Insights in cellular uptake mechanisms of pDNA-polycationic amphiphilic cyclodextrin nanoparticles (CDplexes).
It is generally recognized that the major obstacle to efficient gene delivery is cellular internalization and endosomal escape of the DNA. Recently, we have developed a modular strategy for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) capable of complexing and compacting DNA into homogeneous nanoparticles (<70nm). Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the corresponding pDNA-paCD nanoparticles (CDplexes) might use the cell internalization and endosomal escape mechanisms described for both lipoplexes and polyplexes. To verify this hypothesis, we have now investigated the uptake and transfection efficiencies of CDplexes in the presence of several inhibitors of endocytosis, namely chlorpromazine, genistein, dynasore and methylated beta-cyclodextrin (MbCD). Our data show that CDplexes obtained from paCD 1, which ranks among the most efficient paCD gene vectors reported up to date, are internalized by both clathrin-dependent (CDE) and clathrin-independent endocytosis (CIE), both processes being cholesterol- and dynamin-dependent. We observed that the largest fraction of gene complexes is taken up via CDE, but this fraction is less relevant for transfection. The smaller fraction that is internalized via the CIE pathway is predominantly responsible for successful transfection.
Díaz-Moscoso
Alejandro
A
Instituto de Investigaciones Químicas, CSIC - Universidad de Sevilla, Américo Vespucio 49, Isla de la Cartuja, E-41092 Sevilla, Spain.
Vercauteren
Dries
D
Rejman
Joanna
J
Benito
Juan M
JM
Ortiz Mellet
Carmen
C
De Smedt
Stefaan C
SC
Fernández
José M García
JM
D000818:Animals; D021941:Caveolae / Q000378:metabolism; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D003505:Cyclodextrins / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage*; D004705:Endocytosis / Q000187:drug effects; D053758:Nanoparticles / Q000737:chemistry*; D010957:Plasmids / Q000008:administration & dosage; D014162:Transfection; D014709:Vero Cells
D002966:Clathrin; D003505:Cyclodextrins; D004247:DNA