supplementary data 1. materials and methods 1.1 preparation of … · 2016. 5. 6. · nanosciences,...
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Supplementary Data
Supplementary data that consist of Materials and Methods and supplemental figures can be
found in the online version.
1. Materials and Methods
1.1 Preparation of nanoparticles
Synthesis of 3F peptides All compositions are in weight percent unless otherwise noted. 3F (Ac-
DWFKAFYDKVAEKLKEAF-NH2) was synthesized from D-amino acids by the solid phase
method using a peptide synthesizer (PS3, Protein Technologies). Amino acids blocked by
fluorinylmethyloxycarbonyl (FMOC) group were coupled to a rink amide resin (EMD Millipore).
Piperideine, N-methylmorpholine (NMM), and hexafluorophosphate (HBTU) in
dimethylformamide (DMF, Protein Technologies) were used to remove the FMOC group and
activate amino acids. The peptide was acetylated with acetic anhydride at the N-terminus and
cleaved with trifluoroacetic acid/triisopropylsilane/water (95%/2.5%/2.5%).
Synthesis of targeting peptide Prostate-targeting-peptide (PTP, amino acid sequence =
CQKHHNYLC) was chosen as the targeting moiety of our nanoparticle because it specifically
binds to prostate-specific-membrane-antigen (PSMA) [40], whose overexpression is associated
with malignant transformation of the prostate (Fig. S2) [41, 42]. CQKHHNYLCK(Dde) was
synthesized using FMOC-Lys(Dde)-OH. PTP was conjugated to PEG-phospholipid (PL) as
described below. The 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) side-chain
protecting group was later removed by hydrazine treatment. COOH-PEG-PL (1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000], Avanti Polar Lipids)
was next attached to the ε-amino group of lysine. Cyclization of the peptide was performed in DI
water for 4 days.
Peptides were purified and analyzed by a HPLC (JASCO) which includes a VIVA C18
column (5 µm 250×4.6 mm and 250×21.2 mm, Resteck).
Synthesis of 3F-stabilized nanoparticles encapsulating paclitaxel, SPIO (for MRI) or
fluorophores (for optical detection) Phospholipid based nanoparticles (NP) that are stabilized by
3F peptides and encapsulate paclitaxel (PTX) or imaging probes including super paramagnetic
iron oxide (SPIO) for MRI, DiD, DiR or Cy7.5 for optical detection were prepared (detailed in
SI). All 3F-NP used in this study contain targeting peptides (i.e., PTP); non-targeted 3F-Cy7.5
was compared with 3F-Cy7.5 (with PTP) in Fig S1. The 3F-PTX nanoparticles (NP) were used
in therapeutic studies. The 3F-SPIO NP were used for measuring the kinetics of liver uptake and
blood clearance of 3F-NP by in vivo MRI; 3F-SPIO accumulation in the liver and tumor was
further quantified by transverse relaxation times, i.e., T2 by spin echo and T2 star (T2*) by
gradient echo. 3F-SPIO NP were also visualized by Prussian blue staining of tissue sections. 3F-
DiD, 3F-DiR, and 3F-Cy7.5 nanoparticles were employed in blood clearance and bio-distribution
studies.
Synthesis of 3F-stabilized nanoparticles encapsulating paclitaxel, SPIO, DiD, DiR, or Cy7.5
Briefly, to make 3F-NP encapsulating paclitaxel and decorated by targeting PTP peptides, 78%
of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (phospholipid, PL, purchased from Avanti
Polar Lipids, Alabaster, Alabama ), 10% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-
N-[methoxy(polyethylene glycol)-2000] (PEG-PL, Avanti Polar Lipids), 2% of PTP-PEG-PL in
chloroform and 10% of 3F in methanol were added to paclitaxel (LC Laboratories, Woburn,
Massachusetts) in chloroform and dried in N2 at room temperature to form thin films. Phosphate
buffer solution (PBS) was added to the dried film, followed by sonication at room temperature to
form 3F-PTX. The size and zeta potential of NP were measured by a particle size analyzer
(Zetasizer 3000HS, Malvern).
