Supporting Information

Density-tunable conjugation of cyclic RGD ligands to polyion complex vesicles for the

neovascular imaging of orthotopic glioblastoma

Wataru Kawamuraa, Yutaka Miura*, b, Daisuke Kokuryoc, Kazuko Tohb, Naoki Yamadab, Takahiro

Nomotoa, Yu Matsumotob, Daiki Sueyoshia, Xueying Liub, Ichio Aokic, Mitsunobu R. Kanod, Nobuhiro

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: kataoka@bmw.t.u-tokyo.ac.jp Assistant professor Yutaka Miura Phone: +81-3-5841-1791; Fax: +81-3-5841-7139; Email: miura@bmw.t.u-tokyo.ac.jp

Materials.

α-methoxy-ω-amino polyethylene glycol (MeO-PEG-NH2; Mn = 2400; Mw/Mn

= 1.11; NOF Co., Tokyo, Japan) was purified using an ion-exchange CM Sephadex

C-50 column (GE Healthcare Ltd., Buckinghamshire, UK) before use. Ethylene oxide

(EO; Canon Lifecare Solutions Inc., Oosaka, Japan) was distilled before use.

3,3-Diethoxy-1-propanol (Sigma-Aldrich, St. Louis, Missouri, USA),

1,5-diaminopentane (DAP; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan),

n-butylamine (nBu-NH2; Tokyo Chemical Industry Co.), triethylamine (>99%; TEA;

Wako Pure Chemical Industries, Ltd., Osaka, Japan), and methanesulfonyl chloride

(>99%; MsCl; Wako) were distilled over CaH2 before use. β-benzyl-L-asparate

N-carboxy-anhydride (BLA-NCA; NOF Co., Tokyo, Japan),

1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (EDC; Tokyo Chemical

Industry Co.), cyclo[RGDfk(CX-)] (cRGD peptide, X = 6-aminocaproic acid: ε-Acp;

Peptide Institute Inc., Osaka, Japan), sulfo-Cy3 mono-reactive dye (Lumiprobe Co.,

Orlando, Florida, USA), sulfo-Cy5 mono-reactive dye (Lumiprobe Co.), DyLight488

NHS ester (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), diethyl ether

(Wako), methylamine solution (40%; Wako), N-methyl-2-pyrrolidone (NMP; Koso

Chemical Co., Ltd, Tokyo, Japan), ethanol (EtOH; Koso Chemical Co.), sodium

hydroxide (NaOH; Koso Chemical Co.), hydrochloric acid (HCl; Koso Chemical Co.),

acetic acid (CH3COOH; Nacalai Tesque, Inc. Tokyo, Japan), ammonia solution (25%;

Nacalai Tesque), benzene (Nacalai Tesque), dimethyl sulfoxide (DMSO; Nacalai

Tesque), N-methyl-2-pyrrolidone (99.5%; NMP; Nacalai Tesque), dry tetrahydrofuran

(THF; Kanto Chemical Co. Inc., Tokyo, Japan), dry N,N-dimethylformamide (DMF;

Kanto Chemical Co.), dry dichloromethane (CH2Cl2; Kanto Chemical Co.), and all

other reagents were used without further purification. Dulbecco’s modified Eagle

medium was purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased

from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). EBM™-2 BulletKit™ was

obtained from Lonza (Tokyo, Japan). Dulbecco’s phosphate buffer saline (D-PBS) was

purchased from Wako, and echistatin was obtained from Tocris Bioscience (Bristol,

UK).

Measurements

1H NMR spectra were recorded using a JNM-ECS400 (400 MHz) (JEOL, Tokyo,

Japan) instrument with CDCl3, D2O, and DMSO-d6 containing 1% tetramethylsilane or

trimethylsilyl propanoic acid at 25 or 80 °C . The number-average molecular weight

(Mn) and degree of polymerization (DP) of the all polymers was calculated by 1H NMR

spectra. Size exclusion chromatography (SEC) was performed using a Tosoh HLC-8220

