Drug Delivery

ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: http://www.tandfonline.com/loi/idrd20

Development, optimization and evaluation of

surfactant-based pulmonary nanolipid carrier
system of paclitaxel for the management of drug
resistance lung cancer using Box-Behnken design

Prabhjot Kaur, Tarun Garg, Goutam Rath, R. S. Rayasa Murthy & Amit K.
Goyal

To cite this article: Prabhjot Kaur, Tarun Garg, Goutam Rath, R. S. Rayasa Murthy & Amit K.
Goyal (2016) Development, optimization and evaluation of surfactant-based pulmonary nanolipid
carrier system of paclitaxel for the management of drug resistance lung cancer using Box-Behnken
design, Drug Delivery, 23:6, 1912-1925, DOI: 10.3109/10717544.2014.993486

To link to this article: https://doi.org/10.3109/10717544.2014.993486

Published online: 29 Dec 2014.

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ISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, 2016; 23(6): 1912–1925

! 2014 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/10717544.2014.993486

RESEARCH ARTICLE

Development, optimization and evaluation of surfactant-based

pulmonary nanolipid carrier system of paclitaxel for the management
of drug resistance lung cancer using Box-Behnken design
Prabhjot Kaur, Tarun Garg, Goutam Rath, R. S. Rayasa Murthy, and Amit K. Goyal

Department of Pharmaceutics, I.S.F. College of Pharmacy, Moga, Punjab, India

Abstract Keywords
In the present study, nanostructured lipid carriers (NLCs) along with various surfactants loaded Box-Behnken design, nanolipid carrier,
with paclitaxel (PTX) were prepared by an emulsification technique using a Box-Behnken paclitaxel, pulmonary drug delivery,
design. The Box-Behnken design indicated that the most effective factors on the size and PDI surfactant
were at high surfactant concentration (1.5%), low lipids ratio (6:4) and medium homogenization
speed (6000 rpm). Among all the formulations, Tween 20-loaded NLCs show least particle size History
compared to Tween 80 and Tween 60. Entrapment efficiency of Tween 20, Tween 80 and
Tween 60-loaded formulations were 82.40, 85.60 and 79.78%, respectively. Drug release of Received 13 November 2014
Tween 80, Tween 20 and Tween 60-loaded NLCs is 64.9, 62.3 and 59.7%, respectively (within Revised 25 November 2014
72 h). Maximum cellular uptake was observed with Tween 20 formulation on Caco-2 cell lines. Accepted 26 November 2014
Furthermore, spray drying of resultant NLCs was showed good flow properties and was
selected for drug delivery to deeper airways. In-vivo studies demonstrated the better
localization of drug within the lungs using different surfactant-based pulmonary delivery
systems. From this study, we have concluded that delivering drugs through pulmonary route
is advantageous for local action in lungs as maximum amount of drug concentration was
observed in lungs. The surfactants could prove to be beneficial in treating drug resistance lung
cancer by inhibiting P-gp efflux in the form of nano lipidic carriers.

