See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/49744018

A novel strategy for encapsulating poorly soluble drug into nanostructured lipid
carriers for intravenous administration

Article in Pharmaceutical Development and Technology · January 2011

DOI: 10.3109/10837450.2010.546411 · Source: PubMed

CITATIONS READS

17 315

8 authors, including:

Chunyan Zhao Tingting Fan

National University of Singapore University of Leicester
8 PUBLICATIONS 372 CITATIONS 9 PUBLICATIONS 398 CITATIONS

SEE PROFILE SEE PROFILE

Yang Yang Zhi-Rong Zhang

Sichuan University Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education
117 PUBLICATIONS 3,686 CITATIONS 555 PUBLICATIONS 18,077 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Chunyan Zhao on 23 August 2014.

The user has requested enhancement of the downloaded file.

 Pharmaceutical Development and Technology, 2012; 17(4): 443–456
© 2012 Informa Healthcare USA, Inc.
ISSN 1083-7450 print/ISSN 1097-9867 online
DOI: 10.3109/10837450.2010.546411

RESEARCH ARTICLE

A novel strategy for encapsulating poorly soluble drug into

nanostructured lipid carriers for intravenous administration
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

Chunyan Zhao, Yan Liu, Tingting Fan, Dan Zhou, Yang Yang, Yun Jin, Zhirong Zhang,
and Yuan Huang
Key Laboratory of Drug Targeting and Drug Delivery, Ministry of Education, West China School of Pharmacy, Sichuan
University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China

Abstract
The present study aimed to formulate dexamethasone (DXM), a poorly soluble drug, into nanostructured lipid carriers
(NLCs) for intravenous administration by employing a phospholipids complex. Initially, dexamethasone-phospholipids
complex (DPC) was synthesized and characterized. Subsequently, DPC was entrapped into NLCs and the process was
optimized using spherical symmetric design-surface response methodology. Then, the characteristics, in vitro release
behavior and physical stability of the optimized DPC loaded NLCs (DPC-NLCs) were investigated. Comparison between
DPC-NLCs and free DXM loaded NLCs was also conducted in the aspects of particle size, entrapment efficiency (EE),
drug loading efficiency (DL), initial release and zeta potential. The results showed the optimized DPC-NLCs were
For personal use only.

prepared with an average size of 189.33 ± 0.58 nm, EE of 89.82 ± 1.64%, DL of 2.13 ± 0.13% and good physical stability
for 30 days. In vitro release profile exhibited an initial burst release followed by a prolonged release. Compared with
free DXM loaded NLCs, the EE and DL of DPC-NLCs were higher while the initial release was lower. These advantages of
DPC-NLCs proved the phospholipids complex played an essential role in NLCs formulation and showed the potential
for intravenous administration of poorly soluble drugs.
Keywords: Phospholipids complex, spherical symmetric design-surface response methodology, dexamethasone,
physical stability, nanostructured lipid carriers

1. Introduction Currently, the most direct approach for enhancing

A large number of pharmaceutical substances have poor
solubility which is a limiting factor for clinical use. Over
the last decade, new drug candidates have become more
hydrophobic and less water-soluble.[1] The majority of
water insoluble drugs are found in oncology, anti-infec-
tive, central nervous system and anti-viral therapeutic
indications.[2] With the increased patient population in
these diseases, there has been a strong interest in poorly
soluble drug formulation. Unfortunately, intravenous
administration of those hydrophobic agents could be
associated with serious safety problems. For example,
intravenous administration of relatively large aggregates/
crystals of insoluble drug formed in aqueous media may
embolize blood capillaries. As a result, many promising
drug candidates cannot enter clinic use.
solubility is the use of salt form, pH adjustment and
cosolvent. However, if the compound is non-ionizable
or with high liposolubility, the results will not be satis-
fied. For such molecules, emulsion or lipidic system,
may be feasible. In addition, there are several complex-
ing agents that have been employed to address non-
ionizable or high liposoluble drugs. One of such agents is
cyclodextrin.[3] Nevertheless, the allowable cyclodextrin
used in a formulation is limited by the associated toxic-
ity and high solution viscosity.[2] Micellar drug carriers
can also be explored.[4] Regrettably, problems like insuf-
ficient storage stability and frequent instability in the
body limit the application of micelles.[5] Although some
of these approaches have been successfully utilized,
especially for highly potent compounds with low dose

Address for Correspondence: Prof Yuan Huang, West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, P.R. China
Tel/Fax: +86-28-85501617. E-mail: huangyuan0@yahoo.com.cn
(Received 24 September 2010; revised 01 December 2010; accepted 03 December 2010)

