143

molecular architecture and the structure of polymer/DNA

complexes formed [3].
Amphiphilic block copolymers composed of hy-
drophilic and hydrophobic segments can form a micel-
lar structure with a hydrophobic compact inner core and
a hydrophilic swollen outer shell in selective solvents,
which is thermodynamically favorable for one block but
unfavorable for the others [4]. These micelles are an in-
teresting group of self-assembled nanocarriers for drug
delivery, owing to the prospects of biocompatibility and
large solubilization capacity for poorly watersoluble
molecules. Poly(ethylene glycol)-b- poly( -caprolactone)
(PCL-b-PEG) and poly(ethylene glycol)-b-poly(L-lactide)
(PLL-b-PEG) diblock copolymers are very promising due
to their stability and biocompatibility. Micelles formed
of these copolymers have been utilized in encapsulation
of different lipophilic drugs [5]. Note that it is also pos-
sible to prepare nanocapsules or nanoparticles from these
diblock copolymers in drug or active agent delivery. In
Potential c-myc Antisense Oligonucleotide Carriers: PCl/PEG/PEI
and PLL/PEG/PEI
Sevil Din er
Y ld z Technical University, Bioengineering Department, Davutpasa, Istanbul, Turkey
Mustafa T rk
K r kkale University, Department of Biology, Yah s ihan, K r kkale, Turkey
Ay s e Karag z and G rhan Uzunalan
Y ld z Technical University, Bioengineering Department, Davutpasa, Istanbul, Turkey
Abstract: In this work, positively charged, micelle-forming polymers were synthesized and used as a model vector to deliver antisense
oligodeoxynucleotide (ASODN) into melanoma cells. Polymers and polymer/ASODN complexes were characterized by DLS according
to size, charge, and critical micelle concentration. Nanosize and spherical complexes were observed by AFM. Complexes did not reveal
signicant toxicity to melanoma cells. Antiproliferative effect of the complexes was observed by immunocytochemical staining and
estimated as 56.8% with N/P:9. High amount of apoptosis and very small amount of necrosis were estimated. According to the results,
these positively charged polymers forming micelle-like structures seem promising as ASODN carriers.
Keywords: antisense oligonucleotide , non-viral vectors , c-myc , poly(lactide) , poly(caprolactone) , poly(ethylene glycol) ,
polyethylenemine , micelle
INTRODUCTION
Gene delivery has great potential to cure many genetic
and acquired diseases. Non-viral gene delivery is espe-
cially advantageous because it could avoid unaccept-
able immune responses. Various cationic polymers have
shown promising effects in facilitating gene delivery as
they readily complex with DNA to form polyplexes by
neutralizing the negatively-charged anionic phosphate
groups and hence improve transfection. Poly(ethylene
imine) (PEI) is able to effectively complex even large
DNA molecules [1,2], leading to homogeneous spherical
particles with a size of 100 nm or less that are capable
of transfecting cells efciently in vivo as well as in vitro .
It offers a signicantly more efcient protec tion against
nuclease degradation than other polycations, e.g. poly(L-
lysine), possibly due to their higher charge density and
more efcient complexation. However, further research is
needed to rationalize the relationship between polymeric
This study was supported nancially by TUBITAK- 107T864. We thank Res. Asist. Yeliz Ba s aran for AFM images, and Res. Asist.
Eray Dalgak ran for DLS analysis.
Address correspondence to S. Dincer, Bioengineering Department, Yildiz Technical University, Davutpasa, Istanbul, Turkey. E-mail:
sevild@yildiz.edu.tr
Articial Cells, Blood Substitutes, and Biotechnology, 39: 143154
Copyright 2011 Informa Healthcare USA, Inc.
ISSN: 1073-1199 print / 1532-4184 online
DOI: 10.3109/10731199.2010.506852
144 S. Diner et al.
Chang et al. s study, PEG-PLA in the form of ultrathin
membrane nanocapsules (80150 nm) prepared as red
blood cell (rbc) substitutes containing hemoglobin (Hb)
and enzymes were reported [6].
Copolymers formed between cationic polymers such
as PEI and the aforementioned diblock copolymers offer
the benet of electrostatic complexation while enhanc-
ing solubility and stability of the polyplexes. Insertion
of hydrophilic segments (PEG) into diblock copolymers
provides a hydrophilic corona that creates a steric bar-
rier against self-aggregation, shielding of cationic charge
groups, and inhibits the association with plasma proteins
and other cellular components. Moon et al. [7] described
the synthesis of diblock, triblock, and 4 arm star-shaped
block PEG PTMC copolymers and the characteristics of
micelle as a potential drug delivery vehicle. Addition of
a positively charged unit to these copolymers can allow
their use as gene delivery vehicles.
ASODNs can either hybridize with specic mRNA,
which would inhibit the transcription of genetic informa-
tion into protein synthesis, or activate the RNase H that
can digest the mRNA resulting in the pathway blockage.
These oligonucelotides targeted to oncogenes are widely
used in order to provide specic and effective means to
prevent the growth of cancer cells. c- myc , a member of
the myc gene family, is a key regulator of cell growth
that has been found altered by amplication or point mu-
tation in a large subset of tumors, including leukemia,
melanoma, prostate, breast, and colon carcinomas [8,9].
