protamine increases transfection efficiency and cell

Tenkumo et al., Nano Biomedicine 5(2), 64-74 , 2013

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Introduction As the expectations of gene therapy have been in-creasing in recent years, the development of an efficient gene transfer agent is a very important issue in biology and medicine. Viral vectors have good transfection efficiency, but are associated with cytotoxicity [1], immunogenicity [2] and po-tential recombination or complementation [3]. Systems such as liposomes [4-8], polymers [9-12] and inorganic nanoparticles [13-15] have been in-vestigated as potent non-viral agents for gene transfer. However, most of these suffer from either low gene transfection efficiency or significant cy-totoxicity. For an ideal transfer agent, cellular up-take, protection of nucleic acids from degradation and nuclear delivery should be associated with low cytotoxicity.

Calcium phosphate nanoparticles are an at-tractive carrier system due to their good biocom-patibility, their high biodegradability and their high affinity for nucleic acids [16]. Previously, we demonstrated that the transfection efficiency of DNA-loaded calcium phosphate nanoparticles was considerably higher with incorporation of DNA into multi-shell nanoparticles to prevent its degra-dation within the cell by nucleases [17]. Poly-ethylenimine (PEI) was used for gene delivery as a non-viral transfection agent with high cati-onic-charge density [18, 19]. PEI condenses DNA into positively charged particles (polyplexes), which penetrate through the negatively charged cell membrane by endocytosis. The ability of PEI to destabilize lysosomal membranes enables DNA to efficiently escape the degradation within the

Protamine Increases Transfection Efficiency

and Cell Viability after Transfection with Calcium Phosphate Nanoparticles

Taichi TENKUMO1, Olga ROTAN2, Viktoriya SOKOLOVA2,

and Matthias EPPLE2

. 1Tohoku University Graduate School of Dentistry, Division of Liaison Center for Innovative Dentistry, Sendai, Japan

2Institute for Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Essen, Germany

Synopsis Penetration through the cell membrane, endosomal escape and nuclear entry are the main barriers thattransfection agents have to overcome. We prepared multi-shell calcium phosphate nanoparticles thatwere functionalized with DNA, polyethyleneimine (PEI) and protamine in order to improve transfectionefficiency. PEI-functionalized calcium phosphate nanoparticles showed a high transfection efficiency,which was correlated with cytotoxicity. Additional functionalization with protamine effectively reducedthe cytotoxic effects of PEI, while maintaining the high transfection efficiency. Size, surface charge andmorphology of multi-shell nanoparticles were analyzed by dynamic light scattering and scanning elec-tron microscopy. The influence of nanoparticle concentration on the transfection efficiency and cell vi-ability was tested on HeLa and MG-63 cell lines. Protamine-functionalized multi-shell calcium phos-phate nanoparticles can serve as an efficient and non-toxic gene carrier for cells. Key words: calcium phosphate, nanoparticles, gene transfer, cell viability, fluorescence

ORIGINAL ARTICLE

Tenkumo et al., Protamine Effectively Improve the Gene Transfer with Calcium Phosphate Nanoparticles, Nano Biomedicine 5(2), 64-74, 2013

65

acidic endosomal environment [20]. We previously reported that an outer layer of

PEI in multi-shell calcium phosphate nanoparticles leads to electrostatic and steric colloidal stabiliza-tion and gives the nanoparticles a positive charge, which is helpful for penetration through the nega-tively charged cell membrane [21]. However, PEI clearly exhibits cytotoxicity, depending on its mo-lecular weight [22, 23]. Therefore, an increase in the concentration of PEI leads to higher transfec-tion efficiency, but simultaneously increases the cytotoxicity [21, 24]. To overcome this issue, we selected protamine, which has several positive features. Protamine is a member of a family of di-verse small arginine-rich proteins with a molecular weight of approximately 4-10 kDa. It is a cationic and naturally occurring polypeptide with mem-brane-translocating and nuclear-localizing activity [25-28]. It is generally recognized as being safe by the Food and Drug Administration (FDA) [29]. The biological role of protamine is to efficiently bind negatively charged DNA and to provide a highly compact configuration of chromatin [28, 30, 31]. Protamine contains four nuclear localization sequences (NLS), which specifically direct the complex to the nucleus [8, 25, 32, 33]. Function-alization with protamine may assist a synthetic transfection agent to enter the nucleus. In this study, multi-shell calcium phosphate nanoparticles were functionalized with protamine, and the synergistic effect of protamine was investigated on HeLa and

