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Abstract— In the recent years, gene therapy is on the focus of a

series of studies, since it has a great potential to treat a variety of diseases. To be clinically applicable; vectors must be biocompatible, efficient to overcome cellular barriers and noncytotoxic. Non-viral vectors have become preferable over viral vectors, due to immunogenicity of the last. Up to the date, absence of suitable gene-delivery vector, significantly limits development of genetherapy. This paper reports a novel oligoelectrolyte vector, 83-5, with low cytotoxicity and high success of exceeding barriers. We examined the 83-5 oligoelectrolyte’s critical micellar concentration, DNA condensation state and the physico-chemical properties of DNA/83-5 complexes and the interaction between model membranes by dynamic light scattering and fluorescence spectroscopy. The transfection efficiency was determined by luciferase gene expression by luminometer. Despite the moderate transfection efficiency, since 83-5 was low cytotoxic and successful to exceed obstacles, this oligoelectrolyte represents a potential to take place in non-viral gene delivery approaches.

Keywords— Fluorescence techniques, in vitro transfection, non-viral gene delivery, oligoelectrolyte nanocarrier.

I. INTRODUCTION

ENE therapy offers new approaches for various diseases, especially for cancer [1]. Gene therapy treatments

include to delivery the nucleic acids including plasmids, oligonucleotides and siRNA to affect expression of target genes in specific cells and tissues. To delivery these therapeutic molecules into the targeted site, a variety of carriers have been developed in recent years. The development of the suitable vector is very important. The vectors that are used in gene delivery must be safe for human,

Zeliha Guler1, Semra Zuhal Ficen1 and Sebnem Ercelen Ceylan1

(Corresponding author: [email protected], Tel : +90 262 677 3310, Fax : + 90 262 646 3929) are with the TUBITAK Marmara Research Center Genetic Engineering and Biotechnology Institute, 41470 Gebze Kocaeli Turkey.

Alexander Zaichenko2, Nataliya Mitina2, Lyudmila Ivanitska2, Taras Skorokhoda2 are with Lviv Polytechnic National University, UA-79013 Lviv, Ukraine.

Yevhen Filyak3 and Rostyslav Stoika3 are with Lviv Institute of Cell Biology of NAS of Ukraine UA-79013 Lviv, Ukraine.

S. E.C. Author thanks to TUBITAK for the project grant (109S258

(SBAG-Ukraine-3)).

protect the DNA against the nucleases and degradation and have low toxicity [2, 3]. The gene delivery systems have two subgroups as viral and non-viral [4]. Non-viral vectors have many advantages to viral vectors in terms of immunogenicity, simplicity and amount of production and stability in storage [5]. And these vectors involve cationic polymers, lipids and peptides as polycations that form complex with negative charged DNA through electrostatic interactions [6]. There are a lot of studies have been done about investigation of cationic polymers that have chemically different to obtain transfection efficiency [7]. To obtain efficient transfection results, polycation must condensate pDNA, provide the complex stability and reach to the nucleus where transcription occurs [8]. Under physiologic circumstances such as cell culture environment, cationic pDNA/polymer complexes exhibit different behaviours have been observed in some investigations. Therefore, particle size, zeta potential and complex stability take important place with regard to cellular uptake and also transfection efficiency [9].

Cationic polymer systems have wide variety different agent for using in gene delivery applications. One of them is block copolymers that have been used limitedly in recent studies. In this study, we investigated the biophysical and physicochemical properties of a newly synthesized block copolymer named as 83-5 for gene delivery. 83-5 linear block copolymer consists of (DMAEM-MP) and (NVP-BA-AEM) groups at certain ratio and it is soluble in neutral water.