To make 3F-NP encapsulating SPIO and decorated by PTP, Fe(III) acetylacetonate (2
mmol) was added to phenyl ether (20 mL) with 1,2-hexadecanediol (10 mmol) and cholic acid
(12 mmol) under N2, then heated to 265°C to form superparamagnetic iron oxide (SPIO) coated
with cholesterol acid (chol-SPIO). Using Chol-SPIO as “oil”, NPs of the same shell composition
as described above (78% PL, 10% PEG-PL, 2% PTP-PEG-PL and 10% 3F) were prepared as
above, and referred to as 3F-SPIO NPs or simply, 3F-SPIO. The shell-to-SPIO weight ratio was
15:1 for these NPs. To verify the Fe concentration, 20 µL of IO-containing sample was dissolved
in 980 µL of 5 M hydrochloric acid and the absorbance at 410 nm was measured by an UV-VIS
spectrometer (Evolution 201, Thermo Scientific) with reference to calibration standards [43].
Cy7.5-NHS (Lumiprobe, Florida) was conjugated with 1,2-dihexadecanoyl-sn-glycero-3-
phosphoethanolamine to make Cy7.5-PL. Cy7.5-PL, DiD (1,1'-dioctadecyl-3,3,3',3'-
tetramethylindodicarbocyanine perchlorate, purchased from Invitrogen) and DiR (1,1'-
dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide, purchased from Invitrogen) were
employed to other shell components in methanol and prepared with same procedure as previous
NP preparation. The dye content of NPs was 0.1mg/ml.
1.2 In vivo studies
In vivo studies aim to examine: 1. How liposome-mediated RES-blockade affects the
blood clearance and tissue distribution of subsequently injected testing nanoparticles; 2. Whether
RES-blockade affect liver function and capacity of RES to clear foreign bacteria; 3. To what
degree such blockade can enhance therapeutic efficiency of paclitaxel-nanoparticles on human
prostate xenografts by detection of early cell death and long term tumor growth delay.
All animal studies were approved by Institution Animal Care and Use Committee
(IACUC) of the University of Pennsylvania. Gaseous anesthesia (1- 1.5% isoflurane mixed with
air) administered via a nose cone was used in all procedures required for anesthetizing the mouse.
Human prostate cancer xenografts model: Ten million LNCaP (ATCC) cells were suspended in
75 μl of RPMI 1640 medium mixed with 75 μl Matrigel (BD Biosciences) and were inoculated
subcutaneously into the flank of adult male athymic nude mice (01b74 strain, 4-6 wk old, NCI
Production).
Induction of RES blockade by liposome Commercially available liposome (Encapsula
NanoSciences, Brentwood, TN) that are made of phosphatidylcholine and cholesterol (PC: Chol
= 7: 3 molar ratio) and contain no drug cargo were used for RES-blockade. These liposome have
a broad size distribution (0.3- 3 µm diameter), thus are optimal for rapid phagocyte recognition
and phagocytosis. To induce RES-blockade, the liposome were administered to mice at 376 mg
PL /kg intravenously via tail vein. Animals in control group were given the same volume of
saline.
Effect of RES-blockade on kinetics of liver uptake and blood clearance of testing
nanoparticles: To test the duration and extent of RES-blockade, at 1, 1.5, and 3 h after liposome
injection, testing nanoparticles (3F-SPIO) were injected at 10 mg Fe/kg concentration, and
kinetic data from liver and blood were obtained simultaneously by non-invasive MRI (n=3 for
each time point). Animals in control group (n=3 for each time point) were subjected to the same
MRI protocol (below).
In vivo kinetic study of 3F-SPIO was performed on a 9.4 Tesla /31 cm ID horizontal bore
magnet equipped with a 120 mm ID gradient tube of 20 gauss /cm and interfaced to a Varian
DirectDrive console (Agilent, Palo Alto, CA). A 35 mm ID volume coil was used for all MRI
studies. During imaging, the free-breathing mouse was sedated by isoflurane and its rectal
temperature, electrocardiogram (ECG) and respiration were monitored (SA Inc, Stony Brook,
NY) [44, 45]. The core temperature was maintained at 37°C by directing warm air into the bore
with a feedback loop (SA Inc); meanwhile the respiration signals were used to synchronize (gate)
the image acquisition to minimize motion interference.