Introduction has short-term physical stability upon dilution due to the

precipitation of PTX in the aqueous media. Other problems
Paclitaxel (PTX) is a mitotic inhibitor used in cancer
with ethanol and CrEL include leaching of plasticizer from
chemotherapy. Paclitaxel is now widely used in the treatment
polyvinyl chloride infusion bags and sets during injection of
of different types of cancer including lung, ovarian, breast,
the drug (Kim et al., 2006). Due to the above-aforementioned
head and neck cancer, and advanced forms of Kaposi’s
limitations, there is a need for new alternative formulations
sarcoma (Chaudhary et al., 2014; Gagandeep et al., 2014).
that can overcome poor aqueous solubility of PTX, deliver
Cancer treatment found to be more challenging with the
the drug more specifically to the target organs, and produce
consequences of chemo-resistance (Garg, 2014). Anticancer
fewer side effects.
drugs that cause MDR mostly are hydrophobic in nature
Several approaches, such as parenteral emulsions (Singh
with amphipathic character including taxanes (PTX and
et al., 2014c), liposomes (Garg & Goyal, 2014b), nanopar-
docetaxel), vinca alkaloids (vinorelbine, vincristine and
ticles complex with cyclodextrin (Singh et al., 2014b), water
vinblastine), anthracyclines (doxorubicin, daunorubicin
soluble prodrugs (Singh et al., 2014a), and polymeric micelles
and epirubicin), epipodophyllotoxins (etoposide and tenipo-
(Sharma et al., 2014c) have been employed to deliver PTX
side), etc.
into the body or targeted tissue (Emami et al., 2012; Sharma
Currently, PTX is commercially available as Taxol in a
et al., 2014b). Liposomal encapsulation enables increased
vehicle composed of 1:1 of Cremophor EL (CrEL) and
antitumor efficacy (Sharma et al., 2014a) and decreased
ethanol to enhance water solubility of the drug. Some serious
toxicity (Rohilla et al., 2014) due to higher accumulation of
side effects associated with CrEL include hypersensitivity
drug in tumors (Hao et al., 2005). In spite of these advantages,
reactions, nephrotoxicity and neurotoxicity. In addition, Taxol
liposomes display some storage (Pabreja et al., 2014) and
drug leakage problems (Morie et al., 2014) and a relatively
high cost for large-scale production (Wong et al., 2007;
Modgill et al., 2014). Stable formulations are required to take
Address for correspondence: Dr Amit K. Goyal, Associate
full advantage of the more specific drug distribution into solid
Professor, Department of Pharmaceutics, I.S.F. College of
Pharmacy, Moga 142001, Punjab, India. Tel: 9878286888. Email: tumors (EPR effect) (Marwah et al., 2014) and to maximize
amitkumargoyal1979@gmail.com cell exposure (Drummond et al., 2010; Malik et al., 2014).
DOI: 10.3109/10717544.2014.993486
The use of solid lipids in nanoparticle preparations is a very
attractive alternative for overcoming some of the limitations
associated with liposomes and has attracted increasing
attention to the possible use of solid lipid nanoparticles
(SLN) and related carriers since the 1990s (Muèller et al.,
2000; Mehnert & Mäder, 2001; Kaur et al., 2014i). Solid lipid
nanoparticles are sub-micron particles made from biocom-
patible lipids that are solid at room and body temperature
(Muèller et al., 2000; Kaur et al., 2014h). When compared
with liposomes, SLNs have a slower degradation rate in vivo
(Kaur et al., 2014g), providing better control of drug release
(Kaur et al., 2014f) and superior protection of the incorpo-
rated drug (Vivek et al., 2007; Kaur et al., 2014e).
Furthermore, these systems are easily and cost-effectively
produced on a large scale by high pressure homogenization
techniques (HPH) already available in the pharmaceutical
industry for the production of parenteral O/W emulsions
(Muèller et al., 2000; Kaur et al., 2014d).
However, SLNs are associated with some potential limi-
tations, namely, limited drug loading and drug expulsion
during storage due to the crystallization of lipid matrix or
lipid polymorphism (Kaur et al., 2014c). To overcome these
limitations, nanostructured lipid carriers (NLCs) by mixing
solid lipids with chemically very different liquid lipids (oils)
have been developed to improve drug loading and release
properties of conventional SLNs (Kaur et al., 2014b). By
using special mixtures of solid lipids and liquid lipids the
particles become solid after cooling but do not crystallize,
which leads to more imperfections in the crystal and higher
drug loading (Müller et al., 2002a; Kaur et al., 2014a).
Nanostructured lipid carriers are an improved generation of
SLNs produced by the controlled mixing of solid lipids with
liquid lipids (Müller et al., 2002a; Kataria et al., 2014). These
particles display improved drug incorporation and release
(Joshi et al., 2014). The advantages associated with using
SLN and NLC for cancer chemotherapy were recently
reviewed by Wong et al. (2007).
The objective of the present study was to develop
surfactant-based NLCs for pulmonary delivery of PTX to
the lung cancer cells. In this work for preparation of NLCs,
oleic acid (OA) has been used as liquid lipid and glyceryl
monostearate as solid lipid base. Cremophor EL was used as a
component of marketable formulations of PTX (Taxol);
however, this formulation was found to be toxic. Usually,
non-ionic surfactants can improve dissolution in water
insoluble moieties because of being highly hydrophobic and
comparatively lesser toxicity to biological membranes (Rege
et al., 2002; Johal et al., 2014). Further research reveals the
capability of Tweens to inhibit efflux pumps. Lo (2003) has
established that Tween 20, Tween 80, Myrj 52 and Brij 30
enhance the epirubicin transport and decrease in efflux of
diffusion chambers with excised rat intestinal mucosa. P-gp
inhibition action is based on two essential parameters, i.e.
surfactant concentration and hydrophilic–lipophilic balance
(HLB) (Goyal et al., 2014b; Hussain et al., 2014). Surfactant
concentrations which are non-hazardous to the intestinal
mucosa may be usually preferred for the inhibition of P-gp
(Goyal et al., 2014a). Greater than the crucial micelle
concentration (CMC) surfactants become much beneficial in
the solubilization of lipophilic substrates, while they would
Surfactant-based pulmonary NLC of paclitaxel 1913
gave double achievement of solubilizing lipophilic drugs and
inhibiting efflux (Goyal et al., 2013b). Though, the prototype
of P-gp inhibition alters on the basis of the kind of excipients
used (Garg et al., 2014c). On the basis of research, P-gp
inhibitory activity raises when CMC is achieved and after
CMC there is a failure of the inhibitory activity lead to drug
(P-gp substrate) entrapment in the micelles (Garg et al.,
2012b). In a further development, inhibitory action raises yet
beyond CMC and this could be accredited to the information
that substrate entrapped in micelles avoid P-gp-mediated
efflux. The most advantageous HLB range of surfactant
systems among appropriate hydrocarbon chains and polar part
is a significant issue in designing potential drug formulations.
The most favorable development on the intracellular aggre-
gation of drug was attributing among intermediary HLB range
of 10–17 (Lo, 2003; Goyal et al., 2013a).
Therefore, it is postulated that surfactant containing NLCs
may concentrate in cancerous cells with high level of p-gp in
drug resistance. After inhalation of the NLCs by pulmonary
route, surfactant base of the nanoparticles inhibit p-gp for
reversal of drug resistance (Garg et al., 2013). Nanoemulsions
are in liquid form and undergo instabilities, such as floccu-
lation, drug release, creaming, gelling and aggregation of
particles; whereas, NLCs combine the advantages of poly-
meric nanoparticles, fat emulsions and liposome (Nishiyama
& Kataoka, 2006; Garg et al., 2014b). But in our study, using
surfactant simply inhibits p-gp confirmed by maximum
cellular uptake on Caco-2 cell lines. Maximum cellular
uptake of NLCs due to use on hydrophilic surfactants (Garg
et al., 2014a), because hydrophilic surfactant solubilized the
nanoparticles and gave smaller particle size (Garg et al.,
2012a). Smaller particle size mediates internalization of the
drug to the cancerous cells. In this circumstance, the drug is
largely released inside the cancer cells resulting in enhanced
efficiency (Garg & Goyal, 2014a).
The Box-Behnken design was used to optimize and
evaluate the effect of different processing variables on
characteristics of the NLCs. In aqueous dispersed form,
NLCs show an increase in particle size after a short period of
time. Hence, in a subsequent study, we aimed to increase the
stability of NLCs using spray drying to convert into dry
