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Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA
Biochemical and Biophysical Research Communications 409 (2011) 745–747
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA E.H. Chowdhury ⇑ Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan, Malaysia
a r t i c l e
i n f o
Article history: Received 3 May 2011 Available online 23 May 2011 Keywords: Carbonate apatite Fluoridated apatite Nanocrystal Gene delivery Transgene expression Plasmid DNA Buffering capacity Endosome
a b s t r a c t Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particleforming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Introducing genes or gene-silencing elements, such as small interfering RNA (siRNA) to mammalian cells is a powerful way to modulate the cellular functions. Cytoplasmic delivery of a foreign gene is followed by its nuclear transport enabling its transcription into mRNA which is subsequently transported out to the cytoplasm translating into a specific protein. On the other hand, synthetically designed siRNA after passing through the cell membrane, binds and cleaves its target mRNA, blocking the expression of the particular host gene. Thus, delivery of DNA or RNA, an essential tool to turn on and off the expression of a particular gene, is highly promising in developing new therapeutic concepts, such as, gene therapy and DNA vaccination that are likely to revolutionize the clinical medicine in the future [1]. Although, viral systems are currently the most effective means of DNA delivery, some major limitations, especially those of immunogenicity and carcinogenicity, accelerated research on non-viral vectors, the majority of which are cationic polymers, lipids and peptides [1]. Negatively charged DNA is usually condensed with cationic vectors to allow formation of either positively charged complexes capable of interacting electrostatically with anionic heparan sulfate proteoglycans (syndecans) on cell surface or elec⇑ Fax: +603 8656 7229. E-mail address: [email protected] 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.079
trically neutral complexes that are further modified in order to specifically recognize cell membrane receptors, with the final outcome of internalization across the membrane through endocytosis [2]. DNA that survives degradation by endoplasmic and cytoplasmic nuclease, must dissociate from the condensed complexes either before or after nuclear translocation through nuclear pore or during cell division [3]. Recently, we have developed pH-sensitive carrier for DNA based on some fascinating properties of inorganic nanoparticles of carbonate apatite. By virtue of its unique surface area created predominantly by Ca2+-rich domains, carbonate apatite particle can bind negatively charged DNA molecules (DNA, RNA or proteins) through high affinity interactions whereas due to the existence of other components such as PO43 and CO32 in the crystal structure, the particle tends to be dissolved in acidic compartment of endosome, releasing DNA in the endosome [4,5]. Since the acid-triggered particle dissolution could result in massive proton influx and development of osmotic pressure across the endosomal membrance, endosome swelling followed by its rupture could finally enable release of the DNA in cytosol [5]. Here we demonstrate the dramatic enhancement of transgene expression by inclusion of fluoride in the formulation of carbonate apatite and correlate the higher transfection efficiency to the longer half-life of the plasmid DNA intracellularly delivered by apparently formed fluoridated carbonate apatite in comparison to carbonate apatite only.
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E.H. Chowdhury / Biochemical and Biophysical Research Communications 409 (2011) 745–747
2. Materials and methods 2.1. Cell culture HeLa cells were cultured in 75 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS), 50 lg penicillin ml 1, 50 lg streptomycin ml 1 and 100 lg neomycin ml 1 at 37 °C in a humidified 5% CO2containing atmosphere. 2.2. Transfection of cells Cells from the exponentially growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection. 3 ll of 1 M CaCl2 was mixed with 2 lg of plasmid DNA in 1 ml of fresh serum-free HCO3 -buffered (pH 7.5) medium (DMEM) and incubated for 30 min at 37 °c for complete generation of DNA/carbonate apatite particles. For generation of fluoridated carbonate apatite, 0.1 lM to 3 mM of NaF was maintained along with 3 mM CaCl2 in the media before the incubation period. 10% FBS was adjusted to the particles of various formulations, followed by 4 h incubation with HeLa cells. 100 nM of chloroquine was maintained in one of the samples of carbonate apatite following addition of serum. After replacement of the particle-containing medium with serum-supplemented medium, the cells were cultured for 1 day. Luciferase gene expression was monitored by using a commercial kit (Promega) and photon counting (TD-20/20 Luminometer, USA). Each transfection experiment was done in triplicate and transfection efficiency was expressed as mean light units per mg of cell protein. 