Respiration-gated rapid MR imaging was performed using a two-dimensional gradient-
echo sequence with (TR/TE = 7/3.5 ms, flip angle = 10°, matrix of 128×128), field of view of
30×30 mm and one signal average. Data acquisition occurs only during the expiration plateau
period to reduce motion artifacts. It takes ~3.2 s (4 respiration cycles) to generate one image and
1000 images were acquired continuously in a period of ~3200 s (~53 min). After the first 34
images were obtained, 3F-SPIO nanoparticles were injected intravenously via catheter tubing
over a period of 30-45 s followed by saline flush, while the mouse remained in the scanner and
data acquisition continued without pause. To extract the kinetic data, a region of interest (ROI)
was placed over the liver and vena cava (Fig 1B inset) and the average intensity overtime, S(t),
was normalized to that from average of pre-injection images, S0; -Ln[S(t)/S0] from the liver and
blood ROI was averaged and plotted from saline (blue, n=3) or liposome (red, n=3) treated group,
respectively (Ln is natural logarithm). To quantify the kinetic data, the area under the curve
(AUC) from the time of 3F-SPIO injection up to 3085 s after injection was measured using
ImageJ (http://imagej.nih.gov/ij/ by National Institutes of Health, USA). In addition, to better
view the initial uptake of SPIO nanoparticles by the liver, a sliding window was applied to t ϵ [0,
200] s, in which 5 points were averaged and plotted with 5-fold reduced temporal resolution as
an inset in Fig 1; this approach reduces noise (especially for the red trace).
Effect of RES-blockade on liver and tumor accumulation of testing NP by MRI T2 and T2*
maps for liver and tumor were acquired at pre- and 24 h post-injection of 3F-SPIO (0.5 mg Fe/kg)
using Multi-Echo-Spin-Echo (TR/TE =164.64/5.04 ms, matrix =128×128) and Multi-Echo-
Gradient-Echo-Multi-Slice (TR/TE =200/2.9 ms, matrix = 128×128) pulse sequence,
respectively (n=3 for each group). The data were processed using ImageJ.
Effect of RES-blockade on blood clearance and bio-distribution of testing NP over a 24 h
period To study the bio-distribution in the presence and absence of RES-blockade, mice were
pretreated with 376 mg PL /kg liposome or saline 1.5 h before injection of 3F-Cy7.5 (or 3F-DiR),
containing targeting moiety unless noted otherwise. After 24 h, mice were sacrificed and organs
were collected for optical imaging (IVIS Spectrum Pre-clinical In Vivo Imaging System,
PerkinElmer Inc., USA, excitation/emission filter: 750/788 nm). The fluorescence intensities on
images of each organ were averaged and normalized to its area and the mean intensity was used
to compare the NP uptake among organs (n=3 for each group).
Blood clearance was studied in separate groups of mice: 3F-DiD nanoparticles were
injected 1.5 h post injection of empty liposome or saline. Blood was collected by cardiac
puncture under anesthesia at 0, 15, 60 min, 5 and 24 h post injection of 3F-DiD. Three (3) mice
were used for each time point. Blood was centrifuged at 1000 rpm x 10 min and aliquots of
serum (200 µL for each well for 2-3 wells) were placed in a 96-well microplate and fluorescence
was read from a Microplate Reader (SpectraMax M5, Molecular Devices).
Assessing therapeutic effect of paclitaxel nanoparticles enhanced by RES-blockade Two
therapeutic studies were performed for assessing the short and long term effect of RES-blockade.
The 1st study included 5 groups: 1. RES-Blockade + 3F-PTX; 2. Saline + 3F-PTX; 3. TAXOL; 4.
RES-Blockade only; 5. Saline. To induce RES-blockade, the empty liposome at a dose of 376
mg PL/kg were i.v. injected 1.5 h before injection of 3F-PTX nanoparticles, which were also i.v.
injected at 10 mg paclitaxel per kg. TAXOL is the standard clinical formulation of PTX: 6 mg
PTX is dissolved in 1 mL Cremophor: Ethanol (1:1), and the solution was diluted in saline and
injected i.v. at 10 mg PTX per kg. Diffusion-weighted MRI was performed pre-, 48 h and 9 day
post-treatment to measure apparent diffusion coefficient (ADC) of water in the tumor by MRI
(detailed at the end of this subsection, n=3 for each group).
The 2nd
study examining long term effect on tumor growth delay includes 4 groups: 1.