II. MATERIALS AND METHODS

A. Materials

Block copolymer oligo(DMAEM)-MP- block-oligo(NVP-BA-AEM) 83-5 of a structure presented below (Fig. 1):

Figure1. Schematic representation of 83-5 oligoelectrolyte, block copolymer (contained: links of DMAEM 12.6%, content of MP fragments was 0.6%, links of NVP 70.2%, BA 12.4%, AEM 4.1%. Number average molecular weight Mn was 6500g/mol. Coefficient

Non-viral Gene Delivery to HeLa Cells Using Novel Oligoelectrolyte Vector

Zeliha Guler1, Semra Zuhal Ficen1, Taras Skorokhoda2, Yevhen Filyak3, Lyudmila Ivanitska2, Nataliya Mitina2, Rostyslav Stoika3, Alexander Zaichenko2, Sebnem Ercelen Ceylan1*

G

International Conference on Advances in Biotechnology and Pharmaceutical Sciences (ICABPS'2011) Bangkok Dec., 2011

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polydispersity was 1.29). 83-5 was synthesized according following technique:

monomer mixture NVP-BA-AEM (ratio =80:15:5 % mol) was added to the solution of initiator oligo(DMAEM)-MP (0.015 mol/l) in ethyl acetate. The mixture was stirred at 80 ºC for 24 h under inert atmosphere; the resulting oligo(DMAEM)- MP-block-oligo(NVP-BA-AEM) was precipitated from the solution by petroleum ether multiply, and after the fractioning dried under vacuum at 40 ºC for 8 h.

Fig. 2 represents Macro initiator oligo(DMAEM)- MP of a structure schematically:

Figure2. Schematic representation of oligo(DMAEM)-MP

(contained 95.5% of DMAEM links, 5.5% of MP fragments, Number average molecular weight Mn was 4500 g/mol, coefficient polydispersity was 1.03).

Oligo(DMAEM)-MP synthesis was described earlier [10]. In a typical experiment, AIBN (510-2 mol/l) was dissolved in ethyl acetate, DMAEM (4 mol/l) and MP (1 mol/l) were added and the mixture was purged with argon. Polymerization proceeded at 70 ºC for 6 h under argon atmosphere. Conversion was 85% as determined from dilatometer and gravimetric measurements [11]. After the polymerization, the solution was precipitated by hexane; the DMAEM-MP macro initiator was dissolved in acetone and again precipitated in hexane. The procedure was repeated three times.

Reagents used for the block copolymer 83-5 synthesis: N, N- dimethylaminoethyl methylacrylate (DMAEM) (Fluka) – content of the basic substance 98 %, 1-Isopropyl-3(4)-[1-(tertbutyl peroxy)-1-methylethyl] benzene (IBMB) was synthesized from tertbutyl and 2-(4-isopropylphenyl)-2-propanol in acetic acid solution as described earlier [12, 13]. The constants were d4

20=0.867 (lit. 0.8670); nd20=1.448 (litт.

1.448), N-vinyl-2-pyrrolidinone (NVP) (Merck) was purified by distillation under vacuum; butyl acrylate (BA) (Merck) was purified by distillation under vacuum; 2-Aminoethyl methacrylate hydrochloride (AEM) (Aldrich) - content of the basic substance 95 %, used without further purification. 2,2′-Azobis(2-methylpropionitrile) (AIBN) used as initiator was purified by recrystallization.

9-(diethylamino)- 5H- benzo[R] phenoxazin- 5 -one (Nile red), ethidium bromide (EtBr), Calf thymus DNA, dimyristoylphosphatidyl-DL-glycerol (DMPG), 1,2-dimyristoyl-sn–glycero-3-phosphocholine (DMPC) and In VitroToxicology Assay Kit, (XTT), were purchased from Sigma Aldrich Co. (St. Louis, MO.). 1,1I-(4,4,8,8-tetramethyl-

