European Journal of Pharmaceutical Sciences 46 (2012) 522–529

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European Journal of Pharmaceutical Sciences

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

A one-step process in preparation of cationic nanoparticles

with poly(lactide-co-glycolide)-containing polyethylenimine gives efficient gene
delivery
Min Da Shau a,1, Mei Fen Shih b,1, Chi Cheng Lin a, I Chuan Chuang a, Wei Chih Hung c, Wim E. Hennink d,
Jong Yuh Cherng c,⇑
a
Department of Biotechnology, Chia-Nan University of Pharmacy and Science, 60 Erh-Jen Rd., Sec. 1, Jen-Te, Taiwan
b
Department of Pharmacy, Chia-Nan University of Pharmacy and Science, 60 Erh-Jen Rd., Sec. 1, Jen-Te, Taiwan
c
Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Rd., Chia-yi 621, Taiwan
d
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands

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

Article history: A one-step preparation of nanoparticles with poly(lactide-co-glycolide) (PLGA) pre-modified with poly-
Received 16 September 2011 ethylenimine (PEI) is better in requirements for DNA delivery compared to those prepared in a two-step
Received in revised form 15 March 2012 process (preformed PLGA nanoparticles and subsequently coated with PEI). The particles were prepared
Accepted 4 April 2012
by emulsification of PLGA/ethyl acetate in an aqueous solution of PVA and PEI. DLS, AFM and SEM were
Available online 13 April 2012
used for the size characteristics. The cytotoxicity of PLGA/PEI nanoparticles was detected by MTT assay.
The transfection activity of the particles was measured using pEGFP and pb-gal plasmid DNA. Results
Keywords:
showed that the PLGA/PEI nanoparticles were spherical and non-porous with a size of about 0.2 lm
Cytotoxicity
Transfection
and a small size distribution. These particles had a positive zeta potential demonstrating that PEI was
PLGA attached. Interestingly, the zeta potential of the particles (from one-step procedure) was substantially
PEI higher than that of two-step process and is ascribed to the conjugation of PEI to PLGA via aminolysis.
The PLGA/PEI nanoparticles were able to bind DNA and the formed complexes had a substantially lower
cytotoxicity and a higher transfection activity than PEI polyplexes. In conclusion, given their small size,
stability, low cytotoxicity and good transfection activity, PLGA/PEI–DNA complexes are attractive gene
delivery systems.
Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction categories of nonviral systems for gene delivery (Zhdanov et al.,

The carriers used for gene delivery are commonly classified as
viral or non-viral vectors (Zhang and Godbey, 2006; Mintzer and
Simanek, 2009). Viral-DNA particles normally show a relatively
high level of transgene expression. However, possible immunoge-
nicity, restrictions to the size of inserted DNA and potential local
and systemic toxicity limit their universal applications (Thomas
et al., 2003; Gorecki, 2001). Further, these viral systems have to
be produced in costly facilities (e.g. P2 laboratory). Nonviral-DNA
complexes are a promising alternative for viral nucleic acid deliv-
ery systems because they can more easily pharmaceutically pre-
pared and have less safety concerns. Also, the systems can be
decorated with e.g. PEG coatings, targeting ligand and pH sensitive
peptides to improve circulation kinetics, cell specificity and endo-
somal escape. Cationic lipids and cationic polymers are two major
⇑ Corresponding author. Tel.: +886 5 2720411x66416; fax: +886 5 2721040.
E-mail address: jycherng@yahoo.com (J.Y. Cherng).
1
Equally contributed to this article.
2002; Romøren et al., 2004). Cationic polymers, such as polyethy-
lenimine (PEI) (Boussif et al., 1995; Neu et al., 2005), chitosan
(Borchard, 2001; Kim et al., 2007), poly-L-lysine (PLL) (Kwoh
et al., 1999) and polyurethane (Tseng et al., 2005; Shau et al.,
2006; Hung et al., 2009), have extensively been studied in the last
two decades as gene delivery systems. However, the complexes of
cationic polymers and pDNA (‘polyplexes’) have rather low trans-
fection efficiency compared to viral vectors.
The most frequently studied cationic polymer is PEI. Polypolex-
es based on this polymer generally speaking show high transfec-
tion activity in many cell lines, but this polymer is rather toxic
and together with its non-degradability are major drawbacks for
its in vivo use (Godbey et al., 2001; van de Wetering et al., 1997).
Therefore, there is a need for approaches that reduce PEI’s toxicity
and at simultaneously preserve, or ideally improve, its gene deliv-
ery performance.
Poly(lactide-co-glycolide) (PLGA)-based nanoparticles because
of their excellent biocompatibility and low toxicity have been
extensively studied as delivery vehicles of both low molecular