powder form, so as to easily deliver through pulmonary route.
Moreover, we evaluated the organ distribution of drug
incorporated in NLCs and compared with those of free drug
solutions using the HPLC method.
Materials and methods
Materials
Glyceryl monostearate, oleic acid, Tween 80, Tween 20,
Tween 60, heparin, L-leucine, mannitol sodium hydroxide
were obtained from CDH Laboratory Reagents (New Delhi,
India). Acetic acid glacial, ammonium acetate, fetal bovine
serum, methanol, RPMI 1640 medium, trypsin-EDTA solu-
tion and Potassium dihydrogen phosphate were purchased
from HIMEDIA (Mumbai, India). HPLC grade acetonitrile
and methanol from Rankem Laboratory reagents (New Delhi,
India). Human intestinal cancer cells (Caco-2) were obtained
from NCC (Pune, India). Paclitaxel was purchased from
Emcure Pharmaceuticals (Pune, India).
1914 P. Kaur et al.
Methods
Preparation of NLC using an emulsification and
ultrasonication method
Paclitaxel-loaded nanostructured lipid carriers (NLCs) were
prepared by the emulsification and ultrasonication method
(Vohra et al., 2013). The lipid and aqueous phases were
prepared separately. The solid lipid/liquid lipid phase con-
sisted of stearic acid (or glyceryl monostearate) and oleic acid
at different ratios. Aqueous phase (20 ml) was prepared by
taking desired amount of surfactants (Tween 80, Tween 20,
Tween 40), and it was dissolved in water with continuous
stirring under magnetic stirring (0683, SPINOT Ambala,
Haryana, India) at low revolutions per minute. Lipid phase
was prepared by taking desired amount of lipid (glyceryl
monostearate and oleic acid) that was heated at 70  C.
Paclitaxel being insoluble in water was added to lipid phase
and dissolved. Both aqueous phase and lipid phase were
heated separately at 60–70  C, and then aqueous phase was
poured into lipid phase at the same temperature. This solution
was allowed to homogenize for 3 min at 500 rpm, and this
emulsion was immediately subjected to further size reduction
using a probe sonicator (40050, Steryl Medi Equip System,
Chennai, India). Emulsion was probe sonicated at 60%
amplitude for time 4 min. The period of ultrasound burst was
set 9 s with a pause of 3 s between two ultrasound bursts.
Obtained solution was refrigerated at 2–3  C for 15 min.
Experimental design and analysis
In this study, a 17-run, 3-factor, 3-level Box-Behnken design
was employed to construct polynomial models for the
optimization process, because it requires few runs with 3 or
4 variables. The Box-Behnken design was specifically
selected since it requires fewer runs than a CCD in cases
of three or four variables. This cubic design is characterized
by set of points lying at the midpoint of each edge of a
multidimensional cube and centre point replicates (n¼3)
whereas the ‘‘missing corners’’ help the experimenter
to avoid the combined factor extremes. This property
prevents a potential loss of data in those cases. A design
matrix comprising of 17 experimental runs was constructed.
The non-linear computer-generated quadratic equation is
given as:
Y ¼ b0 þ b1A þ b2B þ b3C þ b12AB þ b13AC
þ b23BC þ b11A2 þb22B2 þb33C 2
where, Y is the measured response associated with each
factor level combination; b0 is an intercept; b1 to b3 are
regression coefficients computed from the observed experi-
mental values of Y from experimental run; and A, B and C are
the coded level of independent variables representing the
linear terms.
The dependent and independent variables selected are
shown in Table 1 along with their low, medium and high
levels for the preparation of final optimized formulation. The
solid lipid:liquid lipid, surfactant concentration and hom-
ogenization speed used for NLCs formation were taken as
independent variables. These three independent variables
were analyzed by the design for two responses which are
Drug Deliv, 2016; 23(6): 1912–1925
Table 1. Variables in the Box-Behnken design.
Levels used
Factor Independent
variables Low (1) Medium (0) High (+1)
X1 Solid lipid:Liquid lipid 6:4 7:3 8:2
X2 Surfactant concentration 0.5% 1.0% 1.5%
X3 Homogenization speed 4000 rpm 6000 rpm 8000 rpm
Dependent variables Constraints
Y1 Size (nm) Minimum
Y2 PDI Minimum
particle size and PDI and these two responses were dependent
variables.
Design matrix comprising of 17 experimental runs with a
range of three variables, i.e. Solid lipid:Liquid lipid (X1),
Surfactant concentration (X2) and Homogenization speed
(X3) and the respective observed responses, i.e. Size and PDI.
The responses obtained after the preparation of these 17
formulations were filled in the design. These data were
analyzed by the design of the experiment (Design ExpertÕ
(Version 9.0.3.1, Stat-Ease Inc., Minneapolis, MN). The best
fitting model was selected. The run which showed the
minimal size range of the NLCs and having minimum range
of PDI was found to be optimized. Finally, optimized was
selected for further characterization.
Determination of particle size and zeta potential of NLCs
Size and zeta potential of NLCs were determined by laser
diffractometry using the Delsa TM Nano C Particle analyzer
(Beckman Coulter India Pvt. Ltd., Mumbai, India). All
samples were diluted appropriately with the aqueous phase
of the formulation for the measurements. Z-Average particle
size, polydispersity index (PI) and zeta potential were
measured in triplicate.
Determination of drug entrapment efficiency (EE) and drug
loading (DL) for NLCs
Paclitaxel-loaded NLCs were separated from the NLCs
suspension by centrifugation at 25–000 rpm for 1 h. Then
1 ml supernatant was taken and diluted up to 100 ml with
methanolic PBS. Finally, drug content was analyzed at 217 nm
using a UV-spectrophotometer (Perkin Elmer, Kyoto, Japan).
EE was calculated using the following formula:
Amount of drug added  Amount of free drug
% EE ¼  100
Total amount of drug added
Amount of drug added  Amount of free drug
% DL ¼  100
Total amount of lipid ðsÞ
In-vitro drug release
In-vitro release was evaluated by using a dialysis bag
diffusion technique under sink condition. The drug release
from PTX-NLCs was performed in methanolic phosphate
buffer saline (PBS) (pH 7.4) using dialysis diffusion bag.
The suspension was placed in dialysis bags (MWCO 12 000
DOI: 10.3109/10717544.2014.993486
Da, Sigma Aldrich, USA) and the dialysis bag was
subsequently placed in flask containing 200 ml dissolution
medium Methanolic-PBS (pH 7.4) were stirred at 150 rpm at
37  C in an incubator shaker. Aliquots of the dissolution
medium were withdrawn at each time interval and the same
volume of fresh dissolution medium was added to the flask
to maintain a constant volume. Drug concentration in the
dissolution medium was determined using a UV/Vis
Spectrophotometry (Shimadzu 1700, Japan) at 217 nm. All
experiments were carried out in triplicates. The release
profile were fitted to kinetic equations (zero order, first
order, Higuchi release and Korsmeyer-Peppas release,
Hixson Crowell) to understand the release mechanisms.
In vitro culture studies
Cell cultures: Caco-2 cell culture. To track the entry of
nanoparticles into the cells, SQV-NLCs were labeled with
the fluorescent dye coumarin-6. Briefly, 5 mg of coumarin-
6 was incorporated in the lipid phase of the formulation
and the preparation continued as aforementioned. Caco-2
cells were grown in RPMI-1640 medium supplemented
with 10% (v/v) inactivated fetal bovine serum, 1% (v/v)
trypsin-EDTA solution, at 37  C under a 10% CO2/90% air
atmosphere. Caco-2 cells were poured into a 24-well plate
and cultivated over 24 h. The permeability of PTX across
intestinal in vitro models was evaluated by comparing free
PTX with all the three surfactant-loaded PTX-NLC formu-
lations, in Caco-2 cells. The experiments were conducted
at 37  C or 4  C by adding a volume of 100 ml at 30 mg/ml
PTX concentrations.
As mentioned previously, surfactants are well-known P-gp
inhibitors. To evaluate the role of surfactant-based NLCs in
the inhibition of P-gp, cells were pre-treated with a solution
of 100 mM verapamil, a well-known P-gp inhibitor, for 1 h and
nanoparticles were subsequently added on the apical side and
incubated for 2 h in the presence of verapamil. The evaluation
of surfactant-based NLCs suspension was also carried out
under P-gp inhibition to confirm that surfactant was a P-gp
substrate in our Caco-2 cell model. In all the assays carried
out in the presence of inhibitors, several inserts were kept as
controls and the transport studies were carried out in transport
buffer instead of in inhibitor solutions.
Intracellular uptake of NLCs by Caco-2 cells. Entry of
nanoparticles into Caco-2 cells was studied qualitatively by
fluorescence microscope for which coumarin-6-loaded nano-