,8-diazaundecamethylene) bis-4-[(3-methylbenz-1,3-oxazol-2-yl)methylidine]- 1,4-dihydroquinolinium] tetraiodide (YOYO-1) were purchased from Molecular Probes (Eugene, OR, USA). Lipofectamine™ DNA transfection reagent and PureLink HiPure Plasmid Maxiprep Kit was from Invitrogen (Löhne, GERMANY). Chloroform and methanol were obtained from MERCK (Darmstadt, GERMANY). All cell culture reagents were purchased from Invitrogen, Gibco Corporation (Carlsbad, CA). HeLa Cervix Epithelloid Carcinoma Cell line was obtained from Sap Institute (Ankara, TURKEY). Luciferase reporter gene encoding plasmid pGL4.51 was purchased from Promega (Madison, USA), then propagated and purified by using Invitrogen PureLink Hipure Plasmid Filter Purification Kit following to transformation of XL1-Blue strain of E. coli with selective growth in ampicillin. The purity and integrity of the plasmid were determined by absorption (A260/A280 ratio) using NanoDrop Spectrometer and electrophoresis on a 1% agarose gel.

83/5 solution was prepared as follows; 10 mg 83-5 molecule was solved in 1ml 3% HCl solution under continuous stirring. 8 ml bidistilled water was added on 1 ml 83-5-HCl solution. Final volume was completed to 10 ml.

B. Methods

All of fluorescence measurements were assessed by PTI Quantamaster Steady State Fluorescence Spectrometer. Dynamic light scattering measurements were performed by Zetamaster 3000 Nanosizer.

C. Fluorescence Spectroscopy and Dynamic Light Scattering Measurements

Determination of Critical Micellar Concentration of 83-5 The determination of Critical Micellar Concentration

(CMC) of 83-5 block copolymer was assessed by using hydrophobic fluorescence probe Nile Red. 83-5 samples in increasing concentrations between 0-500 µg/ml were prepared in 20 mM MES buffer at pH 7.0. The molar concentration of Nile red was 0.02 µM. Nile red was added to samples and then incubated at room temperature, in dark for 30 min. Nile red was excited at 550 nm [14]. The CMC was determined by fluorescence spectrometer (PTI Quantamaster Steady State Fluorescence Spectrometer, New Jersey, USA) through the changes at fluorescence intensity and wavelength.

Particle Size and Zeta Potential Measurements Particle sizes and zeta potential of pDNA/83-5 block

copolymer were measured by using laser tecnique at 25°C (Malvern Nanosizer ZS-3600, Paris, France) [14]. pDNA/83-5 complexes were prepared in 20 mM MES buffer at pH 7.0. at increasing concentrations. The results were analyzed the DTS software included with the instrument.

DNA/83-5 complex Interaction with Model Membranes The interaction between model membranes and pDNA/83-5

complexes was assessed to simulate the cellular internalization and endosomal escape steps of transfection. For monitoring these interactions, EtBr fluorescence properties were used. DNA labeled with EtBr at a molar ratio of 50:1 and incubated


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in room temperature for 0.5 h. pDNA/83-5 complexes were prepared with EtBr labeled plasmid DNA and 83-5 oligoelectroyte at 7.5 µg/ml, 15 µg/ml, 30 µg/ml, 45 µg/ml and 75 µg/ml concentrations and incubated in room temperature, in dark for 0.5 h. Both anionic DMPG and neutral DMPC model membrane vesicles were prepared by extrusion as described previously [14, 15]. Membrane vesicles were added to pDNA/83-5 complex as to Polymer/Lipid ([P/L]) molar ratios was 0.25 and incubated by shaking, at RT, in dark for 15 min. EtBr was excited at 520 nm [15].

