Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2015.
Supporting Information
for Small., DOI: 10.1002/smll.201501902
Direct Observation of Early-Stage High-Dose RadiotherapyInduced Vascular Injury via Basement Membrane-Targeting
Nanoparticles
Kin Man Au, Sayed Nabeel Hyder, Kyle Wagner, Caihong Shi,
Young Seok Kim, Joseph M. Caster, Xi Tian, Yuanzeng Min,
and Andrew Z. Wang*
Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2013.
Supporting Information
Direct Observation of an Early-Stage High-Dose Radiotherapy-Induced Blood Vessel
Injury via Basement Membrane-Targeting Nanoparticles
Kin Man Au, Sayed Nabeel Hyder, Kyle Wagner, Caihong Shi, Young Seok Kim, Joseph M
Caster, Xi Tian, Yuanzeng Min, and Andrew Z. Wang*
Experimental Section
Materials.
Methoxy-functionalized
PEG-PLGA
with
molecular
weight
of
2,000:15,000 (PEG(2K)-PLGA(15K)), non-fluorescet PLGA with molecular weight of 24,000
- 38,000 (PLGA(30K)), collagen IV from human placenta, and trimethylamine (anhydrous, ≥
99.5 %) were purchased from Sigma-Aldrich (St. Louis, MO). Maleimide-functionalized
PEG-PLGA
with
molecular-weight
of
5000:10,000
(Mal-functionalized
PEG(5K)-
PLGA(10K), cat. no. AI053), methoxyl-functionalized PEG-PLGA with molecular-weight of
5000:10,000 (PEG(5K)-PLGA(10K), cat. no. AI053) and rhodamine B-labeled PLGA (M.W.
≈ 30,000, cat. no. AV11) were purchased from Akina, Inc. (West Lafayette, IN). Collagen IVtargeting peptide with an amino acid sequence of KLWVLPKGGGC was purchased from
UNC High-Throughput Peptide Synthesis and Array Facility. The peptide was synthesized via
automated Fmoc solid phase peptide sysnthesis method. The peptide was purified by
preparative HPLC. Peptide homogeneity was confirmed by MALDI-TOF mass spectroscopy
and analytical HPLC. Acetonitrile (HPLC grade), dimethylformamide (anhydrous) and
deionized water (HPLC grade, sub-micron filtered) were purchased from Fischer Scientific
(Hampton, NH). Dulbeco’s phosphate buffer saline (0.1 M, PBS) and fetal bovine serum
(FBS) were purchased from Gibco by Life Technologies (Carlsbad, CA).
Preparation of Rhod-labeled BM-targeting NPs: Collagen IV-targeting peptide was
conjugated to Mal-functionalized PEG(5K)-PLGA(10K) via metal-free “click” reaction in the
presence of trimethylamine as catalyst. Briefly, collagen IV-targeting peptide (3 mg, 2.6
1
µmol) was stirred with Mal-functionalized PEG(5K)-PLGA(10K) (39 mg, 2.60 µmol) in 2
mL of anhydrous DMF in the presence of trimethylamine (1 µL, 7.2 µmol) under nitrogen at
5 °C for 15 h. This resulted in 21 mg/mL of collagen IV-targeting peptide-conjugated
PEG(5K)-PLGA(10K) stock solution, which was used for the preparation of PEG-PLGA NPs
without further purification. 1H NMR spectroscopy confirmed completed conjugation of
collagen IV-targeting peptide. Rhodamine B-labeled collagen IV-targeting NPs were prepared
by nanoprecipitation method. Rhod-PLGA (4 mg/mL) was dissolved in acetonitrile, and
PEG(2K)-PLGA(10K) (15.2 mg/mL) was dissolved in acetonitrile diluted collagen IVconjugated PEG(5K)-PLGA(10K) solution (0.8 mg/mL) via sonication. For the preparation of
10 mg of BM-targeting PEG-PLGA NPs, 500 µL of Rhod-PLGA solution and 500 µL of
PEG-PLGA copolymers mixture solution were mixed together via vortex mixer (3,000 rpm,
30 s) and added dropwise (1 mL/min) to 3 mL of deionized water under constant stirring
(1,000 rpm). The mixture was stirred in darkness under vacuum for 3 h before being purified
by ultrafiltration using an Amicron Ultra ultrafiltration membrane filter with a 100,000
nominal molecular weight cutoff (4 times, 3,000 g for 15 min). After each washing, the NPs
were re-suspended in 0.1 M PBS. The purified NPs were concentrated to 5 mg/mL before
further use. Non-targeting Rhod-labeled PEG-PLGA NPs were prepared and purified via the
same method except methoxy-functionalized PEG(5K)-PLGA(10K). Non-fluorescent BMtargeting and non-targeting NPs were prepared for an in vitro toxicity study and used the same
methods except non-fluorescencet PLGA(30K) was used instead of Rhod-labeled
PLGA(30K).