particles were employed. For this study, Caco-2 cells were
seeded in 24-well cell culture plates at a density of 5  105
cells per well and allowed to adhere for 24 h until confluency.
As for the transport studies, cells were co-incubated with
100 mL of a coumarin-6-loaded nanoparticles suspension in
transport buffer. After 2 h of incubation with fluorescent
nanoparticles, cells were washed three times with PBS and
detached from the plates by trypsinization. Cells were then
centrifuged at 1500  g, the supernatant was discarded, the
cells were resuspended in PBS and fluorescence was
visualized under fluorescence microscope (Olympus-
CKX41, NY, USA). Cell fluorescence was qualified by
capturing the images of fluorescence of coumarin-6.
Surfactant-based pulmonary NLC of paclitaxel 1915
Spray-drying of NLCs
Spray dried powders were prepared by adding mannitol and
leucine as excipients to improve yield and flow properties of
dry powders. The inlet temperature was maintained at 80  C,
outlet temperature at 40–50  C and vacuum at 100–120  C of
WC. 1% mannitol and 0.2% leucine was optimized on the
basis of good flow properties. Incorporation of leucine into
the formulation prior to spray-drying could improve process
yield because of its antiadherent property.
Re-dispersibility of spray dried nanoparticles
A total of 10 mg drug loaded spray dried dry powder of
NLCs samples dispersed in 10 ml of phosphate buffer (7.4)
with continuous vortexing for 20 min, then centrifuged at
10 000 rpm for 30 min. The particles size and PDI of dispersion
was measured with a Delsa-nano Zetasizer, New Delhi, India.
Solid state characterization of DPI
Solid state characterization of DPI formulations like Angle of
repose, Both bulk density (BD) and tapped density (TD),
Carr’s index (C), Hausner’s ratio determined. The
Compressibility Index of the powder blend was determined
by Carr’s compressibility index. The formula for Carr’s Index
is as shown below:
½ðTD  BDÞ  100
Carrs Indexð%Þ ¼
TD
Hausner’s ratio by using the formula:
H ¼ 100/(100C)
The angle of repose was calculated by:
tan  ¼ h/rwhere,  is angle of repose, h is height of the
particles pile and r is distance from the centre of the pile to
the edge.
Particle morphology
Particle morphology of the DPI was performed by Scanning
Electron Microscopy (SEM) and motic microscopy for
determining the surface morphology, size and shape of
formulation and to observe the aggregation property of
NLC with carrier particles.
In-vivo studies
For animal experiments, the protocol complies with the
particular recommendation and approval obtained for their
protocols. Wistar rats of either sex (180–200 g) were used to
study lung deposition of optimized formulations. Organ
distribution studies were obtained after pulmonary adminis-
tration of dry powder of NLCs in comparison to oral
administration of PTX solution in methanolic PBS. Two
groups of rats were taken for pharmacokinetic and lungs
deposition study. Each group had nine rats.
Route of administration and withdrawal of blood
sample. Concentration in organs was obtained from oral
administration of PTX solution, inhalation of DPI of NLCs.
Two groups of rats were taken with nine rats in each group.
Rats were sacrificed and organs (lungs, liver, kidney and
spleen) were removed. For organs distribution studies, organs
1916 P. Kaur et al.
were homogenized separately using phosphate buffer (pH
7.4). Then centrifugation was done at 6000 rpm for 30 min.
Then supernatants were separated and collected in eppendorfs
for further studies. 0.2 ml of the supernatants was taken and
1.8 ml of acetonitrile was added for the purpose of depro-
teinization. Vortexing was done for 5 min and then centrifu-
gation was done for 15 min at 3000 rpm. After that, 1 ml of
supernatant was removed from the centrifuged samples and is
diluted with 1 ml of methanolic PBS and assayed
spectrophotometrically.
Results and discussion
Optimization of NLCs by DOE
The results of the experimental design were analyzed using
Design-Expert software, MN, USA, which provided consid-
erable useful information and reaffirmed the utility of
statistical design for conduct of experiments. The selected
independent variables including the GMS/oleic acid ratio,
surfactants (Tween 80, Tween 20 and Tween 60) concentra-
tion, and homogenization speed, significantly influenced the
observed responses for particle size and PDI. Polynomial
equations involving the main effect and interaction factors
were determined based on estimation of statistical parameters,
such as multiple correlation coefficient, adjusted multiple
correlation coefficient and the predicted residual sum of
squares generated by Design-Expert software.
Analysis of responses
All the responses observed for 17 formulations prepared were
simultaneously fitted to first order, second order and quadratic
models using Design Expert.
Response Y1 (particle size)
Analyzing this response required no transformation, the best-
fitted model was observed to be the linear for Tween 80
loaded NLCs and quadratic for Tween 20 and Tween 60
loaded NLCs and the comparative values of R2, SD and %
C.V. are given in Table 2 along with the regression equation
generated for this response. Only statistically significant
(p50.05) coefficients are included in the equations.
Response Y2 (PDI)
Variation in PDI is a function related to the rate at which
nanostructured lipid carriers are dispersed in the prepared
Table 2. Summary of results of regression analysis for responses Y1, for fitting to linear and quadratic model.
Quadratic model
{Response (Y1) Size} R2 R2 Adjusted
Tween 80 0.9628 0.9151
Tween 20 0.8286 0.6081
Tween 60 0.9617 0.9124
Regression equations of the fitted models (Tween 80)
Particle size (Y1) ¼ 307.02 + 36.54A  17.17B  26.91C + 5.02AB  66.80AC + 59.77BC  7.29A2  5.51B2  119.97C2
Regression equations of the fitted models (Tween 20)
Particle size (Y1) ¼ 383.20 + 72.46A  18.66B + 20.08C + 18.55AB  6.58AC  48.52BC  27.50A2  3.50B2  221.88C2
Regression equations of the fitted models (Tween 60)
Particle size (Y1) ¼ 279.76 + 40.05A + 7.70B  15.35C + 0.87AB  97.88AC + 54.57BC  7.44A2 + 33.16B2 + 232.46C2
Drug Deliv, 2016; 23(6): 1912–1925
suspension. Thus, a transformation is required while analyz-
ing this response and applied, and the best-fitted model was
observed to be quadratic in all the type of surfactant-loaded
NLCs and the comparative values of R2, SD and % C.V. are
given in Table 3 along with the regression equation generated
for this response. Only statistically significant (p50.05)
coefficients are included in the equation.
Response surface analysis for Tween 80 NLCs particle size
and PDI
Contour plot analysis further explained that in all probable
case of interactions, low level of lipids ratio (A), and high
levels of surfactant concentration (B) and medium level of
homogenization speed (C) was providing favorable range of
minimum particle size as well as PDI. Surface response
curve in Figures 1–9 further justified the above made
conclusion.
Effects of variables on particle size
Effect of homogenization speed on particle size. Contour plot
analysis showed that in the case of interaction between the
solid lipid:liquid lipid (A) and surfactant concentration (B),
the effect of increased homogenization speed on the size
response was observed. At low homogenization speed, the
prevalence of red region was high. With the gradient
inclination of speed, the acceptable blue region was increas-
ing, which represents that the size was significantly decreases.
Then, further increase in speed, the prevalence of green
region shows again an increase in size. The phenomenon may
be by keeping the ratio of lipids constant, the homogenization
speed varied and concluded that the particle size was found to
decrease at certain extent then increases with an increase in
homogenization speed. This concluded that at low to medium
speed, particle size decreases due to breakdown in particles,
but drastic change was seen at high speed due to more
breakdowns of vesicles which forms aggregates and ultim-
ately size increases.
Effect of surfactant concentration on particle size. Contour
plot analysis showed that in the case of interaction between
the solid lipid:liquid lipid (A) and homogenization speed (C),
the effect of increased Tween 80 concentrations on the size
response was observed. At lower levels of Tween 80, the
prevalence of acceptable blue region was quite low. With the
R2 Predicted SD % CV
0.6709 24.81 6.94
0.6883 88.93 18.80
0.5438 41.45 10.33
DOI: 10.3109/10717544.2014.993486 Surfactant-based pulmonary NLC of paclitaxel 1917
Table 3. Summary of results of regression analysis for responses Y2, for fitting to quadratic model.

