International Journal of Biological Macromolecules 81 (2015) 718729

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Synthesis, characterization, release kinetics and toxicity prole of

drug-loaded starch nanoparticles
Mehrez E. El-Naggar a, , M.H. El-Rae a , M.A. El-sheikh a , Gina S. El-Feky b,c , A. Hebeish a
a
b
c

Textile Research Division, National Research Centre (Afliation ID: 60014618), 33 Behouth St., Dokki, Giza, Egypt
Pharmaceutical Technology Department Centre (Afliation ID: 60014618), National Research Centre, Egypt
Pharmaceutics Department, Faculty of Pharmacy, October University for Modern Sciences and Arts, Egypt

a r t i c l e

i n f o

Article history:
Received 17 June 2015
Received in revised form 30 August 2015
Accepted 3 September 2015
Available online 7 September 2015
Keywords:
Nanoprecipitation
Cross-linked starch nanoparticles
Drug Delivery
In vitro release
Histopathological study

a b s t r a c t
The current research work focuses on the medical application of the cost-effective cross-linked starch
nanoparticles, for the transdermal delivery using Diclofenac sodium (DS) as a model drug. The prepared
DS-cross-linked starch nanoparticles were synthesized using nanoprecipitation technique at different
concentrations of sodium tripolyphosphate (STPP) in the presence of Tween 80 as a surfactant. The resultant cross-linked starch nanoparticles loaded with DS were characterized using world-class facilities
such as TEM, DLS, FT-IR, XRD, and DSc. The efciency of DS loading was also evaluated via entrapment
efciency as well as in vitro release and histopathological study on rat skin. The optimum nanoparticles
formulation selected by the JMP software was the formula that composed of 5% maize starch, 57.7 mg
DS and 0.5% STPP and 0.4% Tween 80, with particle diameter of about 21.04 nm, polydispersity index of
0.2 and zeta potential of 35.3 mV. It is also worth noting that this selected formula shows an average
entrapment efciency of 95.01 and sustained DS release up to 6 h. The histophathological studies using
the best formula on rat skin advocate the use of designed transdermal DS loaded cross-linked starch
nanoparticles as it is safe and non-irritant to rat skin.
The overall results indicate that, the starch nanoparticles could be considered as a good carrier for
DS drug regarding the enhancement in its controlled release and successful permeation, thus, offering
a promising nanoparticulate system for the transdermal delivery non-steroidal anti-inammatory drug
(NSAID).
2015 Elsevier B.V. All rights reserved.

1. Introduction
Developing suitable drug delivery systems targeted toward
transdermal disease is one of the major focuses of pharmaceutical
scientists. There are several new transdermal drug delivery systems under investigation such as: hydrogels [1]; microparticles [2];
nanoparticles [36]; liposomes [7]; collagen shields [810]; ocular inserts/discs [11]; and dendrimers [12]. The most promising
of all the developed over the past 25 years of intense research in
medical therapeutics are the nanoparticles due to their sustained
release and prolonged therapeutic benet [13]. Nanoparticles for
drug delivery are part of a relatively new research eld called
nanobiotechnology [14]. The role of nanoparticles is to minimize adverse systemic effects of medical treatments by protecting,

Corresponding author. 33 Behouth st. Dokki. Giza, Egypt.

Tel.: +201126642204, 0020233357807.
E-mail addresses: mehrez chem@yahoo.com, melnagg@ncsu.edu
(M.E. El-Naggar).
http://dx.doi.org/10.1016/j.ijbiomac.2015.09.005
0141-8130/ 2015 Elsevier B.V. All rights reserved.

targeting, and nally releasing a drug at a specic site in the body.

For that purpose, nanoparticles are for example designed to interact
with biological interface through specic targetligand interactions. Nanoparticles are solid colloidal particles made of natural
or articial polymers ranging in size from 10 to 1000 nm [15], in
which drug can be dissolved, entrapped, adsorbed or covalently
attached [16]. Colloid size is an important property that affects
the ability of systems to deliver drugs effectively. Particles having
sizes greater than 1 m are too large to diffuse passively through
epithelial membranes; thus, typically, they remain at the site of
administration. If the size of the particles exceeds ca. 250300 nm,
then most will be captured by ltration in the red pulp of the spleen
and phagocytosed within the cells of the reticuloendothelial system. The fate of particles is determined by their size and surface
character [17].
Transdermal delivery for many drugs could avoid many problems associated with the oral route such as the drastic change of
pH, the presence of enzymes, variable transit times and uctuating drug plasma concentrations. In addition, transdermal delivery
offers longer duration of action resulting in a reduction in dosing

M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

frequency, improved bioavailability, reduced side effects as well as

exibility of terminating the drug administration by simply removing the patch from the skin. Regarding to transdermal therapy, the
key point of dosage design is to enhance the controlled release of
soluble drug and improve its permeability via skin without causing
irritation.
Diclofenac sodium (DS) is a non-steroidal anti-inammatory
drug (NSAIDs). Globally, it is the most widely used class of therapeutic drugs and is a suitable candidate for the development of
sustained and/or controlled release products [18]. The NSAID DS
(DS: 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid) is widely
used in the treatment of rheumatoid disorders and other chronic
inammatory diseases [19,20].
Starch is an abundant, non-toxic, biodegradable, edible, and
relatively inexpensive material. Starch has the ability to be crosslinked on contact with polyanions due to the formation of interand intramolecular cross-linking mediated by these polyanions.
Among some polyanions investigated, sodium tripolyphosphate
(STPP) is the most popular as effective cross-linking agent [21,22].
STPP is a non-toxic solid with no reported adverse effects on
humans [23]. The starchSTPP nanosystems exhibit some attractive features. One of these is the ease of preparation under
mild condition which render them promising carriers for drug
delivery [24].
The simplest method to prepare DS loaded starch nanoparticles
is the solvent displacement method also known as nanoprecipitation method, developed by Fessi et al. [25]. The method is based
on the interfacial deposition of a polymer following displacement
of a semi-polar solvent miscible with water. Low complexity and
low energy consumption of this technique as well as being widely
applicable without any additives make it suitable for the manufacturing of dened nanospheres [26]. However, cross-linked starch
nanoparticles loaded with DS fabricated by nanoprecipitation technique for the controlled release in medical applications presents
some challenges, particularly when toxic organic solvents are used
as dissolving system. These organic solvents might affect the stability of the encapsulated bioactive compounds and leave toxic
residues incompatible with medical applications.
Products safety and stability concerns restrict the use of these
toxic organic solvents thereby making it necessary to develop an
alternative safe process. An attractive method based on the phenomenon that in aqueous polymer systems phase can occur. In
this method, an aqueous solution of a water-soluble polymer is
emulsied as a dispersed phase in an aqueous solution of sodium
hydroxide as a continuous phase. Subsequently, the dispersed polymer phase is cross-linked to form nanospheres.
In the present work, cross-linked starch nanoparticles were
selected as the carrier of choice, and these nanoparticles loaded
with DS were prepared by nano-precipitation method without
using toxic organic solvents. The work will be carried out according
to the following steps: Studying the effect of STPP as crosslinking agent and DS concentrations on the prepared starch. A
two-level factorial design experiment will be used for the prediction of optimized formulation for cross-linked starch nanoparticles
loaded with DS. The formulated nanospheres will be systematically characterized by drug content, encapsulation efciency,
size distribution, Transmission electron microscopy (TEM), polydispersity index (PDI), and zeta potential (ZP) for shape and
surface characters, and in vitro release studies. The formulated
nanospheres will be analyzed by making use of physicochemical characterization using Fourier transform infrared spectroscopy
(FT-IR), X-ray diffraction (XRD) and Differential scanning calorimetry (DSC) to nd out the physical nature (crystallinity), thermal
behavior, possible occurrence of interaction between DS and crosslinked starch nanoparticles. In vivo targeting efciency of the
optimum formula of starch cross-linked with STPP and loaded with