NP Characterization. 1H NMR spectrum (2,048 scans) of collagen IV-targeting
peptide-conjugated PEG(5K)-PLGA(10K) was recorded in CDCl3 using a INOVA 500 MHz
NMR spectrometer in the NMR Facility at the UNC Eshelman School of Pharmacy. Purified
BM-targeting NPs and non-targeting NPs were fully characterized by TEM, NTA, DLS, and
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aqueous electrophoresis techniques. TEM images were recorded using a Zeiss TEM 910
Transmission Electron Microscope operated at 80 kV (Carl Zeiss Microscopy, LLC,
Thornwood, NY) in Microscopy Services Laboratory Core Facility at the UNC School of
Medicine. Prior to TEM imaging, concentrated NP samples were diluted to 5 µg/mL by
mixing with deionized water. 5 µL of each diluted sample was mixed with 5 µL of 4 % uranyl
acetate aqueous solution before being added to a 400 mesh carbon filmed copper grid via a
pipette. Excess NP dispersion was removed by filter paper at the edge of copper grid. The
recorded TEM images were processed using ImageJ (NIH). Number-average diameter (Dn)
was determined based on the average diameter of at least 150 particles from a representative
TEM image. The mean number-average diameter (Dn) and particle concentrations of different
NP dispersions were determined by a NP-tracking analysis method recorded on a Nanosight
NS500 instrument (Malvern, Inc.) in Microscopy Services Laboratory Core Facility at the
UNC School of Medicine. All NP dispersions were diluted to 5 µg/mL prior to the NP
tracking analysis. Intensity-average diameter (Dh, also known as hydrodynamic diameter) and
mean zeta potential (mean ζ) of NP dispersions were determined by dynamic light scattering
and an aqueous electrophoresis method using a Zetasizer Nano ZS Instrument (Malvern, Inc.).
Prior to the measurements, NPs were diluted to 1 mg/mL with or 1 mM NaCl electrolyte, or
0.1 M PBS. All measurements were based on the average of 3 separate measurements. UVvisible absorption and emission spectra were of BM-targeting NPs and non-targeting NPs
were recorded usnig a SpectraMax M Series Multiple-Mode Microplate Readers. In vitro
fluorescence image of 0.15 – 1.5 nM of BM-targeting NP dispersions in transparent eppendorf
tubes (1.5 mL) was recorded via an IVIS Kinetic Imaging System (Caliper Life Sciences,
Hopkiton, MA) equipped with an excitation filter of 570 nm and an emission filter of 600 nm
in Small Animal Imaging Facility at UNC School of Medicine. Region-of-interest values were
recorded using Living Image software as photon flux (also known as radiance) in total photon
count per centimeter-squared per steradian (p s-1 cm-2 Sr-1).
3
Cell Culture. KB cells were obtained from American Type Culture Collection
(ATCC). KB cells were cultured in Eagle’s Minimum Essential Medium supplemented with
10% (vol/vol) FBS (Sigma) and penicillin/streptomycin (Sigma). The cell density of
trypsinized cancer cells was determined by an Orflo Moxi Z Mini Automated Cell Counter
(Orflo, Ketchum, ID).
In vitro toxicity study. In a 96-well plate, 1 × 104 KB cells were plated 24 h prior to
treatment with 0 to 10 mg/mL (0 – 15 nM) of non-fluorescent BM-targeting and non-targeting
PEG-PLGA NPs at 37 ⁰C for 24 h. The cells were washed with PBS and allowed to grow in
complete cell culture media for 72 h. Cell viability was then analyzed using a 23-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
assay according to the manufacturer’s (Promega)instructions. The absorbance at 492 nm
(directly reflecting cell viability) was recorded using a 96-well plate reader (Infinite® 200
Pro, Tecan i-control).
In vitro solid-phase binding assay. A quantitative in vitro binding study was
performed in collagen IV-coated wall plate to elevate the binding afinity of the BM-targeting
NPs and non-targeting NPs. Collagen IV-coated well plate was prepared by air-dry 100 µL
per well of collagen IV solution (1 mg/mL, in 0.25 % acetic acid) in a 96-wall plate (96-well
fluorescent imaging plate, Greiner) for 48 h. In the binding study, 100 µL of 0 to 1.5 nM of
BM-targeting NPs (or non-targeting NPs) were generally alligated in the collagen IV-coated
well plate at 20 °C for 30 min before being washed once with PBS and three times with 10 %
FBS to remove non-specifically bound NPs. The amount of Rhod-labeled BM-targeting NPs
or non-targeting NPs bound to collagen IV were determined via a 96-well plate reader
(Infinite® 200 Pro, Tecan i-control) using an excitation wavelength of 535 nm and an
emission wavelength of 595 nm. All binding studies were performed in triplicate.
4
Ex vivo and In vivo studies. Animals were maintained in the Center for Experimental
Animals (an AAALAC-accredited experimental animal facility) under sterile environments at
the University of North Carolina. All procedures involving experimental animals were
performed in accordance with the protocols approved by the University of North Carolina
Institutional Animal Care and Use committee and conformed to the Guide for the Care and
Use of Laboratory Animals (NIH publication no. 86-23, revised 1985).
