 1.08
55.56 6.75
49.04 3.80
41.51 2.33
40.51 2.85
44.64 1.37
42.05 1.80
1.20 0.08
0.99 0.09
1.10 0.08
1.15 0.10
1.15 0.12
1.18 0.07
1.22 0.05
0.034 0.003
0.043 0.005
0.035 0.002
0.030 0.002
0.028 0.004
0.025 0.003
0.031 0.005
22.03 0.81
12.78 0.31
16.52 0.22
18.55 0.33
17.67 0.45
20.44 0.57
21.66 0.32
Toxic
ALA-NE
Standard Drug
Marketed formulation
Dox-NE
f-Dox-NE

Drug Deliv. and Transl. Res.
Fig. 10 Regulation of mitochondrial associated protein signaling in
mammary gland cells. (a) Protein extracted from individual groups
[control, DMBA administered toxic, Standard and f-Dox-NE]. (b)
Desitometric data for expression of individual protein in different
groups. [Values are presented as Mean SD (n = 3). The test groups
were compared to the DMBA treated (Toxic) group (*p < 0.05,
**p < 0.01, ***p < 0.001)]
capillaries. The drug release studies depicted that the standard
drug and the marketed formulation possess immediate type of
drug release profiles. In contrast, the Dox-NE and f-Dox-NE
showed sustained release behavior owing to the slower diffu-
sion of the drug from the lipidic environment thereby
providing therapeutic concentration for a longer time period
i.e., up to 48–72 h (Fig. 5) [35, 37]. The developed formula-
tion was stable against several stresses supporting the theory
of formation of stable nanoemulsion system. During the for-
mulation of NE, both ALA as well as ALA-PEG-NH2 were

Drug Deliv. and Transl. Res.
Fig. 11 Whole mount of the mammary gland tissue of the animal (a) control (normal saline), (b) toxic control, (c) ALA-NE, (d) standard drug solution,
(e) Marketed formulation, (f) Dox-NE, (g) f-Dox-NE. [TEB: Terminal end bud; AB: Alveolar bud]
taken in 20:1 w/w ratio. Since ALA-PEG-NH2 accounts for
only 0.95% w/w of the formulation and ALA accounts for
30.50% w/w, its inherent qualities would nonetheless be
displayed. ALA-PEG-NH2 would naturally align itself on
the surface because of its hydrophilic linker (PEG) on which
ligand (folate) would attach, while unmodified ALA being
hydrophobic in nature, would naturally position itself within
the core of oil droplets. The folate targeting ligand decoration
was successfully performed and conjugation was affirmed by
FTIR, 1H-NMR spectrum and folate content assay which was
in corroboration with previous studies [18, 24–26]. Upon sys-
temic administration, the physical stability of the
nanoemulsions are susceptible to globule aggregation and
physical disruption leading to the rapid clearance, abrupt drug
release and dose dumping from the nanoemulsion. Whereas,
stable delivery systems are conducive to longer systemic
Fig. 12 Effect of Dox treatment
on (a) MDA levels and (b) GSH
levels in rat heart tissue. Control,
pure Dox, ALA-NE and f-Dox-
NE. [All the values are expressed
as mean SD, (n = 6);
***P < 0.001 versus control and
**a p < 0.01 versus Dox
treatment]

