Intrinsically fluorescent PAMAM dendrimer as gene carrier and nanoprobe for nucleic acids delivery: bioimaging and transfection study.
This study successfully evaluated gene delivery and transfection toward rat C6 glioma cell lines mediated by intrinsic blue fluorescent poly(amido amine) (PAMAM) dendrimer. We used three antisense oligonucleotides, (AS-ODN) p75, NGF1, and NGF2 for knocking down specific protein expressions. The three oligonucleotides were electrostatically associated with the photoluminescent amino-terminated PAMAM dendrimer to yield fluorescent complexes at various nitrogen-to-phosphorus (N/P) ratios. Compared with pristine PAMAM dendrimer and hyperbranched polyethylenimine (PEI), the fluorescent PAMAM dendrimer revealed lower in vitro cytotoxicity toward C6 cells, allowing us to transfect the cells with the AS-ODN complexes under a higher N/P ratio. Due to the intrinsic fluorescence, cellular uptake behavior could be directly analyzed by fluorescence microscopy and flow cytometry, without additional fluorescence labeling. As expected, the result clearly suggested that the uptake efficiency increased as the N/P value increased. Furthermore, the quantified data obtained from flow cytometry indicated relatively higher uptake efficiency for the p75 complex, which is mainly due to different association patterns between the fluorescent dendrimer and AS-ODNs. At N/P = 20, atomic force microscopic analysis confirmed that the p75 complex formed well-condensed, spherical particles with dimensions less than 200 nm, but that NGF2 AS-ODN associated poorly with the dendrimer. Finally, Western blot analysis indicated that these complexes were capable of knocking down the specific protein expression to a certain level, being comparable to the hyperbranched PEI-mediated gene transfection. Our preliminary results clearly indicated that intrinsic fluorescent PAMAM dendrimers show promise as gene vehicles that can achieve delivery, transfection, and bioimaging at the same time.
School of Applied Chemistry, Chung Shan Medical University, Taichung, Taiwan.
Ya-Ju
YJ
Tsai
Chao-Chin
CC
Hu
Chih-Chien
CC
Chu
Toyoko
T
Imae
D000818:Animals; D045744:Cell Line, Tumor; D050091:Dendrimers / Q000378:metabolism*; D005434:Flow Cytometry; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods; D005910:Glioma / Q000235:genetics / Q000628:therapy; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D053758:Nanoparticles; D009584:Nitrogen / Q000032:analysis; D009696:Nucleic Acids; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D010758:Phosphorus / Q000032:analysis; D051381:Rats; D014162:Transfection / Q000379:methods*
D050091:Dendrimers; D009696:Nucleic Acids; D016376:Oligonucleotides, Antisense; C104700:PAMAM Starburst; D010758:Phosphorus; D009584:Nitrogen