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Electronic Supplementary Information

of

Tumor targeted gold nanoparticles for FRET-based tumor imaging

and light responsive on-demand drug release

Kai Han,a,b Jing-Yi Zhu,a Shi-Bo Wang,a Zi-Hao Li,a Si-Xue Chenga and Xian-

Zheng Zhanga,*

a Key Laboratory of Biomedical Polymers of Ministry of Education & Department

of Chemistry, Wuhan University, Wuhan 430072, China

b College of Science, Huazhong Agricultural University, Wuhan 430070, China

* Correspoding author. Email address: xz-zhang@whu.edu.cn

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2015

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Experimental

Materials and Methods

Detailed material information was provided in our previous article.[S1] Additionally,

matrix metalloproteinase (MMP-2) was provided by RD-SYSTEMS. Doxorubicin

hydrochloride (DOX) was purchased from Zhejiang Hisun Pharmaceutical Co. (China).

Molecular weight was recorded by electrospray ionization mass spectrometry (ESI-

MS) system (Finnigan LCQadvantage). 1H Nuclear magnetic resonance (1H NMR)

spectra were recorded on a Mercury VX-300 spectrometer (Varian) at 300 MHz by

using CD3OD or D2O as the solvent, and TMS was used as an internal standard.

Transmission electron microscopy (TEM) of samples were observed with an

acceleration voltage of 80 kV (JEM-100CX II microscope). Thermal gravimetric

analysis (TGA) was performed on a TGS-2 thermogravimetric analyzer (Perkin-

Elmer).

Synthesis of Lipoic Acid Modifiedβ-CD (β-CD-SS)

Mono-6-azido-β-CD was synthesized as the previous report.[S2] The lipoic acid

modifiedβ-CD was prepared with the catalysis of PyBOP (benzotriazol-1-yl-

oxytripyrrolidinophosphonium hexafluorophosphate). Briefly, lipoic acid (0.7 g) and

Mono-6-azido-β-CD (0.4 g) were dissolved in 10 mL of DMF (dimethyl formamide),

and then PyBOP (1.7 g) and 0.84 mL of DIEA (diisopropylethylamine) were added.

The mixture was stirred for 24 h under nitrogen atmosphere. The product was purified

by repeated precipitation with acetone. The product was collected by filtration and dried

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under vacuum. ESI-MS (m/z): 1469.7 (M+H+). 1H NMR: (D2O, δ): 4.95-5.01 (m, 7H),

3.35-3.83 (m, 42H), 2.01-3.16 (m, 5H), 1.19-1.82 (m, 8H).

Synthesis of β-CD Modified Au Nanoparticles (Au-β-CD NPs)

100 mg of HAuCl4·4H2O was dissolved in 20 mL of DMSO (dimethylsulfoxide), and

then 20 mL of DMSO solution containing lipoic acid modifiedβ-CD (4 mg) and

NaBH4 (151 mg) was added. After stirring for 24 h at room temperature, 40 mL of

CH3CN (acetonitrile) was added. Then precipitate appeared and was collected via

centrifugation. The precipitate was washed with CH3CN : DMSO (v : v = 1 : 1), ethanol,

and dried under vacuum for 24 h. Au-β-CD NPs were characterized by TEM. 1H NMR:

(D2O, δ): 4.95-5.01 (m, 7H), 3.35-3.83 (m, 42H), 2.01-3.16 (m, 5H), 1.19-1.82 (m, 8H).

Synthesis of PEG-K(PpIX)DPLGVRD3K(Ad)-CONH2

PEG-K(PpIX)DPLGVRD3K(Ad)-CONH2 was synthesized via solid-phase peptide

synthesis (SPPS) method.[S3] The peptide was cleaved from the Rink amide AM resin

via a cleavage cocktail containing trifluoroacetic acid (95%), H2O (2.5%) and

triisopropylsilane (2.5%) for 1.5 h. The product was concentrated under vacuum, and

precipitated in cold ether. Thereafter, the product was collected and freeze-dried.

Synthesis of Ad-PEG-TK

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ROS-cleavable thioketal linker (TK) was synthesized according to a previously

literautre.[S4] 1H NMR: (CD3OD, δ): 2.85 (t, 4H), 2.58 (t, 4H), 1.58 (s, 6H). (PEG8)3-

K(Ad)-CONH2 (Ad-PEG) was prepared via SPPS method. Ad-PEG-TK was

synthesized as below: TK and Ad-PEG were dissolved in anhydrous DMF under

nitrogen atmosphere. 10 min later, N,N’-diisopropylcarbodiimide (DIC) and N-

hydroxysuccinimide (NHS) in anhydrous DMF were added dropwise to the above

solution. After 24 h, the solution was dialyzed (MW: 1000) against deionized water for

48 h. The product was obtained after freeze-dried. Ad-PEG-TK was characterized by

ESI-MS.

Synthesis of Ad-PEG-TK-DOX

Ad-PEG-TK, DOX and triethylamine (TEA) were dissolved in anhydrous DMF to

obtain a clear solution under nitrogen atmosphere. And then DIC and NHS dissolved

in anhydrous DMF were added dropwise to the above solution. The reaction was

finished after 24 h. Then the solution was dialyzed (MW: 1000) against deionized water

for 48 h. Ad-PEG-TK-DOX was obtained after freeze-dried.

