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Intracellular Delivery via Non-Charged Sequence-Defined Cell-Penetrating Oligomers Ngoc Nhu Phan, Connie Li, and Christopher A. Alabi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00336 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 30, 2018
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Bioconjugate Chemistry
Scheme 1. Synthetic methodology utilized for the assembly of sequence-defined oligoTEAs. OligoTEAs are assembled with a series of iterative thiol-ene and thiol-Michael additions interspersed with fluorous solid phase purifications (FSPE). 1299x639mm (72 x 72 DPI)
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Figure 1. (A) Cellular uptake of fluorescein cargo by oligoTEAs in HeLa cells measured by flow cytometry. Cells were treated with 5 µM of fluorescein-oligoTEA conjugates for 1 hour. (B) Live-cell confocal microscopy of fluorescein cargo uptake by PEO2-B and R9 in HeLa cells. Images are the merged green fluorescence of the fluorescein-oligoTEA conjugates and the bright field image of the cell. (C) Cellular uptake of Atto-488PEO2-B (5 µM, 1 hour) in HeLa cells. Cells were treated with a trypan blue solution for cell surface quenching prior to data acquisition. (D) Cellular uptake of PEO2-B in HeLa, SKOV-3 and HEK293 cells measured by flow cytometry. All cells were treated with 5 µM of PEO2-B-fluorescein conjugates for 1 hour. (E) Dose-dependent uptake of PEO2-B in HeLa cells. (F) Uptake kinetics of fluorescein-PEO2-B at 2.5 µM in HeLa cells via flow cytometry.
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Bioconjugate Chemistry
Figure 2. (A) Cellular uptake of PEO2-B and R9 conjugates in HeLa cells with and without serum. (B) Cellular uptake of PEO2-B and R9 in HeLa cells with and without heparin pretreatment at the indicated doses. 230x101mm (300 x 300 DPI)
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Figure 3. Temperature-dependent cellular uptake of (A) PEO2-B and (B) R9 in HeLa, SKOV-3 and HEK293 cells. Effect of endocytosis inhibitors on cellular uptake of (C) PEO2-B and (D) R9 in HeLa, SKOV-3 and HEK293 cells. Cells were pre-treated with chlorpromazine to inhibit clathrin, filipin III to inhibit caveolae, cytochalasin D to inhibit macropinocytosis, and NaN3/2-deoxy-D-glucose to block ATP synthesis. 236x182mm (300 x 300 DPI)
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Bioconjugate Chemistry
Figure 4. Membrane fluidity reflected by the Laurdan GP values of HeLa, SKOV-3 and HEK293 cells. 93x101mm (300 x 300 DPI)
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Figure 5. Confocal microscopy of fluorescein-PEO2-B (green) co-delivered with (A) Transferrin-AlexaFluor 647, (B) Dextran-AlexaFluor 647, and (C) LysoTracker Deep Red. The nuclei are stained blue with Hoescht 33342 at 40X magnification. 778x777mm (72 x 72 DPI)
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