The efcacy of these oligonucleotides in inhibiting gene
expression depends on their ability to penetrate targeted
cells. The uptake of them occurs through active transport,
which, in turn, depends on the temperature, the structure,
and the concentration [10]. Several approaches have been
explored to enhance cellular uptake and permeability in
order to increase their therapeutic efcacy, including con-
jugation to polycations, targeting cell surface receptors,
coupling to cholesterol, and encapsulation into lipid parti-
cles such as microspheres and liposomes. The use of vec-
tors in antisense drug delivery in vivo seems to improve
their biological effects.
Growth inhibition of SK-MEL-30 human melanoma
cells by antisense c-myc oligonucleotides delivered by
poly(N-Isopropylacrylamide)/poly(ethyleneimine) copo-
lymer was reported by our group. By this polymer, we
have only investigated the c-myc inhibition by MTT as-
say [11]. In this study, we have reported the synthesis and
characterization of PEI coupled PCL-mPEG and PLL-
mPEG diblock copolymers having the ability to form mi-
celle-type structures for potential use in delivery of c- myc
antisense oligonucleotide to melanoma cells. All poly-
mers were investigated by Zeta Sizer for their size and
charge characteristics. Protonation ability of polymers
was followed by this technique using different pH values.
Also, complexation ability of ASODNs with prepared
positively charged polymers was investigated by measur-
ing their sizes and charges in different N/P ratios. AFM
was also used to examine the corresponding micelle-type
structures. After physicochemical characterization of
polymer ASODN complexes, we have investigated the
c-myc inhibition efciency of these complexes. Apoptosis
and necrosis indexes were evaluated. Results showed that
these particular carriers would be potential candidates in
the uptake of oligonucleotides to melanoma cells.
MATERIALS AND METHODS
Materials
Methoxy poly(ethylene glycol) (Mn: 5000, Fluka, Germa-
ny; Mn: 2000, Sigma-Aldrich, Germany), poly(ethylene
glycol) (PEG, Mn: 2000 and 5000 Sigma-Aldrich, Ger-
many), -caprolactone ( -CL, Aldrich, USA), stannous
octoate ((SnOct
2
), Sigma, USA), diethyl ether, chloroform,
and dichloromethane were used as received. L-Lactide
(Aldrich, USA) was recrystallized with toluene. Branched
polyethyleneimine (PEI, MW: 2000, Aldrich, Germany)
was used as received. Carbonyldiimidazole (CDI) was
obtained from Sigma (Germany). TNBS (2,4,6-trini-
trobenzene sulfonic acid) and pyrene were purhased
from Sigma-Aldrich (Germany). A 15-mer AS ODN
(5 -AACGTTGAGGGGCAT-3 ) that was complemen-
tary to the translation initiation region of c-myc oncogene
was obtained from Iontek Ltd. (Turkey) and used in all
experiments. Purity was 95% provided by high perfor-
mance liquid chromatograpy.
Methods
Preparation of positively charged PCL-mPEG and PLL-
mPEG diblock copolymers . PCL-mPEG and PLL-mPEG
diblock copolymers carrying two different methoxy PEG
molecules (Mn: 2000 and 5000) were prepared by mi-
crowave irradiaton using Milestone S.r.l MicroSYNTH
microwave synthesis unit. Details for synthesis and char-
acterization were reported elsewhere [12]. For the prepa-
ration of PEI-carrying polymers, diblock copolymers
(PCL-mPEG, PLL-mPEG) in 4:1 ratio were used because
of their water solubility. In the coupling procedure carbo-
nyldiimidazole was used to activate the hydroxyl group
of polymers. The polymer sample was dissolved in di-
oxane in a concentration of 8 10
3
M. Then, 0.08 M
CDI was added to the medium and the reaction mixture
was stirred for 3 h at 37
o
C. At the end of the reaction,
the mixture was precipitated from diethyl ether. This
procedure was repeated three times and the obtained
product was dried in vacuum overnight. Coupling of the
activated polymer to the PEI by amino groups was then
Antisense Oligonucleotide Carriers 145
performed. CDI-activated polymer was dissolved in bo-
rate buffer (pH:8.5) and PEI (2000, branched) was added
to the medium in 1:1 ratio (mole). The reaction mixture
was incubated at room temperature for 24 h. Unreacted
polymers were removed using an ultraltration system
(Millipore) by taking into account the molecular weight of
corresponding polymers as reported elsewhere [12]. The
product was liophilized for further use.
TNBS assay. In gene delivery applications, the compl-
exation of polymer and genetic material (here, oligo-
nucleotide) depends on the amount of amino groups of
polymer and phosphate groups of ODN. It is not possible
to know the exact amount of amino groups of branched
PEI (MW: 2000) molecule used here. Therefore, there is a
need to estimate the amount of primary amine groups for
complexation studies. Amino-group content of PEI-bound
polymers was investigated by the TNBS (2,4,6-trinitroben-
zene sulfonic acid) method [13]. To do this, a calibration
curve was obtained using glycine amino acid in different
concentrations (0.5, 0.1, 0.05, 0.01, 0.001 mg/ml) at pH:
8.5. TNBS was added in a ratio of 0.1% and the reaction
mixture was incubated at 40
o
C for 2 h. Following incuba-
tion, each sample was measured at 335 nm by UV-Visible
Spectrophotometer and recorded. Amine group content of
each polymer was estimated from the calibration curve. In
addition to this method, gel permeation chromatography
(GPC) was also used to conrm polymer formation by
using PEG as standard.
