The role of ganglioside GM1 in cellular internalization mechanisms of poly(amidoamine) dendrimers.
Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher-generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower-generation dendrimers. Dendrimer-based conjugates were characterized by (1)H NMR, UV/vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH(2)) were observed to internalize into KB, Rat2, and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM(1)-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH(2) dendrimers colocalized with CTB; however, experiments with C6 cells indicated that internalization of G7-NH(2) was not ganglioside GM(1) dependent. The G7/CTB colocalization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM(1) in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.
Hong
Seungpyo
S
Program in Macromolecular Science and Engineering and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Rattan
Rahul
R
Majoros
István J
IJ
Mullen
Douglas G
DG
Peters
Jennifer L
JL
Shi
Xiangyang
X
Bielinska
Anna U
AU
Blanco
Luz
L
Orr
Bradford G
BG
Baker
James R
JR
Holl
Mark M Banaszak
MM
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D050091:Dendrimers / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007624:KB Cells; D008051:Lipid Bilayers / Q000378:metabolism*; D008954:Models, Biological; D015394:Molecular Structure; D011073:Polyamines / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013499:Surface Properties
D050091:Dendrimers; D008051:Lipid Bilayers; C104700:PAMAM Starburst; C531249:Poly(amidoamine); D011073:Polyamines; D005677:G(M1) Ganglioside