NANOMEDICINE

Journal of Controlled Release 141 (2010) 85–92

Contents lists available at ScienceDirect

Journal of Controlled Release

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Nanoparticles made from novel starch derivatives for transdermal drug delivery
M.J. Santander-Ortega a,b, T. Stauner c, B. Loretz b, J.L. Ortega-Vinuesa a, D. Bastos-González a, G. Wenz c,
U.F. Schaefer b, C.M. Lehr b,⁎
a
Department of Applied Physics, University of Granada, Granada, Spain
b
Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, Germany
c
Department of Organic Macromolecular Chemistry, Saarland University, Saarbrücken, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The goal of this paper was aimed to the formulation of nanoparticles by using two different propyl-starch
Received 14 May 2009 derivatives – referred to as PS-1 and PS-1.45 – with high degrees of substitution: 1.05 and 1.45 respectively. A
Accepted 11 August 2009 simple o/w emulsion diffusion technique, avoiding the use of hazardous solvents such as dichloromethane or
Available online 21 August 2009
dimethyl sulfoxide, was chosen to formulate nanoparticles with both polymers, producing the PS-1 and PS-1.45
nanoparticles. Once the nanoparticles were prepared, a deep physicochemical characterization was carried out,
Keywords:
Starch
including the evaluation of nanoparticles stability and applicability for lyophilization. Depending on this
Nanoparticles information, rules on the formation of PS-1 and PS-1.45 nanoparticles could be developed. Encapsulation and
Physico-chemical Characterization release properties of these nanoparticles were studied, showing high encapsulation efficiency for three tested
Drug delivery drugs (flufenamic acid, testosterone and caffeine); in addition a close to linear release profile was observed for
Skin hydrophobic drugs with a null initial burst effect. Finally, the potential use of these nanoparticles as transdermal
drug delivery systems was also tested, displaying a clear enhancer effect for flufenamic acid.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction Besides, an increasing number of researchers is investigating on

In the last years, the use of polysaccharides to prepare nanopar-
ticles has increased considerably [1–3]. Polysaccharides possess many
recognition functions, allowing e.g. mucoadhesion or specific receptor
recognition [4], as well as providing neutral coatings with low surface
energy, preventing non specific protein adsorption [5]. On the other
hand, the high amount of hydroxyl groups in the polysaccharide
backbone allows the incorporation of different specific ligands to
obtain polyfunctional colloidal systems [6].
In this context, starch has an interesting potential, which is so far
relatively unexplored. Starch is a biocompatible, biodegradable, non-
toxic polymer, abundantly occurring in nature as the major polysac-
charide storage in higher plants [7,8]. However, despite these
properties, some problems can occur. The hydrophilic nature of
starches is a major constraint that seriously limits the development of
starch-based nanoparticles [9]. A good alternative to solve this
problem is the grafting of hydrophobic side chains to the hydrophilic
starch backbone [10–14]. However, an important restriction usually
arising from the use of these hydrophobic polysaccharides derivatives
to prepare nanoparticles, is the necessity to employ organic solvents,
such as dichloromethane or dimethyl sulfoxide, with considerable
toxicological and other safety risks [10,11].
⁎ Corresponding author. Tel.: +49 681 302 3039; fax: +49 681 302 4671.
E-mail address: lehr@mx.uni-saarland.de (C.M. Lehr).
the improvement of percutaneous drug absorption [15]. However, the
excellent barrier properties of the skin is a handicap that must be
overcome [16]. Usually, to reach a high and constant drug flux through
the skin a change of the skin barrier function is necessary. However, in
the last years, different authors [17–20] have demonstrated that the
use of particles as transdermal drug delivery systems (TDDS) enhance
the rate and extent of transport across skin, without compromising
the skin barrier function. Although such systems were undoubtedly
able to enhance skin penetration and distribution, the mechanism by
which this enhancement was achieved is still unclear [17,21].
The aim of this work was to explore nanoparticulate drug carriers
based on starch derivatives, by using the advantages of hydrophobic
starch derivatives. With this in mind, we have selected propyl-starch
derivatives instead of the well known acetyl-starch derivatives [22–
24] to formulate our nanoparticles. The inclusion of propyl groups,
even with low degree of substitution, would enable good solubility in
low hazardous organic solvents, such as ethyl acetate. Moreover,
propyl-starches may allow a better quality control since degree of
substitution can be determined after acidic degradation to the
respective glucose derivatives by 1H-NMR spectroscopy. Two pro-
pyl-starch derivatives and one un-modified starch polymer were used
as basic constituents for the preparation of nanoparticles, represent-
ing two different degrees of substitutions (Ds) 1.05 (PS-1) and 1.45
(PS-1.45) respectively.
Nanoparticles were formulated by emulsification–diffusion tech-
nique. This method has several advantages such as high yields generally