Preparation of polymer/ASODN complexes . Stock solu-
tions of ODN (5.68.10
6
M) and polymer (1 mg/ml) were
prepared in a phosphate buffer. Complexes were prepared
by adding ASODN solution to the polymer solution in de-
sired amounts to achieve different N/P ratios. Here, poly-
mer amounts were used according to their amine content
estimated by TNBS assay. After mixing of ASODN and
polymer with mild shaking, the sample was left at room
temperature for 30 min to equilibrate before use.
Dynamic Light Scattering (DLS) . DLS measures the in-
tensity correlation function of light scattered from a poly-
meric solution. All DLS measurements were performed
using a Malvern Instruments NanoS Nanosizer. Samples
analyzed were contained in a 1 cm-path length cell, and
the data were analyzed using Malvern Instruments Dis-
persion Technology Software. The polymer refractive
index was taken to be 1.5 with an absorbance of 0.01.
The viscosity and refractive index of water were taken
as 0.8877 cPa and 1.330, respectively. Six measurements
were performed on each sample, with an average of 10
runs taken for each measurement, each within 1 min. We
have estimated the critical micelle concentration by using
data obtained by this instrument. In addition, changes in
zeta potentials and sizes of PEI-coupled polymers were
evaluated in different pHs. Polymer/ASODN interaction
was followed by the same instrument in different N/P
ratios at physiological pH.
Atomic Force Microscopy (AFM). Complex solutions
were deposited onto freshly cleaved mica surfaces and
dried at room temperature for 5 min. The images were
observed by Tapping Mode using the AFM microscope
(SPM9600, Shimadzu, Japan) with 10 nm silicon nitride
tips. Analysis was performed to investigate the size and
images of selected polymers and ASODN complexes.
Cell Culture . SK-MEL30 melanoma cells were obtained
from Hacettepe University, Biochemistry Department.
The cells were cultured in Dulbecco s Modied Medium
without L-glutamine (DMEM, Sigma, Germany) supple-
mented 10% Fetal Bovine Serum (FBS, Sigma, Germany),
and 1% penicilin-streptomycin (Sigma, Germany) at 37
o
C
and 5% CO
2
atmosphere. The cells were subcultured twice
a week, using dissocation medium, trypsin-EDTA, pH 7,4
as buffer system. All tests were triplicated. Statistical sig-
nicance of diferences between test values was estimated
using Nonparametric Kruskal-Wallis test. p 0.1.
MTT assay . For MTT experiments, cells were cultured in
96-well plates at a density of 5 10
3
per well and grown
overnight at 37
o
C, 5% CO
2
atmosphere. The cell culture
medium was removed and then replaced. Copolymer/
ASODN complexes formed at different N/P ratios were
added into each well. Cells were incubated for 72 h. MTT
reagent (5 mg/ml) was added into each well, and the cells
were cultured for a further 4h incubation. After that,
100 l 10% sodium dodecyl sulfate (in 0.01 N HCl) were
added to each well and incubated overnight. Then, plates
were read in Elisa Microplate Reader at 570 nm. Dark
blue formazan crystals are formed if the cells are alive.
Cell transfection . Cells were seeded on cover glasses in
6 well plates at a density of 10 10
3
per well and grown
overnight at 37 C, 5% CO
2
atmosphere. The cell medium
was removed and washed with PBS three times. After
polymer/ASODN complexes formed at N/P ratio: 1, 3,
6, and 9 were added into each well for 24 hours. Only
ASODN was used as a control.
Immunocytochemical detection of c-myc expression in cell
culture . SK-MEL 30 human melanoma cells (1010
3
cells
per well) were placed on cover glasses in DMEM with-
out L-glutamine in 6-well plates and incubated at 37 C in
5% CO
2
atmosphere. Then these cells were washed with
distilled water and polymer/ASODN complexes in dif-
ferent N/P ratio (1, 3, 6, and 9) were applied to cells for
24 hours. Following that, slides stained for c-myc antigen
were cytocentrifuged, xed in cold acetone at 20
o
C for
10 minutes, and treated with hydrogen peroxide (0.3% in
146 S. Diner et al.
methanol) for 10 min to block the endogenous peroxidase
activity. Then they were washed in PBS and incubated
with c-myc antibody (Santa Cruz, 1:100 dilution) at room
temperature for 1 h. As a negative control, preimmune
mouse serum, instead of primary antibody, was used.
For immunohistochemical demonstration of the c-myc
protein, an avidin-biotin-peroxidase technique was used
[14]. The peroxidase-antiperoxidase technique [15] was
used to demonstrate the c-erbB-2 protein. The immu-
noreactivity of the c-myc antibody was conned to the
cytoplasm and nuclear of SKMEL-30 cells. We counted
the number of c-myc positive cytoplasmic and nuclear
staining cells in all elds found at 400 nal magnica-
tion. c-myc positive cells were seen to be brown under a
light microscope. For each slide, three randomly selected
microscopic elds were observed and at least 1000 cells/
eld were evaluated.