GPC system equipped with a TSKgel G3000HHR column, a TSKgel G4000HHR

column, and a TSKgel guard column HHR-L (all from Tosho Co., Tokyo, Japan) in

DMF (10 mM lithium bromide; flow rate, 0.8 mL/min; 45 °C) or NMP (10 mM lithium

bromide; flow rate, 0.3 mL/min; 40 °C). The poly dispersity (Mw/Mn) of the PEG

derivatives was calculated based on calibration with PEG. For PEG-b-poly(aspartic

acid) and PEG-b-poly(aspartic acid) derivatives, SEC was performed at room

temperature using a Jasco high performance liquid chromatography (HPLC) system

(AS-950 intelligent sampler, PU-980 intelligent HPLC pump, DG-980-50 three-line

degasser, 860-CO column oven, RI-930 intelligent RI detector, and UV-1575 intelligent

ultra violet/visible detector; Jasco, Easton, Maryland, USA) equipped with a

SuperdexTM 200 10/300 GL column (GE Healthcare). The procedure used 10 mM PB

containing 150 mM NaCl at a flow rate of 0.75 mL/min, and the Mw/Mn of polymers

were calculated based on the PEG calibration. For

n-butyl-poly([5-aminopentyl]-α,β-aspartamide), SEC was performed on similar system

to that described above using 10 mM acetic acid containing 500 mM NaCl at a flow rate

of 0.75 mL/min. The polymerization of β-benzyl-L-asparate N-carboxy-anhydride was

monitored using an infrared (IR) spectrometer with a Jasco IR report-100 and a NaCl

plate. Dynamic light scattering (DLS) measurements were recorded in water at 25 °C

using a zetasizer Nano-ZS instrument (Malvern Instruments Ltd., Malvern, UK)

equipped with a 4.0 mW He–Ne laser at 633 nm or a 50.0 mW a diode-pumped

solid-state (DPSS) laser at 532 nm with 90° collecting optics. Data were analyzed using

Malvern Dispersion Technology 4.20.

The fluorescence intensities of the polyion complex vesicles (PICsomes) were

determined using a NanoDrop 3300 fluorospectrometer (Thermo Fisher). The iron

concentrations in SPIO-loaded PICsomes were determined using inductively coupled

plasma-mass spectroscopy (ICP-MS) with an Agilent 7700x ICP-MS instrument (RF

power, 1550 W; sampling depth, 8.0 mm; plasma gas current, 15 L/min; carrier gas flow

rate, 1.03 L/min; peristaltic pump, 0.10 rps; monitoring mass, m/z 56 (Fe); integration

interval, 1.0 s; sampling period, 3.0 s) (Agilent Technologies, Inc., Palo Alto, California,

USA). Flow cytometry was performed using a Becton Dickinson LSR II (BD

Biosciences, San Jose, California, USA), and data were analyzed using BD FACS DiVa.

In vitro confocal laser scanning microscopy (CLSM) was performed using an LSM 780

instrument (Carl Zeiss, Oberkochen, Germany) equipped with a plan apochromat 63 ×

1.40 oil DIC M27 objective and two laser sources (blue diode 405 nm, and a DPSS 561

nm). Confocal slices were adjusted to 1.2 mm, and the acquired images were analyzed

using Zen 2010 (ver. 6.2.0.500).

Figure S1. Synthesis of acetal-PEG-b-P(Asp) (4)

Synthesis of acetal-PEG-NH2 (2)

Solutions of 3,3-diethoxypropanol (0.32 mL, 2.1 mmol) and potassium

naphthalene (K+Naph-; 6.8 mL, 2.0 mmol) were mixed with 50 mL THF to form

potassium 3,3-diethoxypropanolate, as reported previously [1]. After the mixture was

stirred for 10 min, liquid EO (5.6 mL, 113 mmol) was chilled to below 0 °C and was

then added; the mixture was the stirred again at room temperature for 2 days. The

reactant polymer was isolated by precipitation with diethyl ether, and was lyophilized

from benzene to obtain α-acetal-ω-alcohol PEG (acetal-PEG-OH (1), 4.99 g, yields

quant, Mn, NMR = 2350, Mw/Mn = 1.04, DPPEG = 50). Based on the 1H NMR

measurements, the DP of the BLA units was calculated to be 50, compared with the

peak intensity ratio of the methyl protons of acetal (δ1.20) and the methylene protons of

