Supporting Information Density-tunable conjugation of cyclic RGD ligands to polyion complex vesicles for the neovascular imaging of orthotopic glioblastoma Wataru Kawamura a , Yutaka Miura *, b , Daisuke Kokuryo c , Kazuko Toh b , Naoki Yamada b , Takahiro Nomoto a , Yu Matsumoto b , Daiki Sueyoshi a , Xueying Liu b , Ichio Aoki c , Mitsunobu R. Kano d , Nobuhiro Nishiyama e , Tsuneo Saga c , Akihiro Kishimura f , Kazunori Kataoka *,a, b, g, h a Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan b Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan c Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan d Department of Pharmaceutical Biomedicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan e Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan f Department of Applied Chemistry, Faculty of Engineering, Kyusyu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan g Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan h Innovation Center of Nanomedicine, Kawasaki Institute of Industry Promotion, 66-20 Horikawa-cho, Saiwai-ku, Kawasaki 212-0013, Japan * To whom correspondence should be addressed; Professor Kazunori Kataoka Phone: +81-3-5841-7138; Fax: +81-3-5841-7139; Email: [email protected]Assistant professor Yutaka Miura Phone: +81-3-5841-1791; Fax: +81-3-5841-7139; Email: [email protected]
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Supporting Information
Density-tunable conjugation of cyclic RGD ligands to polyion complex vesicles for the
Nishiyamae, Tsuneo Sagac, Akihiro Kishimuraf, Kazunori Kataoka*,a, b, g, h
aDepartment of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
bCenter for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
cMolecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
dDepartment of Pharmaceutical Biomedicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
ePolymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan
fDepartment of Applied Chemistry, Faculty of Engineering, Kyusyu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan
gDepartment of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
hInnovation Center of Nanomedicine, Kawasaki Institute of Industry Promotion, 66-20 Horikawa-cho, Saiwai-ku, Kawasaki 212-0013, Japan * To whom correspondence should be addressed; Professor Kazunori Kataoka Phone: +81-3-5841-7138; Fax: +81-3-5841-7139; Email: [email protected] Assistant professor Yutaka Miura Phone: +81-3-5841-1791; Fax: +81-3-5841-7139; Email: [email protected]
(A) 1 h and (B) 6 h after the administration of PICsomes (green, Cy5-labeled
Ctrl-PICsomes; red, DyLight488-labeled 40%-cRGD-PICsomes). Their co-localization
is shown in yellow. Scale bars = 100 m in all images.
Figure S6. Schematic representation of the multivalent binding between single
cRGD-linked nanocarriers and integrin (A) and the required numbers of cRGDs on a
single nanocarrier (B). The values were estimated by the following equation: NLigand =
(4 × π × r2)/(12 × 12), where NLigand is the required number of cRGDs and r is
the radius of nanocarriers.
Table S1 Characterization of cRGD-PICsomes
cRGD
contenta
(mol %)
number of
cRGD on single
PICsomeb
occupied surface
area of cRGDc
(nm2)
distance between
two cRGDd
(nm)
20%-cRGD-PICsome 23.4 1500 20.9 4.6
40%-cRGD-PICsome 40.4 2600 12.1 3.5
100%-cRGD-PICsome 97.5 6400 4.9 2.2 a The cRGD content was determined using 1H NMR. b The numbers of cRGD were determined using fluorescence correlation spectroscopy. c [occupied surface area] = (4πr2)/(number of cRGD), where r is radius of PICsomes. d [distance between two cRGD] = (occupied surface area)0.5
Preparation of SPIO-loaded PICsomes
Briefly, a block aniomer solution [1.0 mg/mL; a mixture of
MeO-PEG-b-P(Asp)/acetal-PEG-b-P(Asp), MeO-PEG-b-P(Asp)-Cy5, and
acetal-PEG-b-P(Asp)-Cy5 at the indicated ratio] was prepared in 10 mM PB without
NaCl (pH 7.4). Bu-P(Asp-AP) solution (1.0 mg/mL) was prepared using 10 mM PB
without NaCl (pH 7.4). Ferucarbotran (Resovist®, Fujifilm RI PharmaCo. Ltd., Tokyo,
Japan) solution (10.0 mg/mL; Fe concentration, 0.516 mg/mL) was prepared in water.
