title: A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells.
abstract: The p53 protein mediated anti-tumor strategy is limited due to the lack of suitable delivery agent with insignificant immunogenic response, serum compatibility, and early and easy detection of the transfected cell population. To overcome these problems, we generated a p53-EGFP-C3 fusion construct which expressed easily detectable green fluorescence protein (GFP) and allowed an estimation of p53 mediated anti-tumor activity. A mixture of cationic cholesterol gemini (Chol-5L) with natural lipid, DOPE (molar ratio 1:4), acronymed as Chol-5LD, formed a nano-liposome as characterized by various physical methods. The prepared clone was evaluated for the expression of GFP and functional p53 in HeLa and two additional cell lines with varied p53 status namely, H1299 (p53(-/-)) and HEK293T (p53(+/+)). Transfected cells were screened using RT-PCR, Western blotting, FACS analysis, MTT, Trypan blue assay and visualized under a fluorescence microscope. The p53-EGFP-C3 fusion protein induced apoptosis in cancer cells as evident from DNA fragmentation, cell cycle analysis, Annexin-V staining and PARP cleavage assays. The transfection and apoptosis induction efficiency of Chol-5LD was significantly higher than commercial reagents Lipofectamine2000 and Effectene irrespective of the cell lines examined. Further it significantly decreases the xenograft tumor volume in nude mice tumors via apoptosis as observed in H&E staining.
authors.affiliation: Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Misra
authors.forename: Sarwat
authors.initials: S
authors.lastname: Naz
authors.forename: Paturu
authors.initials: P
authors.lastname: Kondaiah
authors.forename: Santanu
authors.initials: S
authors.lastname: Bhattacharya
mesh_terms: D000818:Animals; D017209:Apoptosis; D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000737:chemistry*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D049329:Nanostructures / Q000737:chemistry*; D009369:Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D010957:Plasmids / Q000235:genetics / Q000627:therapeutic use*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D002412:Cations; D016159:Tumor Suppressor Protein p53; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002784:Cholesterol
keywords: Cationic cholesterol; Gene therapy; Gene transfer; Nanocomposite; Serum; Xenograft tumor regression