Supplementary information
Methods
Cell lines and primary cell culture
Primary AML cells were obtained with informed consent and IRB approval from
Weill Cornell Medical College-New York Presbyterian Hospital. Primary
cryopreserved AML samples were thawed and prepared for xenotransplants as
described previously 1.
E-selectin aptamer conjugation to porous silicon (pSi) microparticles.
Discoidal porous silicon microparticles were fabricated by electrochemical
etching of silicon wafer and surface modified with 3-aminopropyltriethoxysilane
(APTES) as previously described 2. Ten billions of APTES modified pSi was
dispersed in 0.1 mL of dimethylformamide (DMF) and gently vortexed, 0.5 mL of
10 mM SMCC in DMF and 30 µL of diisopropylethylamine were added with
Argon gas bubbled for 5 Min. Then, the reaction system was end-to-end shook
for 4h. After 3 times of centrifugation and washing by using DMF, the resulted
maleimided-pSi was dried in vacuum at RT. E-selectin apatmer was activated by
using dithiothreitol (DTT). To E-selectin apatmer solution in water (100µL of 10
mM),100 µL of DTT (10 mM) solution was added with short time (1 min) vortex,
and 30 min later activated aptamer was subjected to purification by PD-10
column eluted by PBS buffer (pH7.2, 0.1 M phosphate salts, 0.15 M NaCl, 5mM
EDTA). Last, to 10 B of maleimidated pSi well dispersed in PBS buffer (pH7.2,
0.1 M phosphate salts, 0.15 M NaCl, 5mM EDTA), 5 nmol of activated and
purified aptamer was added, was well dispersed with vortex, and allowed to react
for 4 h at room temperature and then overnight at 4ºC. The excess aptamer was
removed after centrifugation at 4,700 rpm for 12 min, and then washing by using
PBS until no E-selecting aptamer was detected in supernatant by UV spectra.
After dried in vacuum at RT for 12h, stored in -20ºC for the following experiments.
Fabrication and characterization of parthenolide-containing polymeric
micelles and assembly into ESTA-MSV
Discoidal porous silicon microparticles were fabricated by electrochemical
etching of silicon wafer as previously described3. ESTA-MSVs were modified and
conjugated as described above. Co-solvent evaporation method was used to
prepare parthenolide-loaded mPEG-PLA micelles. Briefly, aliquots of mPEG-PLA
with parthenolide were dissolved into acetone. This solution was added dropwise into distilled water and stirred for 2 h. The remaining acetone was removed
in vacuum. The micelle solution was filtered through a 0.45 µm syringe filter to
remove free drug. To load parthenolide-micelles into ESTA-MSV, micelles in
suspension were mixed with dry ESTA-MSVs and sonicated for 3 minutes. The
micelle size and zeta potential were determined by using Malvern Zetasizer
Nano-ZS (Malvern Instruments Ltd., Worcestershire, UK). Parthenolide content in
micelles was detected by HPLC on LaChrom Elite HPLC System (Hitachi, Japan)
equipped with C18 column (150 × 4.6 mm, 3.5 μm, Agilent) and using mobile
phase consists of mixture of acetonitrile: water (55:45, v/v) at a flow rate of 1.5
mL/min and ultraviolet detection at 210 nm.
NOD/SCID xenotransplantation assays
Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were
obtained from the Jackson Laboratory, and sublethally irradiated with 2.7 Gy
(270 Rads) before transplantation. Equal numbers of primary AML cells (2.5 ×
106 cells per mouse) were then injected via the tail vein in a final volume of 0.2
mL of phosphate-buffered saline (PBS) containing 0.5% fetal bovine serum
(FBS). Treatment with MSV or MSV-PTL (one billion particles) was started five
weeks after transplantation via tail vein injection, once every two weeks, for four
weeks. Animals were then sacrificed and the mouse bone marrow cells were
harvested and analyzed for the presence of human cells by flow cytometry. For
the secondary injections, equal numbers of live human CD45+ cells were injected
into sublethally irradiated NOD/SCID using a total volume of 200 l/mouse.
Detection and determination of PTL in plasma and bone samples from
MSV-PTL-treated AML-PDX mice
Plasma and femur bones from MSV-PTL treated AML-PDX mice obtained 1 h
after administration of MSV-particles were removed from the animal and stored
at -80 oC. Prior to analysis, bones were weighed, and pulverized using a pestle
and mortar4. After addition of 1 ml of a mixture of hexane:acetonitrile (1:1), bone
tissues were homogenized using a tissue homogenizer (Biospec Products, Inc.,
Bartlesville, OK), vortex-mixed, sonicated for 1 min and centrifuged at 10,000
rpm for 10 min at room temperature. The cycle of homogenization, vortex-mixing,
sonication and centrifugation was repeated 3 successive times. The pooled
supernatant was evaporated to dryness under nitrogen gas, the residue
reconstituted in 30 µL of acetonitrile/methanol (1:1), and 5 µL of this solution
injected onto an LC/MS/MS spectrometer (Agilent LC/MS/MS Triple Quad 6410
instrument) (Santa Clara, CA, USA). To detect PTL in plasma samples, 50 µL of
control or MSV-PTL-treated plasma samples were extracted with 600 µL of
acetonitrile/methanol (1:1). The mixture was vortexed for 30s, sonicated for 1
min and centrifuged at 10,000 x rpm for 10 min at room temperature.
The
supernatant was then evaporated to dryness at 37 °C under nitrogen gas, the
residue reconstituted with 50 µL of acetonitrile/methanol (1:1) and transferred to
auto-sampler vials, and 5 µL was injected onto the LC/MS/MS spectrometer.
LC/MS/MS Analysis
A sensitive LC/MS/MS method was successfully applied to determine the
concentration of PTL in plasma and bone samples from MSV-PTL-treated mice.