All solutions were purified by filtering through a 0.22-μm membrane filter to remove
any large particles. The block aniomer solutions were mixed with the Bu-P(Asp-AP)
solution to give an equal ratio of COO− and NH3+. The Ferucarbotran solution was
added to the polymer solution, and were vigorously vortexed (Scientific Industries) to
form SPIO-loaded PICs [2]. The PIC solutions were then added to EDC solution (10
mg/mL, 10 eq. per -COOH group in the block aniomers in PB), and mixed gently. After
a 12 h incubation at 4 °C, the solution was purified using a GX-271 liquid handling
system (Gilson, Inc., Middleton, Wisconsin, USA) and a preparative gel permeation
chromatography column (Sephacryl™ S-1000 [linear, 50 mm × 380 mm], GE
Healthcare). The sizes and structures of the obtained PICs were evaluated using DLS.
Preparation of SPIO-loaded 40%-cRGD-PICsomes
A solution of SPIO-loaded 40%-Ace-PICsomes was added to 0.1 N HCl to
reduce the pH to 4, and was then stirred at room temperature for 4 days [3,4]. The
cRGD solution (10 eq. vs. the aldehyde group on the PICsomes) was added to the
PICsome solution, and 0.1 N NaOH was added to adjust the pH to 5.5. The polymer
concentration was adjusted to ~5.0 mg/mL, and the solution was incubated at −20 °C
for 6 h. The resulting frozen solution was then held at 4 °C until it had thawed, and was
then filtered using a polyethersulfone membrane (Vivaspin 6; MWCO, 300,000 Da). To
quench the unreacted aldehyde groups, methylamine [5 eq. vs. acetal-PEG-b-P(Asp)]
was added to the PICsome solution, and stirred at 4 °C for 12 h. Finally, the solution
was purified and concentrated by ultrafiltration using a polyethersulfone membrane
(Vivaspin 6; MWCO, 300,000 Da), and the product was assessed using DLS, TEM, and
inductively coupled plasma-mass spectroscopy (ICP-MS). The encapsulated SPIO was
analyzed using energy-dispersive X-ray spectroscopy (EDS; JEM-2100F field emission
electron microscope, JEOL). The Fe concentrations in the SPIO-loaded PICsomes were
determined using ICP-MS with an Agilent 7700x ICP-MS instrument (Agilent). For
fluorescence imaging, the N termini of the block aniomers were labeled with Cy5 and
then used for PICsome preparation. SPIO-loaded Ctrl-PICsomes were analyzed using
the same methods.
Energy-dispersive X-ray spectroscopy of SPIO-loaded 40%-cRGD-PICsomes
To characterize the SPIO iron nanoparticles within the PICsomes, the
SPIO-loaded PICsome solutions were placed on a 400-mesh copper grid (JEOL) and
dried naturally. The samples were treated with glow discharge in a vacuum to remove
any contamination using an ion cleaner (JIC-410, JEOL). TEM images were then
obtained at 120 kV (JEM-2100F [HC-STEM], JEOL). High-resolution elemental
mapping and analysis were performed (JED-2300, JEOL).
Figure S7. TEM image of SPIO-loaded 40%-cRGD-PICsomes. (A) High resolution
elemental mapping and (B) the same images with labels are shown.
Figure S8. EDS analysis on Figure S5. P1–P6; the points with (black dots) in the
PICsomes (Fe signal, ca. 6.4 keV; red dashed square). P7 and P8; blank points (no Fe
signal).
In vitro R2 and r2 measurements
In vitro magnetic resonance imaging (MRI) measurements were performed to
measure the transverse relaxation rates (R2), which are the reciprocal of the transverse
relaxation time (T2) of water protons (1H) in the presence or absence of SPIO-loaded
cRGD-PICsomes. SPIO-loaded PICsomes without cRGD ligands (Ctrl-PICsomes) and
ferucarbotran (Resovist®) were used as negative and positive controls, respectively. The
SPIO-loaded PICsomes and ferucarbotran samples were diluted using
phosphate-buffered saline, prepared, and aliquoted into 0.2-mL PCR tubes. MR images
were acquired on a 7.0-Tesla, 40-cm bore magnet (Kobelco and Jastec, Kobe, Japan)
interfaced with Advance I system (Bruker-Biospin, Ettlingen, Germany) with a 35-mm
diameter volume coil (Rapid Biomedical, Lymper, Germany). The sample temperature
was maintained at 23 °C using a gradient-coil cooling system and air conditioners.
Two-dimensional multispin-echo images were acquired using the following parameters:
repetition time (TR)/echo time (TE) = 3,000/10–100 ms in steps of 10 ms (10 echoes);
field of view (FOV) = 48.0 × 48.0 mm2; matrix = 256 × 256; resolution = 188 m × 188
m; number of slices = 1; slice thickness = 2.0 mm; slice direction = horizontal; and
number of acquisitions (NEX) = 1. The scanning time was 12 min 48 s. After image
acquisition, the T2 and R2 values were estimated using MRVision image processing