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Antisense oligonucleotide delivery to cancer cell
lines for the treatment of different cancer types
Ebru Kilicay, Ebru Erdal, Baki Hazer, Mustafa Türk & Emir Baki Denkbas
To cite this article: Ebru Kilicay, Ebru Erdal, Baki Hazer, Mustafa Türk & Emir Baki Denkbas (2016) Antisense oligonucleotide delivery to cancer cell lines for the treatment of different cancer types, Artificial Cells, Nanomedicine, and Biotechnology, 44:8, 1938-1948, DOI: 10.3109/21691401.2015.1115409
To link to this article: https://doi.org/10.3109/21691401.2015.1115409
Published online: 27 Nov 2015.
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http://dx.doi.org/10.3109/21691401.2015.1115409
RESEARCH ARTICLE
Antisense oligonucleotide delivery to cancer cell lines for the treatment of
different cancer types
Ebru Kilicaya, Ebru Erdalb, Baki Hazerc, Mustafa Tu¨rkdand Emir Baki Denkbase
a
Department of Electronic and Automation, Zonguldak Vocational School, Bu¨lent Ecevit University, Kilimli, Zonguldak, Turkey;bDepartment of Biology, Faculty of Science, Aksaray University, Aksaray, Turkey;cDepartment of Chemistry, Division of Physical Chemistry, Bu¨lent Ecevit University, Beytepe, Zonguldak, Turkey;dDepartment of Bioengineering, Faculty of Engineering, Kirikkale University, Kirikkale, Turkey;
e
Department of Chemistry, Division of Biochemistry, Hacettepe University, Beytepe, Ankara, Turkey
ABSTRACT
Amphiphilic poly(3-hydroxylalkanoate) (PHA) copolymers find interesting applications in drug delivery. The aim of this study was to prepare nucleic acid adsorbed on (PHB-b-PEG-NH2)
nanoparticle platform for gene delivery. For this purpose, PHB-b-PEG-NH2block copolymers were
synthesized via transesterification reactions. The copolymers obtained were characterized by Proton Nuclear Magnetic Resonance (1H-NMR), Fourier Transform Infrared Spectrometer (FTIR),
Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) techniques. The cytotoxic, apoptotic and necrotic effects of these nanoparticles in the MDA 231 human breast cancer cell, the A549 human lung cancer cell and the L929 fibroblast cell lines were also investigated. ARTICLE HISTORY Received 23 August 2015 Revised 25 October 2015 Accepted 28 October 2015 Published online 26 November2015 KEYWORDS Antisense oligonucleotide; fibroblast cells; human breast cancer cells; human lung cancer cells; PHB-PEG block copolymer; PHB-PEG nanoparticle
Introduction
Oligonucleotides (ODNs), complementary to a specific gene or
RNA message, are short segments (from 13 to25 sequence
bases) of single-stranded DNA. Antisense strategy is to interfere with an expression by preventing the translation of proteins from mRNA. Antisense oligonucleotide (AS-ODN) binds to the single-strand mRNA which prevents the binding of factors that initiate or modulate the translation, by Watson and Crick base pairing and blocks sterically the translation of this transcript
into a protein (Baker et al. 1997). They are therefore a very
attractive approach for the treatment of viral diseases (Kulka
et al. 1989, Smith et al. 1986), cancer (Necker and Whitesell
1993), or other diseases based on an uncontrolled
over-expression of proteins. But, ODNs are poorly stable versus nuclease activity in biological environment and their very poor intracellular penetration has limited their use in therapeutics
(Loke et al. 1989, Yakubov et al. 1989). Some chemical
modifications can be applied to protect ODNs and to facilitate their cellular uptake. Cationic lipids, proteins, dendrimers, polymeric particles, liposomes and peptides have been improved for tide over mentioned above (Lebedeva et al.
2000). Those which, polymeric particles have several
advan-tages for antisense delivery. Particulate carriers can be used to improve the in vivo activity via protecting ODN against degradation, enhancing their intracellular delivery and
pre-venting protein binding. In this present study, PHB-PEG-NH2
nanoparticles have been suggested as ODN carriers.
Polyhydroxyalkanoates (PHAs), biodegradable, natural and biocompatible, are promising bacterial polyesters used for biomedical applications, especially drug delivery systems and tissue engineering. Among them, poly(3-hydroxybutyrate) (PHB) is a well-known hydrophobic bacterial polyester. PHB is too rigid and brittle and lack the superior mechanical proper-ties especially for using biomedical applications. Therefore, its physical, hydrophilic and mechanical properties need to be
improved (Grage et al.2009, Liu et al. 2009). In this manner,
amphiphilic PHA copolymers have been applied to overcome
these drawbacks (Hazer2010a, Hazer et al.2012).
