Chitosan-coated PLGA nanoparticles for DNA/RNA delivery: Effect of the formulation parameters on complexation and transfection of antisense oligonucleotides

Article (PDF Available)inNanomedicine: nanotechnology, biology, and medicine 3(3):173-83 · October 2007with101 Reads
DOI: 10.1016/j.nano.2007.03.006 · Source: PubMed
Abstract
Cationically modified poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles have recently been introduced as novel carriers for DNA/RNA delivery. The colloidal characteristics of the nanoparticles--particle size and surface charge--are considered the most significant determinants in the cellular uptake and trafficking of the nanoparticles. Therefore, our aim was to introduce chitosan-coated PLGA nanoparticles, whose size and charge are tunable to adapt for a specific task. The results showed that biodegradable nanoparticles as small as 130 nm and adjustable surface charge can be tailored controlling the process parameters. As a proof of concept, the overall potential of these particulate carriers to bind the antisense oligonucleotides, 2'-O-methyl-RNA, and improve their cellular uptake was demonstrated. The study proved the efficacy of chitosan-coated PLGA nanoparticles as a flexible and efficient delivery system for antisense oligonucleotides to lung cancer cells.
Genetics
Chitosan-coated PLGA nanoparticles for DNA/RNA delivery:
effect of the formulation parameters on complexation
and transfection of antisense oligonucleotides
Noha Nafee, PharmD,
a,b
Sebastian Taetz, PharmD,
a
Marc Schneider, PhD,
a,
Ulrich F. Schaefer, PhD,
a
Claus-Michael Lehr, PhD
a
a
Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, Germany
b
Faculty of Pharmacy, Department of Pharmaceutics, University of Alexandria, Alexandria, Egypt
Received 22 January 2007; accepted 15 March 2007
Abstract Cationically modified poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles have recently been
int roduced as novel carriers for DNA/RNA delivery. The colloidal characteristics of the
nanoparticlesparticle size and surface chargeare considered the most significant determinants
in the cellular uptake and trafficking of the nanoparticles. Therefore, our aim was to introduce
chitosan-coated PLGA nanoparticles, whose size and charge are tunable to adapt for a specific task.
The results showed that biodegradable nanoparticles as small as 130 nm and adjustable surface
charge can be tailored controlling the process parameters. As a proof of concept, the overall potential
of these particulate carriers to bind the antisense oligonucleotides, 2-O-methyl-RNA, and
improve their cellular uptake was demonstrated. The study proved the efficacy of chitosan-coated
PLGA nanoparticles as a flexible and efficient delivery system for antisense oligonucleotides to lung
cancer cells.
© 2007 Elsevier Inc. All rights reserved.
Key words: PLGA nanoparticles; Chitosan; Gene delivery; Antisense oligonucleotides; Telomerase inhibitors
DNA/RNA delivery is gaining growing attention for the
treatment of genetic deficiencies and is still a hope for
successful future medical treatment [1,2]. Gene therapy can
be defined as the transfer of a genetic material to specific
cells so as to have a therapeutic effect. At the present time,
major gene delivery systems u se either viral or nonviral
vectors [3,4]. Although viral systems are very efficient for in
vivo transfection, as well as immunization, their major
drawback is their possible toxicit y, immunogenicity, and
inflammatory potential [5,6]. Nonviral systems based on
biocompatible polymers are preferred in terms of safety,
stability, relative ease of large-scale production and char-
acterization, and the lack of intrinsic immunogenicity [7].
Nonviral vectors include liposomes, complex es of the
negatively charged plasmid with cationic polymers, and
nanoparticles. Even though liposome vesicles prepared from
lipids may protect the loaded drugs or proteins from
degradation and target them to the site of action [8] , they
have shown a relatively low encapsulation efficiency, poor
storage stability, and rapid clearance from the blood [9]. The
formation of complexes from cationic polymer with anionic
DNA or oligonucleotide solutions was applied by many
research groups [10-12]. The simplicity of these self-
assembling polyelectrolyte complexes is both an advant age
and a drawback. Although such complexes are easy to
generate and can protect DNA from enzymatic degradation,
they are always characterized by a broad size distribution and
variable shape [12]. Solid, biodegradable nanoparticles have
shown their advantage over other carriers by their increased
stability and their controlled-release ability. Furthermore, the
low polydispersity allows better control when used as
delivery vehicles.
Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173 183
www.nanomedjournal.com
No conflict of interest was reported by the authors of this paper.
Corresponding author. Biopharmaceutics and Pharmaceutical Tech-
nology, Saarland University, Campus Saarbrücken, Bldg. A41, P.O. Box
151150, Saarbrucken 66041, Germany.
E-mail address: marc.schneider@mx.un-saarland.de (M. Schneider).
