title: Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives.
abstract: Despite the long history of nanoparticulate calcium phosphate (CaP) as a non-viral transfection agent, there has been limited success in attempts to optimize its properties for transfection comparable in efficiency to that of viral vectors. Here we focus on the optimization of: (a) CaP nanoparticle precipitation conditions, predominantly supersaturation and Ca/P molar ratios; (b) transfection conditions, mainly the concentrations of the carrier and plasmid DNA; (c) the presence of surface additives, including citrate anion and cationic poly(l-lysine) (PLL). CaP nanoparticles significantly improved transfection with plasmid DNA encoding enhanced green fluorescent protein (eGFP) in pre-osteoblastic MC3T3-E1 cells compared to a commercial non-viral carrier. At the same time they elicited significantly lesser cytotoxicity than the commercial carrier. Plasmid DNA acted as a nucleation promoter, decreasing the nucleation lag time of metastable CaP solutions and leading to a higher rate of nucleation and a lower size of the precipitated particles. The degree of supersaturation (DS) of 15 was found to be more optimal for transfection than that of 12.5 or 17.5 and higher. Because CaP particles precipitated at DS 15 were spherical, while DS 17.5 and 21 yielded acicular particles, it was concluded that spherical particle morphologies were more conducive to transfection than the anisotropic ones. Even though the yield at DS 15 was 10 and 100 times lower than that at DS 17.5 and 21, respectively, transfection rates were higher using CaP nanoparticle colloids prepared at DS 15 than using those made at higher or lower DS, indicating that the right particle morphology can outweigh the difference in the amount of the carrier, even when this difference is close to 100×. In contrast to the commercial carrier, the concentration of CaP-pDNA delivered to the cells was directly proportional to the transfection rate. Osteosarcoma K7M2 cells were four times more easily transfectable with CaP nanoparticles than the MC3T3-E1 cells. The addition of citrate increased the transfection rate at lower concentrations; however, a complete redispersal of CaP-pDNA nanoparticles at higher concentrations of citrate coincided with a complete diminishment of transfection, implying the benefits of partial aggregation of CaP nanoparticles carrying pDNA. In contrast, PLL delayed transfection initially, but enhanced it at longer time points (⩾96h), leading to the conclusion that both citrate and PLL could exert positive effects on transfection: citrate if added at low concentrations and PLL to extend transfection over longer periods of time.
authors.affiliation: Advanced Materials and Nanobiotechnology Laboratory, Department of Bioengineering, University of Illinois, Chicago, IL 60607-7052, USA.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Khan
authors.affiliation: Advanced Materials and Nanobiotechnology Laboratory, Department of Bioengineering, University of Illinois, Chicago, IL 60607-7052, USA.
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Wu
authors.affiliation: Advanced Materials and Nanobiotechnology Laboratory, Department of Bioengineering, University of Illinois, Chicago, IL 60607-7052, USA.
authors.forename: Shreya
authors.initials: S
authors.lastname: Ghosh
authors.affiliation: Advanced Materials and Nanobiotechnology Laboratory, Department of Bioengineering, University of Illinois, Chicago, IL 60607-7052, USA. Electronic address: vuk21@yahoo.com.
authors.forename: Vuk
authors.initials: V
authors.lastname: Uskoković
mesh_terms: D000818:Animals; D002130:Calcium Phosphates / Q000737:chemistry*; D045744:Cell Line, Tumor; D019343:Citric Acid / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D011107:Polylysine / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
chemical_list: D002130:Calcium Phosphates; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011107:Polylysine; D019343:Citric Acid; C020243:calcium phosphate
keywords: Calcium phosphate; Citrate; Confocal microscopy; Gene delivery; Hydroxyapatite; Immunofluorescence; Nanoparticles; Plasmid DNA; Poly(l-lysine); Transfection