New Insights into Gene Delivery to Human Neuronal Precursor NT2 Cells: A Comparative Study between Lipoplexes, Nioplexes, and Polyplexes
The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies req...
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| Veröffentlicht in: | Molecular pharmaceutics Jg. 12; H. 11; S. 4056 - 4066 |
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| Format: | Journal Article |
| Sprache: | Englisch |
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02.11.2015
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| ISSN: | 1543-8392 |
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| Abstract | The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies requires a profound knowledge of the whole transfection process. In this work, we elaborated and characterized in terms of size and zeta potential three different nonviral vectors: lipoplexes (144 nm; -29.13 mV), nioplexes (142.5 nm; +35.4 mV), and polyplexes (294.8 nm; +15.1 mV). We compared the transfection efficiency, cellular uptake, and intracellular trafficking of the three vectors in NT2 cell line. Lipoplexes exhibited the highest percentages of EGFP positive cells. The values obtained with polyplexes were lower compared to lipoplexes but higher than the percentages obtained with nioplexes. Cellular uptake results had a clear correlation with respect to the corresponding transfection efficiencies. Regarding the endocytosis mechanism, lipoplexes enter in the cell, mainly, via clathrin-mediated endocytosis (CME) while polyplexes via caveolae-mediated endocytosis (CvME). Nioplexes were discarded for this experiment due to their low cellular uptake. By simulating an artificial endosome, we demonstrated that the vectors were able to release the DNA cargo once inside the late endosome. The data collected from this assay showed that at 6 h the genetic material carried by polyplexes was still located in the late endosome, while DNA carried by lipoplexes was already in the nucleus. This result indicates a faster intracellular traffic of the lipid-based vectors. Overall, our work gives new insights into the transfection process of NT2 cells by different nonviral vectors as a first step in the development of ex vivo gene therapy platform. |
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| AbstractList | The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies requires a profound knowledge of the whole transfection process. In this work, we elaborated and characterized in terms of size and zeta potential three different nonviral vectors: lipoplexes (144 nm; -29.13 mV), nioplexes (142.5 nm; +35.4 mV), and polyplexes (294.8 nm; +15.1 mV). We compared the transfection efficiency, cellular uptake, and intracellular trafficking of the three vectors in NT2 cell line. Lipoplexes exhibited the highest percentages of EGFP positive cells. The values obtained with polyplexes were lower compared to lipoplexes but higher than the percentages obtained with nioplexes. Cellular uptake results had a clear correlation with respect to the corresponding transfection efficiencies. Regarding the endocytosis mechanism, lipoplexes enter in the cell, mainly, via clathrin-mediated endocytosis (CME) while polyplexes via caveolae-mediated endocytosis (CvME). Nioplexes were discarded for this experiment due to their low cellular uptake. By simulating an artificial endosome, we demonstrated that the vectors were able to release the DNA cargo once inside the late endosome. The data collected from this assay showed that at 6 h the genetic material carried by polyplexes was still located in the late endosome, while DNA carried by lipoplexes was already in the nucleus. This result indicates a faster intracellular traffic of the lipid-based vectors. Overall, our work gives new insights into the transfection process of NT2 cells by different nonviral vectors as a first step in the development of ex vivo gene therapy platform. |
| Author | Agirre, Mireia Zarate, Jon Grijalvo, Santiago Eritja, Ramón Ojeda, Edilberto Pedraz, José Luis González-Burguera, Imanol López de Jesús, Maider Sallés, Joan Barrondo, Sergio Puras, Gustavo García del Caño, Gontzal |
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| CitedBy_id | crossref_primary_10_1016_j_colsurfb_2017_01_012 crossref_primary_10_1016_j_ijpharm_2016_02_043 crossref_primary_10_1016_j_ijpharm_2017_02_016 crossref_primary_10_1016_j_ijpharm_2018_09_038 crossref_primary_10_3390_pharmaceutics12030198 crossref_primary_10_1016_j_colsurfb_2017_01_030 crossref_primary_10_1016_j_ejpb_2021_09_011 crossref_primary_10_1002_pola_28376 crossref_primary_10_1111_jphp_12940 crossref_primary_10_1016_j_ijpharm_2023_122968 crossref_primary_10_1007_s12033_017_0044_5 crossref_primary_10_3390_genes10040289 crossref_primary_10_1007_s12672_025_02158_2 crossref_primary_10_1016_j_ijpharm_2018_05_057 crossref_primary_10_1080_10717544_2023_2219420 crossref_primary_10_1016_j_ijpharm_2018_07_035 crossref_primary_10_3390_pharmaceutics13111787 crossref_primary_10_1016_j_apsb_2024_09_009 |
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| Keywords | endocytosis pCMS-EGFP nonviral vectors NTera2/D1 cells transfection |
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| SubjectTerms | Cell Survival Embryonal Carcinoma Stem Cells - metabolism Embryonal Carcinoma Stem Cells - pathology Endocytosis - physiology Gene Transfer Techniques Genetic Therapy - methods Green Fluorescent Proteins - metabolism Humans Lipids - chemistry Liposomes - chemistry Neurons - metabolism Neurons - pathology Plasmids - administration & dosage Polymers - chemistry Transfection |
| Title | New Insights into Gene Delivery to Human Neuronal Precursor NT2 Cells: A Comparative Study between Lipoplexes, Nioplexes, and Polyplexes |
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