Fast, facile and ethidium bromide-free assay based on the use of adsorption indicator for the estimation of polyethylenimine to nucleic acid ratio of complete polyplex assembly for gene delivery.
A new method was developed for the estimation of polyethylenimine (PEI) to nucleic acid ratio at which the polyplex was completely formed. The assay relied on the attraction of dichlorofluoresceinate dye to adsorb on self-assembling particles as counterions, as induced by the surface charge of the polyplex which became positive once PEI associated equivalently with nucleic acid. This phenomenon resulted in the appearance of pink colored pellets of the polyplex after centrifugation. By the other means, sodium hydroxide solution might be added to free the adsorbed dye into the solution, producing conspicuous green fluorescence under UV light (366 nm). The assay was well applied to the polyplex formulations of PEI and plasmid DNA or siRNA with satisfactory detectability and gave the results in agreement with those from gel retardation method and zeta potential analysis. Importantly, the proposed method required no sophisticated instruments, time-consuming gel electrophoresis, carcinogenic ethidium bromide as well as costly dyes and the analysis could be accomplished within less than 10 min. Hence, it was a fast, facile, cost-effective and safe-for-operator alternative method, suited for the investigation of the optimal PEI to nucleic acid ratio for gene delivery.
Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand.
Samarwadee
S
Plianwong
Praneet
P
Opanasopit
Tanasait
T
Ngawhirunpat
Theerasak
T
Rojanarata
D000327:Adsorption; D001681:Biological Assay; D024202:Electrophoretic Mobility Shift Assay; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D010957:Plasmids / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry*; D012972:Sodium Hydroxide / Q000737:chemistry; D055672:Static Electricity; D014466:Ultraviolet Rays
D005452:Fluoresceins; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D012972:Sodium Hydroxide; C037631:2',7'-dichlorofluorescein; D011094:Polyethyleneimine
Dichlorofluorescein; Nucleic acid; Polyethylenimine; Polyplex; Ratio; Surface charge