0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.04.006
 M.D. Shau et al. / European Journal of Pharmaceutical Sciences 46 (2012) 522–529 523

weight drugs as well as biotherapeutics such as pharmaceutical
proteins and pDNA (Romero et al., 2010). However, the low DNA
encapsulation efficiency and capacity hamper their gene therapy
applications (Jong et al., 1997; Ando et al., 1999). Further, the sta-
bility of entrapped DNA in PLGA matrices is a concern. To illustrate,
it his has been shown that the super coiled DNA is converted into
its less active linear form during preparation and/or release (Perez
et al., 2001). Importantly, it is difficult to control the release of the
entrapped DNA from PLGA nanoparticles (Perez et al., 2001; Luten
et al., 2008). As an alternative for entrapment, DNA can be ad-
sorbed onto the surface of PLGA nanoparticles. Because PLGA nano-
particles have a negative surface charge, negatively charged pDNA
does not adsorb spontaneously onto such particles (Chumakova
et al., 2008). To render PLGA nanoparticles adsorbing for DNA, cat-
ionic surface charges have been introduced (Bivas-Benita et al.,
2004; Katas et al., 2009). This has been established by incorporat-
ing or coating of preformed PLGA nanoparticles with cationic poly-
mers such as polylysine (Capan et al., 1999), chitosan (Nafee et al.,
2007) and PEI (Chumakova et al., 2008) or by preparation of PLGA
nanoparticles using cationic surfactants (Basarkar et al., 2007). In
the present study, we prepared positively charged PLGA nanopar-
ticles in a one-step process by addition of PEI to an aqueous PVA
solution in which a PLGA solution in ethyl acetate was emulsified.
In this way it is aimed that PEI is partly present at the surface to
allow DNA binding, but also partly anchored in the PLGA matrix.
The obtained PLGA/PEI nanoparticles were complexed with pDNA
and their physicochemical characteristics and cytotoxicity as well
as transfection efficiency were examined and compared with poly-
plexes based on PEI.
2. Materials and methods
2.1. Materials
PLGA with 40–75 kDa, (lactide:glycolide 65:35 mol/mol) and b-
galactosidase were obtained from Sigma, UK. PVA (30–50 kDa, 87–
89% hydrolyzed) and PEI (branched, 25 kDa) were obtained from
Aldrich, Germany. ONPG (ortho-nitrophenyl b-galactoside) was
bought from Bio Basic Inc., USA. Ethidium bromide and MTT prolif-
eration kit were products of Sigma. COS-7 cells were obtained from
American Type Culture Collection (ATCC).
particles were freeze-dried without adding cryoprotectant (vacu-
umed dried under 60 torr, FD2-6P-M, KingMech, Taiwan) and the
freeze-dried samples were stored at 4 °C when they were not use
immediately.
Comparing to PLGA/PEI nanoparticles (formulation A via one-
step preparation), a control experiment of ‘‘PLGA + PEI cationic
nanoparticles’’(via two-step process) was carried out by coating
of preformed PLGA nanoparticles with PEI. PLGA (54 mg) dissolved
in 4 ml ethyl acetate and subsequently 4 ml of an aqueous solution
(contained 2% PVA without PEI) was mixed under stirring for
10 min and an emulsion was formed. The same procedure as above
was followed to obtain PLGA nanoparticles. Next, the PLGA nano-
particles were washed twice with deionized water to remove
PVA by centrifugation and mixed with PEI (3.6 mg in 11 ml deion-
ized water, pH around 10.5) under stirring condition for 16 h. Two
washing and centrifugation cycles (15,000 rpm; Hermle Z323K,
Germany) for 30 min at 4 °C with deionized water were applied
to remove the excess PEI.
2.3. Preparation of PLGA/PEI–DNA complexes
PLGA/PEI–DNA complexes with various polymer-to-DNA
weight ratios were prepared by adding PLGA/PEI nanoparticles dis-
persion (various amounts in 0.5 ml of plain DMEM) to plasmid
DNA solution (a concentration in 0.5 ml plain DMEM for desire
weight ratios) followed by vortexing for 5 s. The PEI–DNA com-
plexes were prepared likewise. These polyplex dispersions were
kept at room temperature at least 30 min. The final DNA concen-
tration in the complexes was 10 lg/ml for characterization. For
the transfection and cytotoxicity studies, a fixed DNA concentra-
tion (5 lg/ml) was applied to form PLGA/PEI–DNA complexes.
The ratios expressed in figures (Figs. 3–7) are defined as the weight
of PEI (the added amount for nanoparticle preparation) to DNA. For
instance, a polymer-to-DNA weight ratio of 1/1 means that 5 lg
PEI in PLGA/PEI particles (80 lg; calculated from Table 1B) was
mixed with 5 lg DNA in 1 ml medium to obtain the formation A
complexes. For formulation B, 55 lg of PLGA/PEI particles (corre-
sponding with 5 lg PEI) was mixed with DNA. For formulations C
and D, 30 lg and 17.5 lg of PLGA/PEI particles were mixed with
DNA, respectively.