Analysis of apoptosis and necrosis . Double staining was
performed to quantify the number of apoptotic cells in the
culture, based on scoring apoptotic cell nuclei. SKMEL-30
cells (20 10
3
cells per well) were placed in DMEM us-
ing 24-well plates. After treating with polymer/ASODN
complex at N/P ratio of 1,3,6 and 9 (in aqueous solutions)
for a 24- hour period, both attached and detached cells
were collected, washed with PBS, and then stained with
Hoechst dye 33342 (2 g/mL), propodium iodide (PI)
(1 g/mL) and DNAse free-RNAse (100 g/mL) for 15 min
at room temperature. Then, 1050 L of cell suspension
was smeared on the slide and cover slip for examination
using uorescence microscopy. In addition, attached cells
were washed with PBS and stained in plates in the same
manner. While the nuclei of normal cells were stained
light blue, apoptotic cells were stained bright blue by
the Hoechst dye. The apoptotic cells, from their nuclear
morphology, were identied as a nuclear fragmentation or
chromatin condensation. Necrotic cells were stained red
by PI. Necrotic cells lacking plasma membrane integrity
allow PI dye to cross the cell membrane, meaning that PI
dye does not cross the non-necrotic cell membrane. The
results of the counted number of apoptotic and necrotic
cells in 10 randomly chosen microscopic elds were
expressed as a ratio of apoptotic and necrotic to normal
cells. The number of apoptotic and necrotic cells were
determined by Fluorescence Inverted Microscope (Leica,
Germany) using DAPI and FITC lters.
RESULTS
Polymer Characterization
As mentioned in earlier, PCL-mPEG and PLL-mPEG
diblock copolymers were synthesized by microwave
irradiaton and polymer fromation was conrmed by FTIR
and
1
H-NMR. Molecular weight data were obtained from
GPC and also
1
H-NMR spectra. All data were reported else-
where [12]. Here, we have prepared the PEI-bound form of
these diblock copolymers. After a series of purcation steps
by ultraltration, pure PEI-bound polymers were obtained
and characterized by different techniques. Addition of PEI
into the diblock copolymer structure was conrmed by
TNBS assay and GPC as explained below.
Figure 1 shows representative unimodal GPC traces
of PCLPEG5000 and PCLPEG5000/PEI. The addition of
PEI to the diblock copolymer caused higher molecular
weight value as seen here and the obtained polymers after
PEI conjugation seem close to monodispersitiy.
Determination of Amino-group Content
The PEI molecule used in this study has a branched struc-
ture containing tertiary, secondary, and primary-amino
groups, which are not easy to determine with direct meth-
ods. It is important to estimate the amino content of the
corresponding polymers since the quantity of especially
primary amino-groups is required for the complexation
with genetic material by electrostatic interaction. Here,
we have performed TNBS assay and obtained approxi-
mate values. Each sample reacted with TNBS was mea-
sured at 335 nm by UV-Visible Spectrophotometer and
amine group content of each polymer was estimated from
the calibration curve (y 12816x 0.3117, R
2
0.9871).
In this way, the success of PEI-coupling to polymers
was also demonstrated. Amine content of each polymer
was obtained as 0.14, 0.10, 0.15, 0.15 mg/ml for PCL-
mPEG2000/PEI, PCL-mPEG5000/PEI, PLL-mPEG2000/
PEI, PLL-mPEG5000/PEI, respectively.
Figure 1. GPC chromatograms of PCLPEG5000 and PCLPEG
5000/PEI.
Antisense Oligonucleotide Carriers 147
Light Scattering Measurements
The charge and size of the polymer has great inuence
on the stability, DNA-binding, and release efciency of
polyplexes, and thus on targeting efciency. Several stud-
ies reported the importance of positive charge of different
polymers to be used in gene delivery [13,1922].
DLS detects the mean dimension of particles in
aquous soluiton based on the Stocks-Einstein equation.
Measurements were made for positively charged diblock
copolymers and polymer/ASODN conjugates.
Determination of cmc . As explained in the Methods sec-
tion, the micelle forming ability of PCL-mPEG and PLL-
mPEG diblock copolymers and their PEI-bound forms
were investigated by a Zeta Sizer. Figure 2 shows inten-
sity vs. concentration curves of diblock copolymers. Up
to a certain concentration value (0.05 mg/ml), intensity
did not reveal any signicant increase; however, after this
value, a sharp increase in intensity was observed for all
polymers. This certain concentration can be accepted as
criticial micelle concentration ( cmc ) [16]. Note that by
this concentration value a remarkable decrease in size
was also observed, probably caused by ordered micelle
structure formation. This increase was observed for
lactide-based copolymers, which meant that miscellar
structure forms more easily with these polymers. Addi-
tion of PEI to the diblock copolymer structure did not
affect very much the micelle formation for each of the
polymers. Micelle formation is expected to be easy for
diblock copolymers since there is only one hydrophilic
and one hydrophobic block. In the PEI-bound case, the to-
tal polymer chain posseses two hydrophilic chains in both
ends and one hydrophobic in the middle. Therefore, a mi-
celle formation may be suggested, as in Scheme 1. We did
not obtain very different cmc values because all diblock
copolymers and their PEI-bound forms do not possess dif-
ferent structures in terms of hidrophobic and hydrophilic
balance. For the PEI-bound polymers, the concentration
was not different form 0.05 mg/ml. Zhang et al. reported
the CMCs of poly(D,L- Lactide)-b-mPEG copolymers in
the range of 3.50.5 mg/L changing by PDLLA length in
the polymer chain [17]. Gyun et al. found CMC of meth-
oxy propyleneglycol/caprolactone micelles as between
3.470.63.10

7 mol/lt [4].