MG-63 cell lines. Materials and methods 1) Preparation of nanoparticles and polyplexes For preparation of functionalized calcium phos-phate (CaP) nanoparticles (NP), an aqueous solu-tion of calcium nitrate (18 mM) was mixed with an aqueous solution of diammonium hydrogen phos-phate (10.8 mM). The pH of both solutions was adjusted beforehand to 9 with NaOH (0.1 M). Mixing was accomplished by rapidly pumping both solutions into a glass vessel. Of this disper-sion, 36 µL was collected with a syringe and rap-idly mixed with 9.6 µL of an aqueous solution of pcDNA-EGFP (1 mg mL-1) and 4.8 µL of an aqueous solution of protamine (10 mg mL-1), fol-lowed by addition of 18 µL of an aqueous solution of calcium nitrate (18 mM) and 18 µL of an aque-ous solution of diammonium hydrogen phosphate (10.8 mM). Colloidal stabilization was finally ac-complished by addition of either 3.6, 7.2 or 14.4 µL of PEI (2 mg mL-1), dissolved in water, giving three different dispersions of CaP/DNA/ pro-tamine/CaP/PEI nanoparticles (Table 1, A-C). Branched polyethyleneimine (PEI, MW 25 kDa; Sigma-Aldrich Chemie GmbH, Steinheim, Ger-many) and protamine sulfate (Merck, Darmstadt, Germany) were used.

DNA/protamine/PEI polyplexes (control without CaP core) were prepared as follows: 9.6 µL of an aqueous solution of pcDNA-EGFP (1 mg

Table 1 Final concentrations of CaP, DNA-EGFP, protamine and PEI in nanoparticle dispersions.


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mL-1) and 4.8 µL of an aqueous solution of pro-tamine (10 mg mL-1) were mixed, diluted with 72 µL of distilled water and then mixed with 3.6, 7.2 or 14.4 µL of aqueous PEI (2 mg mL-1), respec-tively (Table 1, D-F).

For preparation of CaP/DNA/PEI nanoparti-cles (control without protamine), 36 µL of an aqueous CaP nanoparticle dispersion (prepared as above) was mixed with 9.6 µL of an aqueous solu-tion of pcDNA-EGFP (1 mg mL-1), followed by addition of 18 µL of an aqueous solution of cal-cium nitrate (18 mM), then 18 µL of an aqueous solution of diammonium hydrogen phosphate (10.8 mM) and finally either 3.6, 7.2 or 14.4 µL of PEI solution (2 mg mL-1) (Table 1, G-I).

For preparation of CaP/DNA/protamine nanoparticles (control without PEI), 36 µL of aqueous CaP nanoparticle dispersion (prepared as above) was mixed with 9.6 µL of an aqueous solu-tion of pcDNA-EGFP (1 mg mL-1), followed by addition of 18 µL of aqueous calcium nitrate (18

mM), then 18 µL of aqueous diammonium hydro-gen phosphate (10.8 mM) and finally 4.8 µL of protamine (10 mg mL-1) (Table 1, K).

Characterization of functionalized calcium phosphate nanoparticles was performed by scan-ning electron microscopy (FEI; ESEM Quanta 400 microscope) after gold-palladium sputtering. Light scattering and zeta potential determinations were performed with a Zetasizer nanoseries instrument ( = 532 nm; Malvern Nano-ZS). Particle size data refer to scattering intensity distributions. 2) Cell transfection HeLa cells (human epithelial cervical cancer cell line) and MG-63 cells (human osteosarcoma cell line) were cultivated in DMEM, supplemented with 10% fetal bovine serum (FBS), 100 U mL-1

penicillin, and 100 U mL-1 streptomycin at 37°C under a 5% CO2 atmosphere. Approximately 24 h before transfection, cells were trypsinized and seeded in 96-well plates at a density of 5 × 103

Table 2 Final concentrations of CaP, DNA-EGFP, protamine and PEI in nanoparticle dispersions per 100 µL of cell medium.