443
 444 C. Zhao et al.
requirements, there is a growing need for more effective
and versatile ways to handle formulation issues related to
poorly soluble molecules.
Recently, lipid nanoparticles have been demonstrated
to have the capability to entrap drugs for intravenous
injection.[6] These vehicles are small enough to prevent
embolization of the smallest capillaries and improve the
retention of drug in the body which results in improved
patient compliance owing to reduced frequency of injec-
tion. Additionally, when their surfaces are modified to
avoid being captured by the mononuclear phagocytic
system, targeting to specific tissues or organs can be
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13
achieved.[7,8] Nanostructured lipid carriers (NLCs), the
second generation of lipid nanoparticles, are composed
of solid lipids with spatially incompatible liquid lipids.[9]
In the ideal scenario, the liquid lipids could form drop-
lets within the solid lipid particles matrix. According to
this model, the NLCs nanoparticles would provide a high
incorporation capacity (due to the liquid lipid) as well as
controlled drug release (due to the encapsulating solid
lipid).
Drug phospholipids complex, defined as an integra-
tion of one or more natural active components with
phospholipids, is a frequently-used technique adopted to
increase bioavailability and enhance the pharmacologi-
cal actions of active constituents.[10] In comparison with
its original state, the drug’s lipophilicity can be increased
For personal use only.
in the form of phospholipids complex.
Dexamethasone (DXM) is a potent anti-inflammatory
and immunosuppressive glucocorticoid. It is widely used
for the treatment of inflammatory and autoimmune
conditions like rheumatoid arthritis, edema, multiple
myeloma.[11] Nevertheless, DXM has poor water solubility
and is almost insoluble in most physiologically and phar-
maceutically acceptable solvents, both of which limit its
clinic applications.[12]
The main aim of this work was to formulate DXM
into NLCs for intravenous administration by introduc-
ing phospholipids complex (see Figure 1). Initially,
dexamethasone-phospholipids complex (DPC) was
Phospholipids (PC)
Solvent evaporation
Dxamethasone (DXM) Dexamethasone-phospholipis complex (DPC)
Figure 1. Schematic illustration of synthesis of DPC and preparation of DPC-NLCs.
 Pharmaceutical Development and Technology
synthesized by a solvent evaporation procedure to
improve the liposolubility of DXM. Subsequently,
DPC was entrapped into NLCs and the process was
further optimized by a spherical symmetric design-
surface response methodology to obtain high drug
encapsulation efficiency, high drug loading efficiency,
suitable particle size and sustained drug release pro-
file. Following this, the characteristics, in vitro release
behavior and physical stability of the optimized DPC
loaded NLCs (DPC-NLCs), were investigated. To the
best of our knowledge, this was the first time for DXM
to be encapsulated into NLCs in the form of a phos-
pholipids complex. The comparison between DPC-
NLCs and free DXM loaded NLCs was also conducted
for the first time to explore the importance of DPC in
NLCs formulation. The results showed a new method
to efficiently encapsulate poorly soluble drugs, such
as dexamethasone, was developed. Meanwhile, our
current work provides a promising paradigm to entrap
drug-phospholipids complex into NLCs.
2. Materials and methods
2.1. Materials
Phospholipids (Lipoid E80) was purchased from
Shanghai Toshisun Enterprise Co., Ltd. (Shanghai,
China), and the phosphatidyl content was approxi-
mately 80% (w/w). Dexamethasone was purchased
from Tianyao Pharmaceutical Co., Ltd. (Tianjin, China).
Glycerol tri-caprylate, oleic acid, n-octanoic acid and
glycerol trilaurate were obtained from TCI (Tokyo,
Japan). Medium chain triglycerides was acquired from
Tieling Beiya Pharmaceutical Oil Co., Ltd. (Tieling,
China). Miglyol® 812N was kindly donated by Sasol
(China) Chemical Co., Ltd. (Nanjing, China). Solutol®
H 15 (polyoxyethylene esters of 12-hydroxystearic acid)
was provided by BASF (Luduigshafen, Germany). Myrj
52 (Polyoxyethylene 40 stearate) was from Sigma (St.
Louis, MO, USA). All the other reagents and solvents
were of analytical grade.
Liquid lipid
Solid lipid
O/W emulsion
solvent evaporation
DPC
DPC loaded nanostructured lipid carriers (DPC-NLCs)
 A novel strategy for encapsulating poorly soluble drug into nanostructured lipid carriers for intravenous administration 445
2.2. Preparation of dexamethasone–phospholipids
complex (DPC)
At a molar ratio of 1:2, weighted dexamethasone (DXM)
and phospholipids were placed in a round bottom
flask and dissolved in solvent A (mixture of anhydrous
ethanol and anhydrous acetone at a volume ratio of 1:9).
The mixture was refluxed at 60°C for 7 h. The resulting
clear solution was evaporated and dried under vacuum
(40°C). The product DPC was then collected and stored
at –20°C.
2.3. Characterization of DPC
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13
2.3.1. Thin-layer chromatography (TLC)
Sample solutions were prepared by dissolving DXM,
phospholipids, DPC, the physical mixture of DXM and
phospholipids in methanol respectively. For the physical
mixture, the molar ratio of DXM to phospholipids was 1:
2, which was the same as synthesis of DPC. All sample
solutions were spotted on the TLC plates which were
pre-coated with silica gel at a 0.15−0.2 mm thickness. The
plates were developed with a solvent system comprising
the mixture of ethyl acetate and water.[13] Phospholipids
was visualized by ninhydrin, and the spot of DXM was
observed with a ZF-I ultraviolet analysis instrument
(Shanghai Gucun Optic Instrument Factory, Shanghai,
China) at a wavelength of 254 nm.
For personal use only.
2.3.2. Differential scanning calorimetry (DSC)
The samples sealed in the aluminum crimp cell were
heated at a speed of 5°C /min from 0 to 300°C in a nitro-
gen atmosphere at a flow rate of 50 mL/min. The peak
transition onset temperature of DXM, phospholipids,
DPC and physical mixture were determined and com-
pared with the help of a differential scanning calorimeter
(EXSTAR6000 DSC, Japan).
2.3.3. X-ray powder diffraction (X-Ray) analysis
The X-ray powder diffraction was recorded on an X-ray
diffractometer (PHILIPS X’Pert Pro MPD DY1291, Japan).
Samples were added into the slide for packing prior to
X-ray scanning. Spectra of graphs were plotted from 3.00°
to 65.00° of 2θ angle at room temperature.
2.3.4. Fourier transform infrared spectroscopy (FT-IR)
The Fourier transform infrared spectroscopy (FT-IR)
spectra was recorded on an FT-IR spectrometer (VECTOR
22, Bruker, Germany) after the samples were compressed
into a KBr pellet. The wavenumber range was from 400 to
4000 cm−1.
2.3.5. Solubility studies
Solubility studies were conducted to verify whether the
liposolubility of DXM was enhanced after the forma-
tion of DPC. Briefly, excess amounts of DXM, DPC and
physical mixture were added to 5 mL water or n-octanol
in sealed glass containers at 25 ± 0.5°C, respectively. The
liquids were horizontally shaken (100 rpm) in a constant
temperature culturing shaking incubator (Shanghai
© 2012 Informa Healthcare USA, Inc.
Zhicheng analytical instrument manufacturing Co., Ltd.,
Shanghai, China) for 24 h and centrifuged at 10000rpm
for 10 min. The supernatant was collected and filtered
through a filter membrane (0.45 μm). The filtrate was then
diluted with methanol. Aliquots of the resultant solution
(20 μL) were analyzed by RP-HPLC (1200, Agilent) using
a Dikma C18 column (250 mm×4.6 mm, 5 µm). The detec-
tion wavelength was performed at 240 nm with a mobile
phase composed of 40% acetonitrile and 60% water at
the flow rate of 1 mL/min.
2.4. Preparation of DPC loaded Nanostructured lipid
carriers (DPC-NLCs)
2.4.1. Screening of liquid lipid for NLCs formulation
Solubility studies in oils were carried out to identify
potential liquid lipid ingredient for formulating NLCs.
The solubility of DPC in medium chain triglycerides,
Miglyol® 812 N, glycerol tri-caprylate, oleic acid and
n-octanoic acid was investigated by adding individually
excess DPC to oils (1 g each) in sealed glass containers
at 25 ± 0.5°C. Mixtures were agitated for 24 h and then
centrifuged to remove solid substances (10 000 rpm,
10 min). The supernatant was diluted and the amount
of DXM solubilized in oils was assayed by HPLC as
described above.
2.4.2. Preparation of DPC-NLCs
DPC was formulated into NLCs utilizing the oil-in-water
(O/W) emulsion solvent evaporation technique. A mix-
ture of the solid lipid (glycerol trilaurate (GT)) and the
liquid lipid (medium chain triglycerides (MCT)) (1:1,
w/w) was used as the lipid phase. Briefly, DPC, GT and
MCT were co-dissolved into solvent A (mixture of anhy-
drous ethanol and anhydrous acetone at a volume ratio
of 1:9) in water bath at 50°C. The resultant organic solu-
tion was added into 5 mL aqueous solution containing
Solutol® H 15(1%, w/v) and Myrj 52(1%, w/v) as surfac-
tants. The crude emulsion was further treated by a probe
sonication instrument (TY92-‖, Scientz Biotechnology
Co., LTD. Ningbo, China) at 150W. The organic solvent
was immediately evaporated under reduced pressure
and then purified by filtration through a cellulose acetate
membrane (0.45 μm) to eliminate non-incorporated
drug crystals.
2.4.3. Spherical symmetric design
A 3-factor, 3-level spherical symmetric design was used
to optimize the preparation procedure of DPC-NLCs.[14]
Preliminary investigations of the process parameters
revealed that the DPC to lipid weight ratio (X1), lipid con-
centration (single lipid material) (X2) and ultrasonication
time (X3) were key variables in the NLCs preparation.
Hence, these variables were selected to optimize DPC-
NLCs preparation condition with their ranges and levels
as described in Table 1. The dependent variables were size
(Y1), entrapment efficiency (Y2), initial release at 0.5h (Y3)
and drug loading efficiency (Y4) with constraints applied
as described in Table 1. A statistical model incorporating
 446 C. Zhao et al.
interactive and polynomial terms was used to evaluate of optimized DPC-NLCs (formulation A). For the prepa-
the response employing the equation: ration of free DXM loaded NLCs, the same optimized
manufacturing procedure was applied, but DPC was
Y=b0 +b1 X1 +b2 X 2 +b3 X 3 +b4 X 12 +b5 X 22 + completely replaced with the physical mixture of DXM
b6 X 23 +b7 X1 X 2 +b8 X1 X 3 +b9 X 2 X 3 and phospholipids.