Size and charge characterization of PEI-coupled diblock
copolymers . The following results can be drawn from
size and charge analyses: For diblock copolymer, PCL-
mPEG2000, size was determined as 138 nm without PEI.
Size of PEI-including copolymer ( PCL-mPEG2000-PEI)
at pH: 2 and 10 were determined as 89 and 204 nm, re-
spectively (Figure 3). However, at pH:7.4, the size of
this polymer was 184 nm. By increasing pH, PEI units
(especially between pH: 28) become protonated and
start to swell (proton sponge effect), and this causes an
increase in the hydrodynamic size of the polymer. This
Figure 2. Change in intensity by diblock copolymers con-
centration.
Scheme 1. Micelle formation of PEI-bound diblock copolymers.
Figure 3. Change in size of PEI-bound polymers by pH.
148 S. Diner et al.
behaviour does not occur after pH: 8, which is the up-
per pKa value of PEI. Charge of this polymer was also
evaluated (Figure 4). At acidic pHs, the polymer gained a
positive charge as expected ( 6.57 at pH:2 and 13,17
at pH: 4), since PEI has a great protonation capacity in
acidic medium towards even higher pH values ( 16.8 at
pH: 7.4 and ( 16.1 at pH: 8) [1]. However, at very basic
medium (such as pH: 10), the polymer was not positively
charged any more (close to zero).
For PCL-mPEG5000-PEI , while size was 100 and
195 nm at pH:2 and 10, respectively, it was 175 nm at
pH:7.4. The size of the diblock form of this polymer was
176 nm at pH:7.4, proving no negative effect on particle
formation of PEI-additon to the structure. This poly-
mer has similar behavior to PCL-mPEG2000PEI hav-
ing smaller size and charge in lower pH values. It was
positively charged until pH: 8 ( 10.2), but negatively
charged after this pH (2.38 at pH: 10). PLL-mPEG2000-
PEI was positively charged until pH:7.4 ( 4.86, 24.6,
14.1, 18.8 at pH: 2, 4, 7, 7.4, respectively), but nega-
tively charged at pH: 10 (8.29). Size increased to 163
nm (at pH: 7.4); after that, this increase reached up to
336 nm at pH: 10, probably caused by aggregation. The
size of the diblock form of this polymer was 137 nm at
pH:7.4, which is little different from PEI-bound from
(163 nm). PLL-mPEG5000-PEI was positively charged
until pH:7.4 ( 4.87, 16.6, 16.5, 18.3 at pH: 2, 4,
7.4, 8), but negatively charged at pH 10 (-8.41). Size was
162 nm at pH: 7.4, whereas it was 111, 109, 163 and 235
nm at pH: 2, 4, 8 and 10, respectively. This revealed that
good micelle formation was achieved at physiological
pH. Note that the size of PLL-mPEG5000 was 154 nm.
The particle sizes of all polymers were in a similar range.
Measured sizes here are probably much higher than the
original value because the Zeta Sizer instrument mea-
sures the hydrodynamic size of the particles. PEI addition
to the polymer structure was conrmed by these analyses.
It was also demonstrated that the PEI molecule had high
protonation capacity, which makes PEI very attractive as
an efcient gene delivery vector [1,2].
Polymer/ASODN conjugates . For size and charge of polymer/
ASODN conjugates, polyplexes were prepared in different
N/P ratios (1, 3, 6, 9) by taking into account the amino con-
tent calculated from TNBS analysis. All polymers revealed
sizes in a similar range because of the similarity of resulting
structures. According to Table 1 (PCL-mPEG2000-PEI/
ASODN), mixing of ODN leads to a small increase in the
diameter (polymer s diameter was 184 nm). As the concen-
tration of polymer increases (increase in N/P ratio), the size
of polyplexes decreased to 120 nm (at N/P:9). Thus, N/P:6
seems the best ratio for good complexation because of the
smallest polydispersity. This can be seen also from Figure
5, representing volume intensity distribution proles plotted
against hydrodynamic diameters. Note that size is not the
only determinant in efcient transfection.
PCL-mPEG5000-PEI/ASODN : charge of polymer
showed a similar trend with the previous polymer sample.
Here, the smallest complex size ( 140 nm) was obtained
at N/P:6 and 9, in which polyplexes had an extra positve
charge ( 45 mV). PLL-mPEG2000-PEI/ASODN : in-
creasing of size by N:P ratio may cause aggregation of
complexes, so that up to N:P:6 seems efcient enough.
At this ratio, complex has extra surface charge. PLL-
mPEG5000-PEI/ASODN : size of polyplexes increased
up to N/P: 6. N/P: 9 seems the best ratio at which size is
smallest and charge is highest.
Atomic Force Microscopy (AFM)
Figure 6 shows SPM image of PCL-mPEG2000/PEI/
ASODN complexes in different N/P ratios. Complexes
were prepared by adding ODN solution to the polymer
Table 1. Charge and size of polymer/ASODN complexes
measured by DLS in different N/P ratios.