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cells per well. Plasmid DNA, pcDNA3-EGFP, en-coding for enhanced green fluorescent protein (EGFP) was obtained from Escherichia coli using Nucleobond® endotoxin-free plasmid DNA kit (Macherey-Nagel, Düren. Germany).

In order to test nanoparticles with different concentrations of PEI (Table 1), we added 10 µL of each nanoparticle dispersion to cells and 90 µL of cell culture medium. To find the most efficient amount of functionalized calcium phosphate nanoparticles in each well, transfection experi-ments were carried out as follows: 10, 5, 2.5 or 1.25 µL of CaP/DNA/protamine/CaP/PEI0.05 nanoparticle dispersion were added to cells in cul-ture medium, to give a total volume of 100 µL. Nanoparticles without protamine or PEI and poly-plexes (DNA/protamine/PEI) were used as a con-trol groups. Final concentrations of the CaP, DNA, protamine and PEI in each well for each volume of the nanoparticle dispersion are given in Table 2. The duration of transfection was 7 h. Subsequently, fresh culture medium was added to the cells and they were incubated for 72 h.

Transfection efficiency was determined at 72 h after transfection by transmission light micros-copy and fluorescence microscopy (magnification ×100; Carl Zeiss MicroImaging, Göttingen, Ger-many). Transfection efficiency was calculated based on the ratio of the fluorescing cells (in which

EGFP was expressed) to the total number of ex-amined cells. Dead cells (as recognized by their shape) were not included into the computation. Cell viability was analyzed by MTT assay at 72 h after transfection. MTT (3-(4,5-dimethylthiazol- 2- yl)-2,5-diphenyltetrazoliumbromid; Sigma, Taufkirchen, Germany) was dissolved in PBS (5 mg mL-1) and then diluted to 1 mg mL-1 in culture medium. Culture medium from transfected cells was replaced with 100 µL of MTT solution, fol-lowed by incubation for 1 h at 37°C under 5% CO2 in a humidified atmosphere. After incubation, MTT solution was removed and 100 µL of DMSO were added to the cells. After 30 minutes, a 50-µL ali-quot was taken for spectrophotometric analysis using a Multiscan FC (Thermo Fisher Scientific, Vantaa, Finland) at 570 nm. Absorption of trans-fected cells was normalized to that of control (un-treated cells), thereby indicating the relative level of cell death.

Results and Discussion We previously described transfection by multi-shell CaP/DNA/PEI nanoparticles. In the present study, these nanoparticles were also modi-fied by protamine (Fig. 1) and investigated on HeLa and MG-63 cell lines for transfection effi-ciency and cell viability.

Functionalized CaP/DNA/protamine/CaP/PEI

Fig. 1 Schematic representation of preparation of functionalized calcium phosphate nanoparticles.


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nanoparticles had a diameter of around 200 nm, which is well suited for cellular uptake (Table 3). All nanoparticles carried a strong positive charge because of PEI amino groups on the external layer of nanoparticles and showed a tendency to have smaller size with increasing PEI concentration. Scanning electron microscopy images showed the spherical morphology of nanoparticles (Fig. 2). PEI on the particle surface prevented aggregation. We evaluated the effects of multi-shell CaP/DNA/protamine/CaP/PEI nanoparticles and controls on transfection efficiency on HeLa and MG-63 cells (Table 4). For both cell lines, trans-

fection efficiency was between 39% and 54%, and did not depend on the concentration of PEI (Fig. 3A). However, the viability of HeLa cells de-creased from 94% to 73%. In the case of the more sensitive MG-63 cell line, the viability decreased dramatically from 67% to 8% with an increased concentration of PEI (Fig. 3B; Table 4, Samples A, D, G). In the control groups, transfection efficiency and cell viability were significantly reduced (Table 4). As shown in Table 4 (Sample A), CaP/DNA/protamine/CaP/PEI0.05 nanoparticles have a high transfection efficiency, combined with good viability for both cell lines (Fig. 4).

Fig. 2 Scanning electron micrographs of CaP nanoparticles, loaded with DNA, protamine and polyethyleneimine:A: CaP/DNA/protamine/CaP/PEI0.05; B: CaP/DNA/protamine/CaP/PEI0.1; C: CaP/DNA/protamine/CaP/PEI0.2.

Table 3 Colloid-chemical data of functionalized calcium phosphate (CaP) nanoparticles. PDI = Polydispersity index from dynamic light scattering (DLS).