D. In vitro cell transfection

Transfection efficiency of 83-5 oligoelectrolyte was determined on the basis of luciferase reporter gene expression. The day before transfection, human cervix epithelial carcinoma cells (HeLa) were seeded on 24 well plates at 106 cells/well in a Dulbecco’s modified Eagle’s medium culture medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and incubated at 37ºC in a 5% CO2 humidified atmosphere [16]. pDNA/83-5 complexes were formed with 1 µg luciferase encoding plasmid pGL4.51 and different amound of 83-5 oligoelectrolyte (7.5 µg/ml, 15 µg/ml, 30 µg/ml, 45 µg/ml, 75 µg/ml) and control vector Lipofectamine™. Cells which were reached to 70% confluency were transfected with pDNA/83-5, pDNA/Lipofectamine complexes and naked DNA, incubated for 4 h. Then transfection medium was replaced by refresh complete growth medium and cells were incubated for an additional 24 h at 37ºC in a 5% CO2 incubator. Luciferase gene expression on HeLa cells was determined by Luciferase Activity Assay as manufacturer’s protocol following the lysis of the cells. The cell lysates were centrifuged at +4°C, 12000 g for 2 minutes and then 20 µl of sample was put into the 96 well, white plate. 100 µl of luciferase assay substrate (Promega) was dispenced on samples. The luciferase activity as luminescence (Relative light units, RLU) was measured with chemiluminometer (Thermo Scientific Fluoroskan Ascent FL, Waltham, Massachusetts, USA) and results were expressed as relative light units integrated over 10 s per mg of cell protein lysate (RLU/mg of protein) [17].

E. Cytotoxicity assay

Cytotoxicity of 83-5 oligoelectrolyte were evaluated by the 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-(tetrazolium-5-carboxanilide) XTT assay. HeLa cells were seed at 106 cells/well in 24 well plates and incubated for 24 h at 37ºC in a 5% CO2 incubator. Cells were treated with 83-5 at 7.5 µg/ml, 15 µg/ml, 30 µg/ml, 45 µg/ml, 75 µg/ml concentrations for 4 h and incubated for additional 24 h at incubator following to replacement of oligoelectrolyte containing medium with complete medium. XTT assay was performed as manufacturer’s protocol. Briefly; cells were incubated with XTT solution occupying 20% of growth medium volume for 4h at 37ºC. Absorbance was measured at 450 nm and 690 nm by using Elisa reader (Thermo Scientific Fluoroskan Ascent FC, Waltham, Massachusetts, USA) [18] and the absorbance

at 690 nm was accepted as a background measurement and subtracted from the 450 nm value.

III. RESULTS

A. Results of Fluorescence Spectroscopy and Dynamic Light Scattering Measurements

Nile red is a positive solvatochromic dye [19] which is highly soluble and florescent in organic solvents [20]. The application field of Nile red is common due to presence of photochemical stability and strong fluorescence nature. Because of its hydrophobic character, it tends to bind to the core of micelle structure where the medium has dipolarity [19, 21]. The CMC of 83-5 block copolymer was determined by using of fluorescence emission spectra of Nile red. 83-5 block copolymer forms micelle-like structure at around 100 µg/ml (Fig. 3B).

Figure3. Determination of the CMC value of 83-5

oligoelectrolyte molecule by using of fluorescence emission spectra of Nile red, A) Fluorescence emission of Nile red against changing concentrations (0-500 µg/ml) of 83-5 in 20 mM at pH 7.0, excitation wavelength=550 nm and B) fluorescence intensity (▲) and maximum emission wavelength (■) as of Nile Red at increasing concentrations of 83-5 oligoelectrolyte.

The size and zeta potential measurements of pDNA/83-5 complexes (Table 1) were performed by Nanosizer [22]. The size of plasmid is 297 nm. According to the measurement results, the diameter of complexes is smaller than plasmid DNA suggesting 83-5 condensates pDNA molecules through the electrostatic interactions (p≤95%). The complexes with 83-5 reached a maximum size (~212 nm) at 15 µg/ml concentration and minimum size (~91 nm) at 45 µg/ml concentration. Although the size diameter of the complexes do


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not rise up in direct proportion to increase of polymer concentrations, they are efficient for cellular uptake. In the next step, zeta potential of 83-5 block copolymer was determined in increasing concentrations. The surface charge of the pDNA is -5.45 mV. The zeta potential measurement results showed that the surface charge of pDNA/83-5 complexes is between -0,979 mV and 13 mV. Maximum surface charge of the complexes was 13 mV at 15 µg/ml concentration and minimum surface charge was 2.63 mV at 45 µg/ml concentration.