Polymer N:P Charge (mV) Size (nm)
PCL-mPEG2000/PEI 1
3
6
9
1.97 0.3
4.10 0.7
5.35 1.2
6.71 1.7
212 15
204 25
195 8
123 5
PCL-mPEG5000/PEI 1
3
6
9
1.51 0.6
4.76 0.5
5.37 1.1
4.12 2.3
155 20
145 20
155 15
145 5
PLL-mPEG2000/PEI 1
3
6
9
1.25 0.8
1.83 0.9
4.60 1.6
4.75 2.5
292 15
294 30
242 10
250 10
PLL-mPEG5000/PEI 1
3
6
9
2.15 0.5
5.83 0.9
4.48 1.4
4.52 1.6
180 12
137 10
248 13
209 2
Figure 4. Change in charge of PEI-bound polymers with pH.
Antisense Oligonucleotide Carriers 149
solution in desired amounts to achieve different N/P ra-
tios. After mixing of ASODN, the sample was left at room
temperature for 30 min to equilibrate before use. Sapmles
were dropped onto freshly celaved mica and dried at room
temperature. As clearly seen here, complexes formed
spherical particles, which can be supposed to be micelle.
In some regions, a kind of aggregates were formed, which
can be overcome by preparation of more diluted sample.
However, in all ratios very small structures were obtained
(between 1030nm). More uniform particles were formed
in N/P ratio of 6. In N/P:1, very small particles were
obtained but at the same time they seemed to have a ten-
dency to be aggregated. In N/P ratio 1 and 9, more poly-
dispersity was observed comparing to N/P: 6, as also seen
in DLS proles (Figure 5). The size observed by AFM
seems inconsistent with the ones obtained by DLS, but
this was expected since the DLS instrument measures the
hydrodynamic volume of the sample. The results showed
that formation of spherical structures obtained by polymer
alone was not affected in the presence of ASODN.
MTT Assay
ASODN did not reveal any toxicity towards SKMEL-30
cells. Polymers (100 M) without ASODN showed tox-
icities caused by positive charge on the polymer surface.
Cell viability after polymer/ASODN transfection was ob-
tained as seen in Figure 7 While positive charge enhanced
toxicity directly, size increase did not affect this as much
as charge. MTT assay of polymer/ASODN complexes
depending on time was also evaluated. No signicant
difference was observed in cell viability between 48 and
72 hour incubation time. Similar results were obtained
for the poly(N-Isopropylacrylamide)/poly(ethyleneimine)
complexed with the same ASODN in terms of polymer
toxicity and incubation time [11].
Cell Transfection by Polymer/ASODN Complexes
and c-myc Expression
c-myc expression was followed by immunocytochemical
staining, as mentioned earlier. Polymer/ASODN complex-
es in different N/P ratios were transfected into SKMEL-30
cells seeded on cover glass. Naked oligonucletide was
used as a control and revealed only 5% c-myc inhibition.
By the polymers without ASODN cell inhibition was also
observed (4.37.7%), but this was most likely because of
the toxic effect of unoccupied positively charged polymer
chains. Results are shown in Table 2 and c-myc activites
are illustrated in Figure 8. Most of the complexes showed a
certain amount of cell inhibition as seen in Table 2. As pre-
viously reported in several studies, transfection efciency
decreased with decreasing positive charge density [3]. In
the low N/P ratio range, it is expected that some of the
ASODNs are not complexed with polymer. That means
complexes are not going to be formed and therefore this
will cause reduced transfection. Zhao et al. reported that
lowering the N/P ratio to below 1 resulted in lower trans-
fection efciency [18].
Figure 6. AFM images of PCL-mPEG2000-PEI/ASODN complexes at different N/P ratios (1, 6, 9).
Figure 5. Light scattering proles of PCL-mPEG2000-PEI/
ASODN complexes at different N/P ratios (1, 3, 6, 9).
150 S. Diner et al.
Figure 8A shows c-myc positive cells seen as brown
spots transfected with only naked oligonucleotide. The
following images were taken after transfection by the
complexes formed between polymers and ASODN. As
seen in these images, complex formation caused lower
c-myc activity, especially at lower N/P ratios and higher
particle sizes. The decrease in complex size and increase
in complex charge allowed enhanced oligo uptake into
the cells; therefore inhibited c-myc activity was seen as
blue spots in the Figure 8B. It seems that the charge of the
complexes is more effective on the c-myc expression more
than size difference. Most of the complexes actually are
in a similar size range especially seen in AFM images.
c-myc expression was inhibited as 56.8% and 49.1% with
transfection by PCL-mPEG2000/PEI in N/P: 9 and PLL-
mPEG2000/PEI in N/P: 3, respectively (Figure 8B and
D). When the complex charge reached a very high value,
toxicity was observed. Polymers without oligonucleotide
revealed a similar trend with the control group.
