Figure Legends
Figure 1. The schematic image of novel fusion peptide design, NC formation with siRNAs, specific delivery, and inhibition of cell viability to breast cancer cells. The novel fusion peptide was designed as a combination of polyarginine, GGGG linker, and homing peptide. NCs (278 nm, 10 mV) were self-assembled between peptides and siRNAs via electrostatic attraction and hydrogen bonds. NCs specifically entered into breast cancer cells through specific receptor clathrin-mediated endocytosis. CGI siRNAs repressed the breast cancer cell viability. The image was drawn using the Biorender software.
Figure 2. (A) Agarose gel electrophoresis images using gel retardation assay of RS- and RT-NCs. First, ten picomoles siRNAs were incubated with RS peptide (left) and RT peptide (right) at each N/P ratio for 30 minutes. Next, NCs were loaded into 1% agarose gel wells and electrophoresed at 100V for 35 minutes. The gel documentation system took gel images. (B) Hydrodynamic radius, polydispersity index, and zeta potential of RS-NC at each N/P ratio were measured by zetasizer (n =3). (C) Hydrodynamic radius, polydispersity index, and zeta potential of RT-NC at each N/P ratio were measured by zetasizer (n =3). (D) NC stability in Opti-MEM was assessed by a fluorescence cell counter. The number of three NCs was counted using ImageJ software. The NC count was normalized by the initial NC count.
Figure 3 . Flow cytometry results about cellular delivery of R11-, RS- and RT-NCs with 200 nM Cy3-labeled siRNAs. MDA-MB-231, MCF-7, BT-474, and SK-BR-3 were used as different types of breast cancer cells. RAW264.7 was used as a normal cell line. Red and green populations represented cell numbers of free siRNA and NC-mediated delivery, respectively. The number represented cell percentage with increased fluorescence compared to the control.
Figure 4. (A) Fluorescence imaging about cellular uptake of R11-, RS- and RT-NCs with 200 nM Cy3-labeled siRNAs. Blue, green, and red fluorescences represented the nucleus (Hoechst 33342), actin filament (Phalloidin), and NCs (Cy3), respectively. The fluorescence cell image was observed using fluorescence microscopy at 150× magnification. Scale bar represented 100 μm length. (B) Z-stack imaging about cellular internalization of RS-NC. Red and green fluorescence represented actin filament (Cy3) and RS-NC (FITC). Z-axis images were observed at 900× magnification. Scale bar represented 25 μm length.
Figure 5. The endocytosis pathway is reduced by endocytosis inhibitors when RS-NC application. MDA-MB-231 cells were incubated with each endocytosis inhibitor (Chlorpromazine, Methyl-β-cyclodextrin, Cytochalasin D, and Filipin III) for 30 minutes. Then, cells were incubated with RS NC with 200 nM siRNA for four hours and analyzed using flow cytometry. Red and green populations represented cell counts without endocytosis inhibitor and with endocytosis inhibitor, respectively. The number represented the cell population with decreased fluorescence compared to the control.
Figure 6. (A) Reduced cell proliferation by siRNAs delivered via RS-NC at various N/P ratios. MDA-MB-231 cells were incubated with RS-NC with 200 nM CGI siRNA for 24 hours and repeated once. (B) Reduced cell proliferation by R11-NC of various siRNA concentrations. MDA-MB-231 cells were incubated with R11-NC with various siRNA concentrations for 24 hours and repeated once. (C) Reduced cell proliferation by RS-NC of various siRNA concentrations. MDA-MB-231 cells were incubated with RS-NC with various siRNA concentrations for 24 hours and repeated once. Cell proliferation was measured using a CCK-8 assay kit. The Centerline and error bar represented mean and standard deviation normalized by the value of cell only. (D) Gene silencing by siRNA delivered via RS-NC. MDA-MB-231 cells were incubated with RS-NC with 200 nM for anti-Kaiso siRNA for 24 hours. mRNA concentration was calculated by the ΔΔCt method using quantitative PCR of reversed transcribed cDNA. Centerline and error bar represented mean and standard deviation normalized by the absorbance at 450 nM of the cell only condition. *, **, and *** mean p-value less than 0.05, 0.005, and 0.001 using T-test (n = 12, 2).
Figure 7. Cytocompatibility of RS and RT fusion peptides. (A) MDA-MB-231 cells and (B) HDFn cells were incubated with various concentrations of RS (left) and RT (right) peptides for 4 hours. Cell proliferation was measured using a CCK-8 assay kit. Centerline and error bar represented mean and standard deviation normalized by the absorbance at 450 nM of the cell only condition. * means p-value less than 0.05 using T-test (n = 8).
Figure 8. (A) H&E stained tissue observation of RS-NC-treated skin tissue. DPBS and RS-NC with 200 nM negative control siRNA were injected to epilated back of the Balb/c mouse, respectively. After one day, injected tissue was harvested, fixed, and stained by H&E solution. Stained tissues were observed using a microscope at 20× magnification. Scale bar represented 100 μm length. (B) TNF-alpha and IL-6 levels of RS-NC-treated skin tissue. Conditions of DPBS and RS-NC with 200 nM negative control siRNA were injected to epilated back of the Balb/c mouse, respectively. After solubilization of harvested skin tissue, TNF-alpha (left) and IL-6 (right) levels were measured using each ELISA kit and 4 PL standard curve, respectively. The centerline and error bar represented mean and standard deviation.[35]