1549-9634/$ see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.nano.2007.03.006
For these reasons, biodegradable nanoparticles are an
important area of research in the field of drug and gene
delivery. One of the most widely used polymers for
nanoparticles is the biodegradable and biocompatible poly
(
D, L-lactide-co-glycolide) (PLGA). PLGA nanoparticles
have been extensively investigated for sustained [13] and
targeted [14]/localized [15] delivery of various agents
including anticancer drugs [16], plasmid DNA [17], proteins
and peptides [18,19], and low-molecular-weight compounds
[20]. PLGA nanoparticles have hence shown great efficiency
as drug delivery vehicles, increasing the drug amount
crossing various biological barriers such as the blood-brain
barrier [21,22], gastrointestinal mucosa [23], nasal mucosa
[24], and ocular tissue [25].
In the context of DNA/RNA delivery, the major limitati on
in the application of these nanoparticles is primarily their
negative charge, which limits the interaction with the
negatively charged DNA, in addition to the poor transport
characteristics of the DNA-encapsulated PLGA nanoparti-
cles through the cell membrane. PLGA nanoparticles with
cationic surface modification can overcome these disadvan-
tages and hence readily bind and condense DNA. Several
polycations were used to accomplish this cationic surface
modi fication including polyethyleneimine [26],cetyltri-
methylamonium bromide [27], poly(2-dimethyl-amino)
ethyl methacrylate [28], didodecyl dimethyl ammonium
bromide [29], and chitosan [30].
The naturally occurring linear polysacchari de, chitosan, is
considered to be a good candidate for gene transfection and
expression because of its biodegradability, biocompatibility,
mucoadhesive, and permeability-enhancing properties [31].
In a recent comparative study, chitosan-coated nanoparticles
(cNPs) were found to be best suited for trans fection.
Although nanoparticles coated with pDMAEMA showed
the highest transfection efficiency, cNPs were the only
carrier that released DNA at pH 7, which is a prerequisite for
the successful delivery [28]. The addition of chitosan to the
surface of PLGA nanoparticles was also found to increase
the penetration of the encapsulated macromolecules in
mucosal surfaces [32]. Moreover, cNPs were found to
facilitate gene delivery and expression in vivo with increased
efficiency and without causing inflammation [33].
The transfection efficiency of the cationically modified
particles depends strongly on the particle size, which
determines their cellular uptake, and the surface charge,
which influences the ability of the particles to efficiently
condense plasmid DNA-polynucleotides and to interact
with cell s. Prabha et al [34] investigated the gene transfection
levels of different size fractions of PLGA nanoparticles
and found that the lower size nanoparticle fraction resulted in
a 27-fold higher transfection in COS-7 cells and 4-fol d
higher transfection in HEK293 cells for the same dose
of nanoparticles.
Therefore, the ability to control the colloidal character-
istics of the nanoparticles, most importantly particle size and
surface charge, is central in determining the transfection
efficiency. Hence, the aim of our study was to modulate the
effect of the formulation parameters to tailor the nanocar-
riers' size and charge for their special application, to
optimize payload, and to appropriately address the target
system. Accordingly, many formulation parameters includ-
ing the polyvinyl alcohol (PVA) content, the type and
concentration of PLGA, the type and concentration of
chitosan, and the ratio of the organic to the aqueous phase of
the emulsion were studied. In addition, the effect of other
process parameters on the particle size was also studied, such
as the emulsification and homogenization time and speed.
The overall suitability of these nanoparticles as gene
carriers was then confirmed by studying their binding with
the antisense oligonucleotides 2-O-methyl-RNA (OMR),
which is a 13mer directed against the template region of
human telomerase RNA for the treatment of lung cancer
through telomerase inhibition [35]. The uptake of OMR-
nanoparticle polyplexes into human lung cancer cell line
A549 was demonstrated.
Materials
Poly(
D,L-lactide-co-glycolide) 70:30 (Polysciences Eur-
ope GmbH, Eppelheim, Germany), poly(
D,L-lactide-co-
glycolide) 50:50 (Sigma Chemical Co., St. Louis, MO),
PVA Mowiol 4-88 (Kuraray Specialities Europe GmbH,
Frankfurt, Germany), two types of ultra-pure chitosan
chloride (Protasan UP CL113 and Protasan UP CL213 with
molecular weights of b150 and 150-400 kDa, respectively;
FMC BioPolymer AS, Oslo, Norway), ethyl acetate (Fluka
Chemie GmbH, Buchs, Switzerland) were used as obtained.
The antisense oligonucleotide OMR with a phosphorothioate
(ps) backbone (5-2-O-methyl [C(ps)A(ps)GUUAGGGUU
(ps)A(ps)G]-3) was obtained from (Biomers.net GmbH,
Ulm, Germany). For the uptake studies, the carboxyfluor-
esceinamine-labeled derivative 5-FAM-OMR was used.
RPMI 1640 supplemented with
L-glutamine and 10% fetal
calf serum (PAA Laboratories GmbH, Pasching, Austria) was
used as cell culture medium.