Apoptotic and Necrotic Effect of Complexes
Apoptosis results are in a consistency with c-myc activ-
ity. Apoptotic cell ratio was obtained as 3% and this
ratio was increased by transfection of polymer/ASODN
complexes depending on the charge and particle sizes
(Table 3). As seen in the control experiment, all of the
cell nuclei were stained by Hoechst 33342 as light blue
representing non-apoptotic cells (Figure 9A). Polymer/
ASODN complexes caused apoptosis as seen in bright
blue spots (Figure 9B). The highest apoptotic effect was
observed in high N/P ratios. This might be because of
the free positive charge on the complex facilitating cell
entrance and endosomal escape. For most of the poly-
mers, N/P: 9 seems a good ratio for the efciency. Al-
though toxicity of the polymers without oligonucleotide
were increased by size and charge, apoptotic effect was
observed in lower values. Apoptosis results seem in con-
sistency with MTT assay.
In the necrotic effect study, naked ASODN did not
cause necrosis; however, 100 M polymers revealed in be-
tween 10%15% necrosis. Complexation between poly-
mer/ASODN affected necrosis in a way that was increased
by the N/P ratio and therefore by the charge of the com-
plexes. As seen in Figure 9D, the higher the positive charge,
the more necrotic cells were obtained. Necrotic cells ap-
peared in red (PLL-mPEG2000/PEI/ASODN complex at
N/P:9), while the others were in green (stained by Hoechst
33342). Figure 9C represents the control group not con-
taining polymers and appears in green.
DISCUSSION
This study reported the use of PEI-coupled PCL-mPEG
and PLL-mPEG diblock copolymers in ASODN delivery
to SKMEL-30 cells. Diblock copolymers were synthesized
by microwave irradiation and characterized as reported
elsewhere [12]. Here, the PEI-bound form of these diblock
copolymers was prepared and polymer formation was
demonstrated by TNBS assay and GPC.
DLS was used to investigate the charge and size
characteristics of polymers and also polymer/ASODN
complexes. The micelle forming ability of PCL-mPEG
and PLL-mPEG diblock copolymers and their PEI-
bound forms was investigated by this technique and
it was estimated that a sharp increase in intensity after
0.05 mg/ml was observed for all polymers, which can be
accepted as cmc . It was observed that the charge of the
polymers increased, especially in the acidic medium, be-
cause of the great buffering capacity of the PEI molecule.
Obtained micelle-like structures and complexes were con-
rmed by DLS in terms of polydispersity and optimal N/P
ratio was found as 6. AFM images of polymer/ASODN
complexes showed that spherical structures were formed
also in the presence of ASODN. However, the sizes of
the corresponding structures were in the range of 1030
nm, approximately, which were different from the ones
obtained by DLS. This was because of the DLS technique
Figure 7. MTT assay of polymer/ASODN complexes depending
on N/P ratio. Bars represent mean 3% S.D.
Table 2. c-myc expression index of SKMEL-30 cells obtained
by immunocytochemistry staining method (p0.071).
c-myc negative cell (%)
Polymer
without
ASODN
N/P ratio
1 3 6 9
PCL-mPEG2000/PEI 4.3 14.5 23.3 33.5 56.8
PCL-mPEG5000/PEI 7.7 13.2 22.7 44.6 62.8
PLL-mPEG2000/PEI 6.4 15.7 49.1 32.4 39.6
PLL-mPEG5000/PEI 5.2 11.3 26.6 14.3 21.8
Antisense Oligonucleotide Carriers 151
Figure 8. Immunocytochemistry images of stained SKMEL-30 cell with c-myc antibody. (A) Nucleus of cell (stained with c-myc
antibody), where brown spots indicate c-myc positive cells as a control (non-treated with polymer/ASODN complex). (B) Cells
transfected with PCL-mPEG5000/PEI at N/P: 9; blue spots show c-myc negative cells. (C) Cells transfected with PCL-mPEG2000/
PEI at N/P: 9; arrow shows c-myc positive cells and some of cells nuclei blue as c-myc negative cell. (D) Cells transfected with PLL-
mPEG2000/PEI at N/P: 3; arrow shows c-myc-positive cell. (E) Cells transfected with PCL-mPEG5000/PEI at N/P: 1; arrow showss
c-myc positive cell. (F) Cells transfected with PLL-mPEG5000/PEI at N/P: 3; arrow shows c-myc positive cell. Images were recorded
with 400 magnication and with an Olympus research microscopy.
measuring the hydrodynamic size of the resultant com-
plexes that were higher than the original ones.
According to MTT assay, polymers not carrying
ASODN showed some toxicity because of the free posi-
tive charge of PEI. MTT assay for the polymer/ASODN
complexes depending on charge and incubation time
was consistent with another study performed by poly(N-
Isopropylacrylamide)/poly(ethyleneimine) [11].
Expression of c-myc was followed by immunocy-
tochemical staining. Naked oligonucletide showed only
5% c-myc inhibition. A small amount of inhibiton (4.3
7.7%) was also observed for the polymers without
152 S. Diner et al.
Figure 9. Fluorescence inverted microscopy image of SKMEL-30 carcinoma cells. (A) Nucleus of cells (stained with Hoescht 33342),
where blue spots indicate nucleus of non-apoptotic cells as a control. (B) Shining and smashed nucleus (shown with arrow) of apoptotic
cells in PCL-mPEG2000/PEI (N/P;9) containing medium. (C) Fluorescence microscopy image of cells (stained with Hoescht 33342),
where formation of green cells demonstrates non-necrotic cells (photo taken under FITC lter) as a control. (D) Nucleus of SKMEL-30
cells (stained with PI and Hoescht 33342), where dense red spots indicate nucleus of necrotic cells and green cells indicate non-necrotic
cells. Images were recorded with 400 magnication. Photos A and B were taken under DAPI lter and C and D taken under FITC lter.