Our studies have been focused on the modification reaction of biodegradable polyester for improving mechanical and hydrophilic properties in order to use it in cancer treatment.
In this aim, various hydrophilic polymers have been used for particle coating. In most efforts to synthesize amphiphilic PHAs, PEG has been widely used among them. It has been known as an efficient polymer to limit adsorption of plasma proteins
and cells (Hazer2010b). PEG has promising building blocks for
polymerization in organic environment and aqueous systems because its association in polymers is utility for medical applications. Therefore, due to its hydrophilic character, PEG has been introduced into this PHB to overcome its hydrophobic character. PHB nanoparticles covered with a hydrophilic poly-meric layer have been prepared by double emulsion/solvent
evaporation procedure (Blanco and Alonso1997). The
modifi-cation of PHB with polyethylene glycol (PEG) involved the reduction of opsonization and phagocystoisis by cells of the
CONTACTDr Ebru Kilic¸ay ebru.kilicay@gmail.com Zonguldak Vocational School, Bu¨lent Ecevit University, Kilimli, Zonguldak 67500, Turkey
RES (Donald and Nicholas2006, Dubey et al.2015). Thus, ODN-PHB stayed in the blood circulation for a long time. For this purpose, amine-terminated polyhydroxybutyrate-block-poly-ethylene glycol (PHB-b-PEG) amphiphilic block copolymers were synthesized via transesterification reactions (Erduranli
et al.2008).
AS-ODN has a hydrophilic and polyanionic character.
Therefore, it has attached covalently to PHB-PEG-NH2
nano-particle, allowing the hydrophobic interaction with polymer
surface (Dinc¸er et al. 2010). For this, a double emulsion
technique (Davis and Walker1987, Matsumono1985) was used
to prepare amphiphilic nanoparticles.
In this study, we discussed the performance of the ODN nanoparticles in different cancer cell lines. It was also studied cytotoxic, apoptotic and necrotic effects.
Materials and methods
Materials
Polyethylene glycol-bis-(2-aminopropyl ether) with molecular
weight 2000 g/mol (PEG-NH2) was a gift from Huntsman Co.
(Basel, Switzerland). PHB and microbial polyester was supplied by Biomer (Krailling, Germany). Stannous(II)-ethyl hexanoate
and other chemicals are purchased from Aldrich.
N-Hydroxysuccinimide (NHS) was a gift from University of California, San Diego, CA. AS-ODN was synthesized by Ella Biotech GmbH, Martinsried, Deutschland.
N-(3-dimethylamino-propyl)-N-ethylcarbodiimide hydrochloride (EDC-HCl) was
obtained from AppliChem Biochemica GmbH, Darmstadt, Germany. Hoechst dye 33342 and propidium iodide (PI) were supplied by Roeche (Deutschland, Germany). Tripsin–ethylene-diamine tetra acetic acid (tripsin–EDTA), Dulbecco’s Modified Eagle’s Medium (DMEM F-12), fetal calf serum (FCS) and DMSO (dimethylsulfoxide) were purchased from Biological Industries (Beit Haemek, Israel). 3-(4,5-Dimethylthiazol)-2,5-diphenyltetra-zolium bromide (MTT) was purchased from Serva (USA).
PHB-b-PEG block copolymer synthesis
The block copolymers were carried out by transesterification reactions. The synthesis method was performed from the
previous article cited in the references (Erduranli et al.2008).
PHB was transesterified with PEG-NH2under reflux condition in
chloroform. For the transesterification reaction, 10 g of PHB, 10 g of PEG and 0.1 g of Sn(oct)(II)-ethyl hexanoate were mixed in 300 ml of chloroform. The mixed solution was refluxed for 3 h. The solvent was evaporated by means of rotary evaporator until half of it remained. Then the obtained copolymer was dissolved in a certain amount of chloroform and precipitated in
500 ml of methanol and dried under vacuum overnight at 40C.
The PEG content of PHB-b-PEGN block copolymer was obtained as 5 mol %.
Analytical techniques
Fourier Transform Infrared (FTIR) spectra of the copolymers was obtained by using a Perkin-Elmer SpectrumOne, Nicolet 520 (USA). Since this sample was not dissolved in THF, the
molecular weight of the polymeric sample was not determined by gel permeation chromatography (GPC) but according to the source incoming from the origin that the molecular weight of polymer was 80.000 Da. Differential scanning calorimeter (DSC) was carried out under nitrogen by using Shimadzu DSC-60, Perkin-Elmer Diamond (Houston, TX). Thermogravimetric ana-lysis (TGA) (Shimadzu DTG-60, Perkin-Elmer Pyris, USA) was
carried out to determine thermal degradation. 1H-NMR of the
products was recorded using Bruker 2’ 300, 400 MHz in CDCl3
solvent and tetra methylsilane as the internal standard.
