AIMS: In this work, we have evaluated the ability of targeted lipoplexes to enhance transgene expression in EGF receptor (EGFR) overexpressing tumor cells by using lipoplexes. MATERIALS & METHODS: We prepared DOTAP/cholesterol liposomes modified with EGF at 0.5/1, 1/1, 2/1 and 5/1 lipid/DNA (+/-) charge ratio by sequentially mixing the liposomes with the ligand and adding the reporter or the therapeutic plasmid gene, pCMVLuc (pVR1216) or pCMVIL12, respectively. HepG2, DHDK12proB and SW620 cells were used for in vitro experiments, which were performed in the presence of 60% serum. RESULTS: The characterization of EGF-lipoplexes indicated a size close to 300 nm and a variable net surface charge as a function of the amount of EGF associated to the cationic liposomes. EGF-lipoplexes, which showed an increased transfection activity, were positively charged, noncytotoxic and highly effective in protecting DNA from DNase I attack. Transfection activity in vitro resulted in an enhancement in the luciferase and IL-12 expression by EGF-lipoplexes compared with those without ligand (plain-lipoplexes) and to naked DNA. The results observed in SW620 cells, which are deficient in EGFR, confirmed that DNA uptake was predominantly via EGFR-mediated endocytosis. In vivo transfection activity was confirmed by luciferase imaging in living mice. Bioluminiscence could be detected mainly in the lung with a maximum signal 24 h after application. The resulting EGF-lipoplexes significantly increased the level of gene expression in mice compared with control or naked DNA. CONCLUSION: These findings indicate that these nanovectors may be an adequate alternative to viral vectors for gene therapy.
AIMS: In this work, we have evaluated the ability of targeted lipoplexes to enhance transgene expression in EGF receptor (EGFR) overexpressing tumor cells by using lipoplexes. MATERIALS & METHODS: We prepared DOTAP/cholesterol liposomes modified with EGF at 0.5/1, 1/1, 2/1 and 5/1 lipid/DNA (+/-) charge ratio by sequentially mixing the liposomes with the ligand and adding the reporter or the therapeutic plasmid gene, pCMVLuc (pVR1216) or pCMVIL12, respectively. HepG2, DHDK12proB and SW620 cells were used for in vitro experiments, which were performed in the presence of 60% serum. RESULTS: The characterization of EGF-lipoplexes indicated a size close to 300 nm and a variable net surface charge as a function of the amount of EGF associated to the cationic liposomes. EGF-lipoplexes, which showed an increased transfection activity, were positively charged, noncytotoxic and highly effective in protecting DNA from DNase I attack. Transfection activity in vitro resulted in an enhancement in the luciferase and IL-12 expression by EGF-lipoplexes compared with those without ligand (plain-lipoplexes) and to naked DNA. The results observed in SW620 cells, which are deficient in EGFR, confirmed that DNA uptake was predominantly via EGFR-mediated endocytosis. In vivo transfection activity was confirmed by luciferase imaging in living mice. Bioluminiscence could be detected mainly in the lung with a maximum signal 24 h after application. The resulting EGF-lipoplexes significantly increased the level of gene expression in mice compared with control or naked DNA. CONCLUSION: These findings indicate that these nanovectors may be an adequate alternative to viral vectors for gene therapy.
Authors: Khue G Nguyen; Maura R Vrabel; Siena M Mantooth; Jared J Hopkins; Ethan S Wagner; Taylor A Gabaldon; David A Zaharoff Journal: Front Immunol Date: 2020-10-15 Impact factor: 7.561
Authors: Ramsey N Majzoub; Chia-Ling Chan; Kai K Ewert; Bruno F B Silva; Keng S Liang; Erica L Jacovetty; Bridget Carragher; Clinton S Potter; Cyrus R Safinya Journal: Biomaterials Date: 2014-03-21 Impact factor: 12.479