0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2009.08.012
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86 M.J. Santander-Ortega et al. / Journal of Controlled Release 141 (2010) 85–92
obtained, high batch to batch reproducibility and easy scaling up.
Moreover, it is possible to control the size and polydispersity of nano-
particles by the control of the oil/water phase ratio [25].
Once nanoparticles were formed, a thorough physicochemical char-
acterization was carried out. Afterwards, the capacity of these
nanoparticles as drug delivery systems was explored by the encapsu-
lation and release of three different model drugs, flufenamic acid (FFA),
testosterone (Test) and caffeine (Caff). Finally, considering the nano-
particles characterization results, it was possible to establish the use of
these nanoparticles as TDDS. Hence, the potential application of these
nanoparticles as TDDS was analyzed by studying their influence on the
permeability of human heat-separated epidermis (HSE).
2. Materials and methods
2.1. Materials
Maize Starch polymer with an amylose content of 25% was kindly
donated by BASF, ethyl acetate (Fluka Chemie GmbH, Buchs, Switzer-
land), polyvinyl alcohol (PVA); Mowiol® 4-88 (Kuraray Specialities
Europe GmbH, Frankfurt, Germany), Igepal® CA-630 (Octylphenyl-
polyethylene glycol) (Sigma, St. Louis, MO, USA), cellulose membrane
MW 12–14 kDa (Medicell International Ltd., London, UK) were used
as obtained from the suppliers. Flufenamic acid, testosterone and
caffeine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All
other solvents and chemicals used were of the highest grade
commercially available.
2.2. Preparation of the nanoparticles
Nanoparticles with the different polymers were formulated by a
simple o/w emulsion diffusion method. Briefly, specific starch
derivative (un-modified starch, PS-1 or PS-1.45) was dissolved in
ethyl acetate (1 mg/ml), and 1 ml of this organic solution was poured
on 4 ml of an aqueous phase with different percentages (w/v) of PVA
(0, 0.1, 0.5 and 1). This biphasic system was emulsified with a high
speed homogenizer (Ultra Turrax® Ika®, Staufen, Germany) at
14,000 rpm during 15 min. Then, high purified water was added up
to 10 ml to force the complete diffusion of the organic solvent to the
aqueous phase. Finally, the organic solvent was evaporated under
vacuum at 35 °C (Rotavapor Büchi®, Labortechnik AG, Flawil,
Switzerland), generating stable nanoparticles. After nanoparticles
preparation, high purified water was added to obtain a colloidal
solution with a final volume of 10 ml.
2.3. Characterization of the nanoparticles
Size and ζ-potential of the nanoparticles were analysed by photon
correlation spectroscopy (PCS) using a Nano-ZS (Malvern Instruments,
Malvern, UK). For ζ-potential measurements nanoparticles were diluted
in NaCl 3 mM. AFM images were obtained using an Atomic Force
Microscopy Nanoscope IV Bioscope™ (Veeco Instruments, Santa
Barbara, CA, USA). Imaging was done using Taping mode and a silicon
cantilever with a spring constant of approximately 40 N/m and a
resonance frequency of about 170 kHz. The scan speed applied was
0.2 Hz.
2.4. Colloidal stability
Stability of the nanoparticles was studied as a function of salinity of
the medium using NaCl and CaCl2 as aggregating salts. Particle
aggregation was analyzed by photon correlation spectroscopy (PCS)
using a 4700c System (Malvern Instruments). For more details see
supporting information that can be find in the webpage of this journal.
2.5. Lyophilization
PS-1 and PS-1.45 nanoparticles were lyophilized using a Freeze-drier
Alpha 2-4 LSC (Christ, Osterode, Germany) and sucrose or trehalose as
cryoprotectant agent. Briefly, different volumes of 10% (w/v) sucrose or
trehalose solution were poured onto different aliquots of nanoparticles
in order to obtain a cryoprotectant range between 0 and 1% (w/v).
Previously to the lyophilization step, the different samples were frozen
at −80 °C for 2 h. Hydrodynamic mean size of these aliquots was
measured before and after the lyophilization step.
2.6. Lactat dehydrogenase (LDH) assay
Caco-2 cell line clone C2BBe1 (ATCC No CRL-2102) was used as test
system cultured in DMEM supplemented with 1% Non-essential
amino acids (both from Gibco, Karlsruhe, Germany) and 10% FBS
(F7524, Sigma-Aldrich, Taufkirchen, Germany).
Cytotoxicity detection Kit (LDH) from Roche (Mannheim, Ger-
many) was used basically following the instruction manual with
minor deviations. Caco-2 cells were seeded into 24-well plates and
the toxicity assay was performed at the state of a confluent
monolayer. Samples were prepared by dilution of aqueous suspension
of starch particles with supplemented cell culture medium at ratios
1:3, 1:6, 1:12 and 1:24 resulting in concentrations of 0.0042 to
0.033 mg nanoparticles/ml. As negative control cells were incubated
with cell culture medium and as high control with medium plus 1% of
the detergent Triton X-100. Samples were taken after 4 h and 24 h
incubation time and analysed according to the manufacturer's
protocol. No interaction was found with the detection method and
the nanoparticles.
2.7. MTT assay
Thiazolyl Blue Tetrazolium Bromide (M5655, Sigma-Aldrich,
Taufkirchen, Germany) was dissolved in PBS pH 7.4 to yield a final
concentration of 5 mg/ml for the stock solution.
Caco-2 cells were seeded into 24-well plates and the toxicity assay
was performed at the state of a confluent monolayer by exposure to
particle suspension as reported for LDH assay. After the incubation
period of 4 h the particle suspension was removed. Cells were washed
once with phosphate buffered saline before fresh medium and 50 µl
MTT stock solution per well was added. After further 3 h incubation
500 µl lysis buffer (10% SDS in 0.01 mM HCl) were added. The
absorbance at λ = 550 nm was analysed in a plate reader. Viability
was calculated in comparison to the positive control, untreated cells
as 100% value, and negative control 1%-Triton solution as 0% value.
2.8. Encapsulation of flufenamic acid, testosterone and caffeine
In order to assess the potential use of these nanoparticles as drug
delivery system three different drugs were chosen to study the
encapsulation behaviours of PS-1 and PS-1.45 nanoparticles. These
drugs present different hydrophobicity (expressed as the logarithm of
the octanol–water partition coefficient; Log P) and net charge. Exactly,
the chosen drugs were: flufenamic acid (Log P = 4.80; pKa = 3.90;
Mw = 281.23), testosterone (Log P = 3.47; non-ionizable molecule;
M w = 288.40) and caffeine (Log P = − 0.08; p K b = 10.40;
Mw = 194.20). The preparation method of drug loaded PS-1 and PS-
1.45 nanoparticles was not modified with respect to the un-loaded
nanoparticles. The specific molecule and the PS-1 or PS-1.45 polymer
were dissolved in the organic phase (1:1 ratio), following the same
procedure described in section 2.2. The characterization of these
nanoparticles was performed as it was comment previously for the
un-loaded nanoparticles. Encapsulation efficiency (EE) was calculated
using a Franz diffusion cell. The exact amount of each drug was
determined by HPLC analysis [19,26].