Preparation of nanoparticles
PHB-b-PEG nanoparticles were prepared by double
emulsion-solvent evaporation technique (Cohen-Sela et al. 2009, Song
et al.2006). Briefly, 10 mg of PHB-b-PEG was dissolved in a 10 ml
of chloroform. A dispersion medium was prepared by using 50 ml of 0.5% polyvinyl alcohol The organic phase was added dropwise to the dispersion medium by an ultrasonic probe (IKA T 125 Digital Ultra Turrax Homogenizer, Deutschland, Germany) in an ice bath for 3 min at 90% amplitude. The same volume of polyvinyl alcohol solution was poured into the resultant water-in-oil (w/o) emulsion allowing for the diffusion of the organic solvent into water. In this way, water-in-oil-in-water (w/o/w) emulsion of nanoparticles was obtained. The organic solvents were evaporated by using ultrasonic bath (Alex, Germany) and mechanical stirrer (Heidolph RZR 2021, North America). The nanoparticles were collected by centrifugation (Centrifuge
5810R) at 12,000 rpm for 30 min at 10C and washed three times
with deionized water to remove remaining surfactants and organic solvents. Finally, the nanoparticles were freeze-dried
(Martin Christ GmbH, Osterode am Harz, Germany) (Figure 2).
Physicochemical characterization of NPs
Morphology of nanoparticles was analyzed by atomic force
microscopy (AFM) (Nanomagnetics Instruments, Ankara,
Turkey) and scanning electron microscopy (SEM) (Quanta FEG 450 model SEM, Germany), respectively. The FTIR was per-formed for the structural characterization of the nanoparticles (Perkin-Elmer SpectrumOne, Nicolet 520). Particle size and size distributions were determined by nanodrop (Thermo Scientific Nano Drop 1000 Spectrophotometer, USA). The surface charge of the particles was characterized in terms of zeta potential (Malvern Instruments, Model 3000 HSA, England).
Conjugation of nanoparticles with AS-ODN
The ODN used in this study consisted of 15 nucleotides
(sequence 50-ACC GTT GAG GGG CAT-30, molecular weight
4633). ODN was covalently coupled with the free amino group of PEG present on the surface of nanoparticles using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC)
and the NHS as coupling agent (Dilnawaz et al. 2010),
respectively. Briefly, liophilized nanoparticles were suspended into PBS (pH 7.4). Hundred millimolar of EDC (1 mg/ml) and then NHS (1 mg/ml) were added and incubated for 2 h at room temperature and centrifuged to remove the excess EDC/NHS.
The nanoparticles were redispersed in deionized water and buffered with PBS, pH 7.4. Hundred and twenty five microliter of ODN was then added into the precipitated particles and then incubated again for 2 h at room temperature. The nucleic acid-coupled nanoparticles were seperated from unconjugated ODN using centrifugation at 12,000 rpm for 20 min. All experiments were carried out in sterile conditions for the cell culture studies. The unreacted ODN in the supernatant was determined by using nanodrop and the amount of ODN conjugated on the surface of the nanoparticles was calculated. The existence of ASO on the surface of nanoparticles was determined by using FTIR spectrophotometer (Perkin-Elmer SpectrumOne, Nicolet 520). The amount of ASO bound to the nanoparticles was calculated as the difference between the total amount of the initial ASO added and the amount of ASO determined in the supernatants.
MTT cytotoxicity tests
MTT assay for cytotoxicity, A549 human lung cancer cells, MDA MB 231 human breast cancer cells and L929 fibroblast cells
(5 103 cells/well) were seeded into flat-bottomed 96-well
plates containing DMEM with L-glutamine and 10% FCS
supplemented with 1% FCS and incubated overnight.
Following this incubation, particles with different concentra-tions (50–200 mg/ml) were diluted with cell culture medium
and inoculated into the wells. The plates were kept in the CO2
incubator at 37C in 5% CO2for 24 h. The cell culture medium
was replaced with 100 ml fresh medium and 15 ml of MTT solutions and incubated with the same circumstances for 4 h in a dark condition. After that, 100 ml isopropanol–HCl to dissolve the formed dark blue formazan crsytals was added to each well and incubated for 30 min in a dark condition. The wells were read using the ELISA plate reader at 570 nm. The number of living and dead cells was counted with a cell counter. Each assay was repeated three times.
