fluorescent fatty acid analog bodipy fl c 16  (Thermo Fisher)

Journal: bioRxiv

Article Title: Membrane fusion-based drug delivery liposomes transiently modify the material properties of synthetic and biological membranes

doi: 10.1101/2024.08.14.607934

Figure Lengend Snippet: Fusion efficiency diagram for three-component liposomes. A, FLIM-FRET sketch of the fusion assay. Donor lifetime decreases (red to blue colours) as fusion efficiency increases, as assessed from an increase in FRET efficiency (E FRET ). B, sketch of the content mixing assay in which liposomes encapsulating a water-soluble probe (green) fully fuse with an acceptor GUV (red), transferring its content to the GUV lumen. C, E FRET fusion diagram measured on GUVs incubated with liposomes containing varying fractions of DOPC, DOPE and DOTAP. The sketches show that DOPC alone forms neutral bilayers (lower left), DOTAP alone forms positively charged bilayers (lower right), whereas DOPE alone forms an inverted hexagonal phase (top) due to its molecular geometry. The heat map within the ternary component diagram represents the estimated fusion efficiency based on E FRET measurements. Each green solid circle represents a tested liposomal composition (10-15 GUVs per liposome composition). Insets: GUV FLIM images show representative content mixing results from incubation of GUVs with the respective liposomal compositions. Here, unlabelled liposomes encapsulating 50 μM SRB fuse with Bodipy C 16 -containing GUVs. Bodipy C 16 and SRB are clearly resolved in the FLIM images due to their lifetime differences. Note that in some of the images, docked liposomes can be observed (arrows). Scale bar: 8 μm. The GUVs are made of DOPC:PS:Chol:DOPE (25:25:25:25, mole fraction).

Article Snippet: Glucose, sucrose, Dimethyl sulfoxide (DMSO) and the fluorescent probes Bodipy C 16 (BODIPY™ FL C16; 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid), sulforhodamine B (SRB), Dextran-FITC 3 kDa, Albumin–fluorescein isothiocyanate conjugate (FITC-albumin) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Liposomes, Single Vesicle Fusion Assay, Transferring, Incubation

Journal: bioRxiv

Article Title: Membrane fusion-based drug delivery liposomes transiently modify the material properties of synthetic and biological membranes

doi: 10.1101/2024.08.14.607934

Figure Lengend Snippet: Membrane charge density is the main factor determining fusion with acceptor membranes. A, sketch of liposomes containing the monovalent lipid DOTAP or the multivalent lipid MVL5 in the presence of helper lipids DOPC and DOPE. B, representative FLIM GUV DOPC:PS:Chol:DOPE (25:25:25:25, mole fraction) images upon incubation with CL:DOPC:DOPE (X:50-X:50, mole fraction) 20 μM liposomes (lipid concentration) containing increasing mol% of the CL and labelled with 2 mol% DPPE-Rhodamine. Control GUVs in the absence of liposomes are also shown along with the lifetime map. Scale bar: 8 μm. C, representative lifetime decays, respective fits and residuals from the fits for GUVs labelled with FRET donor Bodipy C 16 upon fusion with several liposome compositions. D and E, fusion efficiency as represented by E FRET as a function of CL mole fraction for liposomes containing DOPE and DOPC or only DOPC as the helper lipid, respectively. Control represents measurements in the absence of liposomes. Each data point represents measurements on 10-15 individual GUVs. Means and s.d. are shown. The lines are a guide to the eye. F, E FRET as a function of the calculated charge density for liposomes containing DOTAP or MVL5 and DOPC:DOPE as the helper lipids. Liposome fusion efficiency strongly depends on σ M and weakly on the chemical identity of the CL. G, E FRET response as a function of charge density for liposomes either containing DOPE or not as the helper lipid and MVL5 as the CL. The response does not have a strong dependence on the helper lipid.

Article Snippet: Glucose, sucrose, Dimethyl sulfoxide (DMSO) and the fluorescent probes Bodipy C 16 (BODIPY™ FL C16; 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid), sulforhodamine B (SRB), Dextran-FITC 3 kDa, Albumin–fluorescein isothiocyanate conjugate (FITC-albumin) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Membrane, Liposomes, Incubation, Concentration Assay, Control

Journal: bioRxiv

Article Title: Membrane fusion-based drug delivery liposomes transiently modify the material properties of synthetic and biological membranes

