- Email: [email protected]
pei ternary complexes: A novel strategy for magnetofection
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
References [1] M.A. Mintzer, E.E. Simanek, Nonviral vectors for gene deliveryChem. Rev. 109 (2009) 259–302. [2] W.T. Godbey, K.K. Wu, A.G. Mikos, Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle, J. Biomed. Mater. Res. 45 (1999) 268–275. [3] X.L. Jiang, M.C. Lok, W.E. Hennink, Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery, Bioconjug. Chem. 18 (2007) 2077–2084. [4] F.-H. Meng, W.E. Hennink, Z.-Y. Zhong, Reduction-sensitive polymers and bioconjugates for biomedical applications, Biomaterials 30 (2009) 2180–2198. [5] F.Y. Dai, P. Sun, Y.J. Liu, W.G. Liu, Redox-cleavable star cationic PDMAEMA by armfirst approach of ATRP as a nonviral vector for gene delivery, Biomaterials 31 (2010) 559–569. [6] S. Bauhuber, C. Hozsa, M. Breunig, A. Göpferich, Delivery of nucleic acids via disulfidebased carrier systems, Adv. Mater. 21 (2009) 3286–3306.
doi:10.1016/j.jconrel.2011.08.060
Dendrimer modified magnetic iron oxide nanoparticle/dna/pei ternary complexes: A novel strategy for magnetofection Wen-Ming Liu, Ya-Nan Xue, Wen-Tao He, Ren-Xi Zhuo, Shi-Wen Huang Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, China E-mail address: [email protected] (S.-W. Huang). Summary Polyamidoamine dendrimer modified magnetic iron oxide nanoparticle/DNA/PEI (25 kDa) ternary complexes were used for the magnetofection of mammalian cells. The results indicated that the transfection efficiencies of COS 7 cells with ternary magnetoplexes significantly increased when a magnetic field was applied, especially in the presence of 10% serum. Further evidence from Prussian blue staining of iron inside the cells and intracellular trafficking of Cy-3 labeled DNA demonstrated that the magnetic field quickly gathered the magnetoplexes to the surface of target cells and enhanced the uptake of the ternary magnetoplexes by the cells. This represents a novel strategy for polycation-based in vitro gene delivery enhanced by a magnetic field. Keywords: Iron oxide nanoparticles, Dendrimer, Polyethylenimine, Magnetofection, Gene delivery Introduction Magnetofection, a recently developed technology, has gained considerable attention because it can quickly gather the magnetoplexes to the surface of target cells in the presence of magnetic field and enhance the transfection efficiency up to several-hundred-fold [1]. In magnetofection, the magnetic particles were generally functionalized with polycations or cationic lipids to condense foreign gene [2–5]. In previous work, cationic magnetic particles were mixed directly with a foreign gene to form positively charged magnetoplexes which were used in gene delivery. In this work, we first mixed dendrimer modified superparamagnetic iron oxide (SPION) with plasmid DNA at low mass ratio to form negatively charged magnetoplexes, and then condensed cationic polymers, such as PEI 25 kDa, to form ternary magnetoplexes with positive surface charge. The effect of magnetic field on the transfection efficiencies, iron uptake, DNA uptake and intracellular trafficking was investigated. Experimental methods Branched PEI 25 kDa was obtained from Sigma-Aldrich (St. Louis, MO). PGL-3 plasmid was purchased from Promega (Madison, WI,
e159
USA). Generation 6 of PAMAM dendrimer modified SPION (G6) was synthesized as described previously [5]. Transfection experiments were performed with COS 7 cells using PGL-3 plasmid as the reporter gene. 100 μL of magnetoplexes or polyplexes were incubated with the cells in the presence or absence of a magnetic field for predetermined times in serum free DMEM or 10% FBS-containing DMEM. The luciferase expression in transfected cells was measured using the luciferase assay kit on a Lumat 9507 luminometer (Berthold, Germany). Cellular uptake of magnetoplexes was visualized by both Prussian blue staining and confocal laser scanning microscope (CLSM). For Prussian blue staining, COS 7 cells were incubated with magnetoplexes for 15 min, 60 min and 4 h in 10% FBS-containing DMEM in the presence or absence of magnetic field. After the incubation, magnetoplexes-containing medium was removed and replaced with fresh 10% FBS-containing DMEM. Cells were further cultured for different times (3.75 h, 3 h or 0 h) and stained with potassium ferrocyanide. The stained cells were observed using a reverse microscope (Ti, Nikon, Japan). For CLMS observation, Cy3labeled pDNA was used in the formation of magnetoplexes. Cells were incubated with magnetoplexes for 15 min in FBS-containing DMEM in the presence or absence of magnetic field and further cultured in fresh FBS-containing DMEM for different time (0 h, 4 h and 24 h). At the end of each culture period, cells were fixed with 4% paraformaldehyde, stained with Hoechst 33258 and visualized with a EZ-C1 confocal laser scanning microscope (CLSM, Nikon, Japan). Results and discussion First, transfection conditions for magnetofection were optimized and we found that the optimal mass ratio of G6/DNA was 2, and the optimal N/P ratio of PEI/DNA was 10. These conditions were used in all following experiments. The results are shown in Fig. 1. The luciferase expression (RLU/mg protein) in