M.J. Santander-Ortega et al. / Journal of Controlled Release 141 (2010) 85–92
Fig. 1. Hydrodynamic size distribution of PS-1 nanoparticles formulated with different
percentages (w/v) of PVA, (dash-dotted line) 0%, (dotted line) 0.1%, (dashed line) 0.5%,
(solid line) 1% (n ≥ 3).
2.9. In-vitro release of flufenamic acid, testosterone and caffeine
Release of FFA, testosterone and caffeine from loaded PS-1 and PS-1.45
nanoparticles was studied using Franz diffusion Cells (FdC) type 6G-01-
00-15 (Perme-Gear, Riegelsville, PA, USA). Briefly, a mixture of 0.5 ml of
nanoparticles dispersion and 1.5 ml of phosphate buffer pH 7 (2 mM) was
poured in the donor compartment of FdC, while the receptor compart-
ment was filled with 12 ml of the same buffer solution. In the case of the
testosterone, the buffer solution was changed due to the low solubility of
this drug. In this case, the medium consisted in phosphate buffer pH 7
(2 mM) with addition of 2% (v/v) Igepal® CA-630 (a non-ionic poly-
ethylene glycol derivative) and 0.4% (v/v) ethanol. In all cases, donor and
receptor compartments were separated by a cellulose membrane with a
molecule cut off of 12–14 kDa. (Medicell Int. Ltd, London, UK.). FdCs were
incubated at 32 °C for a period of time of 72 h. Samples of 0.4 ml were
removed from the receptor compartment at regular time intervals up to
72 h and replaced with an equal volume of fresh buffer. The concentration
of free drug from the receptor was analyzed by HPLC [19,26].
2.10. Skin preparation
Excised human skin from Caucasian female patients, who had
undergone abdominal plastic surgery, was used. Permission of the
Ethical Committee of the Caritas-Traegergesellschaft, Trier, Germany
(July 6, 1998). For more details see supporting information that can be
found in the webpage of this journal.
2.11. Permeation of flufenamic acid, testosterone and caffeine through
human heat-separated epidermis
Permeation studies of the three encapsulated drugs in PS-1 or PS-
1.45 nanoparticles were carried out using FdC. The whole procedures
are described in the supporting information that can be found in the
webpage of this journal.
Table 1
Hydrodynamic mean size (nm), PDI, ζ-potential (mV), ccc (mM) and csc (mM) values in presence of NaCl and CaCl2 of PS-1 and PS-1.45 nanoparticles.
Size (nm) PDI ζ-Pot. ccc (mM)
(mV)
NaCl
PS-1 150.5 ± 3.5 0.12 ± 0.01 − 8.3 ± 0.3 32
PS-1.45 182.7 ± 6.7 0.08 ± 0.02 − 5.8 ± 0.5 36
a
After incubation for 25 days at room temperature.
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2.12. Quantification of flufenamic acid, testosterone and caffeine
2.12.1. HPLC system
Pump: Dionex P580 pump; Autosampler: Dionex ASJ 100 automated
sample injector; detector: UVD 170 S detector; column oven: Dionex
STH 585 column oven; software: chromeleon 6.50 SP2 build 9.68.
A 125 × 4 mm LiChrospher® 100/RP-18 column (Merck-Hitachi,
Darmstadt, Germany) with a 4 × 4-mm LiChrospher® 100/RP-18 guard
column (5 µm) (Merck-Hitachi, Darmstadt, Germany) was used as
stationary phase for all substances.
2.12.2. Solvents and buffers
Methanol (Lichrosolv, Merck, Darmstadt, Germany); Acetonitril
HPLC grade (Merck, Darmstadt, Germany); Deionised water (Millipore
Milli Q Synthesis, Heidelberg, Germany); McIlvaine buffer (pH 2.2):
1 l contains 20.8 g citric acid, 0.4 g (Na2HPO4 × 2 H2O) and deionised
water; Buffer (pH 2.6): 1 l contains 1.15 ml phosphoric acid, 4.08 g
KH2PO4 anhydrous and deionised water.
2.12.3. HPLC analytics of the test compounds
Flufenamic acid: mobile phase: 80:20 (v/v) methanol: McIlvaine
buffer pH 2.2; retention time: 3.1 min ± 0.2 min; detection wavelength:
284 nm; flow rate: 1.2 ml/min; calibration from 25 to 5000 ng/ml
(r 2 = 0.999); injection volume: 50 µl.
Testosterone: mobile phase: methanol/water (v/v); 70:30; retention
time: 4.8 min ± 0.2 min; detection wavelength: 250 nm; flow rate:
1.2 ml/min; calibration from 50 to 5000 ng/ml (r2 = 0.999); injection
volume: 50 µl.
Caffeine: mobile phase: buffer pH 2.6/acetonitrile; 90:10 (v/v);
retention time: 5.1 ± 0.2 min; detection wavelength: 262 nm; flow rate:
1.2 ml/min; calibration from 25 to 10,000 ng/ml (r2 = 0.999); injection
volume: 50 µl.
Drugs in samples were quantified by the external standard method.
3. Results
3.1. Characterization of the nanoparticles
Propyl-starch derivatives, soluble in organic solvents like ethyl
acetate, were synthesized by reaction of starch with propyl bromide
as described in the supporting information that can be found in the
webpage of this journal. Fig. 1 shows the hydrodynamic size dis-
tribution of nanoparticles formulated with PS-1 polymer and different
amounts of PVA. Similar results were obtained for PS-1.45 polymer
(data not shown). However, in the case of un-modified starch the
formation of nanoparticle could not be observed.
As can be observed in Fig. 1, the incorporation of PVA to the
formulation improved the nanoparticle hydrodynamic size distribution.
A mono-modal size distribution was achieved when the aqueous phase
contains a PVA concentration equal or higher than 0.5% (w/v). The
narrowest size distribution was reached when the aqueous phase
presented a 1% (w/v) of PVA. Consequently, nanoparticles formulated
with an aqueous phase of 1% (w/v) of PVA (PS-1 and PS-1.45
nanoparticles) were selected for the next studies. Table 1 summarizes
the mean hydrodynamic size and PDI of these nanoparticles. PS-1
nanoparticles exhibited a lower hydrodynamic mean size than PS-1.45.
If we consider the reactivity of the hydroxyl starch groups (primary
csc (mM) Sizea (nm) PDIa
CaCl2 NaCl CaCl2
6 585 205 180.3 ± 1.1 0.24 ± 0.01
9 185 55 185.5 ± 5.4 0.09 ± 0.01
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alcohol > secondary alcohol), and Ds of both starch derivatives, these