where Y is the dependent variable, b0 is the intercept rep-

2.5.1. Particle size and zeta potential measurement
resenting the arithmetic average of the 15 runs, and b1 to
The particle size and zeta potential of the NLCs were char-
b9 are the estimated coefficients for the factors (Xi, i = 1,
acterized with Malvern Zata-Size Nano ZS90 (Malvern
2, 3). X1, X2, and X3 are the coded levels of the indepen-
Instruments Co., Ltd., Malvern, UK). NLCs suspensions
dent variables. The interaction terms X1X2, X1X3, and X2X3
prepared with different formulations were diluted 8
show how the response changes when two factors are
times with double distilled water for the measurement of
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

changed simultaneously. The polynomial terms Xi2 were

included to investigate nonlinearity. The level values of
three factors and the composition of central composite
design batches 1 to 15 were shown in Table 2. Software
STATISTICA 6.0 was used for the generation and evalua-
tion of the statistical experimental design.
2.5. Characterization of NLCs
In this section, free DXM loaded NLCs (formulation B)
was formulated to compare its characteristics with that
Table 1. Independent variables and their correspondent values
for optimization of DPC-NLCs preparation using the spherical
symmetric design-response surface methodology.
Factor Level
For personal use only.
particle size and zeta potential. All measurements were
carried out at 25°C, in triplicate.
2.5.2. Determination of entrapment efficiency and drug
loading efficiency
After the organic solvent evaporation, crude NLCs
were dispersed in methanol and sonicated. Subse­
quently, the suspensions were centrifuged (10 000 rpm,
10 min) and the supernatants were assayed by HPLC
as described above to gain the initial amount of DXM.
The amount of DXM encapsulated in NLCs was deter-
mined by adding Na2SO4 (10%, w/v) to salt out the
nanoparticles and centrifugation (14 000 rpm, 1 h).
The upper nanoparticles were then added to methanol,

Independent variables –1.732 –1 0 1 1.732 sonicated, and centrifuged (10 000 rpm, 10 min). The
X1 = DPC to lipid weight ratio 0.5 0.6 0.75 0.9 1 supernatant was tested by HPLC for the determination
X2 = Lipid concentration 2 2.6 3.5 4.4 5 of DXM within NLCs. Furthermore, NLCs dispersions
(mg/mL) were freeze-dried to obtain the total weight of NLCs.
X3 = Ultrasonication time (min) 1 1.8 3 4.2 5 Entrapment efficiency (EE%) and drug loading effi-
Dependent variables Constrains ciency (DL%) of DXM loaded NLCs were calculated as
Y1 = Size (nm) ≤200 given below:
Y2 = Entrapment efficiency (%) Maximize
Y3 = Initial release at 0.5 h (%) Minimize amount of DXM encapsulated in NLCs
EE % = ×100%
Y4 = Drug loading efficiency (%) Maximize initial amount of DXM

Table 2. Response values of different variables for the optimization of DPC-NLCs preparation using spherical symmetric design-
response surface methodology.
Factors Responses
Formulation X1 X2 X3 Y1 Y2 Y3 Y4
1 0.60 2.6 1.8 192.23 ± 16.18 82.46 ± 3.90 40.29 ± 11.12 2.05 ± 0.18
2 0.60 2.6 4.2 95.07 ± 4.19 80.36 ± 2.42 50.06 ± 3.28 2.16 ± 0.22
3 0.60 4.4 1.8 203.47 ± 6.43 83.13 ± 4.52 38.48 ± 3.32 2.51 ± 0.24
4 0.60 4.4 4.2 100.4 ± 9.72 77.84 ± 2.61 43.71 ± 4.80 2.54 ± 0.12
5 0.90 2.6 1.8 206.67 ± 4.81 74.67 ± 14.34 38.50 ± 2.80 2.25 ± 0.2
6 0.90 2.6 4.2 95.17 ± 3.30 59.75 ± 1.91 33.65 ± 7.17 1.95 ± 0.08
7 0.90 4.4 1.8 196.57 ± 5.11 37.05 ± 1.22 44.08 ± 6.07 1.65 ± 0.05
8 0.90 4.4 4.2 93.89 ± 1.35 39.68 ± 0.88 47.04 ± 5.18 1.75 ± 0.08
9 0.50 3.5 3.0 164.73 ± 5.77 86.33 ± 0.47 43.35 ± 1.08 2.08 ± 0.16
10 1.00 3.5 3.0 164.67 ± 3.10 43.45 ± 1.25 46.23 ± 7.47 1.83 ± 0.15
11 0.75 2.0 3.0 141.93 ± 7,74 82.85 ± 0.99 38.75 ± 3.58 1.86 ± 0.10
12 0.75 5.0 3.0 141.43 ± 8.11 41.37 ± 3.99 40.01 ± 6.25 1.74 ± 0.11
13 0.75 3.5 1.0 236.6 ± 5.03 58.29 ± 5.31 39.02 ± 2.55 2.02 ± 0.10
14 0.75 3.5 5.0 66.51 ± 9.54 61.72 ± 2.35 39.88 ± 6.61 1.75 ± 0.22
15 0.75 3.5 3.0 149.1 ± 8.707 55.83 ± 5.39 40.07 ± 8.03 1.82 ± 0.16
Values are mean ± S.D. (n = 3).

