pe 850759c  (Croda International Plc)

Journal: ACS nano

Article Title: Prototyping Minimal Extracellular Vesicle Mimetics Using Cell-Free Synthesis

doi: 10.1021/acsnano.5c05047

Figure Lengend Snippet: The VESSEL ( V esicle E ngineering S ystems using S ynthetic E xpression and L oading) platform. (A) Schematic detailing the pipeline of the VESSEL platform. Proteomics data from native EVs are analyzed to extract the list of EV proteins. Next, select proteins are synthesized using cell-free protein synthesis and then displayed on liposomes that mimic the size of native EV. The artificial nanovesicles (ANVs) are investigated using cell cultures. (B) Representative nanoparticle tracking analysis (NTA) data showing the average size of our CY5 positive DOPC liposomes (see ). (C) Super-resolution microscopy imaging showing the expanded field of view of CY5 positive DOPC liposomes and a zoomed-in image of one liposome. Scale bars = 10 μ m and 100 nm, respectively (see ). (D) Cryo-electron microscopy imaging showing our CY5 positive DOPC liposomes. Scale bar = 200 nm (see ). (E) Plate reader analysis of size exclusion chromatography (SEC) flow-through while filtering CY5 positive DOPC liposomes. Peak fluorescence was achieved at 1100 to 1500 μ L (Mean ± SD, n = 3) (see ). (F) Schematic detailing the generation of the in-house cell-free system. BL23 (DE3) Star E. coli are grown and sonicated. The transcription and translation machinery are harvested, generating a whole cell extract (WCE). This WCE is mixed with a plasmid coding for a protein of interest and a supplemental buffer. After an overnight incubation, the desired protein is generated on liposomes (see – ). (G) Plate reader-based fluorescence quantification of cell-free generated green fluorescence protein (GFP) comparing two different whole cell extract batches (WCE 14 = The 14th whole cell extract preparation, WCE 15 = The 15th whole cell extract preparation). (Mean ± SD, n = 3).

Article Snippet: For the liposomes used with Transferrin and Aquaporin-Z, 18:1 (Δ9-Cis) PC (DOPC) (850375P-1g, Avanti Polar) and 18:1 Cy5 PE (850–483, Avanti Polar) were used in a mole fraction of 99.85% and 0.15% respectively.

Techniques: Synthesized, Liposomes, Super-Resolution Microscopy, Imaging, Cryo-Electron Microscopy, Size-exclusion Chromatography, Fluorescence, Sonication, Plasmid Preparation, Incubation, Generated

Journal: ACS nano

Article Title: Prototyping Minimal Extracellular Vesicle Mimetics Using Cell-Free Synthesis

doi: 10.1021/acsnano.5c05047

Figure Lengend Snippet: Aquaporin-Z provides a robust membrane anchor for cell-free protein display on liposomes. (A) Schematic illustrating the three different anchoring techniques used. Transferrin (blue) and Aquaporin-Z (green) utilize cotranslation insertion, while strep-tag (orange) relies on biotin/streptavidin binding. (B) Western blot image of CD9-ANVs post SEC filtration comparing the three anchoring methods (see , , ). (C) Western blot quantification of the three anchoring methods post filtration. All samples were normalized to the liposome only (DOPC or POPC) condition depending on the liposome used for the display method (Strep = POPC, all others = DOPC) (Mean ± SD, ** p < 0.01, **** p < 0.0001, n = 3). (D) ELISA quantification of the three anchoring methods using CY5 fluorescence to quantify expression (Mean ± SD, ** p < 0.01, n = 3) (see ). (E) Quantification of the flow cytometry data comparing the three anchoring methods (vesicles displaying protein % = Number of ANV with displayed protein/Total number of ANV) (Mean ± SD, *** p < 0.001, **** p < 0.0001, n = 3) (see ). (F) Example super-resolution microscopy images showing CY5 labeled ANVs with Alexa488 antibody labeled CD9 protein. Red = Liposome, orange = Strep-tag-CD9, cyan = Transferrin-CD9, green = Aquaporin-Z-CD9 (see ). (G) Quantification of the super-resolution microscopy imaging. Unshaded bars represent the average number of localizations for Alexa 488 found on CY5 liposomes. Shaded bars represent the average number of localizations for CY5 found on liposomes (Mean ± SD, *** p < 0.001, **** p < 0.0001, n = 3). For all statistical tests, a one-way, unpaired t test was used.

Article Snippet: For the liposomes used with Transferrin and Aquaporin-Z, 18:1 (Δ9-Cis) PC (DOPC) (850375P-1g, Avanti Polar) and 18:1 Cy5 PE (850–483, Avanti Polar) were used in a mole fraction of 99.85% and 0.15% respectively.