results suggest that the modification of the primary alcohol is enough for
the nanoparticles formation. But, it is plausible that the increase of Ds
modified the behaviour of starch derivative during the emulsification
step, and then, the final characteristic of nanoparticles will be also
modified. This will be discussed in more detail below. As illustrated by
AFM images (Fig. 2a and b), PS-1 and PS-1.45 nanoparticles showed a
spherical shape with a narrow size distribution.
Regarding to the negative ζ-potential values displayed by both
colloidal systems, it is worthy to remark that neither starch
derivatives nor PVA polymers present ionizable chemical groups.
Hence, the low and negative ζ-potential summarized in Table 1 can be
attributed to the specific interaction of the nanoparticles surface with
the ions present in the medium (as ζ-potential measurement were
developed in an aqueous solution of NaCl 3 mM) [27].

3.2. Colloidal stability
Stability of PS-1 and PS-1.45 nanoparticles as a function of the
electrolyte concentration using NaCl and CaCl2 as aggregating salts was
determined. Fig. 3 illustrates the value of the Fuchs factor (W) as a
function of the salt concentration; the corresponding values of critical
coagulation concentration (ccc) and critical stabilization concentration
(csc) are summarized in Table 1. For both nanoparticle systems ccc
values were lower when CaCl2 was used as aggregating salt. This was
due to the greater screening capacity of Ca2+ ions, which significantly
reduces the repulsive potential among the particles, favoring the
aggregation of the system [28]. Nevertheless, although ccc values
were slightly lower for PS-1 nanoparticles, it was not possible to find a
clear difference between ccc values of both colloidal systems. Moreover,
the low values of ccc for both systems indicate that only a low amount of
PVA must have been incorporated on the nanoparticles surface, that it
was not enough to produce a steric stabilization [29].
Interesting information can be obtained when the csc values are
analyzed. In this case, differences between both salts and both
colloidal systems were clearly found. It should be noted that csc values
depends on hydration forces and they do not only depend on the
Fig. 3. Stability factor as a function of salt concentration for PS-1 (square) and PS-1.45
(circle) nanoparticles. NaCl (closed symbols, solid line); CaCl2 (open symbols, dashed
line).
surface hydrophilicity, but also on the nature and concentration of the
hydrated counterions that surround the particles. Then, as Ca2+ is a
more hydrated cation than Na+, the re-stabilization power of the
former is higher than that of the latter. This explains the lower csc
values obtained for CaCl2 in comparison with NaCl. However, using a
given electrolyte, the lower the csc, the higher the hydrophilicity of
the particle surface [30]. Then, the lower csc values found for PS-1.45
nanoparticles, independently of the employed salt, with respect to PS-
1 nanoparticles, indicate a higher hydrophilic surface of the former.
3.3. Lyophilization
A typical limitation of colloidal systems suspended in aqueous
mediums is their low stability when conserved for a longer time;
lyophilization is a good alternative to obtain long-term stable systems
[31]. The lyophilization of PS-1 and PS-1.45 nanoparticles was studied
as a function of the type and concentration of cryoprotectant agent.
Fig. 4 displays the hydrodynamic mean size of the reconstituted
systems using trehalose as cryoprotectant, similar results were
observed with sucrose (data not shown). To obtain reconstituted
PS-1 nanoparticles with a similar size that the un-lyophilized, it was
necessary to use a cryoprotectant concentration of 0.5% (w/v).
On other hand, reconstituted PS-1.45 nanoparticles displayed a
similar size that the original nanoparticles. This pattern was totally
independent of the concentration and type of cryoprotectant agent.
Fig. 2c depicts an AFM image of reconstituted PS-1.45 nanoparticles
that were lyophilized without cryoprotectant agent. Comparison of

Fig. 2. AFM images of PS-1 (a), PS-1.45 (b), lyophilized PS-1.45 (c) and FFA loaded PS-1 Fig. 4. Hydrodynamic mean size of reconstituted PS-1 (■) and PS-1.45 (●)
(d) nanoparticles. nanoparticles after lyophilization as a function of cryoprotectant concentration.
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Table 2
Cytotoxicity of PS-1 and PS-1.45 nanoparticles after 4 h and 24 h incubation.

Concentration MTT assay LDH assay

(mg NP/ml)⁎ 102 (viability, %) (cytotoxicity, %)

4h 4h 24 h

PS-1 3.33 97.84 ± 1.41 − 0.89 ± 1.65 0.35 ± 4.55

1.66 102.20 ± 3.66 − 1.98 ± 0.53 − 0.97 ± 0.36
0.83 104.57 ± 4.24 − 0.89 ± 1.27 − 0.49 ± 1.40
0.42 102.95 ± 3.74 0.40 ± 0.37 0.00 ± 1.15
PS-1.45 3.33 102.16 ± 3.97 − 1.59 ± 1.72 − 1.51 ± 2.72
1.66 112.46 ± 1.34 − 2.02 ± 1.36 − 3.24 ± 1.25
0.83 108.88 ± 2.22 0.62 ± 0.88 − 2.43 ± 1.13
0.42 108.34 ± 3.02 − 1.88 ± 1.18 − 1.04 ± 0.55

Mean viability/toxicity n = 4 ± SD.

Fig. 2c and b shows that the reconstituted nanoparticles presented a

similar appearance to the un-lyophilised nanoparticles. Fig. 5. Release profile of FFA (■), testosterone (□) and caffeine (⊠) from PS-1
nanoparticles.