719

DS will be tested histopathologically in skin irritation of healthy

rats.
2. Materials and method
2.1. Materials
Starch was kindly supplied by Starch and Glucose Company,
Egypt. Diclofenac sodium (DS) was kindly gifted by Delta Pharma,
10th of Ramadan, Egypt. Tween 80 (polyoxythylene sorbitan
monoleate) > 99.9% was obtained from SigmaAldrich Chemie
GmbH (Germany). Sodium tripolyphosphate (STPP) was purchased
from SigmaAldrich Chemical Co. Ltd. Phosphate buffer solution
(PBS) was prepared using disodium hydrogen orthophosphate, and
potassium dihydrogen orthophosphate. Carbopol powder was supplied by Across Company (New Jersey, USA). Sodium hydroxide and
absolute ethyl alcohol were of analytical grade and were used as
received. All solutions are prepared using deionized water.
2.2. Methods
2.2.1. Preparation of cross-linked starch nanoparticles loaded
with DS
Cross-linked starch nanoparticles loaded with DS were fabricated by nano-precipitation technique modied as follows: 5%
native maize starch was dispersed in 70 ml distilled water containing 30% sodium hydroxide (based on weight of native maize
starch), which performed as dispersed phase under continuous
high speed homogenization for 30 min at 25 C. 0.4 g of Tween
80 dissolved in 20 ml distilled water containing different amount
of DS (20, 50 and 100 mg) was added drop wise to the dispersed
starch under high homogenization speed. After 30 min, 10 ml distilled water containing different amounts of STPP (0.5, 1, and 2 g)
as cross-linking agent was added gently to the previous solution,
keeping in mind that, the total volume of the reaction mixture
is 100 ml. The reaction mixture was kept stand at room temperature for 2 h with agitation to effect cross-linking with constant
agitation rate at 25 C. The resulting DS encapsulated cross-linked
starch nanoparticles were subsequently precipitated by 100 ml of
absolute ethanol. The resultant powder was puried by means
of centrifugation and washing, rinsed twice with 80/20 absolute
ethanol/water to remove unreacted compounds and nally with
absolute ethanol. Then the resultant nanoparticles were isolated by
means of centrifugation for 1 h at 4500 rpm. At the end, the supernatant was then taken for further analysis to determine the loss in
DS. The supernatant was freeze-dried for 12 h and kept in closed
containers for further analysis. The as described freeze-dried DS
loaded cross-linked starch nanoparticles in the solid state can be
easily re-dispersed in distilled water by hand agitation before use
(Scheme 1).
2.2.2. Factorial design
Different DS loaded starch nanoparticle formulations were prepared based on the 32 factorial design [27]. Percentage of the
cross-linking agent (X1) and that of the drug (X2) in the formulation
were selected as two independent variables. Three levels of each
variable were selected and possible batches were prepared using
different levels of variables. JMP (version 7, SAS, USA) was used
to obtain values of the coefcients in the equation and f statistics
were used to identify statistically signicant terms.
2.3. Characterization
2.3.1. Entrapment efciency (EE%) of nanoparticles
The nanoparticles suspension was centrifuged at 4500 rpm
for 60 min. The supernatant solution was separated. 1 ml of

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Scheme 1. Represents the preparation of DS loaded cross-linked starch nanoparticles.

supernatant was distributed in 10 ml distilled water and the

absorbance was measured using UV spectrophotometer (Shimadzu
1601; Shimadzu, Kyoto, Japan) at 277 nm using water as blank. The
amount of drug un-entrapped in the supernatant was calculated.
The amount of drug entrapped was determined by subtracting
amount of free drug in the supernatant from the initial amount
of DS taken. The experiment was performed in triplicate for each
batch and the average was calculated [28,29]. The drug entrapment
efciency (EE) of nanoparticles was calculated as follows:
Entrapment Efciency (EE%)
= (DS given DS loss)/DS given 100%

2.3.2. In vitro drug release study

The in vitro drug release studies [30] of DS-loaded nanoparticles were performed in duplicate. Briey, accurately weighed
twenty milligrams of freeze-dried nanoparticles samples containing DS, Tween 80 and cross-linked starch were introduced in
cellulose acetate dialysis bags (Dialysis tubing, high retention
seamless cellulose tubing, MEMBRA-CEL MC16*100CLR, molecular
weight cut-off of 11,463 D0405-100FT, SigmaAldrich CHEMIE
GmbHH, Steinheim, Germany) which was tied and placed into
100 ml of phosphate buffer of pH 5.5. The glass beaker was placed
in a mechanical shaking bath (50 cycles/min), with temperature
adjusted to 37 C. At selected time intervals (between 30 and
480 min) samples were withdrawn and replaced with fresh phosphate buffer, then the amount of DS released was analyzed by
measuring the absorbance using a single beam UV spectrophotometer (T80 UVvis spectrometer, PG instruments Ltd, Japan) at
277 nm wavelength [31].
2.3.3. Morphology using transmission electron microscopy (TEM)
The morphology of the prepared samples of cross-linked starch
nanoparticles loaded with DS was observed using TEM (JEOL,
JEM-1200, Japan, an acceleration voltage of 120 kV). The solution