PEG (δ3.45–3.81). The 1H NMR results were as follows (400 MHz, CDCl3, 298 K): δ

(ppm) = 1.20 [t, 6H, (CH3-CH2-O)2-CH-)], 1.90 [q, 2H,

(CH3-CH2-O)2-CH-CH2-CH2-O-polymer backbone)], 3.45–3.81 [m, 207H,

(CH3-CH2-O)2-CH-CH2-CH2-(O-CH2-CH2)50-OH)], 4.64 [s, 1H,

(CH3-CH2-O)2-CH-CH2-]. Here and below s, t, q and m stand for singlet, triplet, quartet

and multiplet, respectively.

Acetal-PEG-OH (1) (3.579 g, 1.79 mmol) was dissolved in 30 mL benzene,

and was then freeze-dried. THF (40 mL) and TEA (1.15 mL, 8.06 mmol) were added to

the resulting acetal-PEG-OH solution, which was then mixed in an argon atmosphere.

THF (40 mL) and MsCl (0.43 mL, 5.38 mmol) were added drop-wise to the

acetal-PEG-OH solution over 10 min using a syringe, and the resulting mixture was

stirred in a water bath for 30 min at room temperature followed by 1.5 h in an argon

atmosphere. The resulting mixture was evaporated and reduced to 10 mL. The obtained

acetal-PEG-Ms was precipitated in diethyl ether (200 mL), filtered, and then washed in

ether. The acetal-PEG-Ms were then placed in a 200-mL flask, dissolved in NH3 aq.

(28%, 150 mL) and stirred for 4 days at room temperature. The resulting solution was

evaporated to dryness to yield 4.24 g of yellow solid. The resulting compound was

dissolved in CH2Cl2 and was extracted three times using saturated NaHCO3. The

resulting organic layer was washed twice in water and evaporated to dryness. The

obtained compound was dissolved in a minimum amount of water, flashed using a

diethylaminoethyl column (GE Healthcare Life Science, Buckinghamshire, England),

and freeze-dried to yield Acetal-PEG-NH2 (2) [1.85 g, 8.60 × 10−1 mmol, yield = 67.7%,

Mn,NMR = 2550, Mw/Mn = 1.12, DPPEG = 54]. The 1H NMR data were as follows (400

MHz, CDCl3, 298 K): δ (ppm) = 1.20 [t, 6H, (CH3-CH2-O)2-CH-], 1.90 [q, 2H,

(CH3-CH2-O)2-CH-CH2-CH2-O-polymer backbone], 2.94 (t, 2H, -CH2-NH2), 3.46–3.83

[m, 222H, (CH3-CH2-O)2-CH-CH2-CH2-(O-CH2-CH2)53.5-CH2-], 4.64 [s, 1H,

(CH3-CH2-O)2-CH-CH2-].

Synthesis of acetal-polyethylene glycol-b-poly(benzyl L-asparate)

(Acetal-PEG-b-PBLA) (3)

Acetal-PEG-NH2 (2) (138.2 mg, 5.42 × 10−2 mmol) was freeze-dried from 10 mL

of benzene. The resulting acetal-PEG-NH2 was dissolved in 8 mL CH2Cl2 under an

argon atmosphere. The BLA-NCA solution (1170 mg, 4.70 mmol) was dissolved in 3

mL DMF and 8 mL CH2Cl2, added to the acetal-PEG-NH2 solution, and stirred for 3

days at 35 °C under an argon atmosphere. Polymerization was monitored using IR. The

reaction mixture was poured gently into a mixture of 270 mL hexane and 180 mL ethyl

acetate to obtain a polymer precipitate. The resulting polymer was collected by suction

filtration, and then dried in vacuo to yield Acetal-PEG-b-PBLA (3) (966 mg, 4.95 × 10−2

mmol, yield = 91.0%, Mn,NMR = 19,500, Mw/Mn = 1.05, DPPBLA = 82). The 1H NMR

measurements revealed that the DP of the BLA units was 82, compared with the peak

intensity ratio of the methylene protons of PEG (δ3.34–3.59) and the benzyl protons of