Table 1
2.2. Preparation of PLGA/PEI cationic nanoparticles Formulation compositions and the particle size and zeta potential of formed PLGA/PEI
nanoparticles and PLGA/PEI–DNA complexes (n = 3; PDI = polydispersity index).
The one-step preparation of PLGA/PEI cationic nanoparticles (A) Effect of PVA concentration on the size PLGA/PEI nanoparticles
was performed as follows. PLGA nanoparticles have been prepared PVA (%, w/v) 0.5% 0.6% 1% 2%
by an emulsion–diffusion–evaporation technique (o/w) using eth- Particle size (nm) ± SD 520 ± 15 230 ± 2 213 ± 3 210 ± 2
ylacetate as solvent for PLGA and an aqueous PVA solution as con- (B) Formulation compositions of PLGA/PEI nanoparticles
tinuous phase (Ravi Kumar et al., 2004). We added PEI to this PVA Formulation A B C D
solution to render the particles cationically. In detail: PLGA (9 mg) PLGA (mg) 54 36 18 9
was dissolved in 4 ml ethyl acetate; PEI (3.6 mg) was dissolved in PEI (mg) 3.6 3.6 3.6 3.6
4 ml of an aqueous solution of emulsifier (PVA; concentration
0.5%, 0.6%, 1.0% and 2.0% (w/v)). In another series of experiments, (C) The size and zeta potential of PLGA/PEI nanoparticles from formulations A, B, C,
D
the PVA concentration (2% w/v) and PEI amount (3.6 mg) were
Formulation Size (nm) ± SD PDI ± SD Zeta potential (mV) ± SD
fixed whereas the amount of PLGA dissolved in 4 ml ethyl acetate
A 264 ± 5 0.10 ± 0.03 18.3 ± 1.2
was varied (9, 18, 36, 54 mg) was varied. An aqueous PEI/PVA solu-
B 250 ± 6 0.10 ± 0.06 15.1 ± 1.6
tion (pH around 10.5) was mixed with the PLGA solution in ethyl- C 223 ± 2 0.15 ± 0.05 9.8 ± 2.5
acetate under stirring for 10 min and an emulsion was formed by D 210 ± 2 0.20 ± 0.05 7.5 ± 2.9
homogenizing at 15,000 rpm for 10 min. Next, 7 ml deionized (D) The size and zeta potential of PLGA/PEI–DNA complexes from formulations A,
water was added and homogenization at 15,000 rpm was contin- B, C, D (PLGA/PEI-to-DNA ratio = 2/1)
ued for 2 min. Subsequently the emulsion was stirred overnight Formulation Size (nm) ± SD PDI ± SD Zeta potential (mV) ± SD
at 40 °C to remove the organic solvent. Finally, 18 ml deionized A 273 ± 8 0.15 ± 0.03 1.0 ± 0.9
water was added and the nanoparticles were washed by 2-cycle B 262 ± 12 0.20 ± 0.06 1.3 ± 0.8
centrifugation (15,000 rpm; Hermle Z323K, Germany) for 30 min C 234 ± 11 0.25 ± 0.05 2.0 ± 1.3
D 230 ± 13 0.25 ± 0.05 1.5 ± 1.7
at 4 °C with deionized water to remove PVA and excess PEI. The
524 M.D. Shau et al. / European Journal of Pharmaceutical Sciences 46 (2012) 522–529
2.4. Particle size and zeta potential measurements
The sizes of PLGA/PEI nanoparticles and PLGA/PEI–DNA com-
plexes were determined by DLS using a Malvern Zetasizer Nano
ZS (Malvern, UK). The measurements were performed with a
4 mW, 633 nm He–Ne laser at an angle of 173° and dispersant RI
(1.33) and viscosity (0.8872 cp) were used for the hydrodynamic
diameter calculation. The zeta potential of the different PLGA/PEI
nanoparticles and PLGA/PEI–DNA complexes was determined in
Hepes buffer (20 mM, pH 7) using a Malvern Zetasizer Nano ZS