Drug Deliv. and Transl. Res.
Fig. 13 Amount of Dox in different organs (mammary gland, heart, liver,
spleen, and kidney) after i.v. administration through tail vein (10 mg/kg
Dox equivalent dose) of (a) Standard (Pure Dox solution) (b) Marketed
formulation (c) Dox-NE (Dox loaded NE) (d) f-Dox-NE (folate decorated
Dox loaded NE). [Values are presented as Mean SD (n = 6);
***p < 0.001 as compared to standard and marketed; **p < 0.05 as com-
pared to other groups]
circulation and residence time, thereby increases the possibil-
ity of higher tumor accumulation through enhanced perme-
ability and retention (EPR) effect [38].
components such as phospholipids, proteins, DNA etc. The
damage to the cellular components leads to increase in mito-
chondrial membrane permeability which ultimately pushed
the cancer cell to apoptosis [10, 30]. The loss of MMP is
coupled to the initiation of the intrinsic apoptotic pathway in
the mitochondria. Anticancer agents are known to depolarize
the mitochondria in the cultured MCF-7 cells. These finding
suggested the role of the mitochondria mediated apoptosis in
the cancer cells. Recently, a study in our laboratory demon-
strated the anticancer potential of ALA that promoted the mi-
tochondrial apoptosis in the DMBA induced mice mammary
gland carcinoma [10].
In vivo evaluation of the anticancer efficacy of the system
performed in mammary gland tumor induced in rats using
DMBA as a cancer inducing agent through rat model
established and validated in our laboratory earlier [10]. The
animal weight, survival and total tumor burden suggested
mammary gland tumor progression in DMBA treated animals.
In vitro studies like the MTT assay revealed 36.25% incre-
ment in the cell mortality after Dox was encapsulated into the
nanoemulsion as evident by the IC50 values. Compared with
the Dox and Dox-NE, the f-Dox-NE treatment exhibited sig-
nificantly higher cellular mortality in the MCF-7 cell lines,
i.e., 69.18 and 41.99% (p < 0.01). The cell cycle analysis
depicted marked arrest of the MCF-7 cells in the G1 and
G2/M phase in the f-Dox-NE treated cells while Dox signifi-
cantly arrested cells in the G1 phase. The incorporation of
ALA and Dox in a single system decreased the population
of the cells in G2 phase and increased the cell population in
the G1 and S phase. The cell proliferation, ROS and MMP
data established the superiority of f-Dox-NE over other for-
mulations. The increase in production and accumulation of the
ROS in the MCF-7 cells caused damage of various cellular