Analysis of apoptotic and necrotic cells Double staining
Double staining with Hoechst dye and PI was performed to quantify the number of apoptotic and necrotic cells in culture based on scoring apoptotic and necrotic cell nuclei. A549, MDA
MB-231 cancer cells and L929 fibroblast cells (20 103cells per
well) were seeded in DMEM-F12 with 1% L-glutamine added
10% fetal bovine serum and 1% penicillin–streptomycin at 37C
in a 5% CO2humidified atmosphere using 24-well plates. After
treating with different amounts of ASO, PHB-PEG nanoparticles and ASO-PHB-PEG nanoparticles (50–200 mg/ml) for a 24-h period, attached and detached cells were collected and then washed with PBS. The cells were incubated with Hoechst dye 33342 (2 mg/ml), PI (1 mg/ml) and DNAse free-RNAse (100 mg/ ml) for 15 min at room temperature. Of cell suspension, 10–50 ml was smeared on slide and cover slip for examination under a fluorescence microscope (Leica Microsystems, DMI 6000, Germany). In double-staining method with Hoechst dye, the nuclei of normal cells were stained dark blue but apoptotic cells were stained a stronger blue. The apoptotic cells were
identified via morphological changes in the nucleus based on nuclear fragmentation and chromatin condensation. Nuclei of necrotic cells were stained red by PI since PI dye can cross the cell membrane of necrotic cell lacking plasma membrane integrity. The PI dye can not cross an intact cell membrane. Ten randomly microscopic fields were chosen in order to determine the number of apoptotic and necrotic cells. In this way, the number of apoptotic and necrotic ones were evaluated using a fluorescence inverted microscope (Leica, Germany) with DAPI filter and FITC filter, respectively. Obtained data were expressed as the ratio of the number of apoptotic and necrotic cells to the number normal cells.
Results and discussion
PHB-b-PEG block copolymers characterization
PHB-PEG block copolymers were synthesized by transesterifica-tion reactransesterifica-tions in the presence of PEG with functransesterifica-tional end
groups, NH2. Physicochemical characterization of
PHB-b-PEG-NH2 block copolymers was performed in the previous study
(Erduranli et al.2008, Hazer 2010a, Hazer et al. 2012). As the
amount of PEG into PHB-b-PEG block copolymer increased, the solubility of block copolymer in chloroform decreased. Therefore, our synthesized copolymers contained only 5 mol % PEG in order to solve the copolymers in chloroform during the nanoparticle preparation step. Therefore, it was slightly
different from that of the sample used from Hazer et al. (2012)
in their previous article. For this purpose, we also characterized
the block copolymers prepared newly before using. 1H-NMR
and FTIR were carried out to confirm their chemical structure.
The 1H-NMR spectrum of the block copolymer in Figure 1(A)
shows 3.6 ppm for –O-CH2of PEG and 1.3 ppm for –CH3of PHB.
The FTIR spectrum of the block copolymer in Figure 1(B)
shows the characteristic signals of ether bands of PEG at
1043 cm1and carbonyl and carboxylic acid OH stretch of PHB
at 1720 cm1and at 3000 cm1, respectively.
A thermal analysis of block copolymers was performed by DSC and TGA. DSC and TGA were carried out to determine glass
transition temperatures, Tg, melting temperatures, Tm and
decomposition temperatures, Tdof PHB-PEG block copolymers
(Figure 1C). Samples were heated from ±100C to +200C under
a nitrogen atmosphere at 10C/min rate for DSC analysis and
they were heated again a second time at heating rate. Tgof
pure PHB was 15C and Tmwas about 180C. When PEG was
added, the plasticizing effect of PEG blocks reduced the
melting temperature, Tm to 600C. It was shown that the
copolymer had decomposed above the melting point. All these copolymers transitioned to the gas phase and degraded while
the decomposition temperature, Td was 264C. Tg and Tmof
block copolymers fell below those of the pure PHB due to the plasticizing effect of PEG.
Nanoparticles characterization
Amphiphilic core-shell biodegradable nanoparticles were pre-pared by using the double emulsion-solvent evaporation technique. Chloroform was used as an organic solvent and PVA as a stabilizer yielded uniform spherical nanospheres. The
morphology of the nanoparticles was evaluated by AFM and SEM. Nanoscale particles with uniform spherical shape and smooth in surface and have a size distribution about from 200 to 235 nm were observed by AFM and SEM as shown in
Figure 2, respectively.
PHB-b-PEG nanoparticles were characterized in terms of homogenization rate, polymer and surfactant concentration. The particle size, polydispersity and surface charge are listed in
belowTable I. The average sizes ranged from 200 to 540 nm
and the zeta potential values from22.2 to 29.7 mV. All the
nanoparticles exhibited negative zeta potential values, due to the negative charges of the polymer. The increase of sonication rate reduced the particle size efficiently due to the further energy transferred to the suspension medium. The increase in polymer concentration significantly increased the particle size due to the fact that it was more difficult for the more viscous polymer solution to disperse. The interfacial tension between the aqueous dispersion medium and the polymeric droplets
was decreased by the surfactant (Kilicay et al.2011). However,
the increase in surfactant concentration did not significantly alter the mean particle size.