(Malvern, UK). The measured electrophoretic mobility (l) was
used to obtain the zeta potential (z) by Henry equation (l = 2ezf(-
ka)/3g where e is dielectric constant and g is viscosity). The value
for Henry’s function f(ka) was taken as 1.5.
2.5. Gel electrophoresis of PLGA/PEI–DNA complexes
The electrophoretic mobility of PLGA/PEI–DNA complexes was
measured in 0.8% (w/v) agarose gel with 1% tris–Borate–EDTA
(TBE; pH 8.0). Electrophoresis was done at 100 V for 35 min and
the gels were subsequently immersed into a 0.5 lg/ml ethidium
bromide solution for another 30 min. DNA bands were visualized
by UV irradiation.
2.6. Characterization of PLGA/PEI nanoparticles by AFM and SEM
The surface morphology of PLGA/PEI nanoparticles was ana-
lyzed by SEM and AFM. For SEM, a drop of samples was introduced
onto a carbon strip mounted on an aluminum stage. After air-dry-
ing, the samples were shadowed with gold–palladium at 15 mA for
100 s to minimize surface charging. Observations on their surface
morphology were conducted with a Hitachi S-3000N SEM at an
accelerating voltage of 10 kV. For AFM, the used instrument (Nano-
Scope E, Digital Instruments Inc.) was set in a contact mode using
n+/n silicon (resistivity 0.01–0.02 X cm) cantilever with a spring
constant of 0.07–0.4 N/m and a resonance frequency of 10–17 kHz.
Scanning was performed at a speed of 1.0 Hz with a resolution of
512  512 pixels.
2.7. FT-IR measurements of PLGA/PEI nanoparticles
FTIR spectroscopy of PLGA nanoparticles, PEI, and PLGA/PEI
nanoparticles in KBR pellets was conducted with a Mattson Gale-
rxy Series 5000 spectrometer. Each spectrum was corrected for
the background to obtain the sample vibrational spectrum.
2.8. Quantification of PEI incorporated in PLGA/PEI nanoparticles and
on PEI coated PLGA nanoparticles
The amount of PEI entrapped in PLGA nanoparticles (formula-
tions A–D) and PEI coated PLGA nanoparticles (PLGA + PEI) were
examined by ninhydrin colorimetric assay (Chertok et al., 2010).
Briefly, the ninhydrin reagent (500 ll of 0.2% w/v in 0.1 M buffer
phosphate, pH 9) was added to 200 ll dispersion of 1 mg nanopar-
ticle in buffer phosphate. The samples were subsequently heated in
a boiling water bath for 30 min and then cooled to room tempera-
ture. The absorbance of supernatants was measured at 570 nm on a
UV/Vis spectrophotometer (SP8001, Metertech Inc, Taiwan) and
the amine content was quantified using an established calibration
curve of PEI.
2.9. Cytotoxicity evaluation
The cytotoxicity of different PLGA/PEI–DNA complexes from
four different formulations was evaluated in COS-7 cells using an
MTT assay. This assay is based on an ability of living cells to trans-
form by mitochondrial succinate dehydrogenase a water-soluble
yellow dye, MTT, into purple colored water-insoluble formazan.
The cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) in 5%
CO2 incubator at 37 °C. 4  103 cells were seeded/well in 96-well
microtiter plates followed by incubation for 18 h. The PLGA/PEI–
DNA complexes (with a fixed DNA concentration at 0.5 lg/well