 Pharmaceutical Development and Technology

 A novel strategy for encapsulating poorly soluble drug into nanostructured lipid carriers for intravenous administration 447

amount of DXM encapsulated in NLCs
DL% = ×100%
total weight of NLCs
2.5.3. In vitro release studies
The visking bag method was used to investigate the
in vitro release features of DXM loaded NLCs.[15] Briefly,
DXM loaded NLCs suspensions were placed in dialysis
bags (molecular weight cut off between 8000–14 000).
Samples were dialyzed against 15mL phosphate buffer
saline (pH 7.4) at 37 ± 0.5°C and mechanically shaken at
a speed of 100rpm. At designated time intervals, 0.5mL
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13
enwrap the phospholipids molecule polarity parts,
which decreased the sequence between the phospho-
lipids aliphatic hydrocarbon chains as well as between
the DXM molecules. This highly dispersed state led
to the masking of the endothermal peak of drug and
induced the disappearance of the DXM endothermal
peak in the DPC thermogram. In addition, with the rise
in temperature one may assume that the phospholipids
melted and DXM got dissolved in phospholipids, partly
forming the complex. This could explain why physical
mixture had a similar DSC endothermal curve to that
of the DPC.

sample of the dialysis medium was taken and the same
volume of fresh medium was added. The amount of DXM
in the aliquots was analyzed by HPLC as described pre-
viously. Simultaneously, the same amount of free DXM
was tested as control.
2.6. Morphological observation of DPC-NLCs
An electron microscope (Hitach H-600, Tokyo, Japan)
was used for morphological observation of the optimized
DPC-NLCs colloidal solution. The DPC-NLCs were placed
on a carbon-coated copper grid. The film on the grid was
negatively stained by immediately adding a drop of 2%
(w/w) phosphotungstic acid.
The results of X-ray powder diffraction also confirmed
that DXM was either molecularly dispersed or presented
as an amorphous form in the phospholipids complex.
DXM (Figure 4A) displayed partially sharp crystalline
peaks, which was characteristic of molecules with crys-
tallinity. Phospholipids (Figure 4C) exhibited obvious
diffraction peaks, indicating its crystalline characteristics.
Crystalline signals were still detectable in physical mix-
ture (Figure 4D) due to DXM and phospholipids, but DXM

2.7. Physical stability of DPC-NLCs

For personal use only.

The physical stability of the optimized DPC-NLCs were

determined as follows. Briefly, 0.5 mL DPC-NLCs disper-
sions were filled into glass vials, flushed with nitrogen
and sealed. All samples were protected from light and
stored at different temperatures (4°C and –20°C) for
1 month, and the changes of particle size against storage
time were investigated.

3. Results and discussion

3.1. Preparation and characterization of DPC
A B C D A B C D
In this study, experiments involving TLC, DSC, X-ray
powder diffraction analysis, IR and solubility were car-
Figure 2. TLC of DXM (A), DPC (B), phospholipids (C) and
ried out to prove the formation of DPC. physical mixture (D).
TLC was performed in order to investigate whether a
new substance was formed. It was shown from Figure 2
that the chromatogram of DPC was the same as the 7
physical mixture. Two kinds of spots were observed: one
6
had the same Rf as DXM (Rf = 0.75), and the other one was
similar to phospholipids (on the starting line). No new 5
DSC (mV/mg)

spot was generated. A

4
The thermogram of DXM (Figure 3A) exhibited a peak B
3
at 271.9°C due to crystallization.[16] However, phospho- C
lipids (Figure 3C) showed no sharp endothermal peak, 2
D
indicating it was not pure. The endothermal peak of DXM 1
disappeared from the curves of both DPC (Figure 3B)
and physical mixture (Figure 3D). It was presumed that 0
0 50 100 150 200 250 300
during the period of forming a phospholipids complex, Temperature(°C)
a directional combination happened between DXM
and the polar parts of the phospholipids.[17] The carbon– Figure 3. DSC thermograms of DXM (A), DPC (B), phospholipids
hydrogen chain in phospholipids could turn freely and (C) and physical mixture (D).

© 2012 Informa Healthcare USA, Inc.

