Enhancement of gene transfer activity mediated by mannosylated dendrimer/alpha-cyclodextrin conjugate (generation 3, G3).
To enhance gene transfer activity of dendrimers, we prepared its conjugate (generation 3, G3) with alpha-cyclodextrin bearing mannose (Man-alpha-CDE conjugates) with various degrees of substitution of the mannose moiety (DSM5, 10, 13, 20) and compared their cytotoxicity and gene transfer activity, and elucidated the enhancing mechanism for the activity. Of the various carriers used here, Man-alpha-CDE conjugate (G3, DSM10) provided the highest gene transfer activity in NR8383, A549, NIH3T3 and HepG2 cells, being independent of the expression of mannose receptors. Gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) was not decreased by the addition of 10% serum in A549 cells. Cytotoxicity of the polyplex with Man-alpha-CDE conjugates (G3, DSM10) was not observed in A549 and NIH3T3 cells up to the charge ratio of 200/1 (carrier/pDNA). The gel mobility and particle size of polyplex with Man-alpha-CDE conjugate (G3, DSM10) were relevant to those with alpha-CDE conjugate (G3), but zeta-potential, DNase I stability, pDNA condensation of the former polyplex were somewhat different from those of the latter one. Cellular association of polyplex with Man-alpha-CDE conjugate (G3, DSM10) was almost comparable to that with dendrimer (G3) complex and alpha-CDE conjugate (G3). The addition of mannan and mannose attenuated gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) in A549 cells. Alexa-pDNA complex with TRITC-Man-alpha-CDE conjugate (G3, DSM10), but not the complex with TRITC-alpha-CDE conjugate (G3), was found to translocate to nucleus at 24 h after incubation in A549 cells. HVJ-E vector including mannan, but neither the vector alone nor the vector including dextran, suppressed the nuclear localization of TRITC-Man-alpha-CDE conjugate (G3, DSM10) to a striking degree after 24 h incubation in A549 cells. These results suggest that Man-alpha-CDE conjugate (G3, DSM10) has less cytotoxicity and prominent gene transfer activity through not only its serum resistant and endosome-escaping abilities but also nuclear localization ability.
Arima
Hidetoshi
H
Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
Chihara
Yuko
Y
Arizono
Masayo
M
Yamashita
Shogo
S
Wada
Koki
K
Hirayama
Fumitoshi
F
Uekama
Kaneto
K
D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004563:Electrochemistry; D005079:Excipients; D018014:Gene Transfer Techniques; D006801:Humans; D008358:Mannose / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D010316:Particle Size; D014162:Transfection; D047391:alpha-Cyclodextrins / Q000737:chemistry*
D002241:Carbohydrates; D005079:Excipients; D047391:alpha-Cyclodextrins; D004247:DNA; D008358:Mannose; C032613:alpha-cyclodextrin