Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging

Membrane Fusion Mediated Intracellular Delivery of Lipid Bilayer Coated Mesoporous Silica Nanoparticles

“…[27,[50][51][52][53][54] In this study, wortmannin (Wor) was used as a micropinocytosis inhibitor as it blocks PI3-kinase, [50,52,55,56] while genistein (Gen) inhibits tyrosine-phosphorylation of Cav 1 and caveolin-dependent endocytosis. [57][58][59] Furthermore, chlorpromazine (Chl) was used as it blocks clathrin-dependent endocytosis, [51,60,61] and microtubule formation was inhibited by nocodazole (Noc). Uptake studies in the presence of these inhibitors give insight in the intracellular trafficking and internalization mechanisms involved in the uptake of the lipid bilayer coated MSNs.…”

“…Uptake studies in the presence of these inhibitors give insight in the intracellular trafficking and internalization mechanisms involved in the uptake of the lipid bilayer coated MSNs. [50][51][52][53][54] Finally, as endocytosis of nanoparticles is an energy-dependent process, sodium azide (NaN 3 ) was used to deplete the energy demands for endocytosis and restrict metabolic activity. [62,63] FACS analysis revealed that Gen, Wor, and Noc had no adverse effect on the delivery of fluorescently labeled cytC (Figure 4a), whereas in the presence of Chl and NaN 3 , uptake of nanoparticles containing cytC was slightly lowered to 90% as compared to the initial experiment in the absence of inhibitors.…”

Investigation of Transfection Barriers Involved in Non-Viral Nanoparticulate Gene Delivery in Different Cell Lines

“…Prior to microscopic examination, the cells were fixed with 4 % paraformaldehyde (500 µL, 10 min, RT) and the coverslips were mounted on glass slides with 3 µL MobiGlow (MoBiTec, Goettingen, Germany), an antifading substance to reduce photobleaching effects. Spectral bio-imaging was performed as described previously by Huth et al [8] with a SpectraCube SD-200H system (Applied Spectral Imaging, Migdal HaEmek, Israel). An inverted fluorescence microscope (Axiovert S 100, Zeiss, Jena Germany) equipped with a high-pressure mercury lamp (HBO 100) for excitation and a triple bandpass filter set was used.…”

“…To gain more insight into the intracellular fate of lipoplexes, investigation of their co-localization with a variety of molecules was carried out using spectral bio-imaging. Transferrin alexa fluor 488 (tf) was used as a marker for clathrin-mediated uptake [9][10][11][12][13][14][15][16], cholera toxin B alexa fluor 488 (chltx-B) was used as marker for internalization via caveolae and related membrane structures [8,17], and FITC-dextran was used as a marker for macropinocytosis. It was subsequently investigated whether the failure of the endosomal release of the lipoplex or the desaggregation of the complex was responsible for the low transfection efficiency.…”

Liposomes in Biology and Medicine

“…[42][43][44] Various cell uptake mechanisms for liposomes have been described. 8,45 Due to their particulate properties, phagocytic uptake mechanisms (phagocytose, endocytose, pinocytose) are predominant, however cell membrane adhesion and fusion can also occur. In the phagocytic uptake pathway liposomes are captured at the cell surface followed by endosomal and lysosomal uptake.…”