Binding of nanoparticles to AS-ODN
AS-ODNs are hydrophilic and polyanionic molecules. Therefore, they cannot readily permeate in biological cell membranes and
they are not stable against nuclease degradation. To overcome these disadvantages, various nanoparticle carrier systems were used to provide sustained release of this compound. For this reason, PHB-PEG nanoparticles were prepared. PEG corona included into the nanoparticle can partially protect the ODN
from nucleases (Tang et al.2012).
In this study, conjugation of antisense with nanoparticles was carried out by using the covalent coupling technique
(Dilnawaz et al.2010). ASO was covalently attached to the
PHB-PEG nanoparticle surface due to the electrostatic interaction between phosphate groups of antisense and the –NH2 groups
of PEG on the surface (Figure 3A). The ASO-adsorbed PHB-PEG
nanoparticles were242 nm in diameter, and particles without
ASO binding were220 nm in diameter. The binding efficiency
of ASO-nanoparticles was found at a relatively high level of
85%. ASO-nanoparticles have a zeta potential between30
and35 mV and a particle concentration is 0.0464 mg/ml
(Table II). It was observed that the negative surface charged of particles increased after antisense binding. ASO content of
these particles has 29 mg/mg nanoparticles. The binding of
the negatively charged ASO to the surface of the nanoparticles is possible without the use of any positive compound (Arnedo
et al.2002).
The FTIR spectrum of ASO and ASO-PHB-PEG nanoparticles indicates the characteristic signals of phosphate for nucleic acid
between at 1200 and 1350 cm1(Figure 3B).
Figure 1. (A) The1H-NMR spectrum of the b-PEG block copolymer; (B) The FTIR spectra of b-PEG block copolymer; (C) DSC and TGA thermogram of the PHB-(alone) and PHB-b-PEG-NH2block copolymers.
The morphology of ASO-nanoparticle was obtained by SEM. It was shown that ASO-nanoparticles have a spherical shape with smooth surfaces and sizes about between 230 and 235 nm (Figure 3C).
Evaluation of cytotoxicity
In this study, the cytotoxicities of the all compounds were tested against A549, MDA MB 231 cancerous and normal L929 fibroblast cell lines. The different sample concentrations were incubated with different cell lines for 24 h in cell culture media as mentioned above. In addition, the wells contain only cells specified as control were also tested in cell culture media. After that, the wells were read at 570 nm using an ELISA plate reader. The percentage of viable cells was calculated and the diagrams were drawn at different concentrations (50–200 mg/ml) against
%, cell viability (shown inFigure 4). The different results were
obtained from the graphs. In terms of MTT results, the concentration of samples had a significant impact on cell mortality. An increase of ASO-NPs concentration (especially 200 mg/ml) causes a higher degree of dying cells in comparison to free ASO and unconjugated NPs. Therefore, a lower concen-tration (50 mg/ml) of compounds showed markedly lower toxicity toward different cell lines. As a result of these experiments, ASO-NPs were more toxic than free ASO and bare NPs for each cell lines. The data shows that, cell mortality
values of ASO-NPs (200 mg/ml) were found about as 49.89; 35.34 and 25.51% on A549, MDA MB 231 cancer and L929 fibroblast cell lines, respectively. The lowest mortality rate was obtained with ASO-NPs at a concentration of 50 mg/ml with a decrease of 19.45; 18 and 9.88% on A549, MDA MB 231 cancer
and L929 fibroblast cell lines, respectively (Figure 4). Treatment
with free ASO also induced cytotoxicity in a dose-dependent manner with a reduction of 15.37% (A549); 14.64% (MDA MB 231) and 11.8% (L929) at a concentration of 50 mg/ml,
respectively (Figure 4). Bare NPs did not significantly cause any
observable toxicity effect within a concentration range of 50–200 mg/ml. According to the results, ASO-NPs were found more cytotoxic on A549 than MDA MB 231 and L929 fibroblast cells in a dose dependent manner, in vitro. As a consequence, ASO-NPs can be safely used as a therapeutic agent in the cancer therapy researches. A similar result was found in the literature. Kang et al.