containing nanoparticles was diluted and a drop of it placed on

copper grid without any further modication or coating and
allowed to sit until air-dried.
2.3.4. Hydrodynamic size and particle size distribution
measurements
The particle size and size distribution of the DS loaded crosslinked starch nanoparticles along with its polydispersity (PDI)
were determined using dynamic light scattering (DLS) technique
(Zetasizer nano, Brookhaven Instruments Corporation, Malvern
Instruments, United kingdom). The freeze-dried samples of crosslinked starch nanoparticles was re-dispersed, diluted with ltered
ultra pure water, and analyzed using Zetasizer. The measurements
were performed at scattering angel 90 at 25 C. the particle size
distribution was reported as PDI. The PDI is dened as dispersion
homogeneity, has the range of 01. Values close to 1 indicate heterogeneity and less than 0.5 showed more homogeneity [32].
2.3.5. Zeta potential measurement
Zeta potential is an abbreviation for electrokinetic potential in
colloidal systems. The zeta potential of the synthesized nanoparticles containing different concentrations of STPP and DS was
determined by means of zeta potential analyzer (90 Plus Particle Size Analyzer, Brookhaven Instruments Corporation, using
Zetasizer nano Malvern Instruments, United Kingdom). The measurement of zeta potential was based on the direction and velocity
of particles under the inuence of known electric eld [32].
2.3.6. Fourier transform infrared (FT-IR) spectroscopy
The Fourier transform infrared analysis was conducted to verify the possibility of interaction of chemical bonds between drug
and polymer. FT-IR spectra were recorded for STPP, pure DS
drug, cross-linked starch nanoparticles (inert polymer nanoparticles) and cross-linked starch nanoparticles containing DS (DS
loaded polymer nanoparticles) using a PerkinElmer spectrum
1000 spectrophotometer with a resolution of 2 cm1 . The freezedried sample (0.02 g) was mixed with potassium bromide and

M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

721

Fig. 1. A possible structure of STPP cross-linked maize starch: (a) cross-liking between C2 and C2, (b) cross-liking between C6 and C6 and (c) cross-liking between C6 and C3.

2.3.8. Differential scanning calorimetry (DSC) study

Differential scanning calorimetry (DSC) has been one of the
widely employed calorimetric methods to study the solid state of
the drug in solid dispersions. DSC offers the possibility of detecting
chemical interaction of DS and cross-linked starch nanoparticles.
DSC measurements were carried out on a modulated DSC Instrument: SDT Q600 V20.9 Build 20 equipped with a thermal analysis
data system (TA instrument). Samples of 210 mg were placed in
aluminum pans and sealed. The probes were heated from 25 to
250 C at a rate of 10 K/min under nitrogen atmosphere [33,34].

Fig. 2. FT-IR spectra of STPP and STPP cross-linked starch nanoparticles.

pressed at 15,000 psig using a carver laboratory press to form a

round disk suitable for FT-IR measurements. The IR spectra were
scanned over the wave number range of 4000400 cm1 .

2.3.7. X-ray diffraction (XRD) study

X-ray diffraction analysis was employed to detect the crystallinity state of the pure drug and the drug in the nanoparticle
formulation, which was conducted using a Philips PW 3710 X-ray
diffractometer (XRD) with a copper target and nickel lter (Philips
Electronic Inst, Holland). Powders were mounted on aluminum
stages with glass bottoms and smoothed to a level surface. The XRD
pattern of each sample was measured from 10 to 50 2 using a
step increment of 0.1 2 degrees and a dwell time of 1 s at each
step.

2.3.9. Histological studies

The in vivo experiments were performed on 18 female rats
(weight, 100120 g). The female rats were obtained from animal lab, National Research Centre, Dokki, Cairo. The animals were
housed under a 12-h light12-h dark cycle (07:0019:00 h), with
a room temperature maintained at 23 3 C and humidity at
50 20%. The experiments were conducted in accordance with
the internationally accepted principles for laboratory animal use
and the experimental protocols were approved by the Institutional
Animal Ethical Committee. The rats were housed in polypropylene cages in standard environmental conditions, fed with standard
rodent diet and water ad libitum. All rats were lightly anesthetized
with diethyl ether. Animal rats were divided into two equal groups
as follows: Group 1, animal served as control. Group 2, selected gel
solution of cross-linked starch nanoparticles loaded with 57 mg DS.
These selected gels were applied topically to the skin (2.5 cm2 ) for
successive 5 days. At the end of the experiment, animals were euthanized, the skin were enucleated and immersed in 10% neutralized
buffered formalin solution, embedded in parafn, and vertical sectioned (3 m for histopathological analysis. Images were taken of
the epidermaldermal junction (or the surface of the tissue if there

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M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

Fig. 3. XRD of (a) STPP and (b) STPP cross-linked starch nanoparticles.

was no clear junction due to tissue damage caused by the rashes)

for each animal at each time point at 100 magnication using an
optical microscope with an attached digital camera.
3. Results and discussion
In the present study, proper conditions for the synthesis of
cross-linked nanosized particles of starch was established. Also
established was the feasibility of drug entrapment and release
from these starches nanoparticles as pre-requisite for drug delivery. Emphasis was placed on the formulation of nanoparticulate
systems containing cross-linked starch loaded with DS through a
thorough investigation of two factors, viz., two factors including the
concentration STPP as cross-linking agent and DS as model drug.
Factorial design of the two independent variables; amount of STPP
and amount of DS at three levels were undertaken.
3.1. Possible structure of STPP cross-linked maize starch
The cross-linked starch displayed particular properties, which
were probably associated with its special structure. Cross-linking
can improve granule stability with new covalent bonds, providing desired stability properties [35]. Fig. 1 demonstrates a possible
structure of STPP cross-linked maize starch. Obviously, the reaction
with STPP forms distarch phosphate. Hydroxyl groups of starch and
multiple functional groups of STPP react to form phosphodiester
linkage. Moreover, during the cross-linking reaction, STPP reacts
with hydroxyl groups of the starch molecules in the droplets, to
yield the intra-and inter ester linkages with its phosphate groups
[35,36]. These linkages could possibly occur between C2C2, C2C3,
C6C6, C6C2 or C6C3 of sugar ring (Fig. 1ac). Such kind of
cooperation can connect two or more starch molecules and form
multidimensional space network structure.
Illustration of the presence of newly introduced groups in the
macromolecular structure of starch was made possible by making
use of FT-IR. Fig. 2 depicts the FT-IR of STPP and STPP cross-linked
starch nanoparticles. As is evident, in the FT-IR of STPP, a characteristic band at p = 0 at 1160.72. Whereas in the FT-IR spectra