2.3. Confocal laser scanning microscopy pGL3 vector was labeled with PI at a PI/DNA ratio of 1:1 and the particles generated in different formulations using this labeled plasmid DNA and subsequently supplemented with 10% FBS and (in case-basis) 200 nM bafilomycin A1, were incubated with HeLa cells for 4 h. After replacement of the particle-containing medium with fresh medium and removal of the extracellularly-bound particles with 5 mM EDTA in PBS, the cells were further cultured for an additional 5 h before being observed by confocal scanning microscope, LEICA TCS-NT. 2.4. Southern blot analysis Following 4 h incubation of HeLa cells with carbonate apatite and its highly fluoridated and slightly fluoridated forms, each containing 2 lg of pGL3 vector and in some cases, an additional 5 h of incubation after the cells were washed with fresh medium and treated with 5 mM EDTA in PBS, total DNA (genomic DNA and internalized plasmid DNA) was isolated and quantified spectrophotometrically (A260/A280). One-fifth of the isolated DNA was subjected to electrophoresis on 0.8% agarose gels and transferred onto a positively charged nylon membrane (Hybond-N; Amersham), by capillary force. Hybridization was carried out with radio-labeled probe and the membrane was developed and exposed to an X-ray film in a dark room. 3. Results and discussion Since high concentrations of fluoride added during preparation of carbonate apatite particles is known to reduce the solubility of the endocytosed particles in endosomal acidic vesicles with the consequence of inefficient cytoplasmic release and poor transfection activity of the DNA being carried by the particles [5], the effects of
Fig. 1. Changes in luciferase expression for lM concentrations of F added during formation of DNA/carbonate apatite particles. After incubation of HeLa cells with the particles, cells were washed with EDTA and grown for 1 day. 100 lM of chloroquine was added during incubation of the cells with DNA/carbonate apatite particles.
very low concentrations of fluoride was investigated on the final transgene expression efficiency of carbonate apatite. As shown in Fig. 1, by gradually decreasing fluoride from 100 lM to 1 lM in 1 ml of bicarbonate-buffered DMEM medium having exogenously added Ca2+ and luciferase plasmid so as to facilitate particle formation at 37 °C, the transfection efficacy, following a consecutive 4 h incubation of HeLa cells with the generated particles and additional one day incubation after removal of the extracellular particles, was found dramatically enhanced by almost 100-fold compared to fluoride-free particles. A further decrease of fluoride concentration to 0.1 lM was accompanied by a decline in transgene expression being 50-times more efficient compared to carbonate apatite alone (Fig. 1). Chloroquine which is known as a potent endosome-buffering agent, could similarly increase the transfection efficiency of carbonate apatite when incubated at 100 nM with particles and cells for 4 h (Fig. 1), suggesting that low level of fluoride might enable carbonate apatite to dissolve in endosomal acidic pH at an appropriate rate being neither too fast as for carbonate apatite nor too slow like highly fluoridated apatite, thus buffering the endosome, protecting the DNA against nucleases and facilitating the endosomal escape of the DNA. To elucidate how fluoride inclusion promoted carbonate apatitemediated transgene expression, plasmid DNA was labeled with propidium iodide (PI), a pH-insensitive dye, subjected to complexation with nanoparticles of carbonate apatite either in absence or presence of fluoride and allowed for cellular uptake for 4 h in the first phase and another 5 h either in absence or presence of bafilomycin A1 in the second phase after EDTA-aided removal of cell-associated DNA (Fig. 2A). As shown in Fig. 2, A-a, fluorescence intensity of PI was significantly quenched after 5 h probably due to the nucleasemediated degradation of free plasmid DNA delivered by carbonate apatite particles, but remained sustained for the cells treated with the same particles in presence of bafilomycin A1 after 4 h of DNA uptake, indicating that relatively lower level of transfection efficiency of carbonate apatite might owe to its high acid solubility releasing associated DNA instantly after cellular internalization and resulting in nuclease-mediated cleavage of significant amount of the released DNA. The higher fluorescence intensity in bafilomycin A1-treated cells was possibly due to its endosome-buffering capacity by inhibiting the v-ATPase, a proton pump engaged in endosomal acidification [5]. DNA was also found almost undegraded (Fig. 2, A-b) when uptake of DNA was mediated by (highly or slightly) fluoridated carbonate apatite, suggesting that existence of fluoride in carbonate apatite prevents DNA degradation either by ensuring sustained release of DNA with very lower fluoride level or continuous binding of DNA to the apatite with higher level of fluo-
E.H. Chowdhury / Biochemical and Biophysical Research Communications 409 (2011) 745–747
747