the BLA unit (δ7.16–7.36). The 1H NMR data were as follows (400 MHz, DMSO-d6,

80 °C): δ (ppm) = 1.10 [t, 6H, (CH3-CH2)2-O-], 1.73 [q, 2H,

(CH3-CH2-O)2-CH-CH2-CH2-O-polymer backbone], 2.59–2.89 (m, 176H,

-O-CH2-CH2-NH-polymer backbone and CH-CH2-CO-polymer side chain), 3.34–3.59

[m, 222H, (CH3-CH2-O)2-CH-CH2-CH2-(O-CH2-CH2)53.5-CH2-PEG backbone], 4.34–

4.69 [q, 78H, -CO-CH-NH-polymer backbone and (CH3-CH2-O)2-CH-CH2-], 4.97–5.19

(m, 171H, -CO-O-CH2-Ph-polymer side chain), 7.16–7.36 (m, 412H, -CH2-Ph-PBLA

side chain), and 7.83–8.09 (m, 79H, -CO-CH-NH-polymer backbone).

Synthesis of acetal-polyethylene glycol-b-poly(α,β-aspartic acid)

[Acetal-PEG-b-P(Asp)] (4)

To de-protect the benzyl group, solutions of acetal-PEG-PBLA (3) (966 mg, 4.95

× 10−2 mmol) in NMP (10 mL) and 0.1 N NaOH (200 mL) were mixed, and vigorously

stirred at 25 °C for 3.5 h. The resulting polymer was purified in water using a dialysis

membrane (Spectra/Pro® 1 dialysis membrane; molecular weight cut off (MWCO), 6–

8,000 Da; Spectrum Laboratories Inc., Rancho Dominguez, California, USA) at room

temperature. After 3 days of dialysis, the residue was freeze-dried to yield the

Acetal-PEG-b-P(Asp) (4) [650 mg, 4.74 × 10−2 mmol, yield = 95.8%, Mn,NMR = 13,700,

Mw/Mn = 1.10, DPP(Asp) = 79]. Based on 1H NMR measurements, the DP of the P(Asp)

segment was calculated to be 79, compared with the peak intensity ratio between the

methylene protons of PEG (δ3.46–3.90) and the methylene protons of the α,β-P(Asp)

segment (δ2.41–3.05). The 1H NMR data were as follows (400 MHz, D2O, 80 °C): δ

(ppm) = 1.21 [t, 6H, (CH3-CH2)2-O-], 1.92 [q, 2H,

(CH3-CH2-O)2-CH-CH2-CH2-O-polymer backbone], 2.41–3.05 (m, 160H,

-O-CH2-CH2-NH-polymer backbone, CH-CH2-CO-polymer side chain), 3.46–3.90 [m,

222H, (CH3-CH2-O)2-CH-CH2-CH2-(O-CH2-CH2)53.5-PEG backbone], and 4.39–4.80

[m, 74H, -CO-CH-NH-polymer backbone and (CH3-CH2-O)2-CH-CH2-].

Figure S2. Synthesis of MeO-PEG-b-P(Asp) (6)

Synthesis of methoxy-polyethylene glycol-b-poly(benzyl L-asparate)

(MeO-PEG-b-PBLA) (5)

MeO-PEG-NH2 (168 mg, 7.00 × 10−2 mmol) was freeze-dried from 10 mL of

benzene. The resulting MeO-PEG-NH2 was dissolved in 8 mL CH2Cl2 under an argon

atmosphere. BLA-NCA (1510 mg, 6.07 mmol) in 3 mL DMF and 8 mL CH2Cl2 was

added to the MeO-PEG-NH2 solution, which was then stirred for 3 days at 35 °C under

an argon atmosphere. The product was precipitated into a mixture of 240 mL hexane

and 160 mL ethyl acetate. The obtained precipitate was collected by suction filtration