(2008) prepared indomethacin-loaded poly(L-lactic acid)/poly
(lactide-co-glycolide) (IDMC-PLLA/PLGA) microparticles and they investigated in vitro cytotoxicity and the cellular uptake of drug-loaded microparticles into the A549 human cancer cells. For this purpose, cells were treated with naked and IDMC-loaded microparticles at a concentration of 25, 50, 100 and 200 mM, respectively. It was found that the higher concentration caused
higher cytotoxicity. The IC50of free IDMC and IDMC-PLLA/PLGA
microparticles was 131.52 ± 1.21 and 223.08 ± 2.11 mM at 48 h
exposure. Sahiner et al. (2014) synthesized crosslinked p(sucrose)
microparticles. Cytotoxicity of p(sucrose) particles with varying concentrations (0–200 mg/ml) on MDA MB 231 cancer cells and L929 fibroblast cell was investigated. It was observed that the toxicity of the particles increased with the increase the amounts of particles. At higher p(sucrose) particle concentration, espe-cially at 100 mg/ml and over, the toxicity to both cells increased e.g. the % cell viabilities were 74 and 67.5 for MDA MB-231 cells and L929 fibroblast cells, respectively. It was also found the same result in our previous study about folic acid attached etoposide-loaded PHBHHx. HeLa cells and L929 fibloblast cells were incubated with folic acid attached etoposide-loaded PHBHHx, etoposide-loaded NPs, free etoposide and bare NPs at different
concentrations (5, 10, 25 and 50 mg/ml). The results demon-strated that higher drug concentration caused lower cell viability or equivalently higher mortality of the cells. The cytotoxicity of the folic acid conjugated etoposide-loaded PHBHHX nanoparti-cles to cancer cells was much higher than free etoposide or etoposide-loaded PHBHHX nanoparticles without folic acid
(Kilicay et al.2011).
Apoptotic and necrotic results
In this study, the apoptotic and necrotic effects associated with the morphological changes of ASO, NPs and ASO-PHB-PEG NPs on A549 human lung cancer cells, MDA MB 231 human breast cancer cells and L929 mice fibroblast cells were investigated. Double-staining method was used to determine the apoptotic effect of ASO, PHB-PEG NPs and ASO-PHB-PEG NPs. Hoechst 33342 fluorescent stain was used in this method. For this, 1 mg/ml stock solutions were prepared and dissolved to
Figure 3. (A) Conjugation reaction of AS-ODN with nanoparticles; (B) FTIR spectrum of ASO (a) and ASO-PHB-PEG nanoparticles (b); (C) SEM photograph of ASO-PHB-b-PEG nanoparticles.
Table II. Characterization of PHB-PEG and ASO-PHB-PEG nanoparticles. Samples PHB-b-PEG ASO + PHB-b-PEG
Samples 220 ± 1 242 ± 5.5
Polydispersity 0.116 ± 0.030 0.214 ± 0.047 Zeta Potential (mV) 28.2 ± 4.1 32.7 ± 2.3 Particle content (mg/ml) 0.0464 0.0464 ASO binding efficiency (%) – 85
Table I. Effect of some parameters on size and size distribution of PHB-b-PEG nanoparticles. Polymer concen-tration (mg/ml) PVA concentration (mg/ml) Homogenization rate (% amplitude) Size (nm) Polydispersity (PDI) Zeta Potential (mV) 5 3 1 1.4 1.4 1.4 90 90 90 524 ± 8 323 ± 5 220 ± 1 0.340 ± 0.053 0.251 ± 0.032 0.116 ± 0.030 28.2 mV 24.8 mV 22.2 mV 1 1 1 1.4 1.0 0.4 90 90 90 220 ± 1 225 ± 4 249 ± 2 0.116 ± 0.030 0.135 ± 0.012 0.157 ± 0.023 22.2 mV 23.1 mV 25.9 mV 1 1 1 1.4 1.4 1.4 90 70 50 220 ± 1 341 ± 20 489 ± 11 0.116 ± 0.030 0.325 ± 0.050 0.414 ± 0.019 22.2 mV 24.4 mV 29.7 mV
concentration of 50–200 mg/ml for all samples and applied into the cultured normal and cancer cell cultures. The interaction was observed for each sample with cells within 24 h period of time. The apoptotic and necrotic index percentage diagram and cells photographs based on the double-staining method
are given inFigures 5, 6 and 7, respectively. The Hoechst 33342
fluorescent dye attaches to DNA and results a dark blue color
cell nuclei when observed by the DAPI filter under a fluorescent
microscope (Figure 6A and C). Control group contains only
medium. There was no apoptosis accordingly no morphological difference in the cell nuclei of the control group and bare NPs (Figure 6A and C). An apoptotic effect was highly observed for ASO-PHB-PEG NPs compared to cells treated with free ASO as
seen in Figure 6(D) and (B), respectively. Apototic cell nuclei
0 20 40 60 80 100 120 (A) (B) Cell viability (% ) Control 50µg/ml 100µg/ml 200µg/ml 1=A549 2=MDA MB231 3=L929 2 1 3 1 2 3 Control
Free ASO ASO-NPs
0 20 40 60 80 100 120 Cell viability (% ) Control 50µg/ml 100µg/ml 200µg/ml 1=A549 2=MDA MB231 3=L929 3 1 2
Free ASO NPs ASO-NPs
1 3 2 1 2 3
Control
Figure 4. Cell viability of A549, MDA MB231 cancer cells and L929 fibroblast cells incubated with the free ASO, naked NPs and ASO-NPs at 50, 100 and 200mg/ml concentration at 37C for 24 h. Error bars show standard deviation.