and various PLGA/PEI nanoparticle–DNA ratios, w/w) in 100 ll
supplemented medium were added into wells. After 4 h incuba-
tion, the medium was replaced with fresh medium and the cells
were incubated for another 40 h. Next, 20 ll MTT (from stock
5 mg/ml in PBS, pH 7.4) was added to the cells for further 3 h incu-
bation. Finally, the medium was removed and dimethyl sulfoxide
(DMSO) (100 ll/well) was added to wells. The absorbance of for-
mazan was determined at 570 nm on a microplate reader. Relative
cell viability (%) was expressed as a percentage to untreated cells.
Cytotoxicity studies with PEI–DNA polyplexes were performed
using the same procedure as PLGA/PEI–DNA complexes (with a
fixed DNA concentration at 0.5 lg/well and various PEI–DNA ra-
tios, w/w).
2.10. Transfection studies
COS-7 cells were maintained in supplemented DMEM as for
cytotoxicity studies. The incubation for the PLGA/PEI–DNA com-
plexes with cells in the supplemented medium was 4 h. Then,
the transfection medium was replaced by fresh medium and cells
were incubated for another 43 h. Gene expression was character-
ized by observation of pEGFP-positive cells under a florescent
microscope and by quantification of pb-gal activities with ONPG
assay under an ELISA reader (420 nm). Transfection studies with
PEI–DNA polyplexes were performed using the same procedure
as PLGA/PEI–DNA complexes (with a fixed DNA concentration at
0.5 lg/well and various PEI–DNA ratios, w/w).
3. Results and discussion
3.1. The effect of PVA concentration on the size of PLGA/PEI
nanoparticles
We aimed for the preparation of nanoparticles with a si-
ze < 200 nm because such particles have been shown to be capable
of transfecting cells after DNA complexation (Cherng et al., 1996).
The size of PLGA nanoparticles depends on both the formulation
(e.g. type and concentration of surfactant and the PLGA concentra-
tion of the organic phase) and the processing parameters (e.g.
emulsion method). In this study we particularly focused on the
concentration of the surfactant and the PLGA concentration to tai-
lor the size of the nanoparticles. PVA is a commonly used surfac-
tant to stabilize the formed emulsion and prevents the droplets
against aggregation during preparation of PLGA micro- and nano-
particles (Sahoo et al., 2002). First the influence of the PVA concen-
tration on the size of PLGA/PEI was investigated. Table 1 shows
that the size of PLGA/PEI nanoparticles sharply decreased from
520 to 230 nm when the PVA concentration increased from 0.5%
to 0.6%, respectively. The particle size distribution was rather nar-
row (PDI value around 0.15 (for 0.6% PVA)). When the PVA further
increased to 2% the particle size only slight decreased (from 230 to
210 nm, Table 1). Therefore, the PVA concentration was fixed at 2%
for all subsequent experiments; a concentration also selected in
other studies (Prabha and Labhasetwar, 2004; Feczko et al., 2008).
 M.D. Shau et al. / European Journal of Pharmaceutical Sciences 46 (2012) 522–529 525