Methods
Preparation of the nanoparticles
Nanoparticle formulations were prepared by the emul-
sion-diffusion-evaporation technique [30]. PLGA 70:30
was diss olved in 5 mL ethyl acetate at room temperature
(23-25°C). The aqueous phase was prepared by dissolving
PVA in MilliQ water. In the case of cNPs, chitosan chloride
(Protasan UP CL113) was added to the aqueous phase. The
organic phase was added dropwise to an equal volume of the
aqueous phase under stirring using a magnetic stirrer, at 1000
rpm, for 1 hour, at room temperature. The emulsion was then
homogenized (Ultra-Turrax T25; Janke & Kunkel GmbH &
Co-KG, Germany) at 13,500 rpm for 10 minutes. Nanopre-
cipitation was performed by adding Mil liQ water dropwise
174 N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
under gentle stirring to obtain a final volume of the
suspension of 50 mL. Stirring was continued overnight at
room temperature to get rid of the organic solvent. The so-
prepared nanoparticle suspension contained 2 mg/mL
PLGA, 1 mg/mL PVA, and 0.3 mg/mL chitosan and was
considered as our reference nanoparticle formulation. To
investigate the effect of the formulation parameters on the
colloidal characteristics of the nanoparticles, other formula-
tions with different concentrations and/or compositions were
prepared following the same procedure and compared to the
original particles.
Determination of the colloidal characteristics
The mean particle size and size distribution were
determined in MilliQ water using the Malvern Zetasizer
Nano (Malvern Instruments, Malvern, UK). Measurements
were based on photon correlation spectroscopy at 25°C and
90 deg scattering angle using the contin mode. The surface
charge was estimated by measuring the zeta potential based on
the electrophoretic mobility (Zetasizer Nano, Malvern Instru-
ments, Malvern, UK). All zeta potential measurements were
performed with diluted nanoparticle suspensions (pH 3.5-4).
For each batch, three samples were measured (with 30 subruns
for each sample) and the mean values were calculated.
Zeta potentialpH titration profile
To measure the zeta potential of the nanoparticles as a
function of pH, both chitosan-coated and noncoated
nanoparticle suspensions were diluted with McIlvaine buffer
of different pH values ranging from 2 to 8.
Scanning probe microscopy (SPM)
The surface morphology and the shape of the nanopar-
ticles were examined by SPM with a Bioscop e equipped with
a Nanoscope IV controller (Digital Instruments, Veeco,
Santa Barbara, California). The nanoparticles were investi-
gated under ambient conditions in tapping mode using a
scanning probe with a force constant of 40 N/m at resonant
frequency of 170 kHz (Anfatec , Oelsnitz, Germany).
Complexation of oligonucleotides
Loading of nanoparticles with oligonucleotides and
formation of nanoplexes
To study the ability of chitosan-coated PLGA nanopar-
ticles as a carrier for gene delivery, nanoparticles were loaded
with the antisense oligonucleotide, OMR. The cationic
nanoparticles were mixed with a solution of the oligonucleo-
tides at room temperature and vortexed for 30 seconds
followed by incubation on an orbital shaker at room
temperature. The factors affecting the binding conditions,
including the incubation medium, the incubation time and
the cNP/OMR ratio, were investigated. The binding condi-
tions were optimized to ensure maximum binding ability of
the oligonucleotide to the nanoparticles.
Characterization of nanoplexes
The physicochemical characteristics of cNP-OMR
nanoplexes were determined using the Malvern Zetasizer
Nano (Malvern Instruments, Malvern, UK). The effect of
the change in cNP/OMR ratio, the incubation medium, and
the incubation time on the size and surface charge of the
nanoparticles was studied. The morphology of the nano-
plexes was examined by SPM as described above.
Cellular uptake of cNP-OMR nanoplexes
The uptake of the nanoplexes was investigated using A549
human lung carcinoma cells. The cells were seeded on
LabTec cha mber slides (Nunc GmbH, Wiesbaden, Germany)
at a density of 12,500 cells/mL. Nanoparticles incubated with
fluorescently labeled oligonucleotides 5-FAM- 2-OMR
were mixed with the culture medium RPMI 1640 supple-
mented with 10% fetal calf serum then added to the cells.
After 6 hours of incubation, the medium was replaced by
normal cell culture medium. The uptake was then examined
after 24 hours by confocal laser scanning microscopy.
Confocal laser scanning microscopy
Fluorescence imaging was performed using a BioRad
MRC-1024 confocal laser scanning microscope equipped
with an argon-krypton laser. The objective used was an oil
immersion objective 40× NA = 1.3. The excitation was
performed using λ = 488 nm, and the fluorescence signal
was collected after a band-pass filter (522/35). A step motor
was used to perform 3D sections through the cells. The green
fluorescent particles were located with respect to the cell
membrane using red-stained Ricinus communis agglutinin,
which adsorbs to the cell surface (λ
ex
= 568 nm; fluorescence
detection with LP585). Data evaluation and 3D reconstruc-
tion was done using Volocity software (Improvision,
Tübingen, Germany).