have decomposed and have shapeless borders and a brighter appearance than the non-apoptotic cells (apoptotic cells are
demonstrated with arrow in Figure 6D). The % apoptotic
indices diagram are given inFigure 5. When the free ASO and
PHB-PEG NPs were applied separately to each of the cell lines, ASO and bare nanoparticles displayed a low apoptotic index at 50 mg/ml concentrations. The findings showed that lower concentrations of samples caused lower effect in apoptosis. As the concentration of ASO was increased, the apoptosis
rate was also increased as seen inFigure 5. The samples which
had concentrations ranging between 50 and 200 mg/ml displayed significantly different results. As a consequence, it was demonstrated clearly that the apoptotic effect of ASO and bare NPs was lower than PHB-PEG nanoparticles. ASO-PHB-PEG NPs interacted with each cell line which showed substantial changes in the apoptotic index at low and high concentrations. The increasing of concentration caused an enhancement of the apoptosis. The high penetration of ASO in nanoparticles into the cells led to the increase of the apoptotic effect.
The apoptotic ratio of ASO-NPs varied from 2 ± 1 to 40 ± 3%; 2 ± 1 to 28 ± 3%; 2 ± 1 to 20 ± 2% for A549, MDA-MB 231 and L929 cell lines, respectively when treated with the highest concentration (200 mg/ml). The highest apoptotic effect % 40 ± 3 was observed for A549 cancer cells. The apoptotic effect of ASO-NPs was almost the same for both MDA-MB 231 cancer cells and L929 fibroblast cells. It was clearly observed that A549 cancer cells are more effective than other cancer cell lines.
PI, a fluorescent molecule was used in the double-staining method to determine necrosis in cancer cells. This dye crosses the dead cell membranes led to the cell nuclei staining red color but it does not cross the living cells under fluorescent light (by a FITC filter). The A549, MDA MB 231 cancer cells and L929 fibroblast cells were stained with PI fluorescent dye in double-staining solution. Photographs of necrotic cell
images were given inFigure 7. The healthy cell nuclei appeared
green when it was scanned by an FITC fluorescent filter. The nuclei of necrosed cells was observed in red color in wells treated with ASO-PHB-PEG NPs by use of a fluorescent
microscope (Figure 7H). The images of the control group
and bare NPs show no morphological difference in cell
nuclei (Figure 7E and G). The highest necrotic effect was
observed in ASO-PHB-PEG NPs (Figure 7H) compare to free ASO
(Figure 7F).
ASO and bare nanoparticles had low necrotic effects at 50 mg/ml concentrations. The necrotic effect was increased due to a higher ASO concentration in each type of cells after the toxicity and apoptotic effects were observed. According to the obtained results, the necrotic ratio of ASO-NPs varied from 1 ± 1 to 22 ± 2%; 2 ± 1 to 14 ± 2%; 2 ± 1 to 9 ± 2% for A549, MDA-MB 231 and L929 cell lines, respectively. The highest necrotic effect of % 22 ± 2 was observed for A549 cells compared to L929 fibroblast cells at 200 mg/ml concentration. There is slightly difference between both MDA MB 231 cells and L929 fibroblast cells in terms of necrotic indice
results (Figure 5). 0 5 10 15 20 25 1 2 3 4 % Necr otic index
A549
PHB-PEG NPs ASO ASO-PHB-PEG NPs 0 2 4 6 8 10 12 14 16 1 2 3 4 % Necr otic indexMDA MB 231
PHB-PEG NPs ASO ASO-PHB-PEG NPs 0 2 4 6 8 10 12 1 2 3 4 % Necr otic indexL929
PHB-PEG NPs ASO ASO-PHB-PEG NPs 0 5 10 15 20 25 30 35 40 45 1 2 3 4 % Apoptotic indexA 549
PHB-PEG NPs ASO ASO-PHB-PEG NPs 0 5 10 15 20 25 30 35 1 2 3 4 % Apoptotic indexMDA MB 231
PHB-PEG NPs ASO ASO-PHB-PEG NPs 0 5 10 15 20 25 1 2 3 4 % Apoptotic indexL929
PHB-PEG NPs ASO ASO-PHB-PEG NPsFigure 5. The diagrams of apoptotic–necrotic percentage indexes obtained from A549, MDA MB231 cancer cells and L929 fibroblast cells incubated with different concentrations of samples (0–200mg/ml). The experiments were repeated three times.