RES-Blockade + 3F-PTX; 2. Saline + 3F-PTX; 3. RES-Blockade only; 4. Saline. In this study,
mice were treated at day 0 and 6. Each treatment employs RES-blockade and NP injection the
same dose as the 1st study. In every day (for the first 5 days) and every other day (for the
remaining time), the animal was weighed and the tumor was measured in two orthogonal
dimensions (a, b, b < a) by a caliper. Tumor volume is estimated by formula of πab2/6 [46] (n=5
for each group).
The ADC of the tumor was mapped before treatment and then on day 2 and 9 post-
treatment using diffusion weighted MRI. A multi-slice spin-echo sequence (repetition time/echo
time = 500/ 35.85 msec /, matrix of 128×96 and field of view of 30 x 30 mm2) was employed
along with 12 bipolar gradients (b value = 0 and 939.48 s/mm2). The gradients were applied in
each of six orthogonal (±x, ±y and ±z) directions and were combined to produce a trace data set
to minimize the effects of diffusion anisotropy. ADC map was calculated pixel-wise by IDL
software (Research Systems, Boulder, Colorado) program kindly provided by Dr. Stephen
Pickup (Small Animal Imaging Facility). The region of interest (ROI) of the tumor was drawn on
the ADC map and the necrotic area excluded by visual comparison with T2-weighted images at
the same level. ADC values (mm2/s) pooled from the ROI in multi-slices covering the entire
tumor were used to construct the tumor ADC histogram consisting of 256 bins with a bin width
of 9.57 x 10-6
mm2/s. To validate the MRI-based estimation of ADC, we first measured the ADC
of a water phantom at 37°C using the same pulse sequence and obtained an ADC value almost
identical to that reported in the literature.[47]
Effect of RES-blockade on liver function and host defense To assess the liver function, serum
alanine transaminase (ALT) level was measured 24 h after injection of liposome (or saline) using
a ALT Activity Assay Kit (Cayman Chemical Company, Ann Arbor) on a 96-well microplate
format per manufacturer’s instructions. Measurements were done in triplicate.
To examine whether liposome-based RES-blockade affects RES-mediated clearance of
foreign particles or pathogens, 3 ×108 fluorescence labeled bacteria (Life Technologies) were i.v.
injected per mouse at 24 h after injection of liposome. One hour later, blood was collected by
cardiac puncture upon euthanasia and blood fluorescence was read from a microplate reader
(SpectraMax) at 25℃ with Absorption /Emission set at 494/518 nm. The absolute blood
concentration of fluorescence labeled bacteria was obtained from a calibration curve: 6×100,
6×101, 6×10
2, 6×10
3, 6×10
4, 6×10
5, 6×10
6, 6×10
7 labeled bacteria were suspended in 100 µL of
saline and heparinized blood mixture (1:1) per well in triplicate in 96-well plates; fluorescence
intensities vs. bacteria concentrations were plotted (n=3 for each group).
1.3 In Vitro Studies
In vitro studies were performed on a macrophage cell line, Raw 264.7 cells (ATCC), to
investigate effects of liposome concentration and serum opsonin concentration on macrophage
uptake of testing nanoparticles. Scavenger receptor B1 (SR-B1) knockdown experiment was
performed because SR-B1 binds to high density lipoproteins, which were found on surface of NP
among other serum opsonins [36].
Effect of serum opsonin and liposome concentration To investigate effects of empty liposome
and serum concentrations on blocking or facilitating macrophage uptake of testing nanoparticles
(3F-DiD), 2×106
Raw cells were incubated with liposome of different concentrations (0, 0.047,
0.094, 0.188, 0.564, 1.128 mg/mL) in DMEM medium with 0, 10, and 30% FBS at 37℃. After 1
h, 3F-DiD nanoparticles (80 µl, 100 µg DiD /mL) were added to the medium and incubated for 1
h. The cells were then collected for flow cytometry analysis (BD LSRII, BD Biosciences). Data
were analyzed by FlowJ 7.6.1 software, and the percentage of positive cells was used to quantify
the macrophage uptake of DiD-PTP-NP. The gate for positive cells was set at the level which
excludes 99.98% of PBS treated control cells. The experiments were done in triplicate samples.
Effect of scavenger receptor B-1 (SR-B1) In order to examine whether SR-B1 plays a dominant
role in macrophage uptake of opsonized or naked nanoparticles, SR-B1 was knocked down (KD)
by siRNA (4390771, Ambion, left Technologies); siRNA was mixed with transfection reagent-
Lipofectamine® RNAiMAX (Life Technologies) per manufacturer’s instruction and the mixture
was incubated with Raw 264.7 cells for 24 h at 37℃ and 5% CO2 before the cells were analyzed
for Western blot.