Results
Chitosan-coated versus noncoated PLGA nanoparticles
Chitosan-coated (cNPs) and noncoated nanoparticles
(ncNPs) were prepared with PLGA 70:30 and PVA as
stabilizer using the emulsion-diffusion-evaporation techni-
que. Measurements of the particle size showed a slight
increase in the mean nanoparticle diameter (from 271.1 ±
1.03 nm to 278.95 ± 4.95 nm) by the addition of chitosan to
the aqueous phase. In both cases, the nanoparticles are
monodisperse (polydispersity index 0.04-0.06), spherical,
and have smooth surfaces as revealed by the SPM micro-
graphs (Figure 1). The pH of the standard nanoparticle
suspension was found to be around 4. The addition of chitosan
increases the zeta potential of the particles to 17.1 mV as
compared with 10 mV for the ncNPs in MilliQ water.
The effect of pH on the surface charge of chitosan-coated
and noncoated PLGA nanoparticles was investigated by
175N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
measuring the zeta potential versus pH. As shown in
Figure 2, the zeta potentia l of th e noncoated PLGA
nanoparticles is almost constant (0.4-3.5 mV) at all pH
values test ed. In comparison, cNPs showed a positive
potential of 33.6 mV in acidic medium, which decreas ed
by increasing pH values.
In addition to the surface charge of the nanoparticles, the
particle size is also considered one of the most important
parameters in the mucosal and tissue uptake of the
nanoparticles. Smaller nanoparticles are able to penetrate
through the submucosal layers, whereas larger size particles
were found to be localized in the epithelial lining [36].
Moreover, smaller nanoparticles were f ound to show
significantly higher transfection efficiency as compared
with larger nanoparticles [34]. Therefore, our study aimed
to produce nanoparticles of relatively small(er) size and more
pronounced positive charge on the surface while being able
to adjust these parameters deliberately. This necessitates the
study of the effect of different formulation variables on the
colloidal characteristics of these particles.
Factors affecting the colloidal characteristics
of the nanoparticles
Effect of the stabilizer and type of PLGA
The influence of PVA content on the colloidal properties
of the chitosan-coated PLGA nanoparticles was studied.
Nanoparticles containing various concentrations of PVA
ranging from 1 to 5 mg/mL of the nanoparticle suspension
were prepared using two different types of PLGA copolymer
based on its lactide-to-glycolide ratio (PLGA 70:30 and
PLGA 50:50). In general, it was observed that increasing the
concentration of PVA resulted in a statistically significant
decrease in the mean particle size (P b .05; Figure 3). When
the concentration of PVA was increased from 1 to 2 mg/mL,
the mean particle diameter was significantly reduced,
whereas further increase in the PVA concentration led to
smaller reductions in the particle size (Figure 3). Similar
Fig 1. Morphology of noncoated (A) and chitosan-coated (B) PLGA nanoparticles as observed by the SPM.
Fig 2. Zeta potentialpH profile for noncoated and chitosan-coated PLGA
nanoparticles.
Fig 3. Effect of the concentration of PVA on the particle size and the surface
charge of chitosan-coated PLGA nanoparticles (mean ± SD).
176 N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
results were also reported by Mainardes and Evangelista [37].
The size reduction was significantly more pronounced in the
case of nanoparticles prepared with PLGA 70:30 than those
prepared with PLGA 50:50 (P b .05; Figure 3). Similarly,
measurements of the zeta potential indicate that nanoparticles
containing PLGA 70:30 are characterized by a significantly
higher zeta potential (28.3-35.38 mV) compared with those
containing PLGA 50:50 (16.4-22.9 mV; P b .05; Figure 3).
The ratio of lactide to glycolide content in the PLGA polymer
is known to influence the degradation rate of the polymers
[38,39] as well as their release properties. Therefore, it was
interesting to investigate their effect on the colloidal
characteristics of the nanoparticles. Nevertheless, because
of the biological requirements and goals we did not test all
available ratios of lactide to glycolide units. Only short
degradation times were considered, and therefore only two
types of PLGA copolymers were investigated. No significant
correlation was found between increasing PVA concentration
and the surface charge of the nanoparticles (P b .05).
Formulations containing different amounts of PVA are all
characterized by unimodal size distribution (polydispersity
index = 0.08-0.15).
Effect of PLGA concentration
Nanoparticle suspensions contai ning half and double the
concentration of PLGA 70:30 present in the original
nanoparticle formulation were prepared. It was observed
that increasing the polymer concentration resulted in
production of nanoparticles of larger sizes (Figure 4). This
is in agreement with the findings of Kwon et al [29] and
Chorny et al [20]. However, the change in particle size did
not affect the polydispersity of the nanoparticles; mean
polydispersity index of the three formulations is 0.09 ±
0.012. Measurements of the zeta potential showed that the
surface charge is weakly affected by the variation in the
polymer concentration (Figure 4).