 448 C. Zhao et al.
crystalline peaks were partially covered by phospholip-
ids. This was in agreement with other work that reported
physical mixture of drug and phospholipids could mask
the crystal properties of the drug.[18] No ­crystalline peak
appeared on the XDR graph of DPC (Figure 4B), sug-
gesting that DXM existed in a non-crystalline state. The
disappearance of DXM’s crystalline diffraction peaks
demonstrated the formation of the phospholipids com-
plex. This result was well supported by previous studies
done with the phospholipids complexes of silybin, ber-
genin, and curcumin.[14,17,19]
In order to determine the interaction of DXM and phos-
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13
pholipids, Fourier transform infrared spectral (FT-IR)
analysis was operated. From the FT-IR spectra, the char-
acteristic absorption peaks of DXM (Figure 5A) were pres-
ent at 1708 cm−1(A-1), 1662 cm−1(A-2) and 1617 cm−1(A-3).
Peaks at 1737 cm−1(C-7) and 1650 cm−1 (C-8) were typical
absorption peaks of phospholipids (Figure 5C). However,
Intensity
A
For personal use only.
B chain triglycerides (MCT) was chosen as the liquid lipid
C due to its maximum solubility for DPC among screened
D
0 10 20 30
2–Theta
Figure 4. X-ray diffraction patterns of DXM (A), DPC (B),
phospholipids (C) and physical mixture (D).
A
Transmittance
B 1
C
0
4000
Figure 5. FT-IR spectras of DXM (A), DPC (B), phospholipids (C). The wavenumber of the peaks of 1-8 were 1708, 1662, 1617, 1738, 1664,
1627, 1737 and 1650cm−1 respectively.
 Pharmaceutical Development and Technology
in the spectrum of the complex (Figure 5B), the charac-
teristic absorption peaks of DXM were nearly masked by
that of phospholipids, losing the peaks at 1708 cm−1 and
1617 cm−1. Compared with DXM and phospholipids, the
absorption peak at 1627 cm−1(B-6) in DPC FT-IR spectra
was new. These observations implied that some weak
physical interactions between DXM and phospholipids,
such as hydrogen bonding or Van der Waals force, took
place during the formation of the complex.[14,20]
The solubility of DPC in n-octanol was 10.64 times
greater than that of DXM, while 1.2 times greater in
water (Table 3). For the physical mixture, the addition
of phospholipids did not enhance the drug solubility in
n-octanol. The difference of the solubility between DPC
and the physical mixture indicated an enhancement of
liposolubility of DXM by formation of DPC.
All the above experiments substantiated that the DPC
was not a new chemical compound or simple physical
mixture.
3.2. Preparation of DPC loaded Nanostructured lipid
carriers (DPC-NLCs)
3.2.1. Screening of liquid lipid for NLCs formulation
In comparison to solid lipid nanoparticles (SLNs), NLCs
has higher entrapment efficiency because of the liquid
lipids, which could solubilize drugs in nanoparticles. To
achieve high DPC loading efficiency in NLCs, the medium
oils (Figure 6).
40 50 60
3.2.2. Optimization of experimental data analysis
The response surface methodology using spherical
symmetric design for 3 factors offers an advantage to
minimize the number of tests to be performed.[21] The
3
2
6
5
4
8
7
3500 3000 2500 2000 1500 1000 500
Wavenumber cm−1
 A novel strategy for encapsulating poorly soluble drug into nanostructured lipid carriers for intravenous administration 449
experimental results concerning the tested indepen- reason for selecting nanoparticles with the mean size of
dent variables on the dependent variables were shown less than 200 nm was based on the literature reported by
in Table 2. Four dependent values ranged from 66.51 Kaur et al.[8] They found that particles with size greater
to 236.6 nm (Y1), 37.05 to 86.33% (Y2), 33.65 to 50.06% than 200 nm served as splenotropic agents and were
(Y3) and 1.65 to 2.54%(Y4), respectively. A mathematical later removed by the spleen. Thus, NLCs smaller than
relationship between factors and parameters was gen- 200 nm could increase its circulation time and hence
erated by response surface regression analysis using provide additional time for the drug to be taken up by
the STATISTICA 6.0 software. The polynomial models other organs. Furthermore, the entrapment efficiency
for Y1, Y2, Y3 and Y4 were found to be significant with significantly increased with decreased DPC to lipid
R-value, P-value and F-values (Table 4), indicating a weight ratio and reduced lipid concentration (Figure 8).
good fit to the quadratic model. The fitted models could Whereas, the higher levels of both factors resulted in
be viewed as regression equations. The positive sign more DXM released from NLCs at 0.5 h (Figure 9). The
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

before a factor in polynomial equations represents that two phenomena were not conflicting. Less DPC to lipid
the response increases with the factor, while a negative weight ratio and lower lipid concentration led to more
sign means the response and factors have reciprocal DPC encapsulated in the nanoparticles’core, leaving
relation. For determination of the optimized nanopar- little DPC on the surfaces of particles and finally improv-
ticles formulation, three-dimensional response surface ing entrapment efficiency while retarding initial release
plot graphs from the experimental data were drawn at 0.5 h. As burst effect may cause a peak of drug concen-
(Figure 7–10). tration that can induce adverse and toxic effects, initial
The response surface plot diagrams (Figure 7) showed release at 0.5 h was set as a dependent variable in this
that the longer the ultrasonication time, the smaller the experiment in order to screen an ideal formulation with
particle size. This fact may be induced by the intense minimum initial release. The drug loading efficiency was
structural rearrangement of ingredients during ultra- observed to be reduced on the high levels of DPC to lipid
sonication which reduced the aggregate formation upon weight ratio, lipid concentration and ultrasonication
increase of particle size.[11] On the contrary, higher lipid time (Figure 10).
concentration generated larger particle size. At high lipid Upon comparing various response variables, a pre-
phase concentration, viscosity of inner phase would dicted formulation composition with 0.85 (X1), 2 mg/ml
For personal use only.

increase, decreasing ultrasonication capacity which (X2) and 2 min (X3) was found to fulfill requisites of an
would result in increased particle agglomeration. The optimum formulation.

Table 3. Apparent solubility of samples in water and n-octanol at 3.2.3. Validation of Model Optimization
25°C, n = 3. DPC-NLCs were prepared using the optimum formula-
Apparent solubility (ug/mL) tion in order to evaluate the optimization capability of the
Sample water n-octanol model generated from the results of the surface-response
DXM 68.1 ± 1.1 816.3 ± 109.9 methodology design. Resultant experimental values of
Physical mixture 56.5 ± 2.6 934.0 ± 293.7 the responses were quantitatively compared with the
DPC 81.8 ± 3.8 8684.7 ± 400.4 estimated values for calculating the percentage of the
Values are mean ± S.D. (n = 3). predicted error. The fitted results (Table 5), with low

25
Solubility of DPC in different oils (mg/g)

20

15

10

0
Medium Chain Miglyol® 812N Glycerol Tri- Oleic Acid n-Octanoic
Triglycerides caprylate Acid

Figure 6. Solubility of DPC in medium chain triglycerides, Miglyol® 812N, glycerol tri-caprylate, oleic acid and n-octanoic acid.

© 2012 Informa Healthcare USA, Inc.