of STPP cross-linked starch nanoparticles, a characteristic band at

3447 cm1 which is attributed to OH groups stretching vibration and the band at 2938 cm1 corresponding to C C groups. The
STPP cross-linked starch nanoparticles show a new peak for P O at
1159 cm1 .
The FT-IR of STPP cross-linked starch nanoparticles (Fig. 2)
also shows a new small peak at 1212 cm1 corresponding to
antisymmetric stretching vibrations of PO2 groups implying the
formation of cross-links between hydroxyl groups of starch and
the tripolyphosphate ions of STPP. In STPP cross-linked starch
nanoparticles the peak of 3447 cm1 becomes wider, Moreover, the
absorbance peak in cross-linked starch nanoparticles was not as
sharp as that in St-NPs as shown before in (data not shown) which
was attributed to the broken hydrogen bonds. The salient bands of
starch and STPP were observed for the STPPstarch nanoparticles,
which indicated that the interaction of STPP with hydroxyl groups
of starch molecules was successful.
In order to investigate the physical nature of the cross-linked
starch nanoparticles, the X-ray diffraction technique was used to
illustrate the crystallinity of the cross-linked starch nanoparticles prepared using STPP for the cross-linking reaction. The X-ray
diffraction patterns of STPP and STPP cross-linked starch nanoparticles are displayed in (Fig. 3a and b).
It is seen (Fig. 3a) that the STPP exhibits a crystalline peak at
2 = 19.2 while this crystalline peak disappears in the pattern of the
cross-linked starch nanoparticles (Fig. 3b). The crystalline structure
of starch nanoparticles as well as that of STPP seems to undergo
profound changes through coordination of starch hydroxyls with tri
polyphosphate groups of STPP and, in so doing, results cross-linked
starch nanoparticles having amorphous structure.
In a short, From FT-IR and XRD, it was conrmed the starch
molecules was cross-linked by using STPP.
Our target at this end was not only to optimize the formulation,
but also to ascertain whether the concentration of the cross linking
agent and the drug in separate use or in combination affect particle
diameter, PDI, ZP, polydispersity index and entrapment efciency
of DS in cross-linked starch nanoparticles, in addition to percentage
drug that has been released in 6 h time.

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Table 1
Particle size, polydispersity index, zeta potential and entrapment efciency of the
nine formulations of DS loaded cross-linked starch nanoparticles.
Formula

STPP conc.

DS conc.

Particle size

PDI

ZP (mV)

EE (%)a

F1
F2
F3
F4
F5
F6
F7
F8
F9

0.5
0.5
0.5
1
1
1
2
2
2

20
50
100
20
50
100
20
50
100

50.75
21.04
32.67
58.77
68.06
24.36
68.06
43.82
37.84

0.353
0.295
0.358
0.373
0.272
0.689
0.554
0.349
0.42

16.8
21
6.85
18.1
21.3
13.6
16.7
25.6
20.7

73.32
83.71
77.83
83.47
88.29
89.56
86.21
91.94
85.64

(F1) 0.5 g STPP, 20 mg DS, (F2) 0.5 g STPP, 50 mg DS, (F3) 0.5 g STPP, 100 mg DS, (F4)
1 g STPP, 20 mg DS, (F5) 1 g STPP, 50 mg DS, (F6) 1 g STPP, 100 mg DS, (F7) 2 g STPP,
20 mg DS, (F8) 2 g STPP, 50 mg DS and (F9) 2 g STPP, 100 mg DS.
a
Average of three experiments

The concentrations of each excipient were selected based on

reported values in the literature. Nine batches of different combinations were prepared by taking values of selected variables STPP
and DS at three levels as shown in Table 1.
3.2. Morphology using transmission electron microscopy (TEM)
The morphology of the nine samples of STPP cross-linked starch
nanoparticles loaded with DS was examined using TEM as shown in
Fig. 4. As is evident STPP at low concentration (0.5 g) reacted with
starch to form their cross-linked adduct which is characterized by
narrow diameter with small size and well distribution of the sizes as
conrmed in (Fig. 4ac). Increasing the concentration of STPP to 1 g
increases marginally the diameter of prepared cross-linked starch
nanoparticles as shown in (Fig. 4df) as compared with 0.5 g STPP
but still in good dispersion. Further increase in the concentration of
STPP to 2 g leads to formation of cross-linked starch nanoparticles
with highly aggregated partiicles as displayed in (Fig. 4gi). Magnication of a single particle exhibits the internal cage like structure
in which the drug molecules are dispersed uniformly throughout
the polymer matrix (Fig. 4j).
The above nding could be interpreted in terms of the mode
of interaction of the starch particles with STPP at different concentrations of STPP. At low concentration, all the STPP molecules
are involved cross-linking reaction with starch particles with formation of cross-linked starch nanoparticles having small size
after slowly precipitation with ethyl alcohol. At higher STPP concentration (1 g), on the other hand, involvement of STPP with
starch particles proceeds in two ways: STPP molecules are partly
induced in cross-linking meanwhile they are partly adsorbed on
the surface of starch molecules, and, as consequence, large diameter is observed for the formed cross-linked starch nanoparticles
after precipitation when STPP at 1 g concentration was used for
cross-linking followed by loading the resultant cross-linked starch
particles with DS. Furthermore, the use of higher STPP concentration close the pores network in the starch structures leading to
adsorption of DS on rather than penetrating into the cross-linked
starch particles.
The TEM image in Fig. 4gi discloses that the DS loaded
cross-linked starch nanoparticles are closely darker upon using
higher than lower STPP concentration. Overlapping of cross-linked
starch nanoparticles synthesized using higher STPP concentration
accounts for this. It is further noted that most of the prepared crosslinked nanoparticles are spherical in shape and acquire smooth
surfaces with a diameter of less than 80 nm. The TEM micrograph
exhibits a very little number of cross-linked starch nanoparticles
with irregular and polygonal shapes.
It was observed that there is a signicant correlation between
morphology and particle size and DS content of the resulted