The degree of KD was assessed by western blot: cells were lysed with cell lysis buffer
(RIPA buffer, 1 ml per 107cells, Thermo Fisher Scientific) and Halt protease inhibitor cocktail
(10µL of concentrated cocktail per 1ml of lysis buffer, Thermo Fisher Scientific) to ensure
complete protection of the resulting protein extract. NuPAGE® LDS sample buffer (Life
Technologies) and NuPAGE® reducing agent (Life Technologies) were added to the lysate and
heated to 70℃for 10 minutes. Then samples were loaded to a 4-12% Tris-Glycine-SDS
polyacrylamide gel (Life Technologies), which was run at 120 v for 1 h; the gel was transferred
onto a nitrocellulose membrane (Bio-Rad), which was blocked with 5% milk in Tris Buffer
Saline Tween 20 (TBST) buffer for 1 hour at 4°C and was then blotted with a rabbit monoclonal
antibody against SR-B1 (ab52629, AbCam) in 1: 1000 dilution and β-Actin (ab8227, AbCam) in
1: 2000 dilution overnight at 4°C; the latter servers as house-keeping protein. After incubating
with the HRP-tagged goat anti-rabbit secondary antibodies (ab97051,AbCam) for 1 h at 4°C and
then with ECL detection reagent (Amersham, GE Healthcare) for 1 min at room temperature, the
membrane was exposed for 10 min using ImageQuant LAS 4000 (GE Healthcare). The density
of the band corresponding to SR-B1 was normalized to that of β-Actin by ImageJ software (NIH,
USA).
The effect of SR-B1 knockdown on macrophages’ capability to uptake liposome and
testing nanoparticles (3F-DiD) was assessed by flow cytometry. After 24 h incubation with
siRNA and transfection agent, RAW cells were washed and incubated in fresh media containing
liposome or 3F-DiD nanoparticles in the presence or absence of 30% serum for 2 h before they
were collected for FACS analysis.
Uptake of 3F-PTP nanoparticles into PSMA positive and negative prostate cancer cells
LNCaP cells and DU-145 cells (purchased from ATCC) incubated with FITY-PTP-3F-NP at the
concentrations of 1, 10, 100 and 1000 µM at 4oC for 1 hr. After incubation, cells were fixed with
4% PFA and examined under an epi-fluorescence microscope (Nikon E600 Upright Microscope,
NY).
1.4 Histological analyses
Tissues (tumor, liver and spleen) were excised upon euthanasia, embedded in OTC
medium and snap frozen. Five micrometer sections were then cut and fixed in 10%
paraformaldehyde. For Prussian blue staining [45], tissue sections were incubated for 1 h in dark
with 10% potassium ferrocyanide dissolved in 20% hydrochloric acid; sections were then
counterstained with nuclear fast red.
1.5 Statistical analyses
Numeric data are reported as the mean ± standard deviation. A two-tailed student t-test
was performed and P value of equal or less than 0.05 was considered as statistically significant.
2. Captions for Supplemental Figures
Figure S1 Effect of RES-blockade on bio-distribution of NP
A: Mice were treated with saline (n=3) or liposome (n=3) 1.5h before 3F-cy7.5 NP injection.
After 24hr, mice were sacrificed and organs were collected for optical imaging. RES-blockade
reduced uptake of 3F-cy7.5 in liver (by 1.4 fold) and spleen (1.7 fold) and increased
accumulation in the tumor (1.9 fold). In comparison with 3F-cy7.5 nanoparticle with (red) vs.
without (green) PTP targeting peptides, targeting NP uptake is 1.4 fold of non-targeting ones.
Drug-free (empty) 3F-NP were used to induce RES-blockade for subsequently injected 3F-PTP-
cy7.5. * P<0.05 compared with RES-blockade + 3F-cy7.5 group (in red).
NP were labeled by cy7.5 fluorophore on the surface of the nanoparticle. This leads to high
fluorescence intensity in kidneys in all groups, suggesting the label may be removed from NP
prematurely and excreted via kidney. This problem can be fixed by using a hydrophobic
fluorophore (DiR), which incorporates inside (the hydrophobic core) of the 3F-NP (see B).