doi: 10.1101/2024.08.14.607934

Figure Lengend Snippet: Protein-coated cationic liposomes are unable to fuse with GUVs. A, representative FLIM images of Bodipy C 16 -labelled GUVs (DOPC:DOPG, 1:1 mole fraction) in the absence of liposomes, in the presence of pristine liposomes and in the presence of liposomes pre-incubated with serum (10x diluted). Scale bars: 9 μm. B, Bodipy C 16 fluorescence decays along with the fits and residuals of the fits for the GUVs shown in panel A. The data from the control (- liposomes) and the liposomes pre-incubated in serum (+liposomes/+serum) were well-fitted by a single-exponential decay, and the data for pristine liposomes (+liposomes/-serum) were well-fitted by a double-exponential decay. C, measured E FRET for a number of GUVs upon incubation with pristine or protein-coated liposomes containing DOTAP (DOTAP:DOPE, 33:66 mole fraction) or MVL5 (MVL5:DOPC:DOPE, 5:50:45 mole fraction) as the CL. The control without liposomes is also shown. D, representative GUVs (labelled with 0.5 mol% DPPE-Rhodamine, red) upon incubation with 20 μM (lipid concentration) Atto647-DOPE (cyan, representing fusion) liposomes that have been pre-incubated with increasing concentrations of FITC-labelled albumin (green). The arrows point to protein particles or complexes with liposomes on the GUV surface The overlay composite of liposome and albumin channels is also shown. Scale bar: 10 μm. E, membrane signal measured from the average Atto647-DOPE fluorescence intensity upon liposome fusion for a number of GUVs (10-15 per condition) as a function of albumin-FITC concentration. Mean and standard deviation are shown. A fit of an exponential decay (Y=A 1 e (-X/t1) +y 0 , where A 1 and y 0 are the maximum Atto647-DOPE intensity and offset at 0 intensity, respectively) to the data is also shown. F, sketch of the expected interactions between cationic liposomes and GUVs. In the absence of albumin, the liposomes directly fuse with the GUVs, transferring their (cyan) lipids. At intermediate albumin concentrations, partial liposome coverage reduces liposome binding but those that are bound can still fuse. At high protein concentration, complete protein coverage prevents liposome binding altogether and there is no lipid mixing.

Article Snippet: Glucose, sucrose, Dimethyl sulfoxide (DMSO) and the fluorescent probes Bodipy C 16 (BODIPY™ FL C16; 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid), sulforhodamine B (SRB), Dextran-FITC 3 kDa, Albumin–fluorescein isothiocyanate conjugate (FITC-albumin) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Liposomes, Incubation, Fluorescence, Control, Concentration Assay, Membrane, Standard Deviation, Transferring, Binding Assay, Protein Concentration

Journal: bioRxiv

Article Title: Membrane fusion-based drug delivery liposomes transiently modify the material properties of synthetic and biological membranes

doi: 10.1101/2024.08.14.607934

Figure Lengend Snippet: Intracellular delivery of cargos is associated with plasma membrane permeabilization only at high liposome concentration. A, sketch of the putative delivery process. Liposomes (cyan membrane) deliver the encapsulated water-soluble cargo (green) by direct fusion with the PM of living cells. B, representative confocal images of DOTAP:DOPE liposomes (1:1 mole fraction) incubated with living HEK cells at 300 μM lipid concentration. Exposure results in the transfer of Atto647-DOPE from the liposome membrane to the PM as well as the delivery of Dextran 3 kDa, green (encapsulated at a nominal concentration of 40 μg/ml). Incubation was done in the presence of 10 μM SRB as a cell leakage marker. Arrows in the Dextran channel point at cells whose Dextran 3 kDa signal is below detection sensitivity (and hence not visible in this channel). C, real-time visualization of intracellular delivery of water-soluble cargo. Note that due to its small size (∼ 1 nm) Dextran 3 kDa can locate in the nucleus. D, intracellular Dextran 3 kDa concentration for the cell shown in panel C. The decrease in intracellular Dextran signal at later times is due to the cell moving slightly out of focus. E, single cell intracellular Dextran 3 kDa concentration for different liposome compositions and concentrations after 30 minutes of incubation. Mean and standard error of the mean are shown. Note the log scale. F, fraction of cells that become permeable to SRB as a function of liposome (DOTAP:DOPE, 1:1 mole fraction) concentration (total lipid) during 30 minute incubation. Each data point represents the measured fraction of tens of individual cells. Black and green data represent empty and Dextran 3 kDa-filled liposomes. Inset, image of cells incubated with 100 μM or 500 μM DOTAP:DOPE (1:1 mole fraction) liposomes labelled with Bodipy C 16 . G, measured degree of filling of individual cells for several liposomal (total lipid) concentrations. The dashed line indicates the expected values for complete permeabilization. Values above 1 are a result of non-specific SRB affinity for intracellular structures. Histograms are also shown. H, sketch of protein-coated liposomes co-internalized with SRB by cells. I, overlay of DOTAP:DOPE:DOPC (20:50:30 mole fraction) liposomes (500 μM lipid concentration, labelled with 0.5 mol% Atto647-DOPE, cyan) and SRB after pre-incubation with 10x-diluted serum. Scale in C: 10 μm. All other scale bars: 20 μm. The inset shows the magnified image from the dashed square in panel I.

Article Snippet: Glucose, sucrose, Dimethyl sulfoxide (DMSO) and the fluorescent probes Bodipy C 16 (BODIPY™ FL C16; 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid), sulforhodamine B (SRB), Dextran-FITC 3 kDa, Albumin–fluorescein isothiocyanate conjugate (FITC-albumin) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Membrane, Concentration Assay, Liposomes, Incubation, Marker