Quadratic model
{Response (Y2) PDI} R2 R2 Adjusted R2 Predicted SD % CV
Tween 80 0.8550 0.6687 0.5802 0.048 20.57
Tween 20 0.8288 0.6086 1.1669 0.024 7.66
Tween 60 0.9303 0.8406 0.1090 0.014 4.96
Regression equations of the fitted models (Tween 80)
PDI (Y2) ¼ 0.17 + 0.047A  0.011B  7.875E  003C + 0.011AB  0.022AC + 0.018BC  5.925E  003A2  2.675E  003B2 + 0.13C2
Regression equations of the fitted models (Tween 20)
PDI (Y2) ¼ 0.28 + 0.021A  2.750E  003B + 3.000E  003C + 2.000E  003AB  5.000E  004AC + 8.500E  003BC + 2.000E  004A +
1.700E  003B2  0.059C2
Regression equations of the fitted models (Tween 60)
PDI (Y2) ¼ 0.26  9.00E  003A  4.500E  003B  3.750E  003C  0.013AB  0.029AC + 0.027BC + 2.325E 003A2 + 4.325  003B2 
0.050C2

Figure 1. Response surface plot showing effect of interaction between solid lipid:liquid lipid (A) and surfactant concentration (B) with medium level of
homogenization speed (C) on response (size) and PDI.