and dried in vacuo to yield the MeO-PEG-b-PBLA (5) [1230 mg, 6.65 × 10−2 mmol,

yield = 95.0%, Mn,NMR = 18,500, Mw/Mn = 1.05, DPPBLA = 78]. From the 1H NMR

measurements, the DP of the BLA units was calculated to be 78, compared with the

peak intensity ratio of the methylene protons of PEG (δ3.34–3.59) and the benzyl

protons of the BLA unit (δ7.19–7.46). The 1H NMR peaks were as follows (400 MHz,

DMSO-d6, 80 °C): δ (ppm) = 2.55–2.89 (m, 157H, -O-CH2-CH2-NH-polymer backbone,

CH-CH2-CO-polymer side chain), 3.25 (s, 3H, CH3-O-PEG backbone), 3.34–3.59 [m,

212H, -(O-CH2-CH2)52.5-CH2-PEG backbone], 4.61–4.69 (q, 77H,

-CO-CH-NH-polymer backbone), 4.82–5.19 (m, 161H, -CO-O-CH2-Ph-polymer side

chain), 7.19–7.46 (m, 392H, -CH2-Ph-PBLA side chain), and 7.83–8.09 (m, 75H,

-CO-CH-NH-polymer backbone).

Synthesis of methoxy-polyethylene glycol-b-poly(α,β-aspartic acid)

[MeO-PEG-b-P(Asp)] (6)

To de-protect the benzyl group, solutions of MeO-PEG-b-PBLA (5) (510 mg, 2.76

× 10−2 mmol) in 5 mL NMP and 100 mL 0.1 N NaOH were mixed and stirred

vigorously at 25 °C for 3.5 h. The resulting polymer was then purified in water using

dialysis membrane (Spectra/Pro® 1; MWCO 6–8,000 Da) at room temperature. After 3

days of dialysis, the residue was freeze-dried to yield the MeO-PEG-b-P(Asp) (6) [367

mg, 2.74 × 10-2 mmol, yield = 99.3%, Mn,NMR = 13,400, Mw/Mn = 1.09, DPP(Asp) = 78].

Based on 1H NMR measurements, the DP of the P(Asp) segment was calculated to be

78, compared with the peak intensity ratio of the methoxy protons of PEG (δ3.49–3.89)

and the methylene protons of the α,β-P(Asp) segment (δ2.44–3.05). The 1H NMR peaks

were as follows (400 MHz, DMSO-d6, 80 °C): δ (ppm) = 2.44–3.05 (m, 158H,

-O-CH2-CH2-NH-polymer backbone, CH-CH2-CO-polymer side chain), 3.49–3.89 [m,

212H, CH3-(O-CH2-CH2)52.5-CH2-PEG backbone], and 4.39–4.78 (m, 80H,

-CO-CH-NH-polymer backbone).

Figure S3. Synthesis of Bu-P(Asp-AP) (8)

Synthesis of n-butyl-poly(benzyl L-asparate) (Bu-PBLA) (7)

BLA-NCA (10.0 g, 4.02 × 101 mmol) that had been dissolved in 14 mL DMF

and 140 mL CH2Cl2 was added to a solution of n-Bu-NH2 (47.9 L, 4.85 × 10−1 mmol)

dissolved in CH2Cl2 (4.74 mL) using a syringe, and then stirred for 2 days at 35 °C

under an argon atmosphere. The reaction mixture was gently poured into 600 mL

diethyl ether at 4 °C. The resulting precipitate was collected by suction filtration and

dried in vacuo to yield the Bu-PBLA (7) [7.786 g, 4.21 × 10−1 mmol, yield = 86.8%,

Mn,NMR = 18,500, Mw/Mn = 1.09, DPPBLA = 92]. From the 1H NMR measurements, the

DP of the BLA units was calculated to be 92, compared with the peak intensity ratio of

the methylene protons of the terminal (δ0.79) and the benzyl protons of the BLA unit

(δ7.19–7.36). The 1H NMR data were as follows (400 MHz, DMSO-d6, 80 °C): δ (ppm)

= 0.79 [t, 3H, CH3-(CH2)3-], 1.16–1.24 (m, 2H, CH3-CH2-CH2-CH2-NH-), 1.29–1.36 (m,

2H, CH3-CH2-CH2-CH2-NH-), 2.56–2.92 [m, 200H, CH-CH2-CO-polymer sidechain,

CH3-(CH2)2-CH2-NH-], 4.61–4.69 (q, 91H, -CO-CH-NH-polymer backbone), 4.98–5.07

(m, 177H, -CO-O-CH2-Ph-polymer side chain), 7.19–7.36 (m, 462H, -CH2-Ph-PBLA

side chain), and 7.86–8.13 (m, 93H, -CO-CH-NH-polymer backbone).