Fig. 2. Intracellular stability of plasmid DNA released from apatite crystals, assessed by fluorescence intensity of PI (A) and Southern blotting (B). HeLa cells were incubated with DNA/carbonate apatite particles for 4 h and after EDTA treatment, incubated for an additional 5 h either in absence or presence of 200 nM bafilomycin A1 (A-a). Similarly cells were incubated with DNA/fluoridated carbonate apatite particles for 4 h, treated with EDTA and incubated for an extra 5 h (A-b). White and pink bars indicate 20 and 50 lm, respectively. Cells were incubated with DNA/carbonate apatite and DNA/fluoridated carbonate apatite particles for 4 h, treated with EDTA and incubated for an additional 5 h (B) prior to total DNA isolation and Southern blotting. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
ride. The Southern blot analysis as shown in Fig. 2B based on the plasmid DNA isolated after 4 h as well as the DNA isolated after a total period of 9 h from the cells treated similarly (as described above) proved the notions made by confocal microscopic analysis. Thus an intermediate rate of DNA release from the particles during vesicular acidification could avoid significant DNA degradation, thereby increasing the half-life of the DNA for subsequent nuclear localization and expression. Acknowledgment This work has financially been supported by a research grant (Project ID 02-02-09-SF0013) of the Ministry of Science, Technology and Innovation (MOSTI), Malaysia.
References [1] E.H. Chowdhury, T. Akaike, Bio-functional inorganic materials: an attractive branch of gene-based nan-medicine delivery for 21st century, Curr. Gene Ther. 5 (2005) 669–676. [2] I. Kopatz, J.S. Remy, J.P. Behr, A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin, J. Gene Med. 6 (2004) 769– 776. [3] E.H. Chowdhury, Nuclear targeting of viral and non-viral DNA, Expert Opin. Drug Deliv. 6 (2009) 697–703. [4] S. Hossain, S. Tada, T. Akaike, E.H. Chowdhury, Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells, Anal. Biochem. 397 (2010) 156–161. [5] E.H. Chowdhury, A. Maruyama, A. Kano, M. Nagaoka, M. Kotaka, S. Hirose, M. Kunou, T. Akaike, pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells, Gene 376 (2006) 87–94.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA E.H. Chowdhury ⇑ Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan, Malaysia
a r t i c l e
i n f o
Article history: Received 3 May 2011 Available online 23 May 2011 Keywords: Carbonate apatite Fluoridated apatite Nanocrystal Gene delivery Transgene expression Plasmid DNA Buffering capacity Endosome
a b s t r a c t Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particleforming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Introducing genes or gene-silencing elements, such as small interfering RNA (siRNA) to mammalian cells is a powerful way to modulate the cellular functions. Cytoplasmic delivery of a foreign gene is followed by its nuclear transport enabling its transcription into mRNA which is subsequently transported out to the cytoplasm translating into a specific protein. On the other hand, synthetically designed siRNA after passing through the cell membrane, binds and cleaves its target mRNA, blocking the expression of the particular host gene. Thus, delivery of DNA or RNA, an essential tool to turn on and off the expression of a particular gene, is highly promising in developing new therapeutic concepts, such as, gene therapy and DNA vaccination that are likely to revolutionize the clinical medicine in the future [1]. Although, viral systems are currently the most effective means of DNA delivery, some major limitations, especially those of immunogenicity and carcinogenicity, accelerated research on non-viral vectors, the majority of which are cationic polymers, lipids and peptides [1]. Negatively charged DNA is usually condensed with cationic vectors to allow formation of either positively charged complexes capable of interacting electrostatically with anionic heparan sulfate proteoglycans (syndecans) on cell surface or elec⇑ Fax: +603 8656 7229. E-mail address: [email protected] 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.079
trically neutral complexes that are further modified in order to specifically recognize cell membrane receptors, with the final outcome of internalization across the membrane through endocytosis [2]. DNA that survives degradation by endoplasmic and cytoplasmic nuclease, must dissociate from the condensed complexes either before or after nuclear translocation through nuclear pore or during cell division [3]. Recently, we have developed pH-sensitive carrier for DNA based on some fascinating properties of inorganic nanoparticles of carbonate apatite. By virtue of its unique surface area created predominantly by Ca2+-rich domains, carbonate apatite particle can bind negatively charged DNA molecules (DNA, RNA or proteins) through high affinity interactions whereas due to the existence of other components such as PO43 and CO32 in the crystal structure, the particle tends to be dissolved in acidic compartment of endosome, releasing DNA in the endosome [4,5]. Since the acid-triggered particle dissolution could result in massive proton influx and development of osmotic pressure across the endosomal membrance, endosome swelling followed by its rupture could finally enable release of the DNA in cytosol [5]. Here we demonstrate the dramatic enhancement of transgene expression by inclusion of fluoride in the formulation of carbonate apatite and correlate the higher transfection efficiency to the longer half-life of the plasmid DNA intracellularly delivered by apparently formed fluoridated carbonate apatite in comparison to carbonate apatite only.