COS 7 cells transfected with ternary magnetoplexes, both with and without magnetic field, was higher than that with PEI/pGL-3 polyplexes. When 15 min incubation was applied, the transfection efficiency of magnetoplexes with a magnetic field was 300 fold higher than PEI-mediated transfection. This implied that the magnetic field could rapidly accumulate the magnetoplexes onto the surface of target cells. We also investigated the transfection efficiency in 10% FBS-containing DMEM. The presence of 10% serum significantly reduced the transfection efficiency of PEI-mediated transfection. However, the transfection efficiency of magnetoplexes with magnetic field was found to decrease slightly in the presence of serum. The transfection efficiency of magnetoplexes with magnetic field application and 15 min incubation was even 25 fold higher than PEI mediated transfection with 4 h incubation. This indicated that the fast accumulation of magnetoplexes was not affected by serum. The transfection efficiencies of magnetoplexes in the absence of magnetic field were also higher than PEI mediated transfection, which is due to the “particle sediment effect” [6]. To confirm the fast accumulation of magnetoplexes with a magnetic field and investigate the uptake of magnetoplexes, Prussian blue staining and intracellular trafficking experiments were designed. For Prussian blue staining, transfected cells were stained with potassium ferrocyanide and visualized with a reverse microscope. As shown in Fig. 2, the number of blue-stained cells and blue granules in each cell increased when a magnetic field was applied, this indicated that the uptake of iron in magnetoplexes was significantly enhanced by a magnetic field. We also found that the blue granules in the cells increased a little with increasing incubation time. This observation implied that the magnetoplexes could rapidly gather onto the surface of target cells and be taken up by the cells.
e160
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Conclusion We here described a new strategy to enhance the transfection efficiency of polycation with a magnetic field using dendrimer modified magnetic iron oxide nanoparticle/DNA/PEI ternary magnetoplexes. Prussian blue staining and intracellular trafficking experiments demonstrated that the accumulation of magnetoplexes on the surface of cells and uptake by cells was enhanced by a magnetic field. The advantage of this kind of ternary complexes as magnetic carriers is that only a small amount of magnetic nanoparticles are necessary for highly efficient magnetofection. Furthermore, this ternary system is able to be applied for any kind of polycation, which will be developed as a general method for magnetofection.
Fig. 1. In vitro transfection efficiencies of the magnetoplexes in COS-7 cells in the presence (dashed) or absence (solid) of 10% FBS.
In the intracellular trafficking experiments, transfected cells were fixed, nuclear stained and observed with a confocal laser scanning microscope. The results are shown in Fig. 3. When a magnetic field was used, large numbers of fluorescent particles rapidly attached to the cell surface after 15 min incubation. Four hours after transfection, patches of fluorescence randomly dispersed in cytoplasmic compartments, and some fluorescent particles were observed inside the cell nucleus. 24 h later, more fluorescent dots were observed in the nucleus. In contrast, only a few fluorescent particles were taken up by the cells in the absence of a magnetic field. We can conclude that enhancement of transfection efficiencies of dendrimer modified magnetic iron oxide nanoparticle/ DNA/PEI magnetoplexes with a magnetic field is due to improving the uptake of ternary magnetoplexes in the presence of a magnetic field.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (20874076), National Basic Research Program of China (2005CB623903, 2009CB930300) and Program for New Century Excellent Talents in University (08-0410). References [1] F. Scherer, M. Anton, U. Schillinger, J. Henkel, C. Bergemann, A. Kruger, B. Gansbacher, C. Plank, Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivoGene Ther. 9 (2002) 102–109. [2] R. Namgung, K. Singha, M.K. Yu, S. Jon, Y.S. Kim, Y. Ahn, I. Park, W.J. Kim, Hybrid superparamagnetic iron oxide nanoparticle-branched polyethylenimine magnetoplexes for gene transfection of vascular endothelial cells, Biomaterials 31 (2010) 4204–4213. [3] F.M. Kievit, O. Veiseh, N. Bhattarai, C. Fang, J.W. Gunn, D. Lee, R.G. Ellenbogen, J.M. Olson, M.Q. Zhang, PEI-PEG-chitosan-copolymer-coated iron oxide nanoparticles for safe gene delivery synthesis, complexation and transfection, Adv. Funct. Mater. 19 (2009) 2244–2251. [4] X.G. Pan, J.J. Guan, J.W. Yoo, A.J. Epstein, L.J. Lee, R.J. Lee, Cationic lipid-coated magnetic nanoparticles associated with transferrin for gene delivery, Int. J. Pharm. 358 (2008) 263–270. [5] B.F. Pan, D.X. Cui, Y. Sheng, C. Ozkan, F. Gao, R. He, Q. Li, P. Xu, T. Huang, Dendrimermodified magnetic nanoparticles enhance efficiency of gene delivery system, Cancer Res. 67 (2007) 8156–8163. [6] D. Luo, W.M. Saltzman, Enhancement of transfection by physical concentration of DNA at the cell surface, Nat. Biotechnol. 18 (2000) 893–895.