Effect of chitosan
To study the effect of chitosan concentration on the
colloidal characteristics of the nanoparticles, two ncNP
formulations F1 and F2 of two different particle sizes (249.8
and 148.2 nm) containing increasing concentrations of
chitosan (0.15 to 1.5 mg/mL) were prepared (all other
parameters were unchanged). As observed in Figure 5, A ,
increasing the concentration of chitosan showed a gradual
increase in the particle size of the nanoparticles, which
become significantly more pronounced for nanopar ticles
containing more than 0.9 mg/mL chitosan (P b .05). On the
other hand, measu rement of the zeta potential of the different
formulations indicates a significant increase in the surface
charge with increasing concent ration of chitosan (P b .05).
The connecting lines between the data points were obtained
using y = y
0
+ a(1 e
bx
), (R
2
= 0.999, 0.996) as a fit
function (Figure 5, B). However, the small F2 nanoparticles
are characterized by a higher surface charge using the same
concentration of chitosan; the zeta potential of F1 and F2
both containing 0.6 mg/mL chitosan was found to be 33.03
and 46.43 mV, respectively.
The influence of chitosan properties on the size and
surface charge of the nanoparticles was also investigated.
Fig 4. Effect of the concentration of PLGA on the colloidal characteristics of
chitosan-coated PLGA nanoparticles (mean ± SD).
Fig 5. Effect of the concentration of chitosan on (A) the particle size and (B)
the surface charge of chitosan-coated PLGA nanoparticles (mean ± SD).
177N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
Ultrapure chitosan chloride (Protasan UP CL 213) was used
to prepare nanoparticles. The polymers are characterized by
different molecular weight and viscosity (Table 1). Different
molecular weight of the outer polymer might influence the
overall charge of the particles. The viscosity of chitosan is
expected to influence the formation of the nanoparticles,
which depends on the diffusion and therefore on the viscosity.
However, it was noticed that nanoparticles prepared using
either type of chitosan have the same mean particle size and
surface charge considering the error (Table 1).
Effect of organic to aqueous phase volume ratio
The ratio between the organic and aqueous phase of the
emulsion is of great importance regarding the stability of the
emulsion and is expected to influence the size of the
dispersed globules. Therefore, the organic to aqueous phase
volume ratio was varied among 2:1, 1:1, and 1:2. The results
demonstrate a gradual decrease in particle size by changing
the ratio from 2:1 to 1:2 (Figure 6, A), as well as a
corresponding significant increase in surface charge (P b .05;
Figure 6, B).
Effect of process parameters
Attempts to reduce the particle size by increasing the
speed of magnetic stirrer or the homogenizer or the number
of the homogenization cycles were previously reported
[29,40]. According to Kwon et al [29], an increase in
homogenization speed resulted in a corresponding decrease
in the particle size; however, no significant reduction in
size was observed by increasing the speed above 12,000
rpm. It can be noticed that the modification of the stirring
time of the emulsion, the homog enization speed or time, or
sonication resulted in minor reduction in the particle size
(Table 2). Further increase in the homogenization time and/
or speed was not favorable, because the high energy
provided led to particle fusion and aggregation rather than
particle size reduction.
Based on the aforementioned results, chitosan-coated
PLGA nanoparticles can be produced over a wide range of
size and surface charge. For DNA/RNA delivery purposes,
nanoparticles of small size and high surface charge are
mandatory. Therefore, we selected a chitosan-coa ted PLGA
nanoparticle formulation characterized by a size of 172.3 ±
4.5 nm and a surface charge of 38.6 ± 1.96 mV to study
binding and transfection.
Binding of nanoparticles to oligonucleotides
Antisense oligonucleotides represent a new generation of
drugs with high therapeutic potential. However, the negative
Table 1
Effect of chitosan properties on the colloidal characteristics of nanoparticles
Type of chitosan Chitosan properties Colloidal characteristics of the nanoparticles
Molecular weight (kDa)
Viscosity
(mPa.s)
Mean particle size
(nm ± SD)
Polydispersity
index ± SD
Zeta potential
(mV ± SD)
Protasan UP CL113 b150 b20 166.05 ± 1.5 0.122 ± 0.02 13.32 ± 0.83
Protasan UP CL213 150-400 20-200 167.7 ± 0.5 0.13 ± 0.01 14.4 ± 1.67
* Approximate molecular weight (weight average molecular weight).
Standard viscosity ranges (1% solution, 20°C).
SD denotes the standard deviation of (n = 3).
Fig 6. Effect of the organic-to-aqueous phase volume ratio on (A) the
particle size and (B) the surface charge of chitosan-coated PLGA
nanoparticles (mean ± SD).