746
E.H. Chowdhury / Biochemical and Biophysical Research Communications 409 (2011) 745–747
2. Materials and methods 2.1. Cell culture HeLa cells were cultured in 75 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS), 50 lg penicillin ml 1, 50 lg streptomycin ml 1 and 100 lg neomycin ml 1 at 37 °C in a humidified 5% CO2containing atmosphere. 2.2. Transfection of cells Cells from the exponentially growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection. 3 ll of 1 M CaCl2 was mixed with 2 lg of plasmid DNA in 1 ml of fresh serum-free HCO3 -buffered (pH 7.5) medium (DMEM) and incubated for 30 min at 37 °c for complete generation of DNA/carbonate apatite particles. For generation of fluoridated carbonate apatite, 0.1 lM to 3 mM of NaF was maintained along with 3 mM CaCl2 in the media before the incubation period. 10% FBS was adjusted to the particles of various formulations, followed by 4 h incubation with HeLa cells. 100 nM of chloroquine was maintained in one of the samples of carbonate apatite following addition of serum. After replacement of the particle-containing medium with serum-supplemented medium, the cells were cultured for 1 day. Luciferase gene expression was monitored by using a commercial kit (Promega) and photon counting (TD-20/20 Luminometer, USA). Each transfection experiment was done in triplicate and transfection efficiency was expressed as mean light units per mg of cell protein. 2.3. Confocal laser scanning microscopy pGL3 vector was labeled with PI at a PI/DNA ratio of 1:1 and the particles generated in different formulations using this labeled plasmid DNA and subsequently supplemented with 10% FBS and (in case-basis) 200 nM bafilomycin A1, were incubated with HeLa cells for 4 h. After replacement of the particle-containing medium with fresh medium and removal of the extracellularly-bound particles with 5 mM EDTA in PBS, the cells were further cultured for an additional 5 h before being observed by confocal scanning microscope, LEICA TCS-NT. 2.4. Southern blot analysis Following 4 h incubation of HeLa cells with carbonate apatite and its highly fluoridated and slightly fluoridated forms, each containing 2 lg of pGL3 vector and in some cases, an additional 5 h of incubation after the cells were washed with fresh medium and treated with 5 mM EDTA in PBS, total DNA (genomic DNA and internalized plasmid DNA) was isolated and quantified spectrophotometrically (A260/A280). One-fifth of the isolated DNA was subjected to electrophoresis on 0.8% agarose gels and transferred onto a positively charged nylon membrane (Hybond-N; Amersham), by capillary force. Hybridization was carried out with radio-labeled probe and the membrane was developed and exposed to an X-ray film in a dark room. 3. Results and discussion Since high concentrations of fluoride added during preparation of carbonate apatite particles is known to reduce the solubility of the endocytosed particles in endosomal acidic vesicles with the consequence of inefficient cytoplasmic release and poor transfection activity of the DNA being carried by the particles [5], the effects of
Fig. 1. Changes in luciferase expression for lM concentrations of F added during formation of DNA/carbonate apatite particles. After incubation of HeLa cells with the particles, cells were washed with EDTA and grown for 1 day. 100 lM of chloroquine was added during incubation of the cells with DNA/carbonate apatite particles.