Ex vivo study. Ex vivo binding study was performed on freshly excised Nu mouse
skin. In the ex vivo study, a disease-free Nu mouse (13 weeks old, UNC Animal Services
Core, Chapel Hill, NC) was euthanized by an overdose of carbon dioxide. The skin
immediately removed and cut into small pieces (about 5 mm × 5 mm) without further
fixation. Basement membrane under the mesothelium was expose to the bulk environments by
superficially scratching through the peritoneal surface via a scalpel. The skin samples were
incubated with 1 mL of 0 to 150 pM of BM-targeting NPs or non-targeting NPs in a 24-well
plate under general allegation at 20 °C for 30 min. The skin samples were washed twice with
PBS and 3 times with 10 % FBS to remove unbound NPs. The ex vivo skin samples were
imaged using an Olympus IX 81 Inverted Wide-Field Light Microscope in the Microscopy
Services Laboratory at the UNC Medical School. Fluorescence intensities from representative
fluorescence images were quantified using ImageJ (NIH).
In vivo and histological studies. In vivo study was performed in healthy high-dose Xray irradiated Nu mice. 10 days before the in vivo imaging study, 11 disease-free Nu mice (12
– 14 weeks old, 30 - 31 kg; UNC Animal Services Core, Chapel Hill, NC) were subjected to a
single-dose 30 Gy XRT used a Precision X-RAD 320 (Precision X-ray, Inc.) machine
operating at 320 kvp and 12.5 mA. The source-subject distance was 70 cm and 50 cGy/min.
Only the left flank of the mice was irradiated, as the remaining parts of the body were leadshielded. Full-body fluorescence images were recorded pre-injection and at 24 h after tail-vein
5
intravenous injection of 165 mg/kg of BM-targeting NPs/non-targeting NPs via an IVIS
Kinetic Imaging System (Caliper Life Sciences, Hopkiton, MA) equipped with an excitation
filter of 570 nm and an emission filter of 600 nm in Small Animal Imaging Facility at UNC
School of Medicine. All imaging parameters were kept constant for the whole imaging study.
Region-of-interest values were recorded using Living Image software as photon flux (also
known as radiance) in total photon count per centimeter-squared per steradian (p s-1 cm-2 Sr-1).
All mice were euthanized by an overdose of carbon dioxide after the imaging study. Left and
right legs were collected, and fixed in 4% (v/v) neutral buffered formalin at 5 ⁰C for 2 days
and 70% ethanol at 5 ⁰C for another 2 days. The fixed leg specimens were submitted to
Animal Histopathology Core Facility at UNC Medical School to decalcify the bones via
Immunocal (Decal Chemical Corporation, Congers, NY) at 5 °C for 4 days before sectioning.
The histological sections were imaged using an Olympus IX 81 Inverted Wide-Field Light
Microscope in the Microscopy Services Laboratory at the UNC Medical School.
6
Supporting Figures
Figure S1. Assigned 1H NMR(500 MHz, 2,048 scans) spectra of (i) maleimide-functionalized
PEG(5K)-PLGA(55K) and (ii) collagen IV-targeting peptide conjugated PEG(5K)PLGA(55K). The disappearance of sharp maleimide protons (Ha and Hb) at 6.7 ppm and the
appearance of new broad protons bands at 0.5, 0.8, 1.25, 2.9 - 3.0 and 8.0 - 8.25 ppm (all
contributed from collagen IV-targeting peptide, labeled as *) confirmed the complete
conjugation of collagen IV-targeting peptide to the di-block copolymer. (N.B. The collagen
IV-targeting peptide-conjugated PEG-PLGA was dried under nitrogen for 3 days to vaporize
the DMF (solvent) and trimethylamine (catalyst) before the NMR study.)
7
Figure S2. Intensity-average diameter (Dh) distribution curves recorded for (i) BM-targeting
NPs and (ii) non-targeting NPs dispersed in 0.1 M PBS, as determined by DLS.
Figure S3. In vitro toxicity of 0.02 - 15.00 nM of non-fluorescent BM-targeting NPs and nontargeting NPs against KB epithelial cells. The toxicity study indicate both PEG-PLGA NPs
are non-toxic (cell viability ≥ 90 %). All toxicity study were performed in triplicate.
8
Figure S4. Concentration-dependent fluorescence intensities recorded for 0.0015 – 1.5 nM
(0.00005 to 1 mg/mL) of (i) BM-targeting NPs and (ii) non-targeting NPs via a 96-well plate
reader (Infinite® 200 Pro, Tecan i-control), using an excitation wavelength (λex) of 535 nm
and an emission wavelength (λem) of 595 nm.
Figure S5. Representative in vivo optical and fluorescence overlaid images of high-dose Xray irradiated healthy Nu mice recorded 24 h after i.v. administration of (a) BM-targeting NPs
or (b) non-targeting NPs.
9