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Table 4 Transfection efficiency and cell viability of HeLa and MG-63 cells. * p<0.05, compared toCaP/DNA/protamine/CaP/PEIx (x = 0.05, 0.1 or 0.2) within each group, respectively.

Fig. 3 Comparison of transfection efficiency (A) and cell viability (B) of HeLa and MG-63 cells. Mock representsuntreated cells.


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Fig. 4 Transmission lightmicroscopy and fluorescencemicroscopy of HeLa andMG-63 cells, transfected withCaP/DNA/protamine/CaP/PEI0.05 nanoparticles. Trans-fected cells appear green onfluorescence microscopy.Magnification is ×100 in allcases.

Table 5. Transfection efficiency and cell viability of HeLa and MG-63 cells at different concentrations ofnanoparticles. * p<0.05 compared to CaP/DNA/protamine/CaP/PEI0.05 nanoparticles.


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Fig. 5 Comparison of transfection efficiency (A) and cell viability (B) of HeLa and MG-63 cells, depending on theconcentration of nanoparticles. Mock indicates untreated cells.

Fig. 6 Transmission light microscopy and fluorescence microscopy of HeLa and MG-63 cells transfected withdifferent concentrations of CaP/DNA/protamine/CaP/PEI0.05 nanoparticles. Transfected cells appear green on fluo-rescence microscopy. Magnification is ×100 in all cases.


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Therefore, we selected CaP/DNA/protamine/ CaP/PEI0.05 nanoparticles for further cell experi-ments to improve cell viability by varying the amount of nanoparticle dispersion added per well (or 100 µL cell medium) from 1.25 µL to 10 µL (Table 5). As shown in Figure 5, transfection effi-ciency increased significantly with the increasing of nanoparticle concentration, for HeLa cells from 4% to 38%, and for MG-63 from 5% to 39%. However, cell viability decreased for MG-63 cells from 110% to 59%, but remained unchanged for HeLa cells. Therefore, it is important to note that the optimal amount of CaP/DNA/protamine/CaP/ PEI0.05 nanoparticle dispersion, which results in an optimal combination of transfection efficiency and cell viability, was 10 µL per 100 µL cell me-dium for HeLa cells, and 2.5 µL for MG-63 cells (Fig. 5).

The interaction between nanoparticles and cells depends on cell type [34, 35]. In Figure 6, we show representative light and fluorescence micrographs of HeLa and MG-63 cells after transfection with different amounts of CaP/ DNA/protamine/CaP/PEI0.05 nanoparticles. To summarize the results from our experiments and from the literature, we describe the fate of the functionalized nanoparticles inside the cell as follows. The positively charged functionalized calcium phosphate nanoparticles easily attached to the negatively charged cell membrane via electrostatic interaction and enter the cell by en-docytosis or macropinocytosis [18, 36-38]. The protonated amine groups in the PEI molecules increase the osmotic pressure in the endosome, leading to disruption of the endosomal mem-brane. The cytotoxic effects of PEI can be as-cribed to adsorption of PEI aggregates on the surface of the cell membrane and its rupture due to the high positive charge density of PEI [11, 39, 40]. Nuclear localization sequences (NLS) within protamine may reduce its cytotoxicity [41]. The addition of protamine into CaP/DNA/CaP/PEI nanoparticles probably also enhances the transfection efficiency of both cell lines by improving the nuclear targeting and thus, gene expression [42]. Protamine-DNA com-plexes are able to bind to importins (transport proteins), which attach on cytoplasmic filaments of the nuclear pore complex, resulting in nuclear entry and further transcription and translation through host cell mechanisms of gene expres-

sion [33, 43]. Conclusions In this study, we developed multi-shell nanoparti-cles, loaded with plasmid DNA, PEI and protamine. The polycationic polymer PEI provided high transfection efficiency while protamine reduced the cytotoxicity of PEI, protecting cells from apoptosis and improving entry of plasmid DNA into the cell nucleus. The effects of functionalized nanoparticles were investigated on HeLa and MG-63 cell lines as a function of concentration. Nanoparticles with the smallest concentration of PEI (CaP/DNA/protamine/CaP/PEI0.05) were found to be most effective, showing good transfec-tion efficiency while keeping high viability in both cell lines. The optimal concentration of nanoparti-cle dispersion for both cell lines was determined. Acknowledgements We are grateful to Kateryna Loza for performing SEM imaging. References 1) Herz J, Gerard RD. Adenovirus mediated

transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci USA 1993;90:2812-2816.

