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DNA Nanoparticles
CHEMICAL AND MOLECULAR CONJUGATES II complex stability and colloidal stability for these nanoparticles. In the absence of TPP stabilization, irregular shaped, large aggregates were obtained when BPEI/siRNA complexes were coated with HA. The quaternary nanoparticles showed increased stability in a solution with the physiological ionic strength and serum condition.
a significantly increased transfection efficiency of polybrene/ DNA[4] and calcium phosphate/DNA[5] complexes by DMSO shock. We investigated the effect of DMSO shock on PEI/DNA nanoparticle-mediated transfection in human fibroblasts (FBs), human embryonic stem cell-derived NCSCs and human fetal tissue-derived immortalized SCs, achieving a tunable transfection by adjusting PEI/ DNA nanoparticle dose, shock duration and DMSO concentration (Fig 1). Depending on cell type, varying levels of transfection efficiency and toxicity were observed. Up to ∼95% transfection efficiency was achieved for SCs, maintaining high cell viability. FBs and NCSCs displayed higher sensitivity to DMSO shock, with 53% and 39% of metabolic activity relative to control for conditions yielding ∼70% transfection efficiency. Interestingly, adjusting transfection conditions resulted in tunable transgene expression levels, while a comparable transfection yield was achieved (Fig 1). Controllable transfection of SCs with the neurotrophic factor pleiotrophin illustrates the utility of this method. This method thus composes an efficient non-viral transfection method with an added control over the average transgene expression level.
Using dual luciferase assay, we have shown that gene silencing efficiency of HA-coated siRNA nanoparticles was enhanced by cocondensation with TPP for both BPEI and LPEI in serum-containing media, compared with that of nanoparticles without TPP (Fig. 2). The results highlight the effectiveness of TPP-co-condensation as a useful strategy to improve the gene silencing efficiency of HA-coated siRNA nanoparticles.
Figure 1: Effect of transfection conditions on transfection efficiency (percent GFP positive cells) and average GFP expression level in human Schwann cells. Bars = average + standard deviation. Average GFP expression level was significantly different between all groups (p < 0.01, n=12, REGW test). Cell metabolic activity was between 78-95% relative to the control group for all conditions, as measured by WST-1 assay. (1) Krick K et al, Curr Opin Biotechnol. 2011, 22, 741. (2) Wang AJ et al, Biomaterials 2011, 32, 5023. (3) Kunath K. et al, J. Control. Release 2003, 89, 113. (4) Kawai S and Nishizawa M., Mol. Cell. Biol. 1984, 4, 1172. (5) Lewis WH, et al, Somatic Cell Genet 1980, 6, 333.
648. DMSO Shock Yields Highly Efficient Transient Transfection by PEI/DNA Nanoparticles
Markus Tammia, Yongjuan Ren, Xingyu Liu, Chris R. Cashman,3,4 Ruifa Mi,3 Tiffany Chung,5 Ahmet Höke,3 Hai-Quan Mao.1,2,6 1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore; 2Translational Tissue Engineering Center, Johns Hopkins University, Baltimore; 3Department of Neurology, Johns Hopkins University, Baltimore; 4MSTP/MD-PhD Programs, Johns Hopkins University, Baltimore; 5Department of Biology, Johns Hopkins University, Baltimore; 6Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, MD. 1,2
1,2
1,2
Safe and efficient non-viral transfection methods are desired for growth factor expression from transplanted cells for tissue regeneration. Indeed, neural crest stem cells (NCSCs) and Schwann cells (SCs) can be transfected to overexpress neurotrophic factors prior to transplantation to promote nerve regeneration[1,2]. Low molecular weight linear poly(ethylene imine) (PEI) is amongst the most efficient and compatible gene carriers[3], but with limited success in transfecting primary cells. Previous studies have shown S250
649. Structure-Activity Relationships in a Series of Pyridinium Gemini Surfactants for Gene Delivery Bearing Hydrophilic Linkers
Visnhu D. Sharma,1 Enronmwon A. Aifuwa,1 Marc A. Ilies.1 1 Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, Pa. The success of gene therapy as a revolutionary way to treat diseases at their core level relies on finding efficient and safe delivery systems for the transfer and expression (transfection) of foreign nucleic acids into patient’ cells and tissues affected by various hereditary or acquired diseases, including cancer. For cystic fibrosis or cancer the delivery of large DNA fragments is required. Viral vectors are either unsuitable (plasmid is too large for the virion) or not safe enough, so synthetic transfection systems can represent a benefic alternative with a better therapeutic index. In this context, positively charged gemini surfactants constitute an interesting alternative, borrowing features from both cationic lipids and cationic surfactants. Thus, they have a charge per mass ratio close to surfactants, while their self-assembling properties can be tuned to resemble those of cationic Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
a significantly increased transfection efficiency of polybrene/ DNA[4] and calcium phosphate/DNA[5] complexes by DMSO shock. We investigated the effect of DMSO shock on PEI/DNA nanoparticle-mediated transfection in human fibroblasts (FBs), human embryonic stem cell-derived NCSCs and human fetal tissue-derived immortalized SCs, achieving a tunable transfection by adjusting PEI/ DNA nanoparticle dose, shock duration and DMSO concentration (Fig 1). Depending on cell type, varying levels of transfection efficiency and toxicity were observed. Up to ∼95% transfection efficiency was achieved for SCs, maintaining high cell viability. FBs and NCSCs displayed higher sensitivity to DMSO shock, with 53% and 39% of metabolic activity relative to control for conditions yielding ∼70% transfection efficiency. Interestingly, adjusting transfection conditions resulted in tunable transgene expression levels, while a comparable transfection yield was achieved (Fig 1). Controllable transfection of SCs with the neurotrophic factor pleiotrophin illustrates the utility of this method. This method thus composes an efficient non-viral transfection method with an added control over the average transgene expression level.