3.2. The effect of PLGA concentration on the size of PLGA/PEI

a 20
nanoparticles
PLGA/conjugated PEI (Formulation A)
The effect of the PLGA concentration on the size of PLGA/PEI (PLGA+PEI) (physicially mixed; the control)
nanoparticles was investigated. It was found that smaller PLGA/ 15

zeta potential (mV)

PEI particles (from 264 to 210 nm) were obtained when the
amount of PLGA in a fixed volume of ethyl acetate decreased (from
54 mg to 9 mg; Table 1). When the amount of PLGA in 4 ml of ethyl
10
acetate was 63 mg, no nanoparticle was obtained after emulsifica-
tion of this solution in an aqueous PVA/PEI phase.
It is quite rational that a higher concentration of PLGA in the or-
ganic solvent due to its increasing viscosity increases the size of 5
PLGA/PEI nanoparticles (Hans and Lowman, 2002). Although the
size of polyplexes around 200 nm is slightly too large for i.v. admin-
istration and prolonged circulation (Verbaan et al., 2004), these par-
ticles might have a good size for other routes of administration (e.g. 0
local administration via injection or aerosol; Park et al., 2008). PLGA/PEI PLGA+PEI

3.3. Zeta potential and PEI content of PLGA/PEI nanoparticles b

The zeta potential of nanoparticles is an important characteris-
tic for their stability. It has been reported that the zeta potential of
PLGA nanoparticles in a neutral buffer is around 45 mV; this neg-
ative charge is due to uncapped end carboxyl groups of PLGA pres-
ent on the surface of the particles (Stolnik et al., 1995). However,
such negatively charged particles are unable to complex negatively
charged pDNA. To render the PLGA nanoparticles with a positive
zeta potential, in the present study, we dissolved PEI in the contin-
uous PVA phase used to emulsify the PLGA/ethyl acetate solution.
The results of Table 1 show that indeed positively charged PLGA
nanoparticles were obtained. It was also found that a decrease in
PLGA concentration was associated with a decrease in the zeta po-
tential of the obtained PLGA/PEI nanoparticles (from +18.3 mV to 1 2 3
+7.5 mV, Table 1). When PLGA preformed nanoparticles were
coated with PEI (expressed as PLGA + PEI), they had a zeta-poten- Fig. 1. Zeta potential (A) and electrophoretic pattern (B) of PLGA/PEI nanoparticles
tial of +5 mV (Fig. 1A). The differences in zeta-potential could be prepared in a one-step process (formulation A, Table 1) and PLGA/PEI nanoparticles
obtained by coating of preformed PLGA nanoparticles with PEI (two-step process).
correlated to the extent of PEI incorporation into/onto obtained
(B) Lane 1 = free DNA, lane 2 = (PLGA + PEI)–DNA complexes (nanoparticles
PLGA/PEI nanoparticles. The encapsulation efficiency (% of the prepared in a two-step process), lane 3 = PLGA/PEI–DNA complexes (nanoparticles
added amount) of PEI incorporated in PLGA/PEI nanoparticles is prepared in a one-step process.
75%, 61%, 39%, and 34% for formulations A, B, C and D, respectively.
Moreover, 20% of the added amount of PEI was adsorbed onto the
surface of the PLGA nanoparticles. This means that with the new
process (PEI present in the PVA aqueous phase during emulsifica-
tion of the PLGA/ethyl acetate solution), particles with a higher
zeta-potential and thus higher PEI-loading were obtained. With
this new process, PEI not only interacts with the negatively
charged surface of PLGA nanoparticles by electrostatic interactions,
but possibly PEI also had reacted with PLGA by aminolysis (Croll
et al., 2004). This reaction results in the formation of amide bonds
connecting PEI and PLGA. Indeed, in the FTIR spectrum of the PLGA/
PEI nanoparticles apart from a NH2-vibration of PEI at 1636 cm 1, a
weak but evident absorbance at 1623 cm 1 of amide bonds (Cha-
kravarthi and Robinson, 2011) is detected (Fig. 2). On the other
hand, the spectrum of the spectrum of the PEI coated PLGA nano-
particles showed a broad absorbance at 1628 cm 1 (the Supple-
mentary figure). IR spectra however only give qualitative changes
but not a precise PEI content in the nanoparticles. The ninhydrin
assay showed that a higher PLGA amount present in the formula-
tion leads to a greater extent of PEI conjugation that in turn re-
sulted in higher zeta potential of obtained PLGA/PEI nanoparticles.