 450 C. Zhao et al.

Table 4. Summary of results of regression for response Y1, Y2, Y3, predicted error, were close to the estimated values,
Y4. suggesting the optimized formulation was reliable and
P R F reasonable.
Y1 0.000000 0.9985 1742.89
Y2 0.000016 0.9742 144.23 3.3. Comparison of DPC-NLCs and free DXM
Y3 0.000003 0.9067 301.56
loaded NLCs
Comparing characteristics for each formulation using
Y4 0.000028 0.8661 115.78
SPSS 16.0, significant differences were shown in the
Regression equations of the fitted model
Y1 = 229.998-158.789X1+54.337X2-34.477X3+186.191X12-5.072X22-
results of initial release at 0.5h, entrapment efficiency
0.378X32-25.926X1X2-9.722X1X3+0.324X2X3 and drug loading efficiency (P < 0.05) (Table 6). The ini-
Y2 = 214.893-193.656X1-6.254X2-15.189X3- tial release at 0.5h of formulation A (DPC-NLCs) was
+196.189X12+4.214X22+1.844X32–51.704X1X2-3.403X1X3+1.662X2X3 significantly lower (P < 0.05) than that of formulation B
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

Y3 = 125.061–181.211X1-18.456X2+8.484X3+84.186X12-0.066X22- (free DXM loaded NLCs). The average entrapment effi-

0.020X32+25.120X1X2-11.729X1X3+0.378X2X3
Y4 = 2.76213-2.50351X1+0.52397X2-0.34798X3+5.00356X12+0.07010
ciency and drug loading efficiency of formulation A were
X22+0.06068X32-1.51852X1X2-0.23611X1X3+0.03704X2X3 significantly higher (P < 0.05) than that of formulation B.

260

220
280

180
240
Y1
140
200
For personal use only.

100
Y1 160

60
120
1.1
5.0 1.1
0.9 80
4.0 0.9
0.7 X1
X2 3.0 X1
40 0.7
2.0 0.5
5.0 4.0 0.5
3.0 2.0 1.0
X3

260

220

180

Y1 140

100

60
5.0
20 4.0
X2
3.0

5.0 4.0 2.0

3.0 2.0 1.0
X3

Figure 7. Response surface plot showing the influence of DPC to lipid weight ratio (X1), lipid concentration (X2) and ultrasonication time
(X3) on size (Y1).

 Pharmaceutical Development and Technology

 A novel strategy for encapsulating poorly soluble drug into nanostructured lipid carriers for intravenous administration 451

120
110

100 100

80 90

Y2 Y2 80
60
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

70
2.0
40 1.0
60
20 3.0 2.0
50
X2 3.0
0 4.0 40 X3
4.0
1.0 5.0
0.9 0.8 1.0 5.0
0.7 0.9 0.8
0.6 0.5 0.7
X1 0.6 0.5
X1
For personal use only.

100

90

80
Y2
70

60

50
1.0
40
2.0

5.0 3.0
4.5 X3
4.0 4.0
3.5 3.0
X2 2.5 5.0
2.0

Figure 8. Response surface plot showing the influence of DPC to lipid weight ratio (X1), lipid concentration (X2) and ultrasonication time
(X3) on entrapment efficiency (Y2).

This difference was mainly attributed to the usage Gaete et al. observed that incorporating the physical
of phospholipids complex, and clearly pointed out mixture of DXM and phospholipids failed to improve
phospholipids complex played an essential part in the the encapsulation of DXM in PLGA nanoparticles.[22]
ideal features of formulation A, in terms of low initial In their study, DPPC, a kind of phospholipids, was
release at 0.5h, high entrapment efficiency and drug incorporated in the nanoparticles to favor hydropho-
loading efficiency. From the solubility analysis (Table bic interactions between DXM, phospholipids and
3), the solubility of DPC in n-octanol was found to be polymer matrix. But the amount of DXM loaded in
much higher than the pure drug and physical mixture, nanoparticles was not altered and remained very low
indicating the complex had better lipophilic property. (234.4 ± 4.2 μg/100 mg polymer). Meanwhile, incorpo-
This was also the case when DPC was solubilized in rating the physical mixture of DPPC and DXM did not
medium chain triglycerides (MCT). The saturated con- significantly modify DXM release profile from nano-
centration of DXM in MCT was only 0.189 ± 0.008 mg/g particles, which was similar to the release behavior of
while that was up to 23.213 ± 0.411 mg/g with the formulation B in this study (Figure 11). Nevertheless,
help of phospholipids complex. With this enhanced extra period of time was required for DXM to diffuse
liposolubility in MCT, DXM in complex could be out from formulation A at 0.5 h. This may be raised by
enveloped into NLCs more efficiently. In addition, the enveloping function of the phospholipids complex

© 2012 Informa Healthcare USA, Inc.

 452 C. Zhao et al.

70

60 60

Y3 50 55
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

40 50

Y3
30 45 1.0

2.0
1.0 40
0.9 2.0 3.0
2.5 X3
0.8
3.0 35
0.7 3.5 4.0
X1
0.6 4.0 X2
0.5 4.5 5.0
5.0 1.0 0.9 0.8 0.7 0.6 0.5
X1
For personal use only.

50

46

42
Y3
38

34

30
5.0
5.0 4.5
4.0 4.0
3.5
3.0 3.0 X2
X3
2.0 2.5
2.0
1.0

Figure 9. Response surface plot showing the influence of DPC to lipid weight ratio (X1), lipid concentration (X2) and ultrasonication time
(X3) on initial release at 30 min (Y3).

according to the report that dissolution of drug from Subsequently, delayed release up to 24 h was observed.
lipid matrix was slowly diffused after the formation of The amounts of DXM released from both NLCs disper-
phospholipids complex.[23] sions were within 70% of total drug amount over 24 h,
For both formulations, the mean particle size was whereas about 100% of the free drug was found in release
around 190nm and the zeta potential was within –7 mV medium after approximately 2 hours.
(Table 6) without statistically significant difference The marked biphasic release pattern indicated the
(P > 0.05). drug release model for formulations A and B was a
drug-enriched shell model. This might be because of
3.4. In vitro release studies the accumulation of medium chain triglycerides (MCT)
The ability of NLCs to deliver DXM was examined by at the surfaces of NLCs.[24] When preparing NLCs, the
monitoring the drug release. As shown in Figure 11, lipid core of the particles, composed of MCT dissolved
the two NLCs formulations showed a release pattern in glycerol trilaurate (GT), was recrystallized firstly
characterized by an initial burst release during the first and therefore entrapped within the solid lipid matrix.
sampling period (0.5 h). The percentage of drug burst Subsequently, excess MCT would accumulate in the
release of formulation A (DPC-NLCs) and B (free DXM outer shell of nanoparticles. This kind of oil expulsion
loaded NLCs) were 32.71% and 44.13%, respectively. was typical in the production of NLCs.[25] Even in the

 Pharmaceutical Development and Technology

 A novel strategy for encapsulating poorly soluble drug into nanostructured lipid carriers for intravenous administration 453

3.0

3.5 2.6

Y4
3.0
2.2
2.5

Y4 1.8
2.0
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

1.5 0.5
0.6 1.0
1.0
0.7 2.0
0.8 X1 0.5
3.0
X3 0.6
2.0 0.9 0.7
2.5 4.0
3.0 1.0 0.8
3.5 0.9 X1
4.0 5.0
X2 4.5 1.0
5.0

2.4
For personal use only.

2.2

Y4
2.0

1.8

2.0
1.0 2.5
2.0 3.0
3.5
3.0
X3 4.0 X2
4.0 4.5
5.0 5.0

Figure 10. Response surface plot showing the influence of DPC to lipid weight ratio (X1), lipid concentration (X2) and ultrasonication time
(X3) on drug loading efficiency (Y4).