B: Bio-distribution of 3F-DiR (the treatment timing was the same as A). 3F-DiR distributions in
liver, spleen, tumor and other organs (except for kidney) are similar to A whereas fluorescence in
kidney was much reduced. One mouse was used for each group.
Figure S2 Targeting 3F nanoparticles to PSMA by PTP peptide
FITC-conjugated PTP peptides targeting PSMA were incorporated into 3F-nanoparticles.
PSMA-expressing LNCaP and its negative control, DU-145, cells were used for cell binding
experiments which were conducted at 4oC over a wide range of paclitaxel concentrations from 1
μM (A) to 1 mM (B). Green fluorescence from 3F-NP decorated with FITC-PTP was only
detected in LNCaP cells but not in DU-145 cells.
Figure S3 Effect of scavenger receptor type B1 (SR-B1) knockdown on the uptake of NP
A : Scavenger receptor type B1 (SR-B1) was 87.22% (P<0.05) knocked down in RAW cells by
siRNA
B : SR-B1 knockdown moderately reduced the uptake of 3F-DiD NP by 6.53% (P<0.05) when
opsonins are absent (0% serum). However, the knockdown has little effect on uptake in the
presence of 30% serum, liposome or both.
Figure S4 Time window of liposome-based RES-blockade
A: 3F-SPIO was injected at 40 min after liposome injection. The initial uptake rate and level of
plateau are similar between red and blue trace in the first 20 min (i.e., 60 min after liposome
injection), suggesting RES-blockade has not been induced. After 20 min of 3F-SPIO injection,
two traces started to diverge, suggesting that RES-blockade began to take effect in red trace at 1
h after liposome injection.
B: 3F-SPIO was injected at 3 h after injection of liposome. The red and blue trace showed
difference in the initial uptake and up to 1500s (25 min) after injection; from that time on, there
was no difference between the two traces. This result suggests that RES-blockade was lifted at
3.5 h post liposome injection.
Taking A and B together, we estimate the liposome-mediated RES-blockade window to be ~2.5
h (taking effect at 1 h and ending at 3.5 h post liposome injection).
Flu
ore
sce
nt
inte
nsi
ty [
p/s
/cm
²/sr
]
24h biodistribution of 3F-DIR nanoparticles
Flu
ore
sce
nt
inte
nsi
ty
[p
/s/c
m²/
sr]
0.0E+00
5.0E+08
1.0E+09
1.5E+09
2.0E+09
blood liver spleen tumor kidney brain heart lung skin muscle
RES-blockade + 3F-DiR 3F-DiR
1.5E+08
2.5E+08
3.5E+08
RES-blockade + 3F-cy7.5
RES -blockade + 3F-cy7.5 (non-targeted)
Saline + 3F-cy7.5
empty 3F-NP + 3F-cy7.5
1.0E+07
2.0E+07
3.0E+07
4.0E+07
5.0E+07
6.0E+07
7.0E+07
8.0E+07
9.0E+07
1.0E+08
blood liver spleen tumor kidney brain heart lung skin muscle
24h biodistribution of 3F-cy7.5 nanoparticles *
*
*
*
*
*
*
*
* * *
A
B
Figure S1
A B
LNCaP DU-145 (control) LNCaP DU-145 (control)
Figure S2
A
B
40
50
60
70
80
SR-B1 KDserum+
SR-B1+serum+
SR-B1 KDserum-
SR-B1+serum-
Liposome
% P
osi
tive
ce
lls
40
50
60
70
80
SR-B1 KDserum+
SR-B1+serum+
SR-B1 KDserum-
SR-B1+serum-
3F-DiD
*
SR-B1 KD SR-B1+
SR-B1
β-Actin
0
10
20
30
40
SR-B1+ SR-B1 KD
Re
lati
ve in
ten
sity
*
Figure S3
Time interval between liposome /saline and 3F-SPIO = 3 h
-Ln
( S(
t)/S
0)
Time post 3F-SPIO (s)
Time interval between liposome /saline and 3F-SPIO = 40 minutes
-Ln
( S(
t)/S
0)
Time post 3F-SPIO (s)
Red: Liposome followed by 3F-SPIO Blue: Saline followed by 3F-SPIO
AUC =762±177
AUC=1145±243
AUC =1002±212
AUC=1143±262
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1000 2000 3000 4000
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1000 2000 3000 4000
A
B
Figure S4