Figure 2. Response surface plot showing effect of interaction between solid lipid:liquid lipid (A) and homogenization speed (C) with high level of
surfactant concentration (B) on response (size) and PDI.

gradient inclination of Tween 80, the acceptable blue region
was increasing, which represents that the size was signifi-
cantly decreases. The phenomenon may be by keeping the
amount of lipid constant, the concentration of surfactant
varied and concluded that the particle size was found to
decrease with an increase in surfactant concentration. In
emulsification, due to high shearing droplet size usually gets
reduced and also droplet have tendency to form aggregate in
order to reduce their surface energy. But presence of
surfactant molecule stabilizes the emulsion by providing a
thick protective layer around the droplet to solve aggregation
problem. Similarly, at low concentration of surfactant, amount
1918 P. Kaur et al.
Figure 3. Response surface plot showing effect of interaction between surfactant concentration (B) and homogenization speed (C) with low level of
lipids ratio (A) on response (size) and PDI.
Figure 4. Response surface plot showing effect of interaction between solid lipid:liquid lipid (A) and surfactant concentration (B) with medium level of
homogenization speed (C) on response (size) and PDI.
of lipid increased, size generally increases due to a lack of
ability of surfactant solution to stabilize emulsion at low
concentration.
Effect of solid lipid:liquid lipid on particle size.Contour plot
analysis showed that in the case of interaction between the
surfactant concentration (B) and homogenization speed (C),
the effect of increased ratio of lipids on the size response was
observed. At a low lipid ratio, the prevalence of blue region
was high. With the gradient inclination of lipids ratio, the
acceptable blue region was decreasing, which represents that
the size was significantly increases. The phenomenon may be
by keeping the surfactant ratio constant, the lipids ratio varied
and concluded that the particle size was found to increase
with increase in lipids ratio. This concluded that at low to
high lipid ratio, particle size increases due to an accumulation
Drug Deliv, 2016; 23(6): 1912–1925
of vesicles which forms aggregates and ultimately size
increases.
Effects of variables on PDI
Effect on PDI. Contour plot analysis for response of PDI
showed same results as in particle size, because if large is the
particle size, value of the PDI is too greater. As such, small
particle size shows less PDI. This is because the PI is directly
related to the particle size. Contour plot analysis further
explained that in all probable case of interactions, low level of
lipids ratio (A), and high levels of surfactant concentration
(B) and medium level of homogenization speed (C) was
providing favorable range of minimum PDI.
Although, same trend was followed in design for all the
three cases, i.e. Tween 80, Tween 20 and Tween 60 but still
Tween 20 gave more compact size with narrow range of PDI
DOI: 10.3109/10717544.2014.993486 Surfactant-based pulmonary NLC of paclitaxel 1919

Figure 5. Response surface plot showing effect of interaction between solid lipid:liquid lipid (A) and homogenization speed (C) with high level of
surfactant concentration (B) on response (size) and PDI.

Figure 6. Response surface plot showing effect of interaction between surfactant concentration (B) and homogenization speed (C) with low level of
lipids ratio (A) on response (size) and PDI.

could be better lipid in order to develop the final drug-loaded

optimized formulation. The particle size and particle size
distribution are critical factors in the performance of
nanoparticles, as batches with wide particle size distribution
may show significant variations in drug loading, drug release,
bioavailability and efficacy. Formulation of nanoparticles
with a narrow size distribution will be a challenge if emulsion
cannot be produced with a narrow droplet size distribution.
As nanoparticles are internalized into cells by endocytosis, an
increase in particle size will decrease uptake and potentially
affect bioavailability of the drug.
Particle size and zeta potential of NLCs
The properties of PTX-loaded NLCs, such as particle size, PI,
zeta potential, entrapment efficiency, drug loading and release
percent are listed in Table 4.
It was found that the size of drug-loaded particles was
higher than that of drug-free ones due to the incorporation of
drug in NLC matrix. On the other hand, the most effective
factor on particle size relates to the drug content. Increasing
the concentration of surfactant decreased the particle sizes of
nanoparticles. Müller et al. (2002b) have shown that
1920 P. Kaur et al.
Figure 7. Response surface plot showing effect of interaction between solid lipid: liquid lipid (A) and surfactant concentration (B) with medium level
of homogenization speed (C) on response (size) and PDI.
Figure 8. Response surface plot showing effect of interaction between solid lipid: liquid lipid (A) and homogenization speed (C) with high level of
surfactant concentration (B) on response (size) and PDI.
increasing the poloxamer concentration to 1% was effective in
producing smaller size SLN composed of tripalmitin,
cetylpalmitate and glycerylmonostearate. In the present
study, it seems that 1.5% surfactant is sufficient to cover the
surface of nanoparticles effectively and prevents agglomer-
ation during the process. Zeta potential is often a key factor to
understand the stability of colloidal dispersion. As shown in
Table 4, the absolute value of zeta potential in most
formulation shows the stability of formulations.
In vitro release of PTX from NLC
Figure 10 shows the drug release from NLCs. Surfactant type,
sonication time and homogenization rate did not significantly
affect the release rate. For all formulations, a biphasic drug
Drug Deliv, 2016; 23(6): 1912–1925
release pattern was observed, which is due to the burst release
at the initial stage and is followed by a sustained release in the
later times.
Uptake study of different surfactant-loaded
formulation on Caco-2 cell lines
Different surfactant-loaded NLCs labeled with the fluorescent
dye coumarin-6 were prepared to track the entry of
nanoparticles into the cells. Prepared formulations was
poured into 24-well plates with grown cultured cells and
then visualized by fluorescence B-excitation filter on
20  magnification objective. Tween 20-loaded NLCs show
maximum cellular uptake, which was selected for further
studies, i.e. formulation of dry powder inhaler by spray drying
DOI: 10.3109/10717544.2014.993486 Surfactant-based pulmonary NLC of paclitaxel 1921