The finding of burst release profile was in coinci-

Table 5. Comparison of the observed and predicted values in
dence with other published studies of NLCs.[26,27] Since
DPC-NLCs prepared under predicted optimum conditions.
burst effect may lead to a peak of drug concentration
Response variable predicted value observed value bias (%)
Size (nm) 188.16 189.33 ± 0.58 0.62
that can induce adverse and toxic effects, formulation A
EEa (%) 86.34 89.82 ± 1.64 4.03
with less initial release would be preferred in clinic use.
Burst release (%) 35.84 32.71 ± 1.34 8.73
Furthermore, formulation A could delay the drug release
DLb (%) 2.29 2.13 ± 0.13 –5.68
within 24h comparing to formulation B (Figure 11).
a: EE is the short name of entrapment efficiency; b: DL is the
According to the research of Wei et al.,[23] the interac-
short name of drug loading efficiency tion between drug and phospholipids in complex could
Values are mean ± S.D. (n = 3). slow drug release profile, which advocated the retarded
release of DXM from formulation A. The gradual release
case of 2% oil loaded NLCs, oil expulsion occurred. after initial burst from formulation A would be important
Meanwhile, the MCT enriched outer layers possessed to maintain an effective drug concentration after intrave-
soft and equipped considerably higher solubility for nous administration.
DPC/DXM, easily loading large amount of drug and thus To describe the release kinetics of formulation A,
facilitating release.[7] Therefore, the NLCs showed the diverse mathematical models including zero-order, first
burst release at the initial stage and sustained release order, Higuchi, Hixson-Crowell, Baker-Lonsdale, Weibull
subsequently. and Ritger-Peppas models were applied. The release

© 2012 Informa Healthcare USA, Inc.

 454 C. Zhao et al.

Table 6. Comparison of characteristics of formulation A and B.

Formulation Size (nm) EE (%) Initial release (%) DL (%) Zeta potential (mV)
Aa 189.33 ± 0.58 89.82 ± 1.64* 32.71 ± 1.34* 2.13 ± 0.13* –6.15 ± 1.61
Ba 193.33 ± 1.53 80.33 ± 0.78 44.13 ± 7.12 1.85 ± 0.12 –6.79 ± 0.45
a: A means DPC-NLCs; B means free DXM NLCs
*P < 0.05 versus formulation B
Values are mean ± S.D. (n = 3).

100
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13

DXM cumulative release (%)

80

60

*
*
40 *

* *

20

0
0 5 10 15 20 25
Time (h)
For personal use only.

DPC-NLCs Free DXM NLCs Free DXM

Figure 11. In vitro release profiles of DXM from free DXM, formulation A (DPC-NLCs) and formulation B (free DXM NLCs) in PBS
7.4. *P < 0.05 versus the DXM cumulative release from formulation B at this time point.

kinetics from formulation A could be fitted with Weibull

equation (r = 0.8817).

3.5. Morphological observation of DPC-NLCs

In order to provide information on the morphology of
the optimal DPC–NLCs, TEM was used to take images of
the optimized DPC–NLCs. As shown in Figure 12, NLCs
particles were spherical in shape and non-adherent
to each other. DXM crystals were not observed in the
suspensions. 500nm
100nm

3.6. Physical stability of DPC-NLCs
Lipid nanoparticles have a tendency to lose physical
stability during storage since they are heterogeneous
systems and thermodynamically unstable. To investi-
gate the effect of storage temperature on the physical
stability of optimized DPC–NLCs, the NLCs dispersions
were stored at 4 and –20°C in the dark over a period of
30 days.
As illustrated in Figure 13, after 1 month, the aver-
age size of DPC-NLCs marginally increased from 189 to
197 nm at 4°C. The result reflected good stability of the
nanoparticles at 4°C, which indicated their suitability
for intravenous route. This was further confirmed by the
absence of visible phase separation and flocculation.
Figure 12. Transmission electron micrographs of DPC-NLCs.
However, the mean particle size decreased from 189 to
162 nm at –20°C, which might be the result of particle
surface contraction under –20°C.
In general, particle aggregation is less likely to occur
for charged particles (around −30 mV) due to elec-
tric repulsion. But this rule cannot strictly be applied
for systems which contain steric stabilizers, Solutol® H
15 and Myrj 52 in this study. The adsorption of steric
stabilizers would decrease the zeta potential due to
the shift in the shear plane of the particle.[26,28] In addi-
tion, steric hindrance is another effect which increases
stability.

