Journal
SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS
Volume 13, Issue 1, Pages -Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1088/1468-6996/13/1/015009
Keywords
poly(L-lysine); poly(L-arginine); microinjection; transcription efficiency; endosomal escape
Categories
Funding
- Ministry of Health, Labor and Welfare of Japan
- Grants-in-Aid for Scientific Research [21350093, 23500544] Funding Source: KAKEN
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Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.
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