Figure 9. Response surface plot showing effect of interaction between surfactant concentration (B) and homogenization speed (C) with low level of
lipids ratio (A) on response (size) and PDI.

Table 4. Various parameters of different surfactant-loaded NLCs.

Surfactant loaded Particle size Zeta potential Entrapment In-vitro release

NLCs (nm) PDI (mV) efficiency Drug loading (within 72 h)
Tween 80 243.1 ± 7.47 0.225 ± 1.25 16.12 82.40% 2.36% 62.3 ± 3.45%
Tween 20 178.7 ± 5.38 0.158 ± 0.69 15.22 85.60% 2.37% 64.9 ± 3.40%
Tween 60 398.2 ± 6.20 0.281 ± 0.53 22.23 79.78% 2.16% 59.7 ± 2.8%

80 was size-dependent, i.e. formulation C4B4D. This finding

Tween 80 NLCs
% cumulative drug release

is consistent with Rejman et al. (2004), who also reported a

Tween 20 NLCs tendency to decreased internalization with an increased
60 Tween 60 NLCs particle size. These authors studied the pathway of entry
and subsequent fate of commercial latex nanoparticles inside
40 the cell and concluded that particles with a diameter of less
than 200 nm enter the cell via clathrin-mediated endocytosis
whereas larger particles (200 nm–1 mm) enter preferentially
20
via caveolae-mediated endocytosis. Moreover, the surface
hydrophobicity of the nanoparticles may also determine
0 nanoparticle entrance into Caco-2 cell because the larger
0 20 40 60 80 100 uptake into the cells is correlated with the higher nanoparticle
Time (in hrs.) surface hydrophobicity (formulation C4B4D). In our study,
nanoparticle size and surface hydrophobicity were major
Figure 10. In-vitro release of different surfactant-loaded NLCs.
factors influencing NLC entrance into the cell.

Particle morphology, size, PDI and zeta potential of

using its characterization and in-vivo studies. The fluorescent
images of plain drug and all the surfactant-loaded NLCs
confirming cellular uptake are shown in Figure 11.
From the above results we can conclude that cellular
uptake was more in the form of NLCs as compared with plain
drug solution. Moreover, Tween 20 loaded formulation shows
maximum uptake which successfully confirms the surfactant-
based formulation. More is cellular uptake; greater is p-gp
efflux inhibition. P-gp efflux inhibition is due to the presence
of p-glycoprotein in Caco-2 cell lines. Evidently, inhibition of
p-gp may treat drug resistance.
However, the efflux rate of this treatment was significantly
greater than that of free PTX. The cellular uptake of NLCs
spray dried formulation (DPI)
The size of the PTX-loaded NLCs was found to be
283.4 ± 4.5 nm and PDI was 0.226 ± 0.94. In order to confirm
the size of NLC and to observe surface morphological
characteristics, microscopic images and SEM were carried
out, respectively. Microscopic and SEM images (Figure 12)
showed uniform size; mono-dispersed round shaped NLCs.
Slight rough surface could be observed in majority of NLCs
in SEM photographs. Zeta potential of NLVs estimated by
electrophoretic mobility technique using zetameter was found
to be 25.12 mV indicating the stability of nanoparticle
during storage.
1922 P. Kaur et al. Drug Deliv, 2016; 23(6): 1912–1925

Figure 11. Cellular uptake of (A) Plain drug solution; (B) Tween 80 loaded NLCs; (C) Tween 20 loaded NLCs and (D) Tween 60 loaded NLCs.

Figure 12. Motic (A) and SEM (B) images of optimized formulation.

Table 5. Flow properties of Tween 20 loaded NLCs after spray drying.