Drug Deliv. and Transl. Res.
The ROS are constantly produced in aerobic cells and are
counter balanced by the antioxidant enzymatic bio-machiner-
ies. However, during the stress situations like cancer, the
counter effects of these enzymatic bio-machineries are com-
promised. A profound damage to the cellular lipids and pro-
teins was recorded in DMBA treated animals when monitored
for peroxidative markers of the lipid (TBARS) and the protein
(protein carbonyl) peroxidation. In line with the same, in-
creased ROS production also restrained the antioxidant bio-
machineries of GSH, SOD and catalase as they all work to-
gether to neutralize the ROS through series of peroxidation,
dismutation and oxidation reactions [10, 30].
Among the several anti-apoptotic and pro-apoptotic protein
changes in the expression of Bcl-2 and MMP-9, Caspase-9
and BAX were monitored in the current study. Bcl-2 and
MMP-9, pro-migratory proteins, are known to be
overexpressed in the mammary gland cancer which in turn
promotes the cancer cell proliferation and metastasis [6, 8].
The results of immunoblotting assay suggested that over-
expression of Bcl-2 and MMP-9 in DMBA treated groups
which was successfully restored by f-Dox-NE and standard
treatments. Western blot data suggested the higher anticancer
potential of the modified nanoemulsion [9]. The MMP-9 act
through PKC and ERK1/2 signaling pathways [8] and the Bcl-
2 family proteins viz., Bcl-2 and BAX act through COX Va,
myosin Va and Twist1 [6, 7, 39] and their roles are critical for
the regulation of mammary cancer cell migration, invasion,
and cancer metastasis.
The whole mount preparation is a convenient method
for frequent examination of the small proliferative le-
sions as represented through increase in the number of
AB/TEBs and lobules. The undifferentiated and high
AB/TEBs counts are considered as the sites for the ma-
lignant transformations. The treatment with the f-Dox-NE
was evident with shrinking the AB/TEB count to sizable
amount suggesting positive modulatory effect mediated
by the targeted delivery of Dox and the ameliorative
activity of the ALA against cellular proliferation.
Cardiotoxicity originated from the lipid peroxidation of
Doxorubicin limits its use as a drug of choice [5]. The f-
Dox-NE treated group evidenced no significant alteration of
markers of cardiotoxicity supporting the cardioprotective po-
tential of ALA for cardiac cells and the targeted delivery. The
biodistribution studies further affirmed the selective accumu-
lation of the folate decorated NE owing to the FR over-
expressed in mammary carcinoma (Fig. 13a–d). However,
significant fraction of the Dox from the Dox-NE and f-Dox-
NE was found to be accumulated in the liver and spleen,
indicating involvement of mononuclear phagocytic system.
Even the folate attachment showed no significant change in
biodistribution in liver which might be explained by higher
capture of the folate decorated nanoemulsion by the liver as it
is a major storage organ of folate [40, 41].
As evident from previous discussion, folate mediated de-
livery was safe and effective in controlling the cellular prolif-
eration and migration in mammary gland carcinoma. The rel-
ative selectivity of folate decorated NE for cancer might be
explained on the basis of high folate requirement for rapid cell
growth which happens in cancerous cells. Therefore, folate
receptors (FR) are upregulated in cancers like, lung, colon,
mammary gland, kidney and brain in comparison with normal
cell. Further, the folate has high affinity (Kd~10–10 M) for
FR. Attachment of folate to relatively long PEG chain (MW
3.4 kDa) resulted in effective FR-mediated association and
internalization of folate decorated NE in cancer cells.
Further, superiority of f-Dox-NE and Dox-NE is probably
related to the presence of ALA and better integration of the
lipidic carrier to the cell membrane leading to a higher inter-
nalization better anticancer effect of the drug delivery system.
In line with the earlier findings, the f-Dox-NE was ob-
served to be more efficacious in comparison to the marketed
and the standard treated groups and the same could be attrib-
uted to two facts. Firstly, ALA possesses anticancer effect of
its own towards highly proliferating cancerous cells and being
a PUFA it acts against the ROS (i.e., pro-oxidants) for normal
cells. Secondly, the folate decoration of the Dox loaded ALA
based NE directs the system specifically to mammary gland
cancer cells with over-expressed folate receptors. This facili-
tates the selected binding and endocytosis of the delivery sys-
tems to the tumor cells. Finally, the release of the entrapped
drug in controlled and sustained manner maintains the drug
concentration for a longer period in the cancer cells, leading to
higher apoptosis in these cells. The overexpressed folate re-
ceptors in the mammary cancer cells could be the possible
explanation of higher efficacy of the folate targeted NE as
compared to the non-targeted Dox-NE.
Conclusion
This study reports for the first time the development of ALA-
based nanoemulsion for targeted delivery of doxorubicin to
DMBA-induced mammary cancer. QbD-based concept was
successfully employed for development of a stable
nanoemulsion as revealed by physicochemical characteriza-
tion. ALA showed strong antioxidant, anticancer,
cardioprotective activity and enhanced the anticancer potential
of Dox. In vitro studies using the MCF-7 cell lines demon-
strated the apoptotic and antiproliferative effects of the devel-
oped formulation through ROS and mitochondria mediated
programmed cancer cell death. Immunoblotting successfully
elucidated the regulation of the pro/anti-apoptotic and pro/
anti-migratory proteins by the nanoemulsion. Percent surviv-
al, weight variation and cardiotoxicity data suggested higher
efficacy and safety of the nanoemulsion vis-à-vis marketed
formulation. The folate decoration preferentially delivered

Drug Deliv. and Transl. Res.
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Acknowledgements The authors acknowledge the kind support of
Miracalus Pharma, India, for providing the gift sample of the drug.
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Compliance with ethical standards
Conflict of interest The authors have no conflicts of concern with re-
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