Au-β-CD/mPEG-K(PpIX)DPLGVRD3K(Ad)-CONH2/Ad-PEG-TK-DOX

(Au/PpIX/DOX NPs) System Preparation

Au/DOX/PpIX NPs were obtained through host-guest interaction. To select an optimal

feed ratio, the concentrations of mPEG-K(PpIX)DPLGVRD3K(Ad)-CONH2 and Ad-

PEG-TK-DOX were fixed at 14.5 mg/L and 14.0 mg/L, respectively. the fluorescence

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changes were detected when the concentration of Au-β-CD NPs increased in the

complex solution. To ensure that the fluorescences of PpIX and DOX were quenched,

the reaction condition was fixed (MMP-2 sensitive peptide substrate: TK sensitive

peptide substrate: Au-β-CD NPs = 14.5: 14: 6.5). The mixture of mPEG-

K(PpIX)DPLGVRD3K(Ad)-CONH2, Ad-PEG-TK-DOX and Au-β-CD NPs was

stirred 24 h at room temperature, dialysized (MWCO: 3,000 Da) against DI water for 3

days and lyophilized, then AuNP/PpIX/DOX NPs were prepared. The AuNP/PpIX NPs

and AuNP/DOX NPs were prepared in a similar way.

Fluorescence Recovery Au/PpIX/DOX, Au/PpIX and Au/DOX NPs Under External

Stimuli

MMP-2 enzymatic response of Au/PpIX NPs was studied via fluorescence spetra at 37

°C. Briefly, 4 mg of Au/PpIX NPs was dispersed in 4 mL PBS buffer solution

containing MMP-2 (2 µg/mL, 100 μL). Then the fluorescence spectra of PpIX was

detected at given time intervals (Ex = 409 nm). Similarly, the ROS response of

Au/DOX NPs was studied via incubating 600 μL of Au/DOX NPs with 100 μL of H2O2

(Ex = 488 nm). In order to evaluate the programmed MMP-2 responsive PLGVR

hydrolysis and ROS-responsive DOX release, fluorescence recovery of Au/PpIX/DOX

NPs under MMP-2 enzyme was measured in dark environment. 4 h later, the solution

was irradiated by 630 nm He-Ne laser for preset times (ExPpIX = 409 nm, ExDOX = 488

nm). Both the emission and excitation bandwidths were 5 nm in all fluorescence spectra

detection.

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MMP-2 Responsive Tumor Imaging

SCC-7 cells and COS7 cells (1 × 105 cells/well) were seeded in a 6-well plate,

respectivly. After 24 h, 50 µL Au/PpIX/DOX NPs (PpIX: 41 mg/L) and 950 µL 10%

FBS were added. 5 h later, the sample was replaced with 1 mL fresh medium. The cells

were imaged via CLSM after repeated washing with PBS buffer (C1-Si, Nikon, Japan).

PpIX was excited with a 408 nm laser and the fluorescence was collected at 600-700

nm. To determine the release of PpIX, the cells were harvested and repeatedly freeze-

thawed. Then cells were under ultrasound for 5 min and centrifugated at 3000 r min–1

for 4 min. The fluorescence spetrum of supernate was measured (Ex = 409 nm and Em

= 630 nm). The standard curve of mPEG-K(PpIX)DPLGVRD3K(Ad)-CONH2 was as

below: FL = 20863 C(mg/mL) + 0.833 (R2 = 0.999).

Light Irradiation Triggered DOX Release

SCC-7 cells (1 × 105 cells/well) were seeded in a 6-well plate. After 24 h, the medium

was replaced with 50 µL Au/PpIX/DOX NPs (DOX: 40 mg/L) and 950 µL of 10%

FBS. After incubated for 5 h, the sample was replaced with 1 mL fresh medium and

treated by 30-min light irradiation (band pass: 400-700 nm, 3.3 mW/cm2). Thereafter,

cells were washed with PBS and cell nucleus was stained with 10 µL (2 mg/mL)

Hoechst 33342 for 15 min at 37 °C. After repeated washing with PBS, the cells were

imaged via CLSM with a filter set (Ex = 408 nm, Em = band pass 450-500 nm) for

Hoechst 33342 and a filter set (Ex = 488 nm, Em = band pass 550-600 nm) for DOX.

Cytotoxicity Assay

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The cytotoxicity of Au/PpIX/DOX NPs in SCC-7 and COS7 cells were determined via

MTT assay. Cells were incubated on a 96-well plate with a density of 6000 cells/well.

After 24 h, Au/PpIX/DOX NPs with different concentrations were added to each well.

5 h later, the medium was replaced by 200 µL fresh medium. And then cells were

treated by 30-min light irradiation (band pass: 400-700 nm, 3.3 mW/cm2) and further

incubated for 48 h. Subsequently, 20 µL MTT (5 mg/mL) solution was added. After 4

h, the medium was replaced with 200 µL DMSO. The OD values were measured at 570

nm. The relative cell viability was calculated as: cell viability (%) =

(OD570(samples)/OD570(control)) × 100, where OD570(samples) was obtained in the presence of

Au/PpIX/DOX NPs. OD570(control) was obtained in the absence of Au/PpIX/DOX NPs.

Au/PpIX NPs and Au/DOX NPs were employed as the controls.

Statistical Analysis

Statistical analysis was performed using a Student’s t-test. The differences were

considered to be statistical significant for p value <0.05.

References

[S1] K. Han, Y. Liu, W. N. Yin, S. B. Wang, Q. Xu, R. X. Zhuo and X. Z. Zhang, Adv.

Healthcare Mater., 2014, 3, 1765