178 N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
charge and hydrophilicity of these molecules limit their
cellular uptake. In this study, the ability of chitosan-coated
PLGA nanoparticles as a biocompatible nonviral carrier for
the antisense oligonucleotides, OMR, was investigated.
OMR is a 13mer directed against the template region of
human telomerase RNA for the treatment of lung cancer
through telomerase inhibition [35]. To investigate the
behavior of the nanoplexes during cellular uptake experi-
ments, binding was carried out in various incubation media
including MilliQ water, sodium chloride 10 mM, phosphate-
buffered saline pH 7.4, and Hank's balanced salt solution pH
7.4. The colloidal characteristics of the OMR-loaded nano-
particles were compared to the unloaded nanoparticles. As
shown in Figure 7, A, dynam ic light scattering did not
show a noti ceable increase in size of the nanoparticles due to
oligonucleotide adsorption. In addition, changing the incuba-
tion medium did not result in a corresponding change in the
size of the nanoparticles. On the other hand, binding of the
OMR to particles was confirmed by the surface charge
measurements (Figure 7, B). OMR-loaded particles are
characterized by lower zeta potential values (in MilliQ
water and sodium chloride solution) in comparison to
unloaded particles, whereas in phosphate-buffered saline
and Hank's balanced salt buffers pH 7.4, a weak zeta
potential was recorded with either nonloaded or OMR-
loaded nanoparticles.
The incubation time (t
inc
= 15-60 minutes), referring to
the duration of mixing of the nanoparticles with OMR in
MilliQ water, had no effect on the size and zeta potential
(data not shown).
The binding was investigated as a function of the ratio
between the cNPs and OMR. Various cNP/OMR ratios
(ranging from 200:1 to 10:1) were mixed in MilliQ water and
incubated for 15 minutes. The size and surface charge of the
nanoplexes formed were measured. It could be observed that
increasing the oligonucleotide concentration resulted in
gradual reduction of the surface charge (Figure 8). The
curve was fitted with y = ax
b
, where a and b are constants. b
would be expected, in an ideal case, to represent the surfa ce
dependence of the adsorption process but was found to be
about 0.25 (R
2
= 0.996).
The morphology of cNP-OMR nanoplexes was exam-
ined by SPM. They showed no changes in the surface
Table 2
Effect of various technical parameters on the nanoparticle size
Parameter Duration Mean particle size
(nm ± SD)
Polydispersity
index ± SD
Stirring time 1 hr 277.6 ± 1.3 0.048 ± 0.008
2 hr 273.1 ± 2.08 0.057 ± 0.01
3 hr 271.5 ± 2.02 0.047 ± 0.016
Homogenization time 10 min 273.5 ± 1.15 0.036 ± 0.02
15 min 271.2 ± 3.4 0.049 ± 0.011
Sonication time 1 min 275.7 ± 1.9 0.059 ± 0.01
6 min 274.6 ± 3.4 0.078 ± 0.009
SD denotes the standard deviation of (n = 3).
Fig 7. Effect of the incubation medium on the binding of OMR to cNPs.
A, Particle size and size distribution. B, Zeta potential.
Fig 8. Effect of the OMR concentration on the zeta potential of the
nanoplexes.
179N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
morphology of the particles (Figure 9, B) an d were in the
size range found by photon correlation spectroscopy. In
comparison to nonloaded nanoparticles (Figure 9, A),
smaller structures of around 80-100 nm were observed;
these are suggested to be nanoplexes of the OMR with
excess chitosan in the medium left over from the
nanoparticle preparation procedure.
Applying fluorescently labeled cNP/FAM-OMR to lung
cancer cells (A549) allowed visualization of the uptake of the
nanoplexes. In Figure 10, A, one can see an arbitrary selected
image depicting the x-y plane of the cells after incubation
with the loaded nanoparticles out of a 3D stack. In addition,
cuts along the x-z and the y-z direction are shown. These
cross-sections allow determination of the location of the
nanoplexes relative to the apical cell surface, which was
labeled by adsorption of red dye bound to a lectin. On the
enlarged and focused x-z section the green fluorescence from
the FAM-OMR can be clearly seen inside the cell (toward the
basolateral side; Figure 10, B).
Discussion
Recent studies showed that the adsorption of a cationic
hydrophilic polymer on the nanoparticle surface improves
not only their transmucosal transport but also their efficiency
as gene carriers [28,41]. In this context, both the size and
surface charge of nanoparticles are considered the major
determinants for successful gene delivery. PLGA nanopar-
ticles are known to be negatively charged as a resul t of the
presence of ionized carboxyl groups. The presence of an
amphiphilic polymer such as PVA forms a stable network on
the polymer surface. This network shields the surface charge
and moves the shear plane outward from the particle surface,
which resulted consequently in a slightly negative zeta
potential [42]. Despite this comparatively weak zeta
potential, the nanoparticles wer e stabilized by the layers of
PVA surrounding the nanoparticles by steric hindrance
Fig 9. Morphology and section analysis of (A) cNPs and (B) cNP-OMR nanoplexes as observed by SPM.