2) Simon RH, Engelhardt JF, Yang Y, Zepeda M, Weber-Pendleton S, Grossman M, Wilson JM. Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study. Hum Gene Ther. 1993;4:771-780.

3) Walther W, Stein U. Viral vectors for gene transfer: a review of their use in the treatment of human diseases. Drugs 2000; 60:249-271.

4) Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, Durham SR, Jeffery PK, Hodson ME, Coutelle C, Huang L, Porteous DJ. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fi-brosis. Nature Med 1995;1:39-46.

5) Farhood H, Serbina N, Huang L. The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim Biophys Acta 1995;1235:289-295.

6) Lasic DD, Templeton NS. Liposomes in gene therapy. Adv Drug Deliv Rev 1996;20:221- 266.

7) McNeil SE, Perrie Y. Gene delivery using cationic liposomes. Expert Opin Ther Pat 2006;16:1371-1382.

8) Chen J, Yu Z, Chen H, Gao J, Liang W. Trans-fection efficiency and intracellular fate of polycation liposomes combined with pro-tamine. Biomaterials 2011;32:1412-1418.


73

9) Garnett MC. Gene-delivery systems using cationic polymers. Crit Rev Ther Drug Carrier Syst 2004;16:147-207.

10) Kichler A, Leborgne C, Coeytaux E, Danos O. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gen Med 2001;3:135- 144.

11) Lv HT, Zhang SB, Wang B, Cui SH, Yan J. Toxicity of cationic lipids and cationic poly-mers in gene delivery. J Controlled Release 2006;114:100-109.

12) Pollard H, Remy JS, Loussouarn G, De-molombe S, Behr JP, Escande D. Poly-ethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammal-ian cells. J Biol Chem 1998 March 27, 1998; 273:7507-7511.

13) Xu ZP, Zeng QH, Lu GQ, Yu AB. Inorganic nanoparticles as carriers for efficient cellular delivery. Chem Eng Sci 2006;61:1027-1040.

14) Sokolova V, Epple M. Inorganic nanoparticles as carriers of nucleic acids into cells. Angew Chem Int Ed 2008;47:1382-1395.

15) Liong M, Lu J, Kovochich M, Xia T, Ruehm SG, Nel AE, Tamanoi F, Zink JI. Multifunc-tional inorganic nanoparticles for imaging, targeting and drug delivery. ASC Nano 2008; 2:889-896.

16) Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed 2002;41:3130-3146.

17) Sokolova V, Prymak O, Meyer-Zaika W, Cölfen H, Rehage H, Shukla A, Epple M. Synthesis and characterisation of DNA-functionalised calcium phosphate nanoparticles. Mat-wiss u Werkstofftech 2006;37:441-445.

18) Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: poly-ethylenimine. Proc Natl Acad Sci U S A 1995;92:7297-7301.

19) Wang YQ, Su J, Wu F, Lu P, Yuan LF, Yuan WE, Sheng J, Jin T. Biscarbamate cross-linked polyethylenimine derivative with low molecu-lar weight, low cytotoxicity, and high effi-ciency for gene delivery. Int J Nanomed 2012;7:693-704.

20) Akinc A, Thomas M, Klibanov AM, Langer RS. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hy-pothesis. J Gene Med 2005;7:657-663.

21) Sokolova V, Neumann S, Kovtun A, Chernousova S, Heumann R, Epple M. An outer shell of positively charged poly (ethyle-neimine) strongly increases the transfection efficiency of calcium phosphate-DNA nanoparticles. J Mater Sci 2010;45:4952- 4957.

22) El-Aneed A. An overview of current delivery systems in cancer gene therapy. J Controlled Release 2004;94:1-14.

23) Godbey WT, Wu KK, Mikos AG. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci USA 1999;96(9):5177-5181.

24) Hu J, Kovtun A, Tomaszewski A, Singer BB, Seitz B, Epple M, Steuhl KP, Ergün S, Fuchs-luger TA. A new tool for the transfection of corneal endothelial cells: calcium phosphate nanoparticles. Acta Biomater 2012;8:1156- 1163.

