low molecular weight polyic  (InvivoGen)

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Self-Assembly of Immune Signals Improves Codelivery to Antigen Presenting Cells and Accelerates Signal Internalization, Processing Kinetics, and Immune Activation.

doi: 10.1002/smll.201802202

Figure Lengend Snippet: Figure 1. iPEMs can control immune cell activation by delivering multiple immune signals. a) Schematic of iPEM synthesis and immune activation following treatment with iPEMs. Starting at the top left, iPEMs are syn- thesized through the electrostatic interactions between cationic peptide antigen and anionic nucleic acid-based TLR3a. iPEMs can be used to control the kinetics of APC uptake and activation by delivering signals over time to target receptors in endosomes. b) Fluorescent microcopy images of iPEMs composed of model antigen SIINFEKLR9 (green) and TLR3a, polyIC (red). iPEMs colocalize both signals (yellow) in carrier-free capsules. Scale bar is 10 µm.

Article Snippet: Low molecular weight polyinosinic-polycytidylic acid (polyIC) was purchased from Invivogen. iPEMs were synthesized as previously described with the exception that all cargo and wash solutions were in DI water.

Techniques: Control, Activation Assay, Capsules

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Self-Assembly of Immune Signals Improves Codelivery to Antigen Presenting Cells and Accelerates Signal Internalization, Processing Kinetics, and Immune Activation.

doi: 10.1002/smll.201802202

Figure Lengend Snippet: Figure 2. Time dependent uptake of iPEMs by primary DCs. a) Fluorescence microscopy images of primary CD11c+ DCs incubated with iPEMs for 15 to 60 min reveal an increase in iPEMs taken up over time. Scale bar is 10 µm. b) Image analysis shows quantitatively an increase in the number of iPEMs per cell between 15 and 60 min. c) Flow cytometry of DCs incubated with iPEMs for 0 to 4 h, show an increase in iPEM cargo fluorescent intensity. d) Flow cytometry analysis of cells positive for iPEM cargo show an increase in iPEM cargo uptake (red and green bars) over 4 h. Free polyIC was also taken up over 4 h but to a lesser extent. e) Flow cytometry analysis of DCs incubated with iPEMs for 24 h shows an increase in iPEM signals over the first 4 h followed by a decrease over the course of the next 20 h. DCs incubated with dose-matched soluble polyIC show only a modest increase in polyIC intensity after 24 h. Error bars are standard deviation. Statistical analysis performed by two-way ANOVA; **** = P ≤ 0.0001.

Article Snippet: Low molecular weight polyinosinic-polycytidylic acid (polyIC) was purchased from Invivogen. iPEMs were synthesized as previously described with the exception that all cargo and wash solutions were in DI water.

Techniques: Fluorescence, Microscopy, Incubation, Flow Cytometry, Standard Deviation

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Self-Assembly of Immune Signals Improves Codelivery to Antigen Presenting Cells and Accelerates Signal Internalization, Processing Kinetics, and Immune Activation.

doi: 10.1002/smll.201802202

Figure Lengend Snippet: Figure 3. Codelivery of immune signals to primary DCs with iPEMs. a) Flow cytometry analysis of primary DCs incubated with iPEMs shows that in roughly 40–80% of cells, iPEMs deliver both antigen and adjuvant. b) Flow cytometry analysis of cells positive for both antigen and adjuvant reveals an increase in the frequency of cells over the first 4 h, which was sustained for up to 18 h. c) Image analysis of fluorescence microscopy images of iPEM uptake shows an increase in pixels positive for iPEM signals over time. Each pixel is plotted by the fluorescent intensity of both the FITC signal resulting from antigen and Cy5 signal from polyIC with the highest intensity of both signals in the top right corner. d) Quantifying the fraction of SIIN-R9 signal colocalized with polyIC signal from the image analysis plots in B, shows that over 80% of immune signals are colocalized, which is sustained over 1 h. Error bars are standard deviation.

Article Snippet: Low molecular weight polyinosinic-polycytidylic acid (polyIC) was purchased from Invivogen. iPEMs were synthesized as previously described with the exception that all cargo and wash solutions were in DI water.

Techniques: Flow Cytometry, Incubation, Adjuvant, Fluorescence, Microscopy, Standard Deviation

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Self-Assembly of Immune Signals Improves Codelivery to Antigen Presenting Cells and Accelerates Signal Internalization, Processing Kinetics, and Immune Activation.

doi: 10.1002/smll.201802202

Figure Lengend Snippet: Figure 7. DC activation with iPEMs analyzed by flow cytometry. Flow cytometry analysis of surface markers of DC activation a) CD86 and b) CD40 reveals an increase in DC activation following incubation of DCs with iPEMs over time, to a greater extent than dose-matched soluble delivery of polyIC, especially over the first 4 h. c) Flow cytometry analysis of antigen presentation in MHCI shows an increase in antigen presentation over time following treatment with iPEMs comparable to treatment with dose-matched soluble components. d) TLR3 reporter cell activity shows iPEMs more efficiently activate the TLR3 signaling pathways than a dose-matched treatment with Free polyIC. Error bars are standard deviation. Statistical analysis performed with a T test comparing iPEMs to Free polyIC and SIIN-R9. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001, n.s. = not significant.

Article Snippet: Low molecular weight polyinosinic-polycytidylic acid (polyIC) was purchased from Invivogen. iPEMs were synthesized as previously described with the exception that all cargo and wash solutions were in DI water.

Techniques: Activation Assay, Flow Cytometry, Incubation, Immunopeptidomics, Activity Assay, Protein-Protein interactions, Standard Deviation