3.4. Cytotoxicity
Cytotoxicity was analysed using two diverse in vitro tests. LDH assay
is a test detecting the leakage of cell membrane and is useful in the
investigation of nanoparticle toxicity since the plasma membrane is the
main place of contact between particles and cells. MTT assay monitors
the mitochondrial metabolism of cells as an indicator of their viability.
The results of both tests are consistent and show that propyl-starch
particles up to a concentration of 0.033 mg NPs/ml are not cytotoxic in
the Caco-2 test model (Table 2). The tested samples PS-1 and PS-1.45
differed in their degree of substitution, but toxicological properties of
the particles were not affected by the increase of modification. The same
holds true for LDH assay following 24-h incubation.
3.5. Encapsulation of flufenamic acid, testosterone and caffeine
Table 3 summarizes the main characteristics of PS-1 and PS-1.45
nanoparticles as a function of the encapsulated molecule. When
compared to Table 1, it is easy to see that the encapsulation of FFA,
testosterone or caffeine in PS-1 or PS-1.45 nanoparticles did not alter
their original hydrodynamic mean size and PDI. As can be seen in the
AFM image (Fig. 2d), encapsulation of FFA did not produce any
intelligible change in the spherical shape and soft surface of PS-1
nanoparticles; similar results were found for testosterone and caffeine
and with PS-1.45 nanoparticles (data not shown).
Finally, it is also interesting to note that PS-1 and PS-1.45
nanoparticles exhibited a high EE for the three tested drugs, >95%
for FFA and testosterone; and >80% for caffeine.
3.6. In-vitro release of flufenamic acid, testosterone and caffeine
Once FFA, testosterone and caffeine were encapsulated in PS-1 and
PS-1.45 nanoparticles, next step was to study their release patterns. For
PS-1 nanoparticles (Fig. 5), FFA and testosterone presented a sustained
release without any burst effect and a nearly linear profile. On the other
hand, the hydrophilic drug caffeine showed a much faster but linear
release within the first 10 h before the release reached a plateau phase.
PS-1.45 nanoparticles, Fig. 6 presented different release pattern as
a function of the encapsulated drug. FFA presented the lowest release
rate, followed by testosterone. Both drugs showed a negligible burst
effect and a nearly linear profile. Finally, caffeine showed an initial fast
release, more pronounced than in the case of PS-1, followed by close
to zero release kinetic.
3.7. Permeation of flufenamic acid, testosterone and caffeine
Permeation profiles of FFA are illustrated in Fig. 7a, Papp values
calculated from these profiles are summarized in Table 3. Clear
differences were found between the patterns and Papp values of free
and encapsulated molecules. Moreover, Papp values calculated with
both colloidal systems were significantly different (ANOVA, α < 0.05),
being higher for PS-1.45 nanoparticles. It is worthy to remark the
similarity between the transport across a cellulose membrane (release
experiment, Figs. 5 and 6) and across HSE in the permeation
experiment (Fig. 7a) for this drug, suggesting that release from the
particles rather than skin permeation is rate limiting.
Regarding testosterone, in Fig. 7b, both the free and the
encapsulated drug displayed very similar permeation profiles, proving
that there was no effect of the nanoencapsulation on skin permeation.
No significant differences (ANOVA, α < 0.05) were found among the
different Papp values (Table 3).
Similar results were obtained with caffeine (Fig. 7c); its Papp values
indicate that this molecule showed the lowest permeation rate of the
three drugs. Consequently, rather large differences were observed
between the release patterns (Figs. 5 and 6) and the permeation
experiments (Fig. 7c), suggesting that permeation across the skin
barrier remains rate limiting.
4. Discussion
Results illustrated in Fig. 1 show the clear effect of PVA on the
nanoparticle formation. In this case, the well known surfactant
properties of this polymer [32,33] improved the nanoparticles

Table 3
Hydrodynamic mean size (nm), PDI, ζ-potential (mV), EE (%) and Papp (10− 6 cm/s) values of PS-1 and PS-1.45 nanoparticles with FFA, testosterone or caffeine encapsulated.

Encap. drug Size (nm) PDI ζ-Pot. (mV) EE (%) Papp (10− 6 cm/s) Papp⁎(10— 6 cm/s)

PS-1 FFA 159.7 ± 1.8 0.14 ± 0.02 − 15.6 ± 1.1 >95 2.43† ± 0.42 0.21 ± 0.05
Test 148.4 ± 0.7 0.10 ± 0.01 − 14.1 ± 3.3 >95 0.40 ± 0.10 0.32 ± 0.07
Caff 158.8 ± 1.5 0.11 ± 0.01 − 16.4 ± 2.3 >80 0.08 ± 0.01 0.09 ± 0.01
PS-1.45 FFA 185.5 ± 3.4 0.06 ± 0.02 − 12.3 ± 1.5 >95 3.11† ± 0.41 0.21 ± 0.05
Test 176.6 ± 6.0 0.11 ± 0.04 − 12.7 ± 0.6 >95 0.43 ± 0.11 0.32 ± 0.07
Caff 183.3 ± 5.4 0.11 ± 0.01 − 10.3 ± 1.2 >80 0.11 ± 0.03 0.09 ± 0.01

Papp⁎, Papp value calculated for a solution of un-encapsulated drug. † Indicates statistically significant differences.
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Fig. 6. Release profile of FFA (●), testosterone (○) and caffeine (⊗) from PS-1.45
nanoparticles.

