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  • Title: Intracellular processing and stability of DNA complexed with histidylated polylysine conjugates.
    Author: Gonçalves C, Pichon C, Guérin B, Midoux P.
    Journal: J Gene Med; 2002; 4(3):271-81. PubMed ID: 12112644.
    Abstract:
    BACKGROUND: Glycosylated polylysines and histidylated polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work has been undertaken in two steps to study the uptake and the intracellular processing of pDNA, which are still poorly understood in the polyfection pathway. METHODS AND RESULTS: The kinetics of the uptake and the intracellular processing of pDNA complexed with lactosylated polylysine, histidylated polylysine or histidylated polylysine bearing lactosyl residues (polyplexes) into a CF human airway epithelial cell line were assessed by flow cytometry and confocal microscopy. Complexes formed from histidylated polylysine, even though they were less taken up by cells, show better transfection efficiency with compared with lactosylated complexes. Lactosylated polymers segregated more rapidly when compared with non-lactosylated polymers into compartments different from those containing pDNA on internalization. Intracellular location and pH measurements indicated that polymers ended up in compartments of pH approximately 6.2 while pDNA reached less acidic compartments of pH approximately 6.6. These compartments did not contain the LAMP-1 lysosomal marker. CONCLUSIONS: The present study exhibits that, upon internalization, pDNA and polylysine conjugates underwent segregation with a rate depending on the polylysine substitution and polymer degradation. The better transfection efficiency of polyplexes with histidylated polylysine can be ascribed to their prolonged stability inside the endocytic vesicles that likely favored the pDNA escape in the cytosol.
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