Development of a targeted siRNA delivery system using FOL-PEG-PEI conjugate.
Receptor mediated delivery of siRNA enables silencing of target genes in specific tissues. Folate receptor (FR) is an attractive target for tumor-selective gene delivery. The focus of this study was to deliver the dihydrofolate reductase (DHFR) siRNA expressing plasmid and to silence the DHFR gene in FR positive KB cells, by complexing the plasmid with a folate-polyethylene glycol-polyethylenimine (FOL-PEG-PEI) conjugate, as a gene carrier. A DHFR siRNA sequence was cloned into a pSUPER-RNAi vector and complexed with the FOL-PEG-PEI conjugate. The complex was characterized by particle size analyzer, gel retardation and DNase protection assay. The FOL-PEG-PEI/pSUPER-siDHFR complex was transfected to FR overexpressing (KB) and FR negative (A549) cells. The transfection effiencies and gene inhibition were analyzed by fluorescence microscopy and RT-PCR. The pSUPER-siDHFR/PEI-PEG-FOL complex delivered the siRNA vector and inhibited DHFR gene in KB cells, while A549 cells were unaffected. Lipofectamine mediated transfection of pSUPER-siDHFR, delivered the vector and inhibited the DHFR gene in both KB and A549 cells. FR mediated delivery of siDHFR complexed with PEI-PEG-FOL conjugate inhibits the DHFR expression in FR positive cells alone. This strategy can be extended to deliver a wide range of drugs and post-transcriptional gene silencing therapeutics.
Biswal
Bijesh K
BK
Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India.
Debata
Niladri B
NB
Verma
Rama S
RS
D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000378:metabolism; D005492:Folic Acid / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D023282:Nuclease Protection Assays; D011092:Polyethylene Glycols / Q000378:metabolism*; D011094:Polyethyleneimine / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D034741:RNA, Small Interfering; D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D003850:Deoxyribonuclease I