formation. In addition, the increase of PVA concentration in the external

aqueous phase gave a size reduction and a lower PDI. These results agree
with those published in the 90s by Scholes et al. [34], who found that the
decrease in size and PDI when PVA concentration was increased can be
explained by the increase of the external aqueous phase viscosity.
Regarding to the low and negative ζ-potential shown by both colloidal
systems, it is plausible that, insofar as neither starch derivative nor PVA
present ionizable groups, this value proceed from the specific
interaction of NaCl with the nanoparticles surface. The specific in-
teraction of the ions with the nanoparticle surface depends on their
polarizability, being higher for the anions. Then the results suggest a
higher accumulation of the Cl− anions than Na+ cations although the
nanoparticles surface must be neutral [27].
The analysis of the colloidal stability reveals additional informa-
tion on the role of each polymer during the particle formation. As
mentioned earlier (section 3.2), both colloidal systems showed a low
stability (see ccc values in Table 1). This is indicative of a low
incorporation of PVA in the interface during the emulsification step
[29]. The amount of adsorbed PVA can be enough to stabilize the
emulsion but it was not enough to give a clear steric stabilization. On
the other hand, the csc values are useful to obtain more information of
the superficial composition of both nanoparticles systems. In this case,
PS-1.45 nanoparticles displayed lower csc values than PS-1 with both
salts (see Table 1). This result indicates that the nanoparticles
formulated with the polymer with higher hydrophobic character
(PS-1.45) displayed a more hydrophilic surface. This is because the
adsorption of molecules onto a substrate is mainly controlled by the
hydrophobic forces [35,36]. Hence, the adsorption through the acetate
moieties of the PVA onto the drops of the organic phase during the
emulsion step must be more favourable in the case of the more
hydrophobic starch, PS-1.45, than for PS-1. Concomitantly, due to the Fig. 7. Permeation profile of FFA (a), Testosterone (b) and Caffeine (c) through HSE.
hydrophilic chains of the PVA, the higher accumulation of this Encapsulated in PS-1 (■) or PS-1.45 (●) nanoparticles and free drug (▲).
molecule gives a higher hydrophilic character to the surface of the
nanoparticles. Regarding to the null effect of the different amounts of
PVA incorporated in both colloidal systems on the ccc magnitude, systems [31]. For this reason, lyophilization is advisable. This is why we
studies carried out previously focused on the adsorption of a hydro- studied the freeze-drying behaviour of PS-1 and PS-1.45 nanoparticles.
philic surfactant on PLGA nanoparticles showed that the csc value It is well known that lyophilization may generate a high stress that could
presented a clear dependence with the amount of surfactant placed destabilize colloidal systems. Hence, the use of cryoprotectant agents to
on the nanoparticles surface, while ccc values were less sensitive to protect the nanoparticles integrity from the freezing stress is often
these changes [29]. This observation suggests that although PS-1 and necessary [31]. For this reason, we selected two different disaccharides,
PS-1.45 nanoparticles presented similar ccc values, the obviously sucrose and trehalose, as cryoprotectant agents, as they are well known
different csc values showed by both systems indicate a clearly dif- for their good cryoprotectant properties [37–39].
ferent surface composition. Hence, stability results indicate that the After lyophilization and redispersation in water, the average size of
Ds of the starch had a clear effect in the final properties of these the nanoparticles was analyzed (see Fig. 4). For PS-1, in order to recover
nanoparticles. the initial properties of the un-lyophilized PS-1 nanoparticles, it was
As can be deduced from the previous lines, the long-term colloidal necessary to add at least a concentration of cryoprotectant agent higher
stability is a serious handicap against the clinical use of nanoparticles than 0.5% (w/v), either sucrose or trehalose. Similar behaviour between