doi:10.1016/j.jconrel.2011.08.061
Chitosan/VEGF-sIRNA nanoparticle for gene silencing
Fig. 2. Images of Prussian blue stained cells incubated with magnetoplexes in the absence (A) or presence (B) of magnetic field.
Fig. 3. Images of cells incubated with Cy-3 labeled pDNA magnetoplexes in the absence (A) or presence (B) of magnetic field. The scale bar is 10 μm.
Yan Yang1,2, Xiudong Liu3, Demeng Zhang1,2, Weiting Yu1, Guojun lv1, Hongguo Xie1, Jiani Zheng1,2, Xiaojun Ma1 1 Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 3 College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, China E-mail addresses: [email protected] (X. Liu), [email protected] (X. Ma). Summary The up-regulation of VEGF interrelated to tumor angiogenesis provides a target for tumor treatment. We describe the feasibility of using chitosan nanoparticles for successful VEGF-siRNA delivery to finally reduce the VEGF level in a mouse melanoma model in vitro. The chitosan/VEGF-siRNA (CTS/siRNA) nanoparticles were prepared with a size of 110–200 nm and zeta potential of ~ 20 mV. Moreover, the stable nanoparticles can successfully transport VEGF-siRNA into cells, and release siRNA for VEGF gene silencing.
References [1] M.A. Mintzer, E.E. Simanek, Nonviral vectors for gene deliveryChem. Rev. 109 (2009) 259–302. [2] W.T. Godbey, K.K. Wu, A.G. Mikos, Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle, J. Biomed. Mater. Res. 45 (1999) 268–275. [3] X.L. Jiang, M.C. Lok, W.E. Hennink, Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery, Bioconjug. Chem. 18 (2007) 2077–2084. [4] F.-H. Meng, W.E. Hennink, Z.-Y. Zhong, Reduction-sensitive polymers and bioconjugates for biomedical applications, Biomaterials 30 (2009) 2180–2198. [5] F.Y. Dai, P. Sun, Y.J. Liu, W.G. Liu, Redox-cleavable star cationic PDMAEMA by armfirst approach of ATRP as a nonviral vector for gene delivery, Biomaterials 31 (2010) 559–569. [6] S. Bauhuber, C. Hozsa, M. Breunig, A. Göpferich, Delivery of nucleic acids via disulfidebased carrier systems, Adv. Mater. 21 (2009) 3286–3306.