Fig 10. Confocal microscopic image of A549 cells after incubation with
fluorescently labeled cNP/FAM-OMR. A, Representation of an x-y plane of
a 3D stack in addition to cuts along the x-z and the y-z directions. B,
Representation of the enlarged and focused x-z section.
180 N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
[30,43]. PVA has been extensively used as a promising
stabilizer for PLGA nanoparticles [19,44]. The mechanism
of PVA binding with PLGA has been proposed to be due to
the interpenetration of PVA and PLGA molecules during
nanoparticle formation. PVA is a copolymer of poly(vinyl
acetate) and poly(vinyl alcohol) with considerable block
copolymer character. The hydrophobic segments of PVA, the
vinyl acetate part, penetrate into the organic phase and
remain entrapped into the polymeric matrix of the nano-
particles. The binding of PVA on the nanoparticle surface is
likely to happen when the organic solvent is removed from
the interface in which interpenetration of PVA and PLGA
molecules takes place [42].
Unlike the non-coated PLGA nanoparticles, chitosan-
coated nanoparticles are characterized by a strongly positive
zeta potential. Hence, these nanoparticles are stabilized by
electrostatic repulsion, which prevents their aggregation.
Chitosan is a weak base polysaccharide, consisting of β(1,4)
linked monomers of
D-glucosamine and N-acetyl-D-gluco-
samine. In acidic medium, the amine groups of the
polysaccharide will be positively charged allocating a high
surface charge to the nanoparticles [45]. The pH titrati on
profile (Figure 2) depicts a variation in zeta potential with
pH, which reveals that the surface charge of cNPs is strongly
dependent on pH. Thus, the zeta potential titration provi ded
proof of successful cationic surface modification by the
addition of chitosan. The amount of surfactant plays an
important role as a stabilizing agent in the emulsification
process and in the protection of the droplets. Increasing the
concentration of PVA in the aqueous phase resulted in a
corresponding increase in the viscosity. This contributes in
the formation of a stable emulsion with smaller and uniform
droplet size, leading to the formation of smaller sized
nanoparticles [42]. However, as revealed in Figure 3, the
decrease in particle size with the PVA concentration is not
linear and levels off for higher PVA concentrations.
Furthermore, the reduction of nanoparticle size is a funct ion
of the type of PLGA; PLGA 70:30 f ormed smaller
nanoparticles compared with PLGA 50:50 using the same
concentration of PVA.
In contrast to the effect of PVA, results showed a
significant increase in particle size with higher PLGA
concentration (Figure 4). As the polymer concentration
increases, the viscosity of the organic solution (dispersed
phase) increases, resulting in a poorer dispersibility of the
PLGA solution into the aqueous phase. The molecules are
expected to coalesce in a more concentrated solution, thereby
forming larger particles [37]. The same effect was also
noticed by reducing the volume of the inner aqueous phase in
relation to the organic phase (Figure 6, A and B). Dissolving
the same amount of PVA-chitosan in half the volume of
water resulted in a remarkable increase in the viscosity,
which renders the dispersion of PLGA solution difficult, and
in turn larger nanoparticles were formed. These findings
emphasize the great influence of the viscosity on the
colloidal characteristics of the nanoparticles.
The addition of chitosan to the aqueous phase during
nanoparticles formulation imparts a positive zeta potential to
the nanoparticles, which is a function of chitosan concentra-
tion (Figure 5). However, the charge density on the surface
remains dependent on the nanoparticle size according to the
free surfa ce area with respect to the chitosan amount. As
illustrated in Figure 5, B, the smaller particles show a rapid
increase in surface charge that levels off as the surface
approaches saturation, whereas the same amount of chitosan
leads for larger particles to a partially covered surface.
Finally, a zeta potential of 58-60 mV seems to indicate the
saturation of the nanoparticle surface with the polycation in
both cases. This behavior might be easily exploited for
modulating the particle surface charge to facilitate both the
transport properties as well as the mucoa dhesive properties
of the nanoparticles.
The relation between the particle size and the surface
charge on the nanoparticles is of interest. Smaller particles
acquire a higher zeta potential compared with larger particles
using the same concentration of chitosan (Figure 5, B). This
reflects the dependency of the zeta potential on the charge
density. The smaller the particle size, the larger will be the
relative surface area of the nanoparticles available for
interaction with chitosan. Hence, using constant chitosan
concentration, one can improve the surface charge of the
nanoparticles by varying the mean nanoparticle diam eter.
In theory, mucoadhesion can be promoted by the
positively charged groups on the nanoparticle surface
through their electrical interaction with the negatively
charged mucus [30]. In addition, the positively charged
particle surface may be expected to facilitate adherence to the
negatively charged cellular membranes by localized desta-
bilization of the membrane, thus inducing their intracellular
uptake into the cytoplasmic compartment [34]. Additionally,
nanoparticles carrying positive charge in the acidic solution
of endosomes-lysosomes are more prone to escape into the
cytoplasmic compartment for effective relea se and gene
expression [45]. Thus, by varying the surface charge, one
could potentially be able to direct the nanoparticles either to
lysosomes or to cytoplasm.