 Pharmaceutical Development and Technology

 A novel strategy for encapsulating poorly soluble drug into nanostructured lipid carriers for intravenous administration 455
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13
Figure 13. The changes of particle size against storage time at 4
and –20°C.
4. Conclusion
The results showed that a poorly soluble drug, DXM,
can be efficiently formulated into NLCs by introduction
of phospholipids complex. Initially, DPC was success-
fully prepared by the certification of TLC, DSC, X-ray
powder diffraction analysis, IR and solubility studies.
The optimized formulation was obtained using a 3-fac-
tor, 3-level spherical symmetric design and achieved
with particle size of 189.33 ± 0.58 nm, high entrapment
efficiency of 89.82 ± 1.64%, high drug loading efficiency
For personal use only.
of 2.13 ± 0.13%, spherical shape and good physical sta-
bility for 30 days. In vitro release profile of optimized
DPC-NLCs exhibited an initial burst release followed
by a prolonged release and the release pattern was
found to obey Weibull equation. Upon comparison, the
advantages of DPC-NLCs over free DXM loaded NLCs
proved phospholipids complex played an essential role
in NLCs formulation to encapsulate a poorly soluble
drug. Therefore, phospholipids complex loaded NLCs
have good potential to be exploited as a novel carrier
for intravenous administration to enhance the utility of
poorly soluble drugs.
Declaration of interest
We gratefully acknowledge financial support from National
High-tech R&D Program (863 Program, 2007AA021801),
and National S&T major project (2009zx09310-002). The
authors report no declarations of interest.
References
1. Lipinski CA. Poor aqueous solubility – an industry wide problem
in drug discovery. Am Pharm Rev 2002;5:82–85.
2. Wong J, Brugger A, Khare A, Chaubal M, Papadopoulos P, Rabinow
B, Kipp J, Ning J. Suspensions for intravenous (IV) injection: A
review of development, preclinical and clinical aspects. Adv Drug
Deliver Rev 2008;60:939–954.
3. Brewster ME, Loftsson T. Cyclodextrins as pharmaceutical
solubilizers. Adv Drug Deliver Rev 2007;59:645–666.
4. Blanco E, Kessinger CW, Sumer BD, Gao J. Multifunctional
micellar nanomedicine for cancer therapy. Exp Biol Med 2009;
234(2):123–131.
© 2012 Informa Healthcare USA, Inc.
5. Agarwal A, Lvov Y, Sawant R, Torchilin V. Stable nanocolloids of
poorly soluble drugs with high drug content prepared using the
combination of sonication and layer-by-layer technology. J Contr
Rel 2008;128:255–260.
6. Wissing SA, Kayser O, Müller RH. Solid lipid nanoparticles
for parenteral drug delivery. Adv Drug Deliver Rev 2004;56:
1257–1272.
7. Lu W, He LC, Wang CH, Li YH, Zhang SQ. The use of solid
lipid nanoparticles to target a lipophilic molecule to the liver
after intravenous administration to mice. Int J Biol Macromol
2008;43(3):320–324.
8. Kaur IP, Bhandari R, Bhandari S, Kakkar V. Potential of solid
lipid nanoparticles in brain targeting. J Contr Rel 2008;127:
97–109.
9. Jores K, Mehnert W, Drechsler M, Bunjes H, Johann C, Mäder K.
Investigations on the structure of solid lipid nanoparticles (SLN)
and oil-loaded solid lipid nanoparticles by photon correlation
spectroscopy, field-flow fractionation and transmission electron
microscopy. J Contr Rel 2004;95:217–227.
10. Bialecka M. The Effect of bioflavonoids and lecithin on the course
of experimental atherosclerosis in rabbits. Annu Acad Med Stein
1997;43:41–43.
11. Bhardwaj U, Burgess DJ. Physicochemical properties of extruded
and non-extruded liposomes containing the hydrophobic drug
dexamethasone. Int J Pharma 2010;388:181–189.
12. China Pharmacopeial Convention. Chinese Pharmacopoeia.
Beijing: China medical science and technology press,
2010;2:249.
13. Kang LY. TLC identification for tabella prednisoni acetatis and
tabella dexamethasoni acetates. Strait Pharma J 2005;17:62–63.
14. Qin X, Yang Y, Fan TT, Gong T, Zhang XN, Huang Y. Preparation,
characterization and in vivo evaluation of bergenin-phospholipid
complex. Acta Pharmacol Sin 2010;31:127–136.
15. Zhao XL, Yang CR, Yang KL, Li KX, Hu HY, Chen DW. Preparation
and characterization of nanostructured lipid carriers loaded
traditional Chinese medicine, zedoary turmeric oil. Drug Dev Ind
Pharm. 2010;36(7):773–80.
16. Boddu SHS, Jwala J, Vaishya R, Earla R, Karla PK, Pal D, Mitra
AS. Novel Nanoparticulate Gel Formulations of Steroids
for the Treatment of Macular Edema. J Ocul Pharmacol Th
2010;26:37–48.
17. Xiao YY, Song YM, Chen ZP, Ping QN. The preparation of silybin–
phospholipid complex and the study on its pharmacokinetics in
rats. Int J Pharm 2006;307:77–82.
18. Liang N, Shi K, Wang YS, Yu YW, Feng LL, Cui FD. Preparation and
characterization of ferulic acid-phospholipid complex. J Shenyang
Pharm University 2008;25:163–168.
19. Maiti K, Mukherjee K, Gantait A, Saha BP, Mukherjee P K.
Curcumin–phospholipid complex: Preparation, therapeutic
evaluation and pharmacokinetic study in rats. Int J Pharm
2007;330:155–163.
20. Cui FD, Shi K, Zhang LQ, Tao AJ, Kawashima Y. Biodegradable
nanoparticles loaded with insulin–phospholipid complex for
oral delivery: Preparation, in vitro characterization and in vivo
evaluation. J Contr Rel 2006;114:242–250.
21. Nekahi A, Dehghani K. Modeling the thermomechanical effects
on baking behavior of low carbon steels using response surface
methodology. Mater and Des 2010;31:3845–3851.
22. Gaete CG, Tsapis N, Besnard M, Bochot A, Fattal E. Encapsulation
of dexamethasone into biodegradable polymeric nanoparticles.
Int J Pharm 2007;331:153–159.
23. Wei W, Shi SJ, Liu J, Sun X, Ren K, Zhao D, Zhang XN,
Zhang ZR, Gong T. Lipid nanoparticles loaded with
10-hydroxycamptothecin–phospholipid complex developed for
the treatment of hepatoma in clinical application. J Drug Target
2010;18:557–566.
24. Teeranachaideekul V, Boonme P, Souto EB, Müller RH,
Junyaprasert VB. Influence of oil content on physicochemical
properties and skin distribution of Nile red-loaded NLC. J Contr
Rel 2008;128:134–141.
 456 C. Zhao et al.
25. Jores K, Mehnert W, Mäder K. Physicochemical Investigations
on Solid Lipid Nanoparticles and on Oil-Loaded Solid Lipid
Nanoparticles: A Nuclear Magnetic Resonance and Electron Spin
Resonance Study. Pharm Res 2003;20(8):1274–1283.
26. Yang CR, Zhao XL, Hu HY, Li KX, Sun X, Li L, Cheng DW. Pre­
para­tion, Optimization and Characteristic of Huperzine A Loaded
Nanostructured Lipid Carriers. Chem Pharm Bull 2010;58(5):656–661.
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by National Univ. of Singapore on 02/06/13
For personal use only.
27. Doktorovova S, Souto EB. Nanostructured lipid carrier-based
hydrogel formulations for drug delivery: A comprehensive review.
Expet Opin Drug Deliv 2009;6(2):165–176.
28. Lim SJ, Kim CK. Formulation parameters determining the
physicochemical characteristics of solid lipid nanoparticles
loaded with all-trans retinoic acid. Int J Pharm 2002;243:
135–146.

 Pharmaceutical Development and Technology

View publication stats