RFP gene expression and cell viability status were evaluated using circulation cytometry. GL-NPs showed higher transfection efficiency and comparable viability profile by evaluation using MitoTracker Deep Red in PAM212 cells. Circulation cytometric analysis of PAM212 cells stained with Sytox reddish revealed two cell populations with low and high fluorescent intensity, representing cells with partially-porated and highly-porated membranes, respectively. Additional combined staining with MitoTracker and ethidium homodimer showed that that 18-3-18 GL-NPs disturbed cell membrane integrity, while cells were still alive and experienced mitochondrial activity. Conclusion Taken together, this study exhibited that 18-3-18 GL-NPs have higher transfection efficiency and comparable viability profile to the commercial Lipofectamine Plus, and the conversation of 18-3-18 GL-NPs with PAM212 cell membranes entails a permeability increase, possibly through the formation of nanoscale pores, Pllp which could explain efficient gene delivery. This novel nanoconstruct appears to be a encouraging delivery system for further skin gene therapy studies in vivo. represent LSD post hoc statistical significance compared to Lipofectamine Plus (P?0.05). represent mean??SD, n?=?4 Transfection efficiency of GL-NPs in PAM 212 cells The expression of RFP in PAM212 keratinocytes transfected with the three series of GL-NPs carrying the tdTomato plasmid is usually shown in Fig.?2. GL-NP-mediated RFP expression in PAM212 cells was generally between 0 and 13? % and Lipofectamine Plus produced about 6?% RFP-positive cells. The 18-3-18 GL-NPs induced the highest RFP expression while RFP expression in GL-NPs from 12 and 16 series were significantly lower than Lipofectamine Plus (Fig.?2). The expression of RFP in PAM212 cells transfected with GL-NPs and Lipofectamine Plus were also confirmed by confocal microscopy (Fig.?3). The mean fluorescence intensity (MFI) of RFP expression after Isomalt 18-3-18 GL-NPs transfection was 1.6 fold higher compared to Lipofectamine Plus (Fig.?4) indicating that not only transfection efficiency of GL-NPs was higher based on the number of cells transfected but also on the basis of intensity of gene expression (quantity of protein expressed). Open in a separate windows Fig.?2 RFP expression in PAM212 cells, transfected GL-NPs and Lipofectamine Plus reagent measured by circulation cytometry. Results are expressed as the mean percentage of RFP positive cells??standard deviation. Results are expressed as mean measurements??SD (n?=?4). represent LSD post hoc statistical significance compared to Lipofectamine Plus (P?0.05) Open in a separate window Fig.?3 Confocal microscopic images of PAM212 cells treated with pDNA complexed to Lipofectamine Plus or GL-NPs, prepared using gemini surfactant series 12, 16, and 18. The expression of the tdTomato RFP is usually shown in and nuclei were stained with DRAQ-5 and are shown in and represent control unfavorable and test respectively Circulation cytometry analysis of cell membrane integrity and mitochondrial activity In order to better understand the populations of cells expressing of RFP in PAM212 cells transfected with GL-NPs, we evaluated RFP expression in metabolically active (MitoTracker+) cells, or membrane-porated cells (Sytox reddish+) Isomalt PAM212 cells by circulation cytometry. Viability staining was performed at the same time on the same cell suspension sample that was divided into two microtubes and stained with MitoTracker Deep Red or Sytox reddish. In the case of GL-NP transfected cells, the presence of cell populace that is both MitoTracker and Sytox reddish positive was an indication that cells could be alive while maintaining a compromised membrane. As shown in Fig.?5a, RFP expression was significantly higher in cells transfected with 18-3-18 GL-NPs compared to Lipofectamine Plus (15.5 vs 5.5?%); however, almost half of RFP positive cells (6.62?%) were considered as MitoTracker unfavorable cells since they showed very low mitochondrial activity. Density plots in Fig.?5b also show that the majority of RFP positive cells were also positive for Isomalt Sytox red (14.38?%), indicating that 18-3-18 GL-NPs disturbed cell membrane integrity, while cells were still alive and experienced mitochondrial activity. Open in a separate windows Fig.?5 Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red Isomalt nucleic acid stain. Cells were transfected with either 18-3-18 GL-NPs or Lipofectamine Plus reagent. a A comparison between the proportion of live and lifeless PAM212 cells represented by either MitoTracker or Sytox fluorescent transmission. b The show representative data from one of three individual experiments Further analyses.