M Mar Encabo-Berzosa1,2, Maria Sancho-Albero1,2, Victor Sebastian1,2, Silvia Irusta1,2, Manuel Arruebo1,2, Jesus Santamaria1,2, Pilar Martín Duque3,4. 1. Department of Chemical Engineering. Aragon Institute of Nanoscience (INA), University of Zaragoza, Zaragoza, Spain. 2. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Madrid, Spain. 3. Facultad de Ciencias Biosanitarias, Carretera Pozuelo a Majadahonda, Universidad Francisco de Vitoria, Madrid, Spain. 4. Fundación Araid, Zaragoza, Spain.
Abstract
BACKGROUND: In the present study, we investigated the ability of polyethylene glycol (PEG) functionalized gold nanoparticles to function as nonviral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes. METHODS: Positively-charged gold nanoparticles were synthesized using polyethylenimine (PEI) as a reducing and stabilizer agent and its cytotoxicity was reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). Vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was evaluated indirectly using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase. RESULTS: An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA, together with a reduced size and an elevated electrokinetic potential, are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40 kbp (plasmid size close to the limit of the DNA-carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and Hek293t) and a suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector. CONCLUSIONS: Nonviral vectors based on gold nanoparticles covalently coupled with PEG and PEI can be used as efficient transfection reagents showing expression levels that are the same or greater than those obtained with commercially available lipoplexes.
BACKGROUND: In the present study, we investigated the ability of polyethylene glycol (PEG) functionalized gold nanoparticles to function as nonviral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes. METHODS: Positively-charged gold nanoparticles were synthesized using polyethylenimine (PEI) as a reducing and stabilizer agent and its cytotoxicity was reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). Vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was evaluated indirectly using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase. RESULTS: An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA, together with a reduced size and an elevated electrokinetic potential, are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40 kbp (plasmid size close to the limit of the DNA-carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and Hek293t) and a suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector. CONCLUSIONS: Nonviral vectors based on gold nanoparticles covalently coupled with PEG and PEI can be used as efficient transfection reagents showing expression levels that are the same or greater than those obtained with commercially available lipoplexes.