Synthesis of n-butyl-poly([5-aminopentyl]-α,β-aspartamide) [Bu-P(Asp-AP)] (8)

Bu-PBLA (7; 1030 mg, 6.07 × 10−2 mmol) in 51 mL NMP was stirred at 50 °C for

6 h, and the solution became clear under an argon atmosphere. At a temperature of 4 °C,

DAP (33.7 mL, 2.88 × 102 mmol) in 34 mL NMP was added in a drop-wise manner

over 20 min using a syringe. The resulting solution was stirred at 4 °C for 1.5 h. For

neutralization, the mixture was added drop-wise to 200 mL 5 N HCl at 0 °C. The

polymer was dialyzed against 0.01 N acetic acid for 3 days followed by water for 1 day

using dialysis membrane (Spectra/Pro® 1; MWCO, 6-8,000 Da) at 4 °C. The residue

was freeze-dried to yield the Bu-P(Asp-AP) (8) [1070 mg, 5.32 × 10−2 mmol, yield =

87.6%, Mn,NMR = 20,200, Mw/Mn = 1.10, DPP(Asp) = 81]. From 1H NMR measurements,

the DP of the P(Asp-AP) segment was calculated to be 81, compared with the peak

intensity ratio between the methylene protons of the terminal (δ0.91) and the methylene

protons of the side chain of the P(Asp-AP) segment (δ2.56–3.39). The 1H NMR data

were as follows (400 MHz, D2O, 80 °C): δ (ppm) = 0.91 [t, 3H, CH3-(CH2)3-], 1.22–

1.89 [m, 473H, -CO-NH-CH2-(CH2)3-CH2-NH3Cl-polymer side chain], 2.56–3.39 [m,

490H, CH3-(CH2)2-CH2-NH-polymer backbone, CH-CH2-CO-polymer side chain, and

-CO-NH-CH2-(CH2)3-CH2-NH3Cl-polymer side chain], and 4.52–4.84 (m, 82.3H,

-CO-CH-NH-polymer back bone).

Preparation of 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 ratios] was prepared using 10 mM phosphate

buffer (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). These solutions were purified by filtration

through a 0.22 m membrane filter to remove dust, and the block aniomer solution was

mixed with the Bu-P(Asp-AP) solution to obtain an equal unit ratio of COO− and NH3+

ions; it was then vortexed vigorously (Scientific Industries, Inc., New York, USA) to

form PICs. The PIC solution was 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 ultra-filtered using a polyethersulfone membrane (Vivaspin 6; MWCO,

300,000 D; Satorius Stedim Biotech GmbH, Goettingen, Germany), and the size and

structure of the resulting PICs were evaluated using DLS and transmission electron

microscopy (TEM).

Preparation of cRGD-PICsomes

Acetal functionalized PICsome (Ace-PICsome) solutions were added to 0.1 N

HCl to decrease the pH to <2, and stirred at room temperature. After 1 h, cRGD

solution Cyclo[RGDfK(CX-)] (cRGD peptide, X = 6-aminocaproic acid: -Acp; 10

equivalent vs. aldehyde group onto PICsome) was added to PICsomes, and 0.1 N NaOH

aq. was added to adjust the pH to 5.5. The polymer concentration was then adjusted to

~5.0 mg/mL, and the solution was incubated at −20 °C for 6 h. The resulting frozen

solution was then left at 4 °C until thawed, and was then purified using ultrafiltration

(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 solutions, 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 then analyzed using DLS and TEM.