The interaction of pDNA/83-5 complexes with DMPG

anionic vesicles was monitored by utilizing EtBr fluorescence. EtBr is an intercalation agent that has fluorescent characteristics presence of DNA. EtBr shows little fluorescence in aqueous solution. Its fluorescence quantum yield displays increment when EtBr interacts with phosphate groups in double stranded DNA. It exhibits fluorescence characteristic under U.V. light [23]. EtBr was excited at 520 nm. DMPG anionic vesicles mimics the internal leaflet of the plasma membrane [22]. Fig. 4 (A-E) indicates that according to the fluorescence intensity of EtBr, pDNA/83-5 complexes interacted with DMPG. The interaction degree were close to each other at 7.5 µg/ml (A), 15 µg/ml (B) and 75 µg/ml (E) concentrations of 83-5 but the most efficient interaction was at 75 µg/ml. The interaction degree at 30 µg/ml (C) and 45 µg/ml (E) were lower than at 7-5 µg/ml, 15 µg/ml and 75 µg/ml concentrations.

Figure4. Interaction between DMPG model membrane and

pDNA/83-5 complexes formed with 83-5 at 7.5 µg/ml (A), 15 µg/ml (B), 30 µg/ml (C), 45 µg/ml (D), 75 µg/ml (E) concentrations and EtBr labelled pDNA. EtBr was excited at 520 nm.

TABLE I PARTICLE SIZE AND ZETA POTENTIAL MEASUREMENTS OF 83-5

OLIGOELECTROLYTE COMPLEXES

Size (d.nm) Zeta potential (mV)

Plasmid DNA 297.3 -5.45 pDNA/83-5 7.5 µg/ml 204.5 -0.979 pDNA/83-5 15 µg/ml 212.3 13 pDNA/83-5 30 µg/ml 151.9 11.1 pDNA/83-5 45 µg/ml 145.2 2.63 pDNA/83-5 75 µg/ml 185.1 10.6


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To stimulate the cellular internalization of the complexes,

interaction with DMPC model membrane vesicles which mimics the external leaflet of the plasma membrane [22] was studied. pDNA was labelled with EtBr and pDNA condensation states were determined by means of its fluorescence. When DNA condensates, EtBr is expelled from DNA and its fluorescence significanly decreases [24]. Fig. 5 (A-E) suggests that 83-5 oligoelectrolyte is enable to condensate pDNA at all concentrations but more efficiently at the higher concentrations. According to EtBr fluorescence, pDNA shows no interaction with DMPC vesicles. Compared to fluorescence which was observed when pDNA condansated by 83-5 oligoelectrolyte, there was no significant changes in EtBr fluorescence when DMPC large unilameller vesicles were added to pDNA/83-5 complex samples. This suggests that pDNA/83-5 complexes were not interacted with DMPC model membrane vesicles under these conditions and at these concentrations.

Figure5. Interaction between DMPC model membrane and

pDNA/83-5 complexes formed with 83-5 at 7.5 µg/ml (A), 15 µg/ml (B), 30 µg/ml (C), 45 µg/ml (D), 75 µg/ml (E) concentrations and EtBr labelled pDNA. EtBr was excited at 520 nm.


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B. In vitro cell transfection studies

Transfection efficiency of 83-5 oligoelectrolyte was evaluated by quantifiying the luciferase gene expression on Hela cells after transfection with lusiferase encoding plasmid DNA, pGL4.51. Cells were transfected with both pDNA/83-5, pDNA/Lipofectamine™ complexes and naked plasmid DNA. (Fig. 6). Fig. 5 shows that the 83-5 oligoelectrolyte transfected HeLa cells more efficiently at higher concentrations. Luciferase activity was the highest (~4x104 RLU) on cells which was transfected with the complex which was formed with 75 µg/ml of 83-5. At this concentration, luciferase activity, so the transfection efficiency of 83-5 was almost 13 fold greater than naked plasmid DNA’s efficiency (~400 RLU). The luciferase activity on cells after transfected with control vector Lipofectamine™ was at around 8x104 RLU.