Table 3. Apoptotic and necrotic indexes of SKMEL-30 carcinoma cells; result obtained with double staining method (p0.085 for
Apoptotic indexes, p0.069 for Necrotic indexes).
Apoptotic index (%)* Necrotic index (%)*
N/P ratio 1 3 6 9 1 3 6 9
PCL-mPEG2000/PEI 17.5 26.3 32.4 40.8 5.4 11.3 20.6 25.8
PCL-mPEG5000/PEI 18.7 29.2 37.6 52.3 3.2 7.6 10.9 19.6
PLL-mPEG2000/PEI 18.1 43.7 38.8 36.5 18.3 23.6 16.4 32.7
PLL-mPEG5000/PEI 13.4 22.6 9.7 19.8 9.4 13.7 5.6 12.7
*For naked ASODN apoptotic and necrotic indexes were 3 and 2%, respectively. Polymers without ASODN revealed apoptotic indexes
as 4.3, 6.2, 8.6, 3.1, and necrotic indexes as 10.6, 18.2, 21.7, 8.9 for PCL-mPEG2000/PEI, PCL-mPEG5000/PEI, PLL-mPEG2000/PEI,
PLL-mPEG5000/PEI, respectively.
ASODN, probably because of the toxicity related to
positive charge. Most of the complexes showed a certain
amount of cell inhibition, as seen Table 2. For example,
c-myc inhibiton ratio was estimated as 56.8% and 49.1%
with PCL-mPEG2000/PEI in N/P: 9 and PLL-mPEG2000/
PEI in N/P: 3, respectively. For the efcient inhibition,
lower N/P ratios did not seem as appropriate as the higher
ones because of insufcient complex formation.
Several cationic polymers containing hydrophilic PEG
blocks have been used as transfecting vectors for different
genes [1920]. In the case of PEI as a cationic part, the high
transfecting efciencies observed have been attributed to
Antisense Oligonucleotide Carriers 153
the strong buffering capacity under the physiological pH
conditions. PEI buffers inside the endosomes due to the
transfected polycations pump protons into the endosomes;
this is called the proton sponge effect. This causes an in-
crease in the osmotic pressure inside the endosome, which
destabilizes the endosome, resulting in the escape of the
gene complexes from the degradation inside the lysosomal
environment.
Gebhart et al. [21] and Ochietti et al. [22] have re-
ported that grafting of a hydrophobic polymeric block
to a cationic PEI block can lead to the enhancement of
transfection efciency and control of favorable biodis-
tribution of the complexes in animal models. They have
suggested that by tuning the amphiphilicity of copoly-
mers the binding with gene molecules can be made more
effective, resulting in a favorable size range of the nanop-
olyplexes. They have also speculated that amphiphilicity
together with the variation of block size would allow one
to examine the effect of hydrophobic interaction on the
nanostructure and net charge density of the oligonucle-
otide polyplexes. The relationship between such informa-
tion with cell transfection and cytotoxicity needs to be
studied for more efcient results [19].
In this study, c-myc activity was not demonstrated by
only MTT and immunocytochemical staining. Apoptosis
and necrosis indexes were also evaluated. Here, com-
plexes caused apoptosis in the cells while it was obtained
only 3% by the polymer alone. For most of the polymers,
N/P: 9 seems a good ratio for the efciency. On the other
hand, necrosis caused by the polymers increased by the
N/P ratio depending on the unoccupied positively charged
chains. It was concluded that the optimal N/P ratio has to
be estimated in order not to cause necrosis but at the same
time good apoptosis related to c-myc inhibition. Accord-
ing to the results, the most efcient N/P ratio seems to be
between 6 and 9.
CONCLUSIONS
In this study we have prepared polymers and polymer/
ASODN complexes and used DLS and AFM to charac-
terize the size and charge characteristics. We have inves-
tigated these propeties in different N/P ratios. After all,
these complexes were evaluated by their c-myc activity
towards SK-MEL 30 cells together with apoptotic and
necrotic effect.
Since a very small oligonucleotide was used in this
study, shrinkage of genetic material was not expected that
much, which was in the case of using large DNA chains.
DLS experiments showed that the N/P ratio affected the
uniformity of the complexes, as also seen in AFM images.
All polymers seem to have the ability of micelle forming as
demonstrated by DLS and AFM. We obtained very small
particles after complexation, but the most uniform ones
were obtained especially in N/P:6. The cell culture studies
showed that N/P:9 was also accepable in terms of c-myc
transfection and inhibition. Complexes showed apoptotic
activity, while a small amount of necrotic behavior was
observed, possibly because of positive charges. Since all
polymers are similar in terms of structural properties, they
did not reveal a great difference in corresponding inhi-
bition activity. According to the results, these polymers
seems to have a potential to improve intracellular avail-
ability for c-myc directed antisense strategies.
Declaration of interest: The authors report no conicts of
interest. The authors alone are responsible for the content
and writing of the paper.
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