TY - JOUR
T1 - Polymorphic assembly of virus-capsid proteins around DNA and the cellular uptake of the resulting particles
AU - de Ruiter, M. V.
AU - van der Hee, R. M.
AU - Driessen, A. J.M.
AU - Keurhorst, E. D.
AU - Hamid, M.
AU - Cornelissen, J. J.L.M.
N1 - Elsevier deal
PY - 2019/8/10
Y1 - 2019/8/10
N2 - Virus-like particles (VLPs), i.e. molecular assemblies that resemble the geometry and organization of viruses, are promising platforms for therapeutics and imaging. Understanding the assembly and cellular uptake pathways of VLPs can contribute to the development of new antiviral drugs and new virus-based materials for the delivery of drugs or nucleic acid-based therapies. Here we report the assembly of capsid proteins of the cowpea chlorotic mottle virus (CCMV) around DNA into defined structures at neutral pH. Depending on the type of DNA used, we are able to create spherical structures of various diameters and rods of various lengths. In order to determine the shape dependency, the cellular uptake routes and intracellular positioning of these formed polymorphic VLPs in RAW264.7, HeLa and HEK 293 cells are evaluated using flow cytometry analysis with specific chemical inhibitors for different uptake routes. We observed particular uptake routes for the various CCMV-based nanostructures, but the experiments point to clathrin-mediated endocytosis as the major route for cell entry for the studied VLPs. Confocal microscopy reveals that the formed VLPs enter the cells, with clear colocalization in the endosomes. The obtained results provide insight in the cargo dependent VLP morphology and increase the understanding of shape dependent uptake into cells, which is relevant in the design of new virus-based structures with applications in drug and gene delivery.
AB - Virus-like particles (VLPs), i.e. molecular assemblies that resemble the geometry and organization of viruses, are promising platforms for therapeutics and imaging. Understanding the assembly and cellular uptake pathways of VLPs can contribute to the development of new antiviral drugs and new virus-based materials for the delivery of drugs or nucleic acid-based therapies. Here we report the assembly of capsid proteins of the cowpea chlorotic mottle virus (CCMV) around DNA into defined structures at neutral pH. Depending on the type of DNA used, we are able to create spherical structures of various diameters and rods of various lengths. In order to determine the shape dependency, the cellular uptake routes and intracellular positioning of these formed polymorphic VLPs in RAW264.7, HeLa and HEK 293 cells are evaluated using flow cytometry analysis with specific chemical inhibitors for different uptake routes. We observed particular uptake routes for the various CCMV-based nanostructures, but the experiments point to clathrin-mediated endocytosis as the major route for cell entry for the studied VLPs. Confocal microscopy reveals that the formed VLPs enter the cells, with clear colocalization in the endosomes. The obtained results provide insight in the cargo dependent VLP morphology and increase the understanding of shape dependent uptake into cells, which is relevant in the design of new virus-based structures with applications in drug and gene delivery.
KW - UT-Hybrid-D
KW - Plant virus
KW - Transfection
KW - Virus-like particles
KW - Cargo controlled assembly
UR - http://www.scopus.com/inward/record.url?scp=85068511996&partnerID=8YFLogxK
U2 - 10.1016/j.jconrel.2019.06.019
DO - 10.1016/j.jconrel.2019.06.019
M3 - Article
C2 - 31228473
AN - SCOPUS:85068511996
VL - 307
SP - 342
EP - 354
JO - Journal of controlled release
JF - Journal of controlled release
SN - 0168-3659
ER -