Preparation of cRGD-PICsomes in D2O for 1H NMR analysis

To evaluate the attachment of cRGD onto Ace-PICsomes, cross-linked

100%-Ace-PICsomes were prepared in D2O. All synthesis and purification steps were

performed using D2O. The obtained 100%-Ace-PICsomes in D2O were added to 0.1 N

DCl to lower the pD of the solution to <2.0. After 1 h, the cRGD solution (10 eq. vs. the

aldehyde groups on the PICsomes) was added to the PICsome solution, and 0.1 N

NaOD aq. was added to increase the pD 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 held at 4 °C until it had thawed, and was then purified using ultrafiltration

(Vivaspin 6; MWCO, 300,000 Da). The resulting solution (470 μL) was added to 30 μL

of 1% TMS to yield a final volume of 500 μL, and was then analyzed using 1H NMR.

The Ace-PICsomes, 20%-, and 40%-cRGD-PICsomes were prepared in the same

manner using homogeneous systems with stirring at pD 5.5 at room temperature for 12

h.

Fluorescence correlation spectroscopy (FCS)

To confirm the number of cRGD peptides on a single PICsome (NcRGD),

Cy3-labeled PICsomes consisting of a polyanion mixture [Cy3-labeled polyanion (Cy3

introduction rate [Irate] was confirmed by 1H NMR and estimated to be 96 %) and

none-labeled polyanion at 50%:50% (mol/mol)] were prepare in the same manner as

described in Section 2.3., and the aggregation number of polyanion on PICsomes (Nagg)

was estimated by FCS using a Zeiss LSM 510 META equipped with the FCS setup

ConfoCor 3 (Carl Zeiss, Germany). Cy3-labeled polyanion was used as the control. The

numbers of Cy3-labeled polyanions (Npoly) and Cy3-labeled PICsomes (NPIC) were

determined as 0.056±0.024 and 176±10, respectively. The Nagg and NcRGD were

calculated with the following equations:

Nagg = 2 × (100 × NPIC)/(Npoly × Irate) (1)

NcRGD = Nagg × (NcRGDcont/100) (2)

where NcRGDcont is the molar-based cRGD content on a single cRGD-linked PICsome as

shown in Figure 1C. The values of NcRGD were estimated to be ca. 6400 for

100%-cRGD PICsomes, ca. 2600 for 40%-cRGD PICsomes, and ca. 1500 for

20%-cRGD PICsomes.

Transmission electron microscopy (TEM)

TEM was performed using a JEM-1400 electron microscope (JEOL) at 100 kV.

Copper grids of 400-mesh (JEOL) were coated with a thin Formvar film, and then

coated with carbon and glow-discharged (2 mA, 5 s) using an Eiko IB-3 ion coater

(Eiko Engineering Co. Ltd., Tokyo, Japan). Two-microliter aliquots of the sample

solution were placed on the grid, stained using a drop of 50% ethanol solution

containing 2% (w/v) uranyl acetate, left to rest for two minutes, and then dried at room

temperature after the removal of surplus water.

Figure S4. Area under the curve (AUC) ratios between the tumor and blood (A) at 3h,

(B) at 6h, and (C) at 24h after administration of 20%- and 40%-cRGD-PICsomes. The

data were analyzed using the Student’s t-test, and presented as the mean ± s.e.m., n = 4.

*P < 0.05. NS = not significant.

Real-time observation of the neovascular targeting of cRGD-conjugated PICsomes

using intravital confocal laser scanning microscopy (IVCLSM)

Figure S5. Intravital confocal laser scanning microscopy images of tumor blood vessels

(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

software (version 1.6.8, MR vision Co., Massachusetts, USA). Transverse relaxation

(r2) was calculated using the equation r2 = (R2obs − R2d)/[Fe], where R2obs = the R2 of the

sample, R2d = the R2 of the aqueous solution, and [Fe] = the Fe concentration measured

using ICP-MS.

Table S2. Characterization of SPIO-loaded PICsomes

Sizea (nm) PDIaFe conc.b

(mM)*Zeta potentialc

(mV)

SPIO-loaded Ctrl-PICsomes 97 0.034 3.05 −27.7

SPIO-loaded 40%-cRGD-PICsomes 109 0.065 3.37 −26.1

a Determined using DLS; b Determined using ICP-MS; c Determined using a zetasizer.

Figure S9. (A) Concentration dependence of R2s. (B) Representative R2 mapping of

SPIO-loaded PICsomes, ferucarbotran, and D-PBS.

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