Figure 6. Transfection efficiency of 83-5 oligoelectrolyte on HeLa cells transfected with luciferase encoding pDNA, pGL4.51. Cells were incubated in complete medium and transfected with naked pDNA, pDNA/83-5 complexes formed with 7.5 µg/ml, 15 µg/ml, 30 µg/ml, 45 µg/ml, 75 µg/ml 83-5 and pDNA/Lipofectamine™ complex as control. Luciferase expression determined from the luciferase assay was expressed as luciferase activity as RLU.

C. Cytotoxicity of 83-5

Cytotoxicity effect of 83-5 oligoelectrolyte in HeLa cells was determined after incubation with the increasing concentraion (7.5 µg/ml, 15 µg/ml, 30 µg/ml, 45 µg/ml and 75 µg/ml) of oligoelectrolyte for 4 h at 37 ºC by XTT assay and the relative cell viability was calculated as:

100690450690450 controlnmnmsamplenmnm ODOD

Fig. 7 indicates that 83-5 have no toxic effects (> 80% cell

viability) [18] in HeLa cells up to concentration of 45 µg/ml oligoelectrolyte. When cells were treated with 75 µg/ml of 83-5, cell viability was slightly decreased (68%).

Figure7. Cytotoxicity of 83-5 was determined by XTT assay after

4 h incubation at 37ºC with increasing concentrations of oligoelectrolyte.

IV. DISCUSSION

Gene therapy is a promising method for treatment of variable genetic or acquired diseases [25]. This method is based on expression of therapeutic genes following to their transfer to the cells [26]. The success of gene therapy depends on the selection of an appropriate vector which is used as DNA carrier [27]. Vector should be biologically safe and capable of exceeding cellular barriers which limit the efficiency of cell transfection [28]. Non-viral vectors have become preferable over viral vectors, because of the safety concerns, as well ads because they have many advantages such as having the capacity of carrying huge amounts of DNA, easy and cheap production and storage [29]. For these reasons, researchers have focused on non-viral gene delivery vectors which have low cytotoxicity and high transfection efficiency. Block copolymers have been limitedly used as gene delivery vectors in recent studies inspite of their many unique advantages. They have hydrophilic and hydrophobic groups thus, they show self-assembly characteristic in water solution [30]. In current study we synthesized novel oligoelectrolyte DNA nanocarrier (called as 83-5) and characterized its gene delivery properties by different biophysical methods.

We analysed its in vitro transfection efficiency and cytotoxicity in HeLa cells. To futher understand the transfection modality of 83-5 oligoelectrolyte we characterized the physicochemical, biophysical properties of both 83-5 itself and pDNA/83-5 complexes, since it is known the low cellular internalization, deficient release of DNA with limited stability and lack of nuclear entry were major cellular barriers which effects the transfection efficiency [31] and the success of gene transfer is depending on overcoming of these barriers [32]. We also examined the interaction between pDNA/83-5 complexes and model membranes which mimics the cellular internalization and endosomal escape.

Primarily, we determined the CMC. Generally, block copolymers form micelle-like structure as mentioned above certain critical micellar concentration. If dilution occurs in


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certain concentration, it causes micelle dissociation and release of carried substance such as plasmid and drug [33]. The core of micelle-like structure is hydrophobic and the corona is hydrophilic in copolymers [34]. Baines et al. (1996) reported a study with PDMAEMA-b-MMA block copolymer and they showed interesting results about micelle structure of block copolymer. We determined the critical micelle concentration of 83-5 by Nile red fluorescence emission. According to the fluorescence profile of Nile red, the 83-5 block copolymer forms micelle-like structure at around 100 µg/ml.