doi:10.1016/j.jconrel.2011.08.060
Dendrimer modified magnetic iron oxide nanoparticle/dna/pei ternary complexes: A novel strategy for magnetofection Wen-Ming Liu, Ya-Nan Xue, Wen-Tao He, Ren-Xi Zhuo, Shi-Wen Huang Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, China E-mail address: [email protected] (S.-W. Huang). Summary Polyamidoamine dendrimer modified magnetic iron oxide nanoparticle/DNA/PEI (25 kDa) ternary complexes were used for the magnetofection of mammalian cells. The results indicated that the transfection efficiencies of COS 7 cells with ternary magnetoplexes significantly increased when a magnetic field was applied, especially in the presence of 10% serum. Further evidence from Prussian blue staining of iron inside the cells and intracellular trafficking of Cy-3 labeled DNA demonstrated that the magnetic field quickly gathered the magnetoplexes to the surface of target cells and enhanced the uptake of the ternary magnetoplexes by the cells. This represents a novel strategy for polycation-based in vitro gene delivery enhanced by a magnetic field. Keywords: Iron oxide nanoparticles, Dendrimer, Polyethylenimine, Magnetofection, Gene delivery Introduction Magnetofection, a recently developed technology, has gained considerable attention because it can quickly gather the magnetoplexes to the surface of target cells in the presence of magnetic field and enhance the transfection efficiency up to several-hundred-fold [1]. In magnetofection, the magnetic particles were generally functionalized with polycations or cationic lipids to condense foreign gene [2–5]. In previous work, cationic magnetic particles were mixed directly with a foreign gene to form positively charged magnetoplexes which were used in gene delivery. In this work, we first mixed dendrimer modified superparamagnetic iron oxide (SPION) with plasmid DNA at low mass ratio to form negatively charged magnetoplexes, and then condensed cationic polymers, such as PEI 25 kDa, to form ternary magnetoplexes with positive surface charge. The effect of magnetic field on the transfection efficiencies, iron uptake, DNA uptake and intracellular trafficking was investigated. Experimental methods Branched PEI 25 kDa was obtained from Sigma-Aldrich (St. Louis, MO). PGL-3 plasmid was purchased from Promega (Madison, WI,
e159
USA). Generation 6 of PAMAM dendrimer modified SPION (G6) was synthesized as described previously [5]. Transfection experiments were performed with COS 7 cells using PGL-3 plasmid as the reporter gene. 100 μL of magnetoplexes or polyplexes were incubated with the cells in the presence or absence of a magnetic field for predetermined times in serum free DMEM or 10% FBS-containing DMEM. The luciferase expression in transfected cells was measured using the luciferase assay kit on a Lumat 9507 luminometer (Berthold, Germany). Cellular uptake of magnetoplexes was visualized by both Prussian blue staining and confocal laser scanning microscope (CLSM). For Prussian blue staining, COS 7 cells were incubated with magnetoplexes for 15 min, 60 min and 4 h in 10% FBS-containing DMEM in the presence or absence of magnetic field. After the incubation, magnetoplexes-containing medium was removed and replaced with fresh 10% FBS-containing DMEM. Cells were further cultured for different times (3.75 h, 3 h or 0 h) and stained with potassium ferrocyanide. The stained cells were observed using a reverse microscope (Ti, Nikon, Japan). For CLMS observation, Cy3labeled pDNA was used in the formation of magnetoplexes. Cells were incubated with magnetoplexes for 15 min in FBS-containing DMEM in the presence or absence of magnetic field and further cultured in fresh FBS-containing DMEM for different time (0 h, 4 h and 24 h). At the end of each culture period, cells were fixed with 4% paraformaldehyde, stained with Hoechst 33258 and visualized with a EZ-C1 confocal laser scanning microscope (CLSM, Nikon, Japan). Results and discussion First, transfection conditions for magnetofection were optimized and we found that the optimal mass ratio of G6/DNA was 2, and the optimal N/P ratio of PEI/DNA was 10. These conditions were used in all following experiments. The results are shown in Fig. 1. The luciferase expression (RLU/mg protein) in COS 7 cells transfected with ternary magnetoplexes, both with and without magnetic field, was higher than that with PEI/pGL-3 polyplexes. When 15 min incubation was applied, the transfection efficiency of magnetoplexes with a magnetic field was 300 fold higher than PEI-mediated transfection. This implied that the magnetic field could rapidly accumulate the magnetoplexes onto the surface of target cells. We also investigated the transfection efficiency in 10% FBS-containing DMEM. The presence of 10% serum significantly reduced the transfection efficiency of PEI-mediated transfection. However, the transfection efficiency of magnetoplexes with magnetic field was found to decrease slightly in the presence of serum. The transfection efficiency of magnetoplexes with magnetic field application and 15 min incubation was even 25 fold higher than PEI mediated transfection with 4 h incubation. This indicated that the fast accumulation of magnetoplexes was not affected by serum. The transfection efficiencies of magnetoplexes in the absence of magnetic field were also higher than PEI mediated transfection, which is due to the “particle sediment effect” [6]. To confirm the fast accumulation of magnetoplexes with a magnetic field and investigate the uptake of magnetoplexes, Prussian blue staining and intracellular trafficking experiments were designed. For Prussian blue staining, transfected cells were stained with potassium ferrocyanide and visualized with a reverse microscope. As shown in Fig. 2, the number of blue-stained cells and blue granules in each cell increased when a magnetic field was applied, this indicated that the uptake of iron in magnetoplexes was significantly enhanced by a magnetic field. We also found that the blue granules in the cells increased a little with increasing incubation time. This observation implied that the magnetoplexes could rapidly gather onto the surface of target cells and be taken up by the cells.
e160
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Conclusion We here described a new strategy to enhance the transfection efficiency of polycation with a magnetic field using dendrimer modified magnetic iron oxide nanoparticle/DNA/PEI ternary magnetoplexes. Prussian blue staining and intracellular trafficking experiments demonstrated that the accumulation of magnetoplexes on the surface of cells and uptake by cells was enhanced by a magnetic field. The advantage of this kind of ternary complexes as magnetic carriers is that only a small amount of magnetic nanoparticles are necessary for highly efficient magnetofection. Furthermore, this ternary system is able to be applied for any kind of polycation, which will be developed as a general method for magnetofection.