Conclusions
In this study, amine-terminated PHB-PEG block copolymers were synthesized and characterized. PHB-co-PEG copolymer nanoparticles were prepared by use of the double emulsion/ solvent evaporation technique. The obtained nanoparticles are spherical in shape and have small size distribution and better DNA binding capacity. Thus, the ASO was covalently attached to the PHB-PEG nanoparticle surface. The binding of the ODN to the co-polymeric nanoparticles protected the ASO from enzymatic degradation, increased the intracellular capture of these molecules and also improved the delivery of the drug.
PHB-PEG nanoparticles were evaluated for their use as potential drug carrier systems. For this purpose, the anticancer activity of compounds was investigated. Therefore, these
samples were evaluated towards A549, MDA MB 231 cancer cells and L929 fibroblast cells with cytotoxic, apoptotic and necrotic effects.
Cytotoxicity of NPs was carried out by using the MTT assay. The results indicated that the cytotoxicity of ASO-NPs against A549 and MDA MB 231 cancer cells (as compared with L929 fibroblast cells) was much higher than that of free ASO and naked NPs. ASO-NPs showed strong cytotoxicity to A549 human lung cancer cell lines especially at 200 mg/ml concentration.
Detection of the apoptotic and necrotic index was realized with fluorescent light microscope (by DAPI and FITC filter, respectively) at a wavelength of 480–520 nm. According to the results, the apoptotic and necrotic ratio increased in a dose dependent manner. Thus, the apoptotic and necrotic effects were highly concentration dependent. The apoptotic and Figure 6. Apoptotic cell photographs obtained from the double-staining method using Hoechst 33342 fluorescent stain. Fluorescence microscopy image of (A) nucleus of A549, MDA MB 231 and L929 cells stained by Hoechst 33342 fluorescent stain. Each cell not treated with ASO and PHB-PEG NPs. The whole pale blue spots show the nucleus of non apoptotic cells. Nucleus borders are normal and the nucleus did not decompose (control group); (B) Cells interacted with only free ASO at a concentration of 200mg/mL; (C) Cells exposed to the PHB-PEG NPs at a concentration of 200 mg/ml; (D) Cells treated with ASO-PHB-PEG NPs at a concentration of 200 mg/ml. The arrows show some of the apoptotic cell nuclei look bright and decomposed. Photographs were taken under DAPI filter by using Leica inverted fluorescent microscope 400 magnification, scale bar shows 70.5 mm.
necrotic cell percentages for A549 human lung cancer cell were 12 ± 1%; 40 ± 3% (at 50 and 200 mg/ml) and 5 ± 1%; 22 ± 2% (at 50 and 200 mg/ml, respectively). The apoptotic effect induced by ASO-NPs on A549, MDA MB 231 and L929 were observed even at a concentration as low as 50 mg/ml. However, the necrotic index was not very high at 50 mg/ml concentration. This study shows that the apoptotic and necrotic effects are low in low concentration (50 mg/ml) and are high in high concentration (200 mg/mL). Especially in A549 with the concentration of 200 mg/ml, toxic, apoptotic and necrotic effects were remarkable. In addition, the apoptotic and necrotic activities seem in consistency with the MTT assay. In vitro studies demonstrated that obtained particulate system is an effective uptake enhancer for ODNs. According to cytotoxicity and results of the apoptotic and necrotic effects, nanoparticu-late systems based on antisense have therapeutic potential in cancer therapy researches. In conclusion, the particles based on
ODN can be safely used for in vivo applications in the near future.
Acknowledgements
The authors thank Tug˘ba Toraman for her contributions as regards to copolymer preparation stage and Evren Sean Kilicay for editing the article.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
This work was financially supported by the Bulent Ecevit University Research Fund (Grant no. BEU-2012-M6-00-03).
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Figure 7. Necrotic cell images obtained from the double-staining method using PI fluorescent stain. (E) A549, MDA MB 231 and L929 cells stained by double-staining method. Each cells not treated with ASO and PHB-PEG NPs (control group). Cells did not necrose. Green spots shows nucleus of non-necrotic cells (stained by Hoechst 33342); (F) Cells exposed to free ASO at a concentration of 200mg/ml; (G) Cells exposed to the PHB-PEG NPs at a concentration of 200 mg/ml; (H) Photograph of all cells transfected with ASO-adsorbed PHB-PEG NPs. The arrow shows some of the necrotic cells where dense red spots. Photographs were taken under FITC filter by using Leica inverted fluorescent microscope at 400 magnification. The scale shows a distance of 70.5 mm.
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