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A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation.

Gharagozloo M, Rafiee A, Chen DW, Foldvari M - J Nanobiotechnology (2015)

Bottom Line: Gemini-lipid nanoparticles have been received major attention recently as non-viral delivery systems due to their successful non-invasive gene delivery through tough barriers such as eye and skin.No significant viability loss was detected in cells transfected with 18-3-18, 18-7-18, 18-7NH-18, and 18-7NCH3-18 NPs, whereas a significant reduction of viability was detected in cells treated with 12-3-12, 12-7-12, 12-7NH-12, 16-7NH-16, or 16-7NCH3-16 GL-NPs.This novel nanoconstruct appears to be a promising delivery system for further skin gene therapy studies in vivo.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada. marjangh@gmail.com.

ABSTRACT

Background: Gemini-lipid nanoparticles have been received major attention recently as non-viral delivery systems due to their successful non-invasive gene delivery through tough barriers such as eye and skin. The aim of this study was to evaluate non-viral gene delivery by a series of dicationic gemini surfactant-phospholipid nanoparticles (GL-NPs) and to explore their mechanism of interaction with cellular membranes of murine PAM212 epidermal keratinocytes.

Methods: NPs containing pCMV-tdTomato plasmid encoding red fluorescent protein (RFP) were prepared using 12 different gemini surfactants (m-s-m, with m = 12, 16 and 18C alkyl tail and s = 3 and 7C polymethylene spacer group and 7C substituted spacers with 7NH and 7NCH3) and dioleoylphosphatidylethanolamine helper lipid. RFP gene expression and cell viability status were evaluated using flow cytometry. MitoTracker Deep Red mitochondrial stain and the cell impermeable Sytox red nuclear stain were used as indicators of cell viability and cell membrane integrity, respectively.

Results: No significant viability loss was detected in cells transfected with 18-3-18, 18-7-18, 18-7NH-18, and 18-7NCH3-18 NPs, whereas a significant reduction of viability was detected in cells treated with 12-3-12, 12-7-12, 12-7NH-12, 16-7NH-16, or 16-7NCH3-16 GL-NPs. Compared to Lipofectamine Plus, 18-3-18 GL-NPs showed higher transfection efficiency and comparable viability profile by evaluation using MitoTracker Deep Red in PAM212 cells. Flow cytometric analysis of PAM212 cells stained with Sytox red 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 had mitochondrial activity.

Conclusion: Taken together, this study demonstrated that 18-3-18 GL-NPs have higher transfection efficiency and comparable viability profile to the commercial Lipofectamine Plus, and the interaction of 18-3-18 GL-NPs with PAM212 cell membranes involves a permeability increase, possibly through the formation of nanoscale pores, which could explain efficient gene delivery. This novel nanoconstruct appears to be a promising delivery system for further skin gene therapy studies in vivo.

No MeSH data available.


Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red 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 dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments
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Fig5: Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red 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 dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments

Mentions: 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 red+) PAM212 cells by flow 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 red. In the case of GL-NP transfected cells, the presence of cell population that is both MitoTracker and Sytox red 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 negative 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 Sytox red (14.38 %), indicating that 18-3-18 GL-NPs disturbed cell membrane integrity, while cells were still alive and had mitochondrial activity.Fig. 5


A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation.

Gharagozloo M, Rafiee A, Chen DW, Foldvari M - J Nanobiotechnology (2015)

Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red 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 dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587676&req=5

Fig5: Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red 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 dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments
Mentions: 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 red+) PAM212 cells by flow 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 red. In the case of GL-NP transfected cells, the presence of cell population that is both MitoTracker and Sytox red 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 negative 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 Sytox red (14.38 %), indicating that 18-3-18 GL-NPs disturbed cell membrane integrity, while cells were still alive and had mitochondrial activity.Fig. 5

Bottom Line: Gemini-lipid nanoparticles have been received major attention recently as non-viral delivery systems due to their successful non-invasive gene delivery through tough barriers such as eye and skin.No significant viability loss was detected in cells transfected with 18-3-18, 18-7-18, 18-7NH-18, and 18-7NCH3-18 NPs, whereas a significant reduction of viability was detected in cells treated with 12-3-12, 12-7-12, 12-7NH-12, 16-7NH-16, or 16-7NCH3-16 GL-NPs.This novel nanoconstruct appears to be a promising delivery system for further skin gene therapy studies in vivo.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada. marjangh@gmail.com.

ABSTRACT

Background: Gemini-lipid nanoparticles have been received major attention recently as non-viral delivery systems due to their successful non-invasive gene delivery through tough barriers such as eye and skin. The aim of this study was to evaluate non-viral gene delivery by a series of dicationic gemini surfactant-phospholipid nanoparticles (GL-NPs) and to explore their mechanism of interaction with cellular membranes of murine PAM212 epidermal keratinocytes.

Methods: NPs containing pCMV-tdTomato plasmid encoding red fluorescent protein (RFP) were prepared using 12 different gemini surfactants (m-s-m, with m = 12, 16 and 18C alkyl tail and s = 3 and 7C polymethylene spacer group and 7C substituted spacers with 7NH and 7NCH3) and dioleoylphosphatidylethanolamine helper lipid. RFP gene expression and cell viability status were evaluated using flow cytometry. MitoTracker Deep Red mitochondrial stain and the cell impermeable Sytox red nuclear stain were used as indicators of cell viability and cell membrane integrity, respectively.

Results: No significant viability loss was detected in cells transfected with 18-3-18, 18-7-18, 18-7NH-18, and 18-7NCH3-18 NPs, whereas a significant reduction of viability was detected in cells treated with 12-3-12, 12-7-12, 12-7NH-12, 16-7NH-16, or 16-7NCH3-16 GL-NPs. Compared to Lipofectamine Plus, 18-3-18 GL-NPs showed higher transfection efficiency and comparable viability profile by evaluation using MitoTracker Deep Red in PAM212 cells. Flow cytometric analysis of PAM212 cells stained with Sytox red 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 had mitochondrial activity.

Conclusion: Taken together, this study demonstrated that 18-3-18 GL-NPs have higher transfection efficiency and comparable viability profile to the commercial Lipofectamine Plus, and the interaction of 18-3-18 GL-NPs with PAM212 cell membranes involves a permeability increase, possibly through the formation of nanoscale pores, which could explain efficient gene delivery. This novel nanoconstruct appears to be a promising delivery system for further skin gene therapy studies in vivo.

No MeSH data available.