25) Balhorn R, Brewer L, Corzett M. DNA con-densation by protamine and arginine-rich pep-tides: analysis of thoroid stability using single DNA molecules. Molecular reproduction and development 2000;56:230-234.

26) Lochmann D, Weyermann J, Georgens C, Prassl R, Zimmer A. Albumin-protamine- oligonucleotide nanoparticles as a a new an-tisense delivery system. Part 1: Physico-chemical characterization. Eur J Pharm Bio-pharm 2005;59:419-429.

27) Masuda T, Akita H, Harashima H. Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microin-jection: The use of a nuclear transfer score. FEBS Letters 2005;579:2143-2148.

28) Reynolds F, Weissleder R, Josephson L. Pro-tamine as an efficient membrane-translocating peptide. Bioconjugate Chem 2005;16:1240- 1245.

29) Kerkmann M, Lochmann D, Weyermann J, Marschner A, Poeck H, Wagner M, Battiany J, Zimmer A, Endres S, Hartmann G. Immu-nostimulatory properties of CpG-oligonucleotides are enhanced by the use of protamine nanoparticles. Oligonucleotides 2006;16:313-322.

30) Futaki S, Suzuki T, Ohashi W, Yagami T, Ta-naka S, Ueda K, Sugiura Y. Arginine-rich pep-tides. An abundant source of mem-brane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem 2001;276:5836-5840.

31) Meistrich ML, Mohapatra B, Shirley CR, Zhao M. Roles of transition nuclear proteins in spermiogenesis. Chromosoma 2003;111:483- 488.

32) Sorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther 1997;4:961-968.

33) Noguchi A, Hirashima N, Nakanishi M. Cati-onic cholesterol promotes gene transfection using the nuclear localization signal in pro-tamine. Pharm Res 2002;19:933-938.

34) Sokolova V. Synthesis, characterization and application of calcium phosphate nanoparticles for the transfection of cells. Universität Duis-burg-Essen 2006;1-156.


74

35) Morille M, Passirani C, Vonarbourg A, Clavreul A, Benoit JP. Progress in developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials 2008;29: 3477-3496.

36) Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC. Nanoparticles in medicine: Therapeutic applications and developments. Clin Pharmacol Ther 2008;83:761-769.

37) Rotan O, Sokolova V, Gilles P, Hu W, Dutt S, Schrader T, Epple M. Transport of su-pramolecular drugs across the cell membrane by calcium phosphate nanoparticles. Mat-wiss u Werkstofftech 2013;44:176-182.

38) Sokolova V, Kozlova D, Knuschke T, Buer J, Westendorf AM, Epple M. Mechanism of the uptake of cationic and anionic calcium phos-phate nanoparticles by cells. Acta Biomater 2013;9:7527-7535.

39) Godbey WT, Barry MA, Saggau P, Wu KK, Mikos AG. Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. J Biomed Mater Res 2000 Sep 5;51:321-328.

40) Lee M. Apoptosis induced by polyethylen-imine/DNA complex in polymer mediated gene delivery. Bull Korean Chem Soc 2007;28:95-98.

41) Hu Q, Wang J, Shen J, Liu M, Jin X, Tang G, Chu PK. Intracellular pathways and nuclear localization signal peptide-mediated gene transfection by cationic polymeric nanovectors. Biomaterials 2012;33:1135-1145.

42) Tachibana R, Harashima H, Ide N, Ukitsu S, Ohta Y, Suzuki N, Kikuchi H, Shinohara Y, Kiwada H. Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes. Pharm Res 2002;19: 377-381.

43) Zhou R, Geiger RC, Dean DA. Intracellular trafficking of nucleic acids. Expert Opin Drug Deliv 2004;1:127-140.

(Received: August 3, 2013/ Accepted: August 31, 2013)

Corresponding author: Taichi Tenkumo, D.D.S.,Ph.D Division of Liaison Center for Innovative Dentistry Tohoku University Graduate School of Dentistry 4-1 Seiryo machi, Aoba-ku, Sendai 980-8575, Japan Tel: +81-22-717-8368 Fax: +81-22-717-8371 E-mail: [email protected]

protamine increases transfection efficiency and cell

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