Fig. 1. In vitro transfection efficiencies of the magnetoplexes in COS-7 cells in the presence (dashed) or absence (solid) of 10% FBS.
In the intracellular trafficking experiments, transfected cells were fixed, nuclear stained and observed with a confocal laser scanning microscope. The results are shown in Fig. 3. When a magnetic field was used, large numbers of fluorescent particles rapidly attached to the cell surface after 15 min incubation. Four hours after transfection, patches of fluorescence randomly dispersed in cytoplasmic compartments, and some fluorescent particles were observed inside the cell nucleus. 24 h later, more fluorescent dots were observed in the nucleus. In contrast, only a few fluorescent particles were taken up by the cells in the absence of a magnetic field. We can conclude that enhancement of transfection efficiencies of dendrimer modified magnetic iron oxide nanoparticle/ DNA/PEI magnetoplexes with a magnetic field is due to improving the uptake of ternary magnetoplexes in the presence of a magnetic field.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (20874076), National Basic Research Program of China (2005CB623903, 2009CB930300) and Program for New Century Excellent Talents in University (08-0410). References [1] F. Scherer, M. Anton, U. Schillinger, J. Henkel, C. Bergemann, A. Kruger, B. Gansbacher, C. Plank, Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivoGene Ther. 9 (2002) 102–109. [2] R. Namgung, K. Singha, M.K. Yu, S. Jon, Y.S. Kim, Y. Ahn, I. Park, W.J. Kim, Hybrid superparamagnetic iron oxide nanoparticle-branched polyethylenimine magnetoplexes for gene transfection of vascular endothelial cells, Biomaterials 31 (2010) 4204–4213. [3] F.M. Kievit, O. Veiseh, N. Bhattarai, C. Fang, J.W. Gunn, D. Lee, R.G. Ellenbogen, J.M. Olson, M.Q. Zhang, PEI-PEG-chitosan-copolymer-coated iron oxide nanoparticles for safe gene delivery synthesis, complexation and transfection, Adv. Funct. Mater. 19 (2009) 2244–2251. [4] X.G. Pan, J.J. Guan, J.W. Yoo, A.J. Epstein, L.J. Lee, R.J. Lee, Cationic lipid-coated magnetic nanoparticles associated with transferrin for gene delivery, Int. J. Pharm. 358 (2008) 263–270. [5] B.F. Pan, D.X. Cui, Y. Sheng, C. Ozkan, F. Gao, R. He, Q. Li, P. Xu, T. Huang, Dendrimermodified magnetic nanoparticles enhance efficiency of gene delivery system, Cancer Res. 67 (2007) 8156–8163. [6] D. Luo, W.M. Saltzman, Enhancement of transfection by physical concentration of DNA at the cell surface, Nat. Biotechnol. 18 (2000) 893–895.
doi:10.1016/j.jconrel.2011.08.061
Chitosan/VEGF-sIRNA nanoparticle for gene silencing
Fig. 2. Images of Prussian blue stained cells incubated with magnetoplexes in the absence (A) or presence (B) of magnetic field.
Fig. 3. Images of cells incubated with Cy-3 labeled pDNA magnetoplexes in the absence (A) or presence (B) of magnetic field. The scale bar is 10 μm.
Yan Yang1,2, Xiudong Liu3, Demeng Zhang1,2, Weiting Yu1, Guojun lv1, Hongguo Xie1, Jiani Zheng1,2, Xiaojun Ma1 1 Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 3 College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, China E-mail addresses: [email protected] (X. Liu), [email protected] (X. Ma). Summary The up-regulation of VEGF interrelated to tumor angiogenesis provides a target for tumor treatment. We describe the feasibility of using chitosan nanoparticles for successful VEGF-siRNA delivery to finally reduce the VEGF level in a mouse melanoma model in vitro. The chitosan/VEGF-siRNA (CTS/siRNA) nanoparticles were prepared with a size of 110–200 nm and zeta potential of ~ 20 mV. Moreover, the stable nanoparticles can successfully transport VEGF-siRNA into cells, and release siRNA for VEGF gene silencing.