3.4. Complexation and condensation of DNA by PLGA/PEI nanoparticles

Gel electrophoresis was used to study the ability of the cation-

ically charged PLGA/PEI nanoparticles to bind DNA. Fig. 3 shows
that the different PLGA/PEI nanoparticles in formulations A–D Fig. 2. FT IR spectra of PLGA, PLGA/PEI and PEI.
526 M.D. Shau et al. / European Journal of Pharmaceutical Sciences 46 (2012) 522–529

Formulation A Formulation B
1 2 3 4 5 6 1 2 3 4 5 6

Formulation C Formulation D
1 2 3 4 5 6 1 2 3 4 5 6

DNA 8/1 4/1 2/1 1/1 0.5/1 DNA 8/1 4/1 2/1 1/1 0.5/1

Fig. 3. Electrophoretic patterns of PLGA/PEI–DNA formulations. Lane 1: 400 ng DNA and lanes 2–6: PLGA/PEI–DNA complexes were prepared at Hepes (20 mM, pH 7);
formulations A–D refers to Table 1.

Fig. 4a. SEM micrographs of PLGA/PEI nanoparticles of formulations A–D.

were able to quantitatively complex DNA at PLGA/PEI-to-DNA ra-
tios (w/w) above 2/1. At lower ratios free DNA was detected. In
Fig. 1B, the PLGA nanoparticles were coated with PEI had much
lower DNA binding capacity (at a PLGA/PEI-to-DNA weight ratio
of 2/1). Free DNA was observed present, whereas the PLGA/PEI
nanoparticles (from formulation A) showed complete DNA binding.
The lower DNA binding capacity of PEI-coated PLGA nanoparticles
is likely caused by their lower zeta-potential (+5 mV).
Binding of DNA to PLGA conjugated PEI nanoparticles (PLGA/
PEI–DNA complexes) resulted in a slight increase in size (from
230 to 273 nm) whereas the zeta potential of these particles was
close to neutral. This indicates that the surface of the particles is
 M.D. Shau et al. / European Journal of Pharmaceutical Sciences 46 (2012) 522–529 527

Fig. 4b. AFM images of PLGA/PEI nanoparticles (formulation A, upper graph; and formulation D, lower graph).

70
100
CD 60
B
Beta-galactosidase (mU/mg protein)

A
Relative cell viability (%)

80 BC C
PEI
A AB D 50 A
B
A
D B
60
40

PEI CD
40 CD
PEI 30
AB
PEI
20 A
20 CD
PEI PEI B
PEI 10
0 CD C
1/1 2/1 4/1 8/1 AB D PEI
0
PLGA/PEI-to-DNA ratios 1/1 2/1 4/1 8/1
PLGA/PEI-to-DNA ratios
Fig. 5. Cytotoxicity of PLGA/PEI–DNA complexes prepared using different PLGA/PEI
nanoparticles (formulations A–D, Table 1 and different PLGA/PEI-to-DNA ratios, w/ Fig. 6. Transfection efficiency of PLGA/PEI–DNA (pb-gal) complexes (prepared at
w) and PEI–DNA polyplexes analyzed by MTT assay, 47 h after transfection in COS-7 plain DMEM at different PLGA/PEI-to-DNA ratios, w/w) and PEI–DNA polyplexes in
cells in the presence of 10% serum. Results are presented as mean ± SD (n = 3). the presence of 10% serum. The complexes were incubated for 4 h with COS cells
after which the cells were washed and incubated for another 43 h. Protein
coated with DNA as shown in Fig. 3, the binding capability of PLGA/ expression was measured with ONPG assay under an ELISA reader (420 nm); n=3.
PEI nanoparticles from formulations A to D.
zeta potential (e.g. formulations C and D, Table 1) showed some fu-
3.5. SEM and AFM observations sion of particles (Fig. 4A, pointed with arrows), whereas individual,
non-fused/agglomerated, particles were observed in formulation A
The surface morphology of PLGA/PEI nanoparticles was ana- and B. Clearly, a higher zeta potential (e.g. formulation A and B) of
lyzed by SEM and AFM. The PLGA/PEI nanoparticles with lower the PLGA/PEI nanoparticles prevented agglomeration/fusion due to
528 M.D. Shau et al. / European Journal of Pharmaceutical Sciences 46 (2012) 522–529