Techniques: Membrane, Liposomes, Strep-tag, Binding Assay, Western Blot, Filtration, Enzyme-linked Immunosorbent Assay, Fluorescence, Expressing, Flow Cytometry, Super-Resolution Microscopy, Labeling, Imaging

Journal: ACS nano

Article Title: Prototyping Minimal Extracellular Vesicle Mimetics Using Cell-Free Synthesis

doi: 10.1021/acsnano.5c05047

Figure Lengend Snippet: ANV exhibits a differential uptake profile into HEK293FT cells. (A) Schematic detailing the workflow of studying ANV uptake into HEK293FT cells. After a 4-h incubation, the cells are imaged, trypsinized, then analyzed by flow cytometry and microscopy (see , ). (B) Representative image of HEK293FT cells with uptaken LAMP1-ANVs. Scale bar = 100 μ m . (C) Geometric mean ( Y -axis) vs the percent uptake ( X -axis) from the cell-based flow cytometry (Protein-ANV Uptake % = number of CY5 positive HEK293FT cells/Total number of HEK293FT cells). Dotted regions are proteins that show statistically significant differences for only the percent uptake. Shaded regions are proteins that show statistically significant differences for both uptake percentage and geometric mean . All protein-ANVs were statistically significant when compared to the cell-free (no protein) condition at the medium dose, except for 7 proteins (Mean, * p < 0.05, n = 3) (see , ). Proteins that are not significant but within the shaded region are colored orange . (D) Single nanoparticle flow cytometry (left) and cell-based flow cytometry (right) show the effect of reducing the plasmid concentration on both protein display and uptake percentage. (Protein Display % = Number of protein displaying ANV/Total number of ANV, Protein-ANV Uptake % = Number of CY5 positive HEK293FT cells/Total number of HEK293FT cells). Significance values on the top represent ITM2C-ANV compared to 1X plasmid concentration. Significance values on the bottom represent CADM1-ANV compared to 1X plasmid concentration (Mean, * p < 0.05, ** p < 0.01, n = 3). These proteins were selected due to their “robust-uptake” classification. (E–G) Dose response curve based on cell-based flow cytometry. The fold change of protein-ANV uptake percentage decreases with a decrease in ANV dose. All samples normalized to the cell-uptake-percentage at 20,000 ANV/cell. Three categories were created from trends observed: “robust-uptake” (red) = supra-linear changes, “standard-uptake” (black) = linear changes, and “sensitive-uptake” (green) = sublinear changes with decreasing ANV dose. Graph E contains 5 proteins, graph F contains 13 proteins, and graph G contains 21 proteins. (H) Heatmap comparing different characteristics of the proteins. U = Uptake percentage at medium dose, C = net charge, S = size of the full Aquaporin-Z-protein construct in kDa, and L = frequency of mention in literature for publications that feature both the protein name and the term “vesicle uptake” (see ). Proteins are ordered by the lowest HEK293FT uptake percentage on the left, with the highest uptake percentage on the right. Net charge and size of the proteins do not correlate with the HEK293FT uptake of these proteins. Finally, frequency in literature (L) shows that a vast majority of the proteins have not been tested for uptake effectiveness. Some proteins, like the three tetraspanins and LAMP1, are mentioned frequently due to their use as markers for EVs or lysosome uptake respectively (see ). For all statistical tests, a one-way, unpaired t test was used.

Article Snippet: For the liposomes used with Transferrin and Aquaporin-Z, 18:1 (Δ9-Cis) PC (DOPC) (850375P-1g, Avanti Polar) and 18:1 Cy5 PE (850–483, Avanti Polar) were used in a mole fraction of 99.85% and 0.15% respectively.

Techniques: Incubation, Flow Cytometry, Microscopy, Plasmid Preparation, Concentration Assay, Construct

Journal: ACS nano

Article Title: Prototyping Minimal Extracellular Vesicle Mimetics Using Cell-Free Synthesis

doi: 10.1021/acsnano.5c05047

Figure Lengend Snippet: VESSEL mimetics promote neuroprotection using cell-free generated protein. (A) Schematic detailing the SH-SY5Y neuroprotection assay. A culture of SH-SY5Y cells will be treated with staurosporine for 4-h. This staurosporine will be removed, and the protein-ANVs will be added. After a 5-day incubation, Calcein-AM is used to quantify growth (see ). (B) Representative calcein-AM images comparing SH-SY5Y growth after 5 days. Scale bar = 200 μ m. (C) Quantification of Calcein-AM branch length fold change at the medium dose. Branch length calculation found in . All samples normalized to the blank (PBS only) condition (Mean ± SD, * p < 0.05, n = 3) (see , ). These proteins were selected due to their reference in the literature with neuroprotection/neurite outgrowth function. (D) Cell-based flow cytometry analysis showing geometric mean ( Y -axis) vs percent uptake ( X -axis) for SH-SY5Y cells at the medium (4,000 ANV/cell) dose. (Uptake % = Number of CY5 positive SH-SY5Y cells/Total number of SH-SY5Y cells). Starred points represent significant differences when compared to cell-free for uptake % (Mean, * p < 0.05, n = 3) (see ). The proteins were selected based on the cell-uptake classifications, with two proteins representing each group (“robust-uptake” – LAMP1 and CADM1, “standard-uptake” – MMP14 and SDC2, and “sensitive-uptake” – PDPN and MPZL1). (E) Cell-based flow cytometry quantification showing uptake fold change decreasing as dose decreases for the proteins found in D. All samples were normalized to their 20,000 protein-ANV/cell uptake value.

Article Snippet: For the liposomes used with Transferrin and Aquaporin-Z, 18:1 (Δ9-Cis) PC (DOPC) (850375P-1g, Avanti Polar) and 18:1 Cy5 PE (850–483, Avanti Polar) were used in a mole fraction of 99.85% and 0.15% respectively.

Techniques: Generated, Incubation, Flow Cytometry