Studying the influence of chitosan properties on the
colloidal characteristics of the nanoparticles, it was expected
that the use of chitosan of a relatively higher molecular
weight and higher viscosity would have a more pronounced
impact on the particle size and the surface charge of the
nanoparticles. However, the effect of chitosan proper ties was
found to be without impact. This might be attributed to the
relatively low concentration of chitosan included in the
nanoparticle suspension. Therefore, the viscosity is not
significantly altered and the particle's size is not influenced.
Because every monomer may have a charge, the amount of
monomers on the surface of the nanoparticles determines the
ζ-potential, and the size of the molecule itself plays a minor
role. On the contrary, a remarkable increase in the size and
surface charge of the nanoparticles was observed by Gan
et al [45] when higher mol ecular weig ht chitosan -
181N. Nafee / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173183
tripolyphosphate was used. In the aforementioned study,
nanoparticles were produced via tripolyphosphate-initiated
ionic gelation mechanism, whereas in our study chitosan is
thought to form a thin coat around the PLGA nanoparticles
as suggested by the linear increase in the surface charge with
increasing chitosan concentration previously discussed.
Therefore, these Protasan derivatives will be substituted
with chitosan oligomers and high-molecular-weight chit-
osan, and their effect on the colloidal characteristics of the
particles will be investigated in the future.
Binding to antisense oligonucleotides
Although the improvement of the colloidal characteristics
of the nanoparticles was an essential part, the ability of these
nanoparticles to b ind antisense oligonucleotides an d
enhance their cellular uptake required investigation. Our
binding experiments reflected that the addition of oligonu-
cleotides to the nanoparticles caused a reduction in the
surface charge especially in MilliQ water and sodium
chloride solution 10 mM, indicating electrostatic binding of
the negatively charged oligonucleotides to the positively
charged nanoparticles. This effect was more pronounced
when MilliQ water was used as incubation medium than in
sodium chloride, reflecting the ionic strength of the medium,
which in turns affects the surface charge and hence the
binding efficiency of the OMR to the nanoparticles. The pH
of the incubation medium also plays an important role in the
OMR binding to the nanoparticles. The electrostatic forces
involved in the binding proces s are relatively weak at pH
7.4, as deduced from the low zeta potential of the
nanoparticles in phosphate-buffered saline and Hank's
balanced salt buffers. Thus, fewer OMR molecules can be
attracted to the surface at this pH.
From the decrease in surface charge of the nanoparticles by
increasing the oligonucleotides concentration, it could be
noted that the reduction in surface charge due to OMR
binding would reach a plateau. However, the positive values
of the zeta potential indicate that the surface charge is not fully
compensated. This is also reflected in the power dependence
that was found to be 0.25 instead of the expected value of
2. However, comparing the two power dependences reveals
that for b = 2 the charge would be completely compensated,
whereas the found value describes a similar behavior but
including the plateau resulting from charges that can
obviously not be compensated. Therefore, further binding
of the OMR is assumed to be prohibited by steric hindrance.
Our future work will focus on the investigation of the
conformation of chitosan on the nanoparticle surface and
possible conformational changes during binding to oligonu-
cleotides. In general, the net positive surface charge of cNP-
OMR nanoplexes is expected to facilitate their adherence to
the negatively charged cellular membranes and consequently
increase their intracellular uptake, which was demonstrated
by uptake experiments; the cNP-OMR nanoplexes were able
to transfect A549 cells as was shown with confocal laser
scanning microscopy (Figure 10).
These results provide evidence that these chitosan-coated
PLGA nanoparticles are well suited as transporters for
antisense oligonucleotides such as OMR. Further studies
concerning the cytotoxicity, the transfection efficiency, as well
as the dependence of the size and charge of the nanoparticles
on the oligonucleotides delivery are currently underway.
Conclusions
Chitosan-coated PLGA nanoparticles offer a flexible
technology platform for DNA/RNA delivery. By varying the
formulation parameters, a wide range of particle sizes
(135.95-514.3 nm) and surface charges (13.5-60.4 mV)
can be adjusted to adapt the carrier system for the envisaged
task. The results clearly demonstrate that these particles
effectively bind the antisense RNA and are taken up into the
cells, which are essential requests for DNA/RNA delivery.
Acknowledgments
The present work was financially supported by
Deutscher Akademischer Austausch Dienst (DAAD) and
the Deutsche Krebshilfe e.V. (Project no.: 10-2035-Kl I).
Prof. Dr. U. Bakowsky and J. Schaefer (Philipps University,
Marburg, Germany) are acknowledged for the provision of
the Malvern Zetasizer Nano.
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