Fig. 7. Transfection efficiency of PLGA/PEI–DNA (pEGFP) complexes (prepared at plain DMEM at different PLGA/PEI-to-DNA ratios, w/w) and PEI–DNA polyplexes in the
presence of serum (10%). The complexes were incubated for 4 h with COS cells after which the cells were washed and incubated for another 43 h. The efficiency of PLGA/PEI–
DNA (pEGFP) complexes was analyzed by the fluorescence-expressing cells.

higher repulsion forces. AFM was applied to study the PLGA/PEI
nanoparticles in solution. In line with the SEM observations, AFM
micrographs of nanoparticles of formulation A showed that these
nanoparticles were more uniform in size than those of formulation
D (Fig. 4B).
3.6. Cytotoxicity of PLGA/PEI–DNA complexes
Cationic gene delivery polymers are known for their cytotoxic-
ity (Fisher et al., 2003); e.g. PEI induces cell death by membrane
destabilization prior to cellular internalization (Godbey et al.,
2001). In line herewith, Fig. 5 shows that PEI–DNA polyplexes
(fixed amount of DNA = 0.5 lg/well) in the presence of serum
showed a dose-dependent toxicity (IC50 between 1/1 and 2/1, w/
w) towards COS 7 cells. This figure also shows that the PLGA/
PEI–DNA formulations showed a substantial lower cytotoxicity
(IC 50 was around 8/1, w/w) than PEI–DNA polyplexes in the
examined range (from w/w = 1/1 to 8/1) meaning that less non-
degradable PEI is required for delivering DNA.
Fig. 6 shows that the PLGA/PEI–DNA complexes have a substan-
tially better transfection activity than PEI polyplexes. Moreover,
the gene expression was found dependent to a PLGA/PEI-to-DNA
ratio. At higher ratios (e.g. w/w = 2/1 or 4/1), PLGA/PEI–DNA com-
plexes showed a higher transfection efficiency till a ratio (w/w > 8/
1) where a decrease in transfection activity was observed, likely
due to the cytotoxicity of the formulations (Fig. 5) as we previous
observed (Cherng et al., 2011).
Fig. 6 also shows that the formulation A and B have higher
transfection efficiency than C and D which might be explained by
the higher zeta potential of the naked particles which give better
DNA retention and thus better intracellular delivery of the nucleic
acid.
The transfection ability of PLGA/PEI nanoparticles complexed
with DNA encoding for green fluorescent protein (pEGFP) was also
examined in COS-7 cells. In line with the results of Fig. 6, the com-
plexes made at PLGA/PEI-to-DNA ratios of 2/1 and 4/1 resulted in
the highest fluorescence (Fig. 7) and thus higher protein expres-
sion. Also, the PLGA/PEI–DNA complexes had a higher transfection
activity than the PEI polyplexes.

3.7. Transfection activity of PLGA/PEI–DNA complexes

The different PLGA/PEI nanoparticle DNA formulations were
evaluated for their transfection ability in COS-7 cells in the pres-
ence of serum and compared with that of PEI polyplexes using
pDNA encoding for b-galactosidase (pb-gal) as reporter genes.
4. Conclusion
In this study, a substantial advantage PLGA/PEI nanoparticles
prepared in a one-step process has been shown over the prepara-
tion of PLGA nanoparticles that were coated with PEI in a two-step
 M.D. Shau et al. / European Journal of Pharmaceutical Sciences 46 (2012) 522–529
process. The nanoparticles prepared using the one-step procedure
are spherical in morphology, have a small size distribution and
have a much better DNA binding capacity than the particles pre-
pared in a two step process. Importantly, they have a relatively
low cytotoxicity and a substantial better transfection activity of
PEI polyplexes even in the presence of serum.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejps.2012.04.006.
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