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]