Analysis of PTL was carried out using an Agilent LC/MS/MS Triple Quad 6410
instrument operated in the positive electrospray ionization (ESI) mode with
optimal ion source settings determined by a standard of PTL. A curtain gas of 20
psi, an ion spray voltage of 4000 V, an ion source gas1/gas2 of 35 psi and
temperature of 300 °C were also employed in the collection of chromatographic
data. Chromatographic separations were performed on an EC-C18 column, 2.7
µm, 4.6 X 50 mm (Agilent technologies, Santa Clara, CA) eluted with a mobile
phase composed of 0.005 % formic acid and acetonitrile/methanol (1:1)
containing 0.005% formic acid. PTL was eluted at 8.5 min with good resolution
(Fig. 1g). Separation was achieved using a gradient of 10 to 90 % solvent B in
6.8 min, which was then equilibrated back to the initial conditions over 3.2 min.
The flow rate was 0.5 mL/min with a column temperature of 30 οC. PTL MRM
transitions monitored were as follows: PTL, m/z 249.1/231.1; m/z 249.1/185.1;
249.1/91.0.
Flow cytometric assays
The percentage of human AML cells was determined by staining the cells
obtained from the BM from NOD/SCID xenotransplanted with antibodies for PECy5 conjugated rat anti-mouse CD45 (mCD45; eBiosciences) and APC-H7
conjugated anti-human CD45 (hCD45; BD Biosciences) in FACS buffer (PBS +
0.5% FBS) for 20 minutes at room temperature, washed once with FACS buffer,
cells were stained with mCD45 and hCD45 antibodies as mentioned above,
washed once with FACS buffer, and resuspended in Annexin V-FITC (BD
Biosciences) and 7-aminoactinomycin (7-AAD; Molecular Probes-Invitrogen).
Percent viability was determined by gating annexin V negative and 7-AAD
negative populations. For intracellular assays, cells were stained as described
above for surface markers, then cells were fixed with 4% formaldehyde (EMgrade; Electron Microscopy Sciences) at room temperature for 10 minutes,
pelleted by centrifugation, and permeabilized by resuspending in cold 100%
methanol on ice for 20 minutes. Cells were then washed with FACS buffer twice
(0.5% FBS in PBS), and stained for 1 hour with antibodies against phospho-NF-H2AX (Cell signaling). At least 1 × 105 events were recorded
per sample using a LSR II flow cytometer (BD Biosciences). Data analysis was
conducted using FlowJo 9.3 software for Mac OS X (TreeStar).
Confocal microscopy
Cells were fixed in methanol at −20°C, and then permeabilized using blocking
buffer (10% FBS and 0.1% Tween 20 in 1× phosphate-buffered saline [PBS; pH
7.4]) as described previously5. Cells were stained using rabbit anti-γH2AX (Cell
Signaling Technologies) in blocking buffer for 2 hours at room temperature. Cells
were washed and stained with goat anti-rabbit Alexa 488 (Invitrogen) secondary
antibody. Slides were mounted using Fluoromount with DAPI (Southern Biotech).
Fluorescence was observed using a 100× objective on a Zeiss confocal
microscope.
Immunoblotting.
Cells were lysed in protein lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1%
Triton X-
-glycerophosphate, 1 mM Na3VO4,
1 mM NaF, 1 × protease inhibitor cocktail (Calbiochem)) on ice for 30 minutes.
Protein lysates were run in 10% SDS-PAGE (Invitrogen) and transferred to an
Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore). The
membrane was incubated with antibodies for phosphorylated p65, phosphohistone H2A.X Ser139 (H2AX) (Cell Signaling), and -actin (Sigma-Aldrich) at
4°C overnight, washed and incubated with IRDye 680 goat anti-rabbit or IRDye
800CW goat anti-mouse secondary antibodies (Li-COR) at room temperature for
30 minutes. The membrane was then scanned using the Odyssey imaging
system (Li-COR).
Statistical analysis
Analyses and graphs were performed using GraphPad Prism software to
evaluate significance. The specific test utilized is indicated in the figure legends
*p<0.05, **p<0.01.
References for supplemental information
1.
Guzman ML, Yang N, Sharma KK, Balys M, Corbett CA, Jordan CT, et al.
Selective activity of the histone deacetylase inhibitor AR-42 against
leukemia stem cells: a novel potential strategy in acute myelogenous
leukemia. Molecular cancer therapeutics 2014 Aug; 13(8): 1979-1990.
2.
Godin B, Chiappini C, Srinivasan S, Alexander JF, Yokoi K, Ferrari M, et
al. Discoidal Porous Silicon Particles: Fabrication and Biodistribution in
Breast Cancer Bearing Mice. Adv Funct Mater 2012 Oct 23; 22(20): 42254235.
3.
Ananta JS, Godin B, Sethi R, Moriggi L, Liu X, Serda RE, et al.
Geometrical confinement of gadolinium-based contrast agents in
nanoporous particles enhances T1 contrast. Nat Nanotechnol 2010 Nov;
5(11): 815-821.
4.
Safarpour H, Connolly P, Tong X, Bielawski M, Wilcox E. Overcoming
extractability hurdles of a 14C labeled taxane analogue milataxel and its
metabolite from xenograft mouse tumor and brain tissues. Journal of
pharmaceutical and biomedical analysis 2009 Apr 5; 49(3): 774-779.
5.
Topisirovic I, Guzman ML, McConnell MJ, Licht JD, Culjkovic B, Neering
SJ, et al. Aberrant eukaryotic translation initiation factor 4E-dependent
mRNA transport impedes hematopoietic differentiation and contributes to
leukemogenesis. Mol Cell Biol 2003 Dec; 23(24): 8992-9002.