very low concentrations of fluoride was investigated on the final transgene expression efficiency of carbonate apatite. As shown in Fig. 1, by gradually decreasing fluoride from 100 lM to 1 lM in 1 ml of bicarbonate-buffered DMEM medium having exogenously added Ca2+ and luciferase plasmid so as to facilitate particle formation at 37 °C, the transfection efficacy, following a consecutive 4 h incubation of HeLa cells with the generated particles and additional one day incubation after removal of the extracellular particles, was found dramatically enhanced by almost 100-fold compared to fluoride-free particles. A further decrease of fluoride concentration to 0.1 lM was accompanied by a decline in transgene expression being 50-times more efficient compared to carbonate apatite alone (Fig. 1). Chloroquine which is known as a potent endosome-buffering agent, could similarly increase the transfection efficiency of carbonate apatite when incubated at 100 nM with particles and cells for 4 h (Fig. 1), suggesting that low level of fluoride might enable carbonate apatite to dissolve in endosomal acidic pH at an appropriate rate being neither too fast as for carbonate apatite nor too slow like highly fluoridated apatite, thus buffering the endosome, protecting the DNA against nucleases and facilitating the endosomal escape of the DNA. To elucidate how fluoride inclusion promoted carbonate apatitemediated transgene expression, plasmid DNA was labeled with propidium iodide (PI), a pH-insensitive dye, subjected to complexation with nanoparticles of carbonate apatite either in absence or presence of fluoride and allowed for cellular uptake for 4 h in the first phase and another 5 h either in absence or presence of bafilomycin A1 in the second phase after EDTA-aided removal of cell-associated DNA (Fig. 2A). As shown in Fig. 2, A-a, fluorescence intensity of PI was significantly quenched after 5 h probably due to the nucleasemediated degradation of free plasmid DNA delivered by carbonate apatite particles, but remained sustained for the cells treated with the same particles in presence of bafilomycin A1 after 4 h of DNA uptake, indicating that relatively lower level of transfection efficiency of carbonate apatite might owe to its high acid solubility releasing associated DNA instantly after cellular internalization and resulting in nuclease-mediated cleavage of significant amount of the released DNA. The higher fluorescence intensity in bafilomycin A1-treated cells was possibly due to its endosome-buffering capacity by inhibiting the v-ATPase, a proton pump engaged in endosomal acidification [5]. DNA was also found almost undegraded (Fig. 2, A-b) when uptake of DNA was mediated by (highly or slightly) fluoridated carbonate apatite, suggesting that existence of fluoride in carbonate apatite prevents DNA degradation either by ensuring sustained release of DNA with very lower fluoride level or continuous binding of DNA to the apatite with higher level of fluo-
E.H. Chowdhury / Biochemical and Biophysical Research Communications 409 (2011) 745–747
747
Fig. 2. Intracellular stability of plasmid DNA released from apatite crystals, assessed by fluorescence intensity of PI (A) and Southern blotting (B). HeLa cells were incubated with DNA/carbonate apatite particles for 4 h and after EDTA treatment, incubated for an additional 5 h either in absence or presence of 200 nM bafilomycin A1 (A-a). Similarly cells were incubated with DNA/fluoridated carbonate apatite particles for 4 h, treated with EDTA and incubated for an extra 5 h (A-b). White and pink bars indicate 20 and 50 lm, respectively. Cells were incubated with DNA/carbonate apatite and DNA/fluoridated carbonate apatite particles for 4 h, treated with EDTA and incubated for an additional 5 h (B) prior to total DNA isolation and Southern blotting. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
ride. The Southern blot analysis as shown in Fig. 2B based on the plasmid DNA isolated after 4 h as well as the DNA isolated after a total period of 9 h from the cells treated similarly (as described above) proved the notions made by confocal microscopic analysis. Thus an intermediate rate of DNA release from the particles during vesicular acidification could avoid significant DNA degradation, thereby increasing the half-life of the DNA for subsequent nuclear localization and expression. Acknowledgment This work has financially been supported by a research grant (Project ID 02-02-09-SF0013) of the Ministry of Science, Technology and Innovation (MOSTI), Malaysia.
References [1] E.H. Chowdhury, T. Akaike, Bio-functional inorganic materials: an attractive branch of gene-based nan-medicine delivery for 21st century, Curr. Gene Ther. 5 (2005) 669–676. [2] I. Kopatz, J.S. Remy, J.P. Behr, A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin, J. Gene Med. 6 (2004) 769– 776. [3] E.H. Chowdhury, Nuclear targeting of viral and non-viral DNA, Expert Opin. Drug Deliv. 6 (2009) 697–703. [4] S. Hossain, S. Tada, T. Akaike, E.H. Chowdhury, Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells, Anal. Biochem. 397 (2010) 156–161. [5] E.H. Chowdhury, A. Maruyama, A. Kano, M. Nagaoka, M. Kotaka, S. Hirose, M. Kunou, T. Akaike, pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells, Gene 376 (2006) 87–94.