Flow Bulk density Tapped density Carr’s Hausner’s Angle of Percentage Aerodynamic
properties (g/cm3) (g/cm3) index ratio repose yield (%) diameter (mm)
Results obtained 1.36 1.60 18.35 1.22 22.29 33.9 3.53

Solid state characteristics
Solid state characteristics like angle of repose, bulk and
tapped density, Carr’s index, Hausner’s ratio, percentage yield
and aerodynamic diameter are essential parameters that
determine the delivery efficiency of DPI formulations.
These characteristics determined for DPI formulations are
given in Table 5.
Improved aerodynamic properties of nanoparticulate for-
mulations have confirmed the finding to previous reports that
mannitol have been used to improve the yield and fine particle
fraction of the aerosol powder. Similarly, leucine was used
because of its dispersibility enhancing properties, which
improved the aerosolization characteristic of powders. The
effect of leucine on particle surface morphology of spray
dried powders containing mannitol was obvious. SEM
photographs of these powders presented spherical particles
with rough surface. This surface roughness could improve the
aerosolization capability of produced spray dried powders.
By adding leucine to the excipients, particle size of spray
dried powders decreased significantly. It has been reported
that incorporation of leucine into the formulation prior to
spray-drying could improve process yield because of its
antiadherent property. The data tabulted in Table 5 indicates
good flow property of the powders.
In-vitro release studies of PTX NLCs and DPI
In-vitro drug release profile of optimized formulation after
and before spray drying using dialysis bag in methanolic PBS
(pH 7.4) was shown in Figure 13.
Drug release study on optimized formulation after
redispersion showed 67.7% drug release from NLCs and
64.9% drug release from DPI. As shown in Figure 5, there is
DOI: 10.3109/10717544.2014.993486 Surfactant-based pulmonary NLC of paclitaxel 1923

no significant change in the burst effect and drug release In-vivo studies
content from NLCs before and after lyophilization. Drug
Organ distribution studies of optimized formulations at
release profile shows the Higuchi model having R2 ¼ 0.871,
different time intervals were done on male Wistar rats
which shows release of drug from formulations by diffusion.
(180–220 g). Doses were delivered through pulmonary route
The initial drug release (up to 8–10 h) is because of the burst
by using insufflators (Penny Century, PA, USA). The bio-
effect that does not correspond to the real mechanism of the
analytical method was developed by HPLC spectroscopy. The
drug release from NLCs. It is due to immediate dissolution of
chromatographic conditions were optimized as WATERS,
drug particles adsorbed on the surface of NLCs. About 50% of
C-18 column (250 mm*4.6 mm*5 m) as stationary phase,
drug was released from NLCs within the first 48 h and the
Buffer (Na2HPO4): ACN (43:57) as mobile phase and flow
70% drug was released up to 72 h. Drug release followed the
rate was 1 ml/min. The amount of drug distributed in different
Higuchi kinetics indicating the release predominantly by
organs (lungs, liver, spleen and kidney) is shown in Figure 14
diffusion. Increased diffusional distance due to the formation
and Table 6.
of drug depletion zone at particle surface and presence
Lungs uptake of the drug from the DPI was higher as
on the surrounding lipid shell barrier might have slowed
compared to plain drug suspension. Because in case of
down the release rate resulting in a sustained drug release
lyophilized NLC, clearance of the drug from the organ was
profile.
less due to slow release of the drug from the NLC. PTX-DPI
longevity in lung tissue was a direct consequence of the
80 retention of the lipidic nanocarriers at the site and the
% cumulative drug release

NLC of PTX retention of the drug in lipid nanoparticles. The potential for
DPI of PTX pulmonary delivery of may have clinical significance in the
60 light of recent data demonstrating that PTX-NLC-DPI was
highly effective, following inhalation in rat lungs. Drug
concentration in lungs was higher because of the direct
40
exposure of the PTX-DPI to lungs.

20 Conclusion
In this study, drug-loaded NLCs were successfully prepared
0 using different surfactants. NLCs were prepared by the
0 20 40 60 80 100 emulsification technique using a Box-Behnken design and
Time (in hrs.) displayed small, homogeneous size, high entrapment effi-
ciency and satisfactory drug loading. The Box-Behnken
Figure 13. Relese profile of NLCS before and after spray drying. design indicated that the most effective factors on the size
and PDI were at high (1.5%) surfactant concentration, low
lipids ratio (6:4) and medium homogenization speed
Lungs Spleen (6000 rpm). Maximum cellular uptake was observed with
400 Liver Plain drug solution
Tween 20 formulation was shown on Caco-2 cell lines.
Drug concentration (in µg)

Furthermore, spray drying of resultant NLCs showed good

Kidney
aerodynamic behavior and were found suitable for drug
300
delivery to deeper airways. In-vivo studies confirmed the
therapeutic efficacy of developed formulation and demon-
200 strated the better localization of drug within the lungs
using different surfactant-based pulmonary delivery sys-
100
tems. We can conclude that delivering drugs through
pulmonary route for local action is advantageous. The
surfactants were helpful in treating drug resistance lung
0 cancer by inhibiting P-gp efflux in the form of nano
hr

hr

hr

hr

lipidic carriers. This work can thus give us the better idea
1

Time (in hrs) to work in the direction of surfactant-based delivery system

by inhibiting P-gp efflux inhibition for treating lung cancer
Figure 14. Organ distribution studies of PTX-loaded NLC-based DPI. via pulmonary route.

Table 6. Amount of drug present in organs (lungs, liver, kidney, spleen), homogenate of tested and control animals.

Time (h) Lungs (Formulation) Liver (Formulation) Kidney (Formulation) Spleen (Formulation) Lungs (Plain Drug Solution)
AFTER 1 h 287.33 ± 3.75 52.33 ± 3.17 34.00 ± 2.08 25.00 ± 1.55 51.66 ± 3.17
AFTER 4 h 266.66 ± 2.60 47.00 ± 2.30 28.66 ± 2.33 17.66 ± 0.88 46.00 ± 2.08
AFTER 8 h 263.00 ± 3.46 39.00 ± 1.52 25.00 ± 0.57 16.33 ± 1.45 40.06 ± 2.49
1924 P. Kaur et al.
Declaration of interest
The authors confirm that this article content has no conflicts
of interest.
Author Dr Amit K. Goyal is thankful to the Department of
Biotechnology (DBT), New Delhi (under IYBA scheme; BT/
01/IYBA/2009 dated 24 May 2010), for providing financial
assistance to carry out research.
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