M.J. Santander-Ortega et al. / Journal of Controlled Release 141 (2010) 85–92
both disaccharides was previously published by others authors [40].
However, it is easy to find in the literature that trehalose seems to be the
preferable cryoprotectant due to its better properties in comparison
with other sugars. Among these properties, it is remarkable its higher
glass transition temperature Tg [41,42]. Crowe et al. [43] found that, for
long storage times trehalose is better cryoprotectant than sucrose, due
to its higher Tg. At this moment it is necessary to remark that PS-1
nanoparticles were resuspended in purified water just after lyophiliza-
tion and may be, for this reason, it was not possible to find a clear
difference between sucrose and trehalose.
For PS-1.45 nanoparticles, from Figs. 4 and 2c, it is possible to
conclude that it was not necessary to use any cryoprotectant to obtain
reconstituted nanoparticles with similar properties as the original
nanoparticles. Experimental results commented in the previous
section indicated that both starch derivative nanoparticles present
different surface composition, being the PS-1.45 nanoparticles
enriched in PVA in comparison with the PS-1. Different publications
have shown that a fraction of this PVA is strongly attached to the
nanoparticle surface [33,44]. Moreover, several authors have demon-
strated that this PVA shell can enhance the nanoparticles stability
during freeze-drying, allowing to omit additional cryoprotectants
[31]. Accordingly, our data corroborate that the excess of PVA
presented in the surface of the PS-1.45 nanoparticles, in comparison
with PS-1, leads to a better stability.
To complete the characterization of these systems we have also
evaluated their cytotoxicity. At least in the tested concentration range,
none of the two polymers showed any measurable cytotoxic effect,
neither with the LDH nor with the MTT assay.
Finally, encapsulation and release properties of PS-1 and PS-1.45
nanoparticles were analyzed. Encapsulation of FFA, testosterone or
caffeine did not produce a clear effect in the size and PDI of both
colloidal systems, see Table 3 and Fig. 2a–d. On the other hand,
considering the chemical characteristics of the starch derivatives and
encapsulated drugs, EE summarized in Table 2 can be classified as a
function of the hydrophobic character of the encapsulated macro-
molecule, being higher and very similar for FFA and testosterone (Log
P ~5 and ~ 3.5, respectively) and lower for the less hydrophobic drug,
caffeine (Log P ~− 0.1).
Figs. 5 and 6 depict the release profile of the three drugs from the PS-1
and PS-1.45 nanoparticles respectively. To simplify the discussion, PS-1
nanoparticles will be firstly analyzed. The three release profiles
illustrated in Fig. 5 can be clearly separated in two different groups.
The first group was composed by FFA and testosterone, while caffeine
forms the second one. As was commented above, FFA and testosterone
are relatively hydrophobic (Log P ~5 and ~3.5, respectively), while
caffeine is much more hydrophilic (Log P ~−0.1) and moreover has a
positive net charge under release conditions (pkb ~10.5). Hence, from
Fig. 5 can be concluded that the release pattern from PS-1 nanoparticles
was mainly controlled by the hydrophobic interaction between the
encapsulated macromolecule and the nanoparticle matrix. Considering
the starch derivative structure, without any ionisable groups, it appears
that the degree of drug ionisation did not much affect drug release,
which instead seemed to be mainly governed by drug-matrix hydro-
phobic interactions [45].
Fig. 6 illustrates the release profiles of PS-1.45 nanoparticles. In this
case three drugs displayed three different release patterns. Release
velocity followed the sequence caffeine>>testosterone>FFA. Consider-
ing the drug characteristics described above, this sequence fits perfectly
with the drug's hydrophilic character, caffeine>>testosterone>FFA,
independently of the ionic drug state. Hence, for both nanoparticles
suspensions the control of the release pattern can be explained on the