In the second step, we determined the size and zeta potential of pDNA/83-5 complexes at different 83-5 concentrations. The size of polycation/pDNA complex is the important factor affects the cellular uptake and pathway during transfection process [35, 36]. There have been many studies about the size diameter of different gene delivery agents but the major problem in comparing the results obtained from these studies is consist of various conditions at each other. The most important factor is cell lines which are used in experiments [36, 37] reported those different sized complexes between a few nanometers and 3 µm can enter to the cell at various cell lines. Win et al. (2005) showed that nanoparticles of 100-200 nm around are the most efficient for cellular uptake [38]. The complex sizes of 83-5 block copolymer are under 250 nm. According to the studies mentioned above, 83-5 condensates pDNA as small size at various concentrations and the size of pDNA/83-5 complexes is convenient to get efficient transfection. The results of zeta potential measurements support the results of size measurement. Since 83-5 molecule condensates pDNA, pDNA/83-5 complexes carry positive (+) charge. Positive (+) charged 83-5 molecule neutralizes the phosphate groups of the negative charged pDNA.

Cellular uptake of complexes varies according to the cell membrane environment [39]. Understanding the interaction between complex and cell membrane, phospholipids vesicles are used as model membranes for mimicking the biomembranes [40]. We used negative charged lipid vesicles DMPG and neutral lipid vesicles DMPC as model membranes. We utilized the fluorescence intensity of EtBr molecule for studying the complex-membrane interaction [14]. As discussed earlier, neutral DMPC vesicles mimicking the external leaflet of the cell membrane and their interaction with complexes stimulates the interaction with cell membrane, thus cellular internalization. Although it is important for complex to internalize to the cell, strong interaction with DMPC vesicles is not desired since it could cause the dissociation of the complex. According to EtBr fluorescence intensity, we showed that neither pDNA nor pDNA/83-5 complexes were interacted with DMPC model membrane vesicles. As a consequence of this result, internalization of pDNA/83-5 complexes might be limited but at least their stability was protected. DMPG anionic vesicles mimic the internal leaflet of plasma membrane [14]. According to the EtBr fluorescence intensity, we observed the interaction between complex and

DMPG at various concentration of 83-5 molecule but the most efficient interaction was at 75 µg/ml. The interaction degree at 7.5 µg/ml and 15 µg/ml was close to 75 µg/ml but the interaction degree at the other concentration was not sufficient for internalization.

Transfection efficiency of 83-5 oligoelectrolyte was determined by luciferase activity after Hela cells were transfected with luciferase encoding plasmid DNA. Compared to control vector Lipofectamin™, the transfection efficiency of 83-5 oligoelectrolyte was low but it was almost 13 fold higher than naked DNA. According to this result, it was thought that 83-5 was quite successful to transfer luciferase gene to the cells. The oligoelectrolyte was more efficient at its higher concentrations (75 µl/ml). This was not surprising because its CMC was at around 100 µg/ml and it was reported that polymers at under [42, 43] or close concentrations [44] of CMC showed higher transfection efficiency. Since pDNA condensation states and the particle size of pDNA/83-5 complexes were as it refered to be [45], and considering the results of model membrane interaction assays the relatively low transfection efficiency might because of limited cellular internalization or endosomal trapping.

An efficient transfection is not possible, unless the vector is non or less cytotoxic. For this reason, 83-5 oligoelectrolyte’s cytotoxicity was determined by XTT assay and the relative cell viability was calculated. 83-5 did not show any toxic effect on HeLa cells at 7.5 µg/ml, 15 µg/ml and 30 µg/ml concentrations because the relative cell viability was higher than 80% [18]. When concentration of 83-5 was increased to 45 µg/ml and 75 µg/ml, relative viability was slightly decrease. These results were expected because 2-dimethylaminoethylamino (DMAEA) containing carriers are low cytotoxic [46] in HeLa cell line. Since 83-5 oligoelectrolyte has quite positive biophysical properties and strong interactions with DNA and the formal importance of being non cytotoxic, it is possible to improve the transfection efficiency of 83-5 with enhancing cellular internalization and endosomal escape.

V. CONCLUSION

The data obtained with 83-5 block copolymer molecule are quite promising for its use in gene delivery. Despite the moderate transfection efficiency of 83-5, since it was low cytotoxic and successful to exceed obstacles, this oligoelectrolyte represents a huge potential to take place in non-viral gene delivery approaches after optimization of its in vitro and in vivo transfection efficiency.

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