Cationic shell-crosslinked knedel-like nanoparticles for highly efficient gene and oligonucleotide transfection of mammalian cells.
In this work, a robust synthetic nanostructure was designed for the effective packaging of DNA and it was shown to be an efficient agent for cell transfection. An amphiphilic block copolymer, poly(acrylamidoethylamine)(128)-b-polystyrene(40) (PAEA(128)-b-PS(40)), was synthesized, micellized in water and shell-crosslinked using a diacid-derivatized crosslinker, to give cationic shell-crosslinked nanoparticles (cSCKs) with a mean hydrodynamic diameter of 14 +/- 2 nm. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) was able to be formed over a broad range of polymer amine:pDNA phosphate ratios (N/P ratio), 2:1-20:1. The sizes of the complexes and their ability to fully bind the pDNA were dependent upon the N/P ratio, as characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and gel retardation assay. A luciferase activity assay and EGFP expression were used to evaluate intracellular delivery of a splice-correcting phosphorothioate and genetic material, respectively, by the cSCKs, which indicated that an N/P ratio of 6:1 gave the highest transfection. It was shown by both luciferase activity assay (48 h) and EGFP transfection data that high transfection efficiencies were achieved for HeLa cells transfected by cSCK/CCUCUUACCUCAGUUACA and cSCK/pEGFP-N1 plasmid, respectively. The cSCK/pEGFP-N1 plasmid transfection efficiency of 27% far exceeded the performance of Polyfect (PAMAM dendrimers), which achieved only 12% transfection efficiency, under the same conditions. Cytotoxicities for the cSCKs were evaluated for HeLa and CHO cells.
Zhang
Ke
K
Department of Chemistry, Washington University, 1 Brookings Drive, St. Louis, MO 63130, USA.
Fang
Huafeng
H
Wang
Zhenghui
Z
Taylor
John-Stephen A
JS
Wooley
Karen L
KL
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000009:adverse effects / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D014162:Transfection / Q000379:methods*
D009841:Oligonucleotides; D011108:Polymers; D004247:DNA