basis of the drug-polymer hydrophobic interactions, which were more
intense for the PS-1.45 case. This can be attributed to the higher
hydrophobic character of PS-1.45 matrix with respect to PS-1 [45]. This
allows discrimination between FFA and testosterone, in spite of their
similar Log P, being more favourable the FFA-PS-1.45 than the
NANOMEDICINE
91
testosterone-PS-1.45 interaction. Concomitantly, it is also logic the faster
release of caffeine as observed for PS-1.45 nanoparticles due to the weaker
interaction within the matrix caffeine-PS-1.45 than the caffeine-PS-1.
As a function of the stability results discussed previously, topical
application seems to be most promising route for these nanoparticles
and hence was also addressed in the last section of this study.
For FFA, encapsulation in starch nanoparticles caused a significant
(more than 10-fold, see Table 3) increase in its Papp value across human
skin. A similar skin penetration enhancement by polymeric nanopar-
ticles had been previously observed by us using PLGA [19], and by other
authors with other drugs [17,20,21]. For testosterone, no such
enhancement was observed after nanoencapsulation in starch poly-
mers, suggesting that transport across the skin barrier remained rate
limiting also for the encapsulated drug. Similar results were obtained
with caffeine, which are in agreement with those previously reported by
Schäfer-Korting et al. [26]. The low Papp values of particle-bound and
free caffeine can be due to its inherent physicochemical characteristics
[16,46]. Considering Log P and Mw of caffeine the expression calculated
by Potts and Guy [46] estimates a Papp ~0.03·10− 6 cm/s for this
molecule, which is very close to our result.
Overall, the skin permeation data for the three drugs suggest that
starch nanoparticles have potential for transdermal drug delivery
applications. Although no attempt was made to further investigate the
interaction of the nanoparticles with the skin barrier itself, the low drug
permeation obtained with testosterone and caffeine and the physico-
chemical characteristics of PS-1 and PS-1.45 nanoparticles hint that
these colloidal systems themselves did not cross the HSE [16].
5. Conclusions
In this study, the formulation of nanoparticles by the emulsion
diffusion technique using propyl-starch derivatives with two different
Ds was analyzed. From the colloidal stability, the different surface
characteristics were determined, indicating a higher amount of PVA on
the nanoparticles surface prepared with the more hydrophobic
polymer, PS-1.45. Superficial differences between PS-1 and PS-1.45
nanoparticles determined their lyophilization, since PS-1.45, the system
with a PVA enriched surface, recovered better their initial properties.
In addition, we explored the potential of nanoparticles formulated
with propyl-starch derivatives for transdermal drug delivery. Both
nanoparticle systems, PS-1 and PS-1.45, showed high encapsulation
efficiency for a wide range of chemically different drugs. The drug
release was driven by hydrophobic character of both the polymer and
the drug. Both systems showed a close to linear release pattern for
hydrophobic drugs. Finally, PS-1 and PS-1.45 nanoparticles showed a
remarkable enhancer effect for FFA across the skin barrier.
Acknowledgements
The authors wish to thank to Dr. Kostka (Caritas-Krankenhaus
Lebach, Germany) for the supply with human skin samples as well as the
financial support given by Galenos Fellowship in the Framework of the
EU Project “Towards a European PhD in Advanced Drug Delivery”, Marie
Curie Contract MEST-CT-2004-404992; the German Bundesminister-
ium für Bildung und Forschung (BMBF) program “Nanochance” project
number 13N9133 and the companies Toroma Organics Ltd, and BASF
and the Spanish “Ministerio de Ciencia e Innovación” (MICINN) project
number MAT2007-66662-C02-01.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jconrel.2009.08.012.
NANOMEDICINE
92 M.J. Santander-Ortega et al. / Journal of Controlled Release 141 (2010) 85–92
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