Using dual luciferase assay, we have shown that gene silencing efficiency of HA-coated siRNA nanoparticles was enhanced by cocondensation with TPP for both BPEI and LPEI in serum-containing media, compared with that of nanoparticles without TPP (Fig. 2). The results highlight the effectiveness of TPP-co-condensation as a useful strategy to improve the gene silencing efficiency of HA-coated siRNA nanoparticles.
Figure 1: Effect of transfection conditions on transfection efficiency (percent GFP positive cells) and average GFP expression level in human Schwann cells. Bars = average + standard deviation. Average GFP expression level was significantly different between all groups (p < 0.01, n=12, REGW test). Cell metabolic activity was between 78-95% relative to the control group for all conditions, as measured by WST-1 assay. (1) Krick K et al, Curr Opin Biotechnol. 2011, 22, 741. (2) Wang AJ et al, Biomaterials 2011, 32, 5023. (3) Kunath K. et al, J. Control. Release 2003, 89, 113. (4) Kawai S and Nishizawa M., Mol. Cell. Biol. 1984, 4, 1172. (5) Lewis WH, et al, Somatic Cell Genet 1980, 6, 333.
648. DMSO Shock Yields Highly Efficient Transient Transfection by PEI/DNA Nanoparticles
Markus Tammia, Yongjuan Ren, Xingyu Liu, Chris R. Cashman,3,4 Ruifa Mi,3 Tiffany Chung,5 Ahmet Höke,3 Hai-Quan Mao.1,2,6 1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore; 2Translational Tissue Engineering Center, Johns Hopkins University, Baltimore; 3Department of Neurology, Johns Hopkins University, Baltimore; 4MSTP/MD-PhD Programs, Johns Hopkins University, Baltimore; 5Department of Biology, Johns Hopkins University, Baltimore; 6Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, MD. 1,2
1,2
1,2
Safe and efficient non-viral transfection methods are desired for growth factor expression from transplanted cells for tissue regeneration. Indeed, neural crest stem cells (NCSCs) and Schwann cells (SCs) can be transfected to overexpress neurotrophic factors prior to transplantation to promote nerve regeneration[1,2]. Low molecular weight linear poly(ethylene imine) (PEI) is amongst the most efficient and compatible gene carriers[3], but with limited success in transfecting primary cells. Previous studies have shown S250
649. Structure-Activity Relationships in a Series of Pyridinium Gemini Surfactants for Gene Delivery Bearing Hydrophilic Linkers
Visnhu D. Sharma,1 Enronmwon A. Aifuwa,1 Marc A. Ilies.1 1 Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, Pa. The success of gene therapy as a revolutionary way to treat diseases at their core level relies on finding efficient and safe delivery systems for the transfer and expression (transfection) of foreign nucleic acids into patient’ cells and tissues affected by various hereditary or acquired diseases, including cancer. For cystic fibrosis or cancer the delivery of large DNA fragments is required. Viral vectors are either unsuitable (plasmid is too large for the virion) or not safe enough, so synthetic transfection systems can represent a benefic alternative with a better therapeutic index. In this context, positively charged gemini surfactants constitute an interesting alternative, borrowing features from both cationic lipids and cationic surfactants. Thus, they have a charge per mass ratio close to surfactants, while their self-assembling properties can be tuned to resemble those of cationic Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy