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Supplementary Information
Supplementary materials and methods
Materials. Tetramethylrhodamine-5-isothiocyanate (TRITC) and black hole quencher
(BHQ-1) were obtained from Life Technologies (Carlsbad, CA, USA) and Biosearch
Technologies (Novato, CA, USA), respectively. Cell counting kit-8 (CCK-8) was
from Dojindo Laboratories (Kumamoto, Japan). Millicell hanging culture inserts
(pore size: 8 μm) were obtained from Merck Millipore (Darmstadt, Germany).
Matrigel basement membrane matrix and biocoat matrigel invasion chamber were
purchased from BD Biosciences (San Jose, CA, USA). Paraformaldehyde (4%) and
crystal violet were from Solarbio (Beijing, China). Rabbit polyclonal antibodies
against focal adhesion kinase (FAK), phosphorylated FAK (p-FAK, phospho-Y397),
extracellular signal regulated kinases (ERK1/2), phosphorylated ERK1/2 (p-ERK,
phospho-T202/Y204), phosphoinositide 3-kinase (PI3K) p85/p55, phosphorylated
PI3K (p-PI3K, phospho-Y467/Y199), protein kinase B (Akt), phosphorylated Akt
(p-Akt, phospho-S473) , membrane type 1 matrix metalloproteinase (MT1-MMP),
cortactin and GAPDH were obtained from Bioworld Technology (St. Louis Park, MN,
USA). Anti-CD31 rabbit polyclonal antibody was from Abcam (Cambridge, UK).
Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG and mouse
monoclonal antibody against proliferating cell nuclear antigen (PCNA) were from
Santa Cruz Biotechnology (Dallas, TX, USA). Doxorubicin was from Huafeng United
Technology (Beijing, China).
Evaluation of the drug loading and release profiles of DEAP-C16Y-Dox.
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Doxorubicin (Dox; 0.1-1.0 mg) together with DEAP-16Y or a fragment of human
serum albumin conjugated with DEAP (DEAP-HSAF; 1.0 mg) were dissolved in 10
μL DMSO followed by dilution to 1 mL of PBS (pH 7.4) under ultrasonication (600
W) for 2 min. After incubation at the room temperature for 2 h, the supernatant was
collected and lyophilized. The lyophilized materials were dissolved in DMSO and
examined using UV-Vis spectrophotometry (LAMBDA650, PerkinElmer, USA). The
encapsulation efficiency was calculated using the formula: (mass of drug
encapsulated/mass of drug added)×100%. The drug loading ratio was derived from
the formula: [mass of drug encapsulated/(mass of peptide + mass of drug
encapsulated)] ×100%.
The Dox release profile from DEAP-C16Y-Dox was measured using dialysis. Briefly,
DEAP-C16Y-Dox solution was injected into dialysis cartridges with a molecular
weight cutoff value of 2000, followed by dialysis against 10 mL of PBS at different
pHs. The concentration of Dox remaining in the dialysis cartridge at different time
points was measured by UV-Vis spectrophotometry.
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DEAP-C16Y-Dox
DEAP-HSAF-Dox
Dox: Peptide (mass ratio)
1: 10
3: 10
1: 2
4: 5
1: 1
Encapsulation efficiency (%)
56.1
44.0
44.6
26.9
21.6
Drug loading ratio (%)
5.31
11.7
18.2
17.7
17.8
Encapsulation efficiency (%)
55.7
47.3
43.0
26.6
21.0
Drug loading ratio (%)
5.28
12.4
17.7
17.6
17.4
Supplementary Table S1. Encapsulation and loading efficiency of doxorubicin
(Dox) in DEAP-C16Y-Dox or DEAP-HSAF-Dox assemblies
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Supplementary Figure S1. Characterization of the DEAP-C16Y self-assembling
nanoprobe. (A) TEM images of the DEAP-C16Y self-assembling nanoprobe at
different pHs in PBS. A mixture of TRITC and BHQ-1 in PBS (pH=7.4) was also
examined. Scale bars, 100 nm. (B) Fluorescence intensity of the DEAP-C16Y
self-assembling nanoprobe at different pHs in PBS. A mixture of TRITC and BHQ-1
(molar ratio, 1:1) in PBS (pH=6.5) was used as control. (C) Fluorescence intensity of
TRITC and BHQ-1 mixture at different pHs in PBS. No significant differences were
observed among different pHs.
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Supplementary Figure S2. Characterization of DEAP-HSAF self-assembling
nanostructures and effect of DEAP-16Y peptide on endothelial cells and tumor
cells. (A) Schematic structure of the DEAP-HSAF peptide. (B) TEM images of
DEAP-HSAF nanostructures at pH 7.4 and 6.8. Loss of the nanoparticulate structure
was observed at pH 6.8. Scale bars, 50 nm. (C) Representative images of the HUVEC
transwell migration assay. Migrated cells were stained and captured. Scale bars, 25
μm. (D) Representative images of the HUVEC tubule formation assay. Scale bars, 50
μm. (E) Quantification of the gelatin degradation areas based on Fig. 4C. The overlap
of F-actin with gelatin degraded areas per cell in each group was quantified using
Image J, and normalized to that in PBS group. Eight fields in each group were utilized
for quantification. **, p< 0.01 compared with the PBS group.
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Supplementary Figure S3. Biodistribution and antitumor efficacy of
DEAP-C16Y nanostructures in mice bearing MDA-MB-231 tumors. (A) The
biodistribution of Cy5.5-DEAP-C16Y nanostructure in tumors and major organs 12 h
after systemic injection into tumor-bearing mice. Control mice were injected with
PBS. The average fluorescence intensity (FI) in each organ was quantified and
normalized to the total fluorescence intensity in all organs (right panel). (B) Growth
of MDA-MB-231 tumors. Tumor-bearing mice were intravenously injected with PBS,
DEAP-HSAF nanostructures, C16Y or DEAP-C16Y nanostructures with the peptide
concentration of 6.5 μmol/kg for indicated period. Tumor volume was calculated
every other day. (C) Weight of above MDA-MB-231 tumors on day 27 when mice
were sacrificed. (D) Microvessel density in MDA-MB-231 tumors. MDA-MB-231
tumors were sectioned and stained with CD31 antibody for blood vessels. Tumor
sections were captured by light microscopy (magnification: 200×). Scale bars, 25 μm.
The average number of microvessels per field was quantified in (E). **, p< 0.01.
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Supplementary Figure S4. Evaluation of the toxicity of DEAP-C16Y
nanostructures. (A) H&E staining of major tissues. BALB/c mice (female, 8 weeks)
were treated by C16Y (6.5 μmol/kg each day) or DEAP-C16Y nanostructures (6.5
μmol/kg every other day) for 14 days (five mice for each treatment). Sections of
major organs were analyzed by H&E staining. Tissues from normal BALB/c mice
served as control. Scale bars, 25 μm. (B) The body weight of mice during the
treatment period.
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Supplementary Figure S5. Combination therapy of DEAP-C16Y and Dox. (A)
TEM images of DEAP-HSAF-Dox and DEAP-C16Y-Dox in PBS at pH 7.4 or 6.8.
Scale bars, 200 nm. (B) The release profiles of Dox from DEAP-C16Y-Dox
nanostructures at different pHs. (C) Staining of lung sections from mice in Fig. 6A by
H&E or PCNA. T indicates metastatic foci. Scale bars, 200 μm. (D) The lung weight
of 4T1-bearing mice on day 27. **, p<0.01 compared with the DEAP-HSAF group.
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