723

cross-linked starch nanoparticles loaded with DS drug. Decreasing

the DS content (100 mg) leads to decreasing the particle diameter which is may be attributed to decreasing the encapsulated DS
in the cross-linked starch nanoparticles may lead to a more compact matrix structure which increasing STPPstarch interaction and
nally leads to decreasing the so prepared nanoparticles size.
3.3. Particle size and size distribution analysis of DS loaded
cross-linked starch nanoparticles
All the as prepared nine samples were analyzed for their particle size and size distribution (PDI) and were shown in Table 1. A
close examination of the results would reveal that the mean particle
diameter for all tested DS loaded cross-linked starch nanoparticles
formulations is less than 60 nm, which makes such particles suitable for medical applications. Current results exhibit also that the
mean PDI values for the DS loaded cross-linked starch nanoparticles ranges from 0.272 to 0.689. PDI is less than 0.5, indicating the
uniformity of DS loaded nanoparticle systems.
From the data obtained from particle size analysis and PDI, it is
logical to accept that this polymer (starch) with suitable amount of
cross-linking agent can entrap particulate DS at nano range and in
amorphous state for a long lasting time. It was also found that the
particle size determined using DLS was larger when compared with
Tem images which may be attributed to in the case of DLS measurements the analysis occurred in a solution and there is tendency for
the particles to agglomerate and hence increasing the particle size
while TEM measurements represent a dried layer of nanoparticles
on TEM grid and there no tendency to agglomerate.
3.4. Zeta potential determination of the resulted nanocomposite
containing DS drug
Zeta potential determination for all the said nine-cross-linked
starch nanoparticles loaded with DS formulae containing different concentrations of STPP and Model drug (DS) were assessed as
shown in Table 1.
As is evident, the zeta potential remains in the range of negative
values for all formulae and vary between 6.85 and 25.6 mV. The
zeta potential value of the colloidal solution having zeta potential
values less than 20 may not be stable and may lead to aggregation
as may be realized from Table 1 (F1, F3, F4, F6 and F7). On the
contrary, Table 1 (F2, F5, F8 and F9) contains zeta potential value
of colloidal solution having highly surface charge. Such colloidal
solution is more stable without appreciable aggregation.
The negative zeta potential value for the nanoparticles could be
attributed to the presence of hydroxyl groups of starch molecules
entangled onto the surface of the formed nanoparticles of DSloaded STPPstarch.
3.5. Drug encapsulation by the cross-linked starch nanoparticles
The entrapment efciency of DS in the prepared formulations
was determined and recorded in Table 1. It is seen that the entrapment efciency ranges of 73.3291.94%. Moreover, the highest
entrapment is obtained with formula F8 containing 2 g of STPP and
100 mg of DS whereas the lowest entrapment efciency is observed
with formula F1 containing the lowest concentration of both the
cross linker and the drug (0.5 g and 20 mg, respectively).
The low entrapment efciency values indicate relatively low
afnity of the DS for the cross-linked starch nanoparticles matrix.
The poor entrapment efciency is most probably associated with
the solubility and ionization of the drug. The zeta potential values
of the cross-linked starch nanoparticles were all greatly affected by
the absorption of DS.

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Fig. 4. TEM images of nine formula of DS loaded cross-linked starch nanoparticles at 5% NS, 1.5 g NaOH, 0.4% Tween 80, 100 ml H2 O, 100 absolute ethanol.

The increase of cross-linking content is expected to improve the

EE% by providing more space to incorporate the drug. Increment of
the cross-linking agent also enlarges the particle size and reduces
the possibility of drugs to enter the external phase. As the concentration of STPP increases percentage entrapment efciency of DS
increases. The holding capacity of STPPStarch nanoparticles to DS
drug increases with increasing the concentration of STPP.
The in vitro drug represents a very important parameter in the
prediction of drug bioavailability from different formulations. The
in vitro drug release of DS drug from cross-linked starch nanoparticles was carried out in a PBS (pH 5.5) at 37 C. All the tested
formulations exhibited similar release proles. They were characterized by an initial fast release phase followed by a slower and
more gradual drug release. The burst release phase within the rst
30 min was mainly caused by desorption of the surface-bound or
adsorbed drug. The second phase was a relatively slow release up to
360 min, which attributed to the drug diffusion through the pores,
channels of the polymeric nanoparticles and nanoparticle matrix
erosion or degradation. STPP has been used to cross-link starch in
the concentration of 0.5, 1, and 2 g in the feed mixture.
As indicated in Fig. 5, release of DS showed a greater rate and
extent from F3 containing 0.5 g STPP and 50 mg drug compared
to other prepared formulations. Approximately 20% of DS were
released from the nanoformulation in 4 h time. The enhancement of
dissolution rate of DS from the nanoformulation could be attributed
to increased surface area of the nano-sized preparations. With the
decrease in particle size, a high energy state is achieved which
increases the extent to which the particle can dissolve due to the
increase in dissolution pressure. These parameters increase the
hydrophilic character and improve the wettability of the drug [37].
The results of Fig. 5 depict an increase in drug release when
the concentration of STPP was 2 g. Also, the DS release was slow
when the concentration of STPP was 0.5 g. The observed increase in
released drug is due to the fact that STPP is a low molecular weight

25
FF1
F2
F
F3
F
F4
F
F5
F
F6
F
F7
F
F8
F
F9
F

20
15
10
5
0
0

Fig. 5. In vitro release prole of nine formulae of DS loaded cross-linked starch

nanoparticles of different nanoparticles formulations in phosphate buffer pH 5.5 at
37 C. Data are the mean of three determination.

cross-linking agent for starch; STPP at its two terminals undergoes

cross-linking with the hydroxyl groups of starch. Thus, a crosslinked starch network could be considered as ultrahigh molecular
weight starch molecules that contain wide pore sizes in its structure
and, therefore, possesses an abnormal capacity of accommodating
into the network. Thus, capacity to imbibe increases the number of
DS molecules and results in an increased release as shown in Fig. 5.
Results of Fig. 5 make it evident that as the STPP density
increases to 2 g, the network of starch is highly cross-linked, which,
as a consequence, reduces the free volume accessible to the penetrant DS molecules and the DS will be adsorbed on the surface
of starch molecules. Hence, the obtained increase in the release
permits a greater number of molecules to diffuse out of material
and pass into the release medium. When the concentration of STPP
reaches 0.5 g for 5% starch, the pores of starch molecules crosslinked starch molecules become suitable for holding large number
of drug molecules in its network. Therefore, the amount of DS in
the dispersed phase at pH 5.5 is very little.

M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

Table 2
The value of regression coefcients (R2 ) for each formula.
Formula

Zero order (R2 )

Higuchi (R2 )

First order (R2 )

Order

F1
F2
F3
F4
F5
F6
F7
F8
F9

0.9676
0.9805
0.7435
0.9491
0.9962
0.7251
0.9888
0.9959
0.8242

0.9082
0.9698
0.9457
0.9951
0.9370
0.9289
0.9258
0.9239
0.967

0.8219
0.7878
0.5101
0.6803
0.8537
0.4612
0.8523
0.8817
0.5148

Zero
Zero
Higuchi
Higuchi
Zero
Higuchi
Zero
Zero
Higuchi

Table 3
Analysis of variance for a 32 Factorial design experiment for evaluation of variables
effect on the DS NPs formulations.
Response

Variable

Sum of squares

dfa

F value

Prob > F

Particle Diameter

X1
X2
X1*X2
X1
X2
X1*X2
X1
X2
X1*X2
X1
X2
X1*X2
X1
X2
X1*X2

382.6269
1057.8029
32.5020
0.00729906
0.07049
0.0176
37.294
55.802
50.433
6.77
118.41
7.12
2.430556
11.680556
0.670635

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

1.2055
3.3328
0.1024
0.332
3.209
0.801
2.118
3.1695
2.8646
0.2447
4.2787
0.2574
3.9112
18.7963
1.0792

0.3223
0.1275
0.7619
0.589
0.1332
0.4120
0.205
0.1351
0.1513
0.6418
0.0934
0.6335
0.1049
0.0075b
0.3465

PDI

Zeta Potential

Entrapment efciency (%)

With the above in mind, it is obvious that the decreased percentage of DS release is due to the formation of a more compact
wall around the DS by the cross-linked nanostarch polymer. Significance of this is that the cross-linked starch nanoparticles possess
sustained drug release properties for a prolonged period of time
as shown in the charts of F1, F2, F5, F7, and F8. Data obtained for
in vitro release studies were utilized for release kinetics.
3.6. Kinetic analysis of release data: modeling of release
mechanism
The drug (DS) loaded cross-linked starch nanoparticles may
be visualized as a three dimensional network of starch macromolecules containing drug molecules which occupy the space
available between the network chains. When such drug-loaded
nanoparticles are allowed to swell in a release medium the solvent
(normally water) molecules enter into the nanoparticles network
as a result of their diffusion into the nanoparticles matrix and subsequent relaxation of polymer chains. The drug molecules dissolve
into water and release out through water permeation channels
present in the macromolecular network. The diffusion of drug
molecules and relaxation of nanoparticle chains determine the type
of release mechanism being followed by the drug molecules. It has
been laid down by Higuchi equation that if n = 0.43, the release
is diffusion controlled (Fickian), when n = 0.84 the release is nonFickian (or case II), and for when n is in between 0.43 and 0.84, the
mechanism becomes anomalous. In some cases n has been found
to exceed 0.84 and the mechanism is known as super case II [38].
The respective values of diffusion coefcient (D) and release
exponents (n) have been calculated as described above and are
summarized in Table 2. Along with the values of D and n are the
respective values of regression coefcients (R2 ) have also been
expressed. It is clear from the data that the values of R2 are always
greater than 0.9 and, suggest therefore, a good applicability of
release data.
It is as well to emphasize that the release kinetics ranged from
zero order prole in formulae F1, F2, F5, F7, and F8 to Higuchi
release prole with the other formulations. This indicates that the
nanoformulation under study is capable of providing a highly controlled release prole.
3.7. Factorial design
The results of analysis of variance (ANOVA) of the factorial
design are presented in Table 3. Based on the results of ANOVA, the
release percentage was found to be signicantly (p < 0.01) affected
by DS concentration. In contrast to the release percentage, other
tested responses seemed to be inuenced by each of the tested
factors.
The optimum formulation selected by the JMP software was
composed of 57.7 mg drug and 0.5 g cross-linking agent. The calculated desirability factor offered for the formulation was 0.622573.
No signicant deviations between theoretical and experimental

725

Release percentage (%)

a
b

Degrees of freedom.
Signicance level based on 1 df; p < 0.01.

values of tested responses were found. Fig. 6a and b shows the PS

and zeta potential of DS loaded cross-linked starch nanoparticles
after factorial design at the optimum conditions for STPP and DS
(0.5 g and 57 mg, respectively).
The selected formula of DS loaded cross-linked starch nanoparticles by the JMP software showed a mean particle diameter of
21.04 nm with a polydispersity index of 0.200 and a zeta potential of 35.3 mV and an average entrapment efciency of 95.01%.
It was found that the mean article size decreases and have smallest size compared with those prepared using 50 mg DS under the
same condition. In addition, the PDI acquires have small value
indicating the more homogeneity of the formed nanoparticulate
system as shown in Fig. 6a. Moreover, the surface charge (zeta
potential) of DS loaded cross-linked starch nanoparticles (Fig. 6b)
has 35.3 mV indicating the well stability of the nanoparticulate
system.

3.8. FTIR spectrum of DS and DS loaded starch cross-linked

nanospheres
Fig. 7 shows FT-IR, which was carried out to conrm the
compatibility between the DS and DS loaded cross-linked starch
nanoparticles at the previously established optimum condition.
Obviously, the IR spectra of DS exhibit distinctive peaks at
3386.39 cm1 due to N H stretching of the secondary amine, aromatic stretching, CH stretching at 2965 cm1 . The characteristic
peak at 1574.59 cm1 is due to the aromatic stretching and the
COO asymmetric stretching, the peaks observed at 1504.2 and
1452.14 cm1 are due to N H deformation and C N stretching,
respectively; and a sharp peak observed at 768.494 cm1 is due to
the C Cl group attached to the aromatic moiety. In case of the FT-IR
spectra of the DS loaded cross-linked starch nanoparticles the peaks
of DS are present at 3396, 1561.09, 1452, 1158, 1452, 1084, and
765.601 cm1 . Based on these results, it is conrmed that no major
shifting and no loss of fundamental peaks between the spectra of DS
and DS loaded cross-linked starch nanoparticles in addition to all
other peaks normally present in the cross-linked starch nanoparticles can be seen. Therefore, it can be concluded that there is
no strong chemical interaction between DS and STPP-cross-linked
starch nanoparticles.
It was found that the data obtained from FT-IR analysis could be
correlated to those of the TEM analysis that shows the encapsulation of DS into nanoparticles of STPP cross-linked starch molecules
(Fig. 4j).

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M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

Fig. 6. (a) Particle size analyzer and PDI, (b) zeta potential of DS loaded STPP-cross-linked starch nanoparticles.

3.9. XRD of DS and DS loaded cross-linked starch nanoparticles

In order to study further the interaction between the crosslinked starch nanoparticles and DS, the XRD patterns and typical
diffraction peaks of DS, and starch cross-linked nanoparticles
loaded with DS at the aforementioned optimum condition are
shown in (Fig. 8a and b). As can be seen, The crystalline nature of
the drug was clearly demonstrated by the characteristic XRD pattern with peaks appearing at about 2 = 6.62 , 8.52 , 11.1 , 15.2 ,
17.2, 19.9 , 23.47 , 25.05, 27.11 , and 27.91 indicating crystalline
structure of DS (Fig. 8a). After DS was incorporated (Fig. 8b), typical diffraction peaks of starch cross-linked nanoparticles at 13.04 ,
20.96 , 25.96 , and 31.70 2 values are observed.
It was observed from XRD that indicates the DS is still present
in its STPPstarch nanoparticles structure in the physical mixture
whereas it is completely amorphous inside the nanoparticulate formula.
Particularly notable is the weakening of the diffraction peaks
of cross-linked starch nanoparticles loaded with DS which may be
attributed to the presence of DS in the amorphous phase or may
be due to the limited amount of DS containing STPP cross-linked
starch nanoparticles. While the slight shift of the diffraction peak of

DS is intimately may be only because of the electrostatic interaction

between cross-linked starch and DS, instead of chemical reaction
of DS with cross-linked starch nanoparticles.

3.10. DSC of DS and cross-linked starch nanoparticles loaded

with DS
The thermogram of the DS and DS incorporated into STPP
cross-linked starch nanoparticles showed in Fig. 9. It has been
observed that the DS exhibited a sharp endotherm peak at 303 C
indicating the melting of drug crystals [39]. The thermograms of
cross-linked starch nanoparticles loaded with DS showed no peak
at 303 C and indicated amorphous nature of DS in the formulation [30]. The disappearance of DS peak may also be related to the
insignicant amount of DS entrapped onto STPPstarch nanoparticles.
Apart from abroad signal around 6085 C due to a partial loss
of residual humidity of DS loaded cross-linked starch nanoparticles. This clearly indicated that the drug present in these systems
remained mostly in amorphous state and was dispersed homogeneously throughout the polymer nanoparticles.

M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

DS

33900

3400
3

727

DS looaded nanopparticles

22900

22400

11900

11400

900

400

Waavelength (nnm)

Fig. 7. FT-IR of diclofenac sodium and DS loaded STPP-cross-linked starch nanoparticles.

Fig. 8. XRD of (a) diclofenac sodium, (b) DS loaded STPP-cross-linked starch nanoparticles.

DS

H e at Flow W/g

1
0
-1
-2
-3

D S lo aded po lymer nanoparticles

-4
0

50

100

150
Te mperature (C)

200

250

Fig. 9. DSC of diclofenac sodium and DS loaded STPP-cross-linked starch nanoparticles.

300

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M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

Fig. 10. (a) Section of skin of rat, (b) section of skin of rat treated with St-NPs loaded
with DS (scale bar 20 m).

3.11. Histopathological study for skin irritation evaluation

The systematic experiments of the factorial design enabled the
successful identication of cross-links starch nanoparticles loaded
with DS as a formula with improved particle diameter, zeta potential, entrapment efciency as well as drug release. The next logical
step was to conrm that F4 formulation is perfectly tolerated with
no evidenced symptoms of skin irritation.
Dissolve about 3 g of Carbopol 940 in 100 ml distilled water to
form a gel under magnetic stirrer. Then triethanol amine was added
drop wise to adjust the pH to 6.57.5. Finally 2 g of gel was accurately weighed and mixed with 20 mg of cross-linked nanoparticles.
The samples rat were coated with the gel having DS loaded with
cross-linked St-NPs and compared with the uncoated rats.
The histological features of untreated skin rat show normal
structure of epidermis and dermis as displayed in Fig. 10a. While
in Fig. 10b which represents the skin rat treated with cross-linked
starch nanoparticles loaded with DS, showing the epidermis layer
appeared in normal arrangement and the dermis that includes
sebaceous gland and hair follicle with other vascular features of
the dermis layer. The histological studies on the rat skin containing
epidermis and dermis after treatment with the optimized DS loaded
starch nanoparticles revealed normal structure, which means that
the prepared St-NPs loaded with DS did not exhibit any harmful
effect on the rat skin. Therefore, the potential clinical interest of
the cross-linked starch nanoparticles loaded with DS is supported
because of the absence of irritant effect in vivo.

4. Conclusion
Current work presents a manifold study aiming at development
of a promising and controlled release transdermal delivery system to enhance the therapeutic efciency of diclofenac sodium
(DS). The system is based on cross-linked starch nanoparticles,
which were synthesized using native starch (NS). Cross-linking
was effected by reacting sodium tripolyphosphate (TPP) at different concentrations. Cross-linked starch nanoparticles loaded
with DS were synthesized according to the nanoprecipitation
method using different DS concentrations. A two-level factorial
design was practiced for the prediction of optimized formulation
for DS loaded cross-linked starch nanoparticles. The formulated
nanospheres were systematically studied by monitoring drug
loading, entrapment efciency, transmission electron microscopy

(TEM), particle size analyzer, polydispersity index (PDI) and zeta

potential for shape and surface characteristics and in vitro release
studies. Physicochemical characterization and analysis of the
formulated nanospheres were also exercized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and
Differential scanning calorimetry (DSC) to determine the ne physical nature (crystallinity), thermal behavior and possible occurrence
of interaction between DS and the cross-linked starch nanoparticles. Interaction of cross-linked starch nanoparticles with DS
causes profound changes in the crystalline structure of DS; DS
is completely converted to amorphous structure. Application of
experimental in vivo targeting efciency of the optimum formula
of cross-linked nanoparticles of starch with TPP and loaded with
DS was examined histopathologically in skin irritation of healthy
rats. Obviously, then, the essential target of the work could be
successfully achieved. Application of experimental design allowed
the optimization of different factors to yield spherical nanoparticles with small particle size, low polydispersity index and high
entrapment efciency and sustained release for DS drug. The
histophathological studies on rat skin advocate the use of the
designed transdermal DS loaded cross-linked starch and medium
oxidized starch nanoparticles formulations as they are safe and
non-irritant to rat skin. This would render the designed formulation of a safe, highly effective, controlled and convenient
mean of therapy with the non-steroidal anti-inammatory drug
(NSAID).
References
[1] L. Xinming, C. Yingde, A.W. Lloyd, S.V. Mikhalovsky, S.R. Sandeman, C.A.
Howel, L. Liewen, Cont. Lens Anterior Eye 31 (2) (2008) 5764.
[2] S. Ding, Pharm. Sci. Technol. Today 1 (8) (1998) 328335.
[3] R.C. Nagarwal, S. Kant, P.N. Singh, P. Maiti, J.K. Pandit, J. Control. Release 136
(1) (2009) 213.
[4] R. Gurny, T. Boye, H. Ibrahim, J. Control. Release 2 (1985) 353361.
[5] K. Subramani, W. Ahmed, Nanoparticulate drug delivery systems for oral
cancer treatment, in: Emerging Nanotechnologies in Dentistry, William
Andrew Publishing, Boston, 2012, pp. 333345 (Chapter 19).
[6] B. Mandal, K.S. Alexander, A.T. Riga, J. Pharm. Sci. 13 (4) (2010) 510523.
[7] M.S. Nagarsenker, V.Y. Londhe, G.D. Nadkarni, Int. J. Pharm. 190 (1) (1999)
6371.
[8] M.C. Callegan, J.A. Hobden, R.J. OCallaghan, J.M. Hill, Int. J. Pharm. 123 (2)
(1995) 173179.
[9] S.R. Unterman, D.S. Rootman, J.M. Hill, J.J. Parelman, H.W. Thompson, H.E.
Kaufman, J. Cataract Refract. Surg. 14 (5) (1988) 500504.
[10] W. Friess, Collagen biomaterial for drug delivery, Eur. J. Pharm. Biopharm.
45 (2) (1998) 113136.
[11] V. Baeyens, V. Kaltsatos, B. Boisram, E. Varesio, J.L. Veuthey, M. Fathi, L.P.
Balant, M. Gex-Fabry, R. Gurny, J. Control. Release 52 (1998) 215220.
[12] T.F. Vandamme, L. Brobeck, J. Control. Release 102 (1) (2005) 2338.
[13] B. Sahu, M. Das, Appl. Nanosci. (2013) 19.
[14] K.K. Jain, Drug Discov. Today 10 (21) (2005) 14351442.
[15] M.H. El-Rae, M.E. El-Naggar, M.A. Ramadan, M.M.G. Fouda, S.S. Al-Deyab, A.
Hebeish, Carbohydr. Polym. 86 (2011) 630635.
[16] W.L.F. Armarego, C. Chai, Nanomaterials and nanotechnology, in: Purication
of Laboratory Chemicals, 7th ed., Butterworth-Heinemann, Boston, 2013, pp.
908942 (Chapter 8).
[17] A. Hebeish, M.H. El-Rae, M.A. EL-Sheikh, A.A. Seleem, Mehrez E. EL-Naggar,
Int. J. Biol. Macromol. 65 (2014) 509515.
[18] M.V. Bagal, P.R. Gogate, Ultrason Sonochem. Sonochem 21 (3) (2014)
10351043.
[19] M.L. Gonzlez-Rodrguez, M.A. Holgado, C. Snchez-Lafuente, A.M. Rabasco, A.
Fini, Int. J. Pharm 232 (2002) 225234.
[20] S.M. Agnihotri, P.R. Vavia, Nanomedicine 5 (1) (2009) 9095.
[21] A. Shi, D. Li, L. Wang, B. Li, B. Adhikari, Carbohydr. Polym 83 (4) (2011)
16041610.
[22] B. Li, L. Wang, D. Li, Y.L. Chiu, Z. Zhang, J. Shi, X.D. Chen, Z. Mao, J. Food Eng 92
(3) (2009) 255260.
[23] K. Woo, P.A. Seib, Carbohydr. Polym 33 (4) (1997) 263271.
[24] T. Higuchi, J. Pharm. Sci 50 (10) (1961) 874875.
[25] H. Fessi, F. Puisieux, J.P. Devissaguet, N. Ammoury, S. Benita, Int. J. Pharm. 55
(1989) R1R4.
[26] A. Hebeish, M.H. El-Rae, M.A. EL-Sheikh, Mehrez E. El-Naggar, J. Inorg.
Organomet. Polym. Mater. 24 (3) (2014) 515524.
[27] U. Bhosale, D.V. Kusum, N. Jain, J. Young Pharm. 3 (4) (2011) 275283.
[28] Q. Wang, J. Wu, W. Wang, A. Wang, J. Biomater. Nanobiotechnol. 2 (2011)
250257.

M.E. El-Naggar et al. / International Journal of Biological Macromolecules 81 (2015) 718729

[29] J. Alonso, Nanoparticulate drug carrier technology, in: Microparticulate
Systems for the Delivery of Proteins and Vaccines, Marcel Dekker, New York,
1996, pp. 203242.
[30] M. Saravanan, K. Bhaskar, G. Maharajan, K.S. Pillai, Int. J. Pharm. 283 (2004)
7182.
[31] G.S. El-Feky, M.H. El-Rae, M.A. El-Sheikh, Mehrez E. El-Naggar, A. Hebeish, J.
Nanomed. Nanotechnol. 6 (1) (2015) 254.
[32] R. Pignatello, N. Ricupero, C. Bucolo, F. Maugeri, A. Maltese, G. Puglisi, AAPS
PharmSciTech 7 (1) (2006) E27.
[33] R. Gurny, N.A. Peppas, D.D. Harrington, G.S. Banker, Drug Dev. Ind. Pharm. 7
(1981) 125.

729

[34] S. Magdassi, K. Margulis, Nanomedicine 5 (2009) 274281.

[35] J. Singh, L. Kaur, O.J. McCarthy, Food Hydrocoll. 21 (1) (2007) 122.

[36] M.L. Rodrguez-Marn, C. Nnez-Santiago,

Y.-J. Wang, L.A. Bello-Prez, Strke
62 (10) (2010) 530537.
[37] G.S. El-Feky, G. Zayed, A.R.H. Farrag, Int. J. Pharm. Pharm. Sci. 5 (2013)
213219.
[38] J. Siepmann, N.A. Peppas, Adv. Drug Deliv. Rev. 48 (23) (2001) 139157.
[39] S. Puttipipatkhachorn, T. Pongjanyakul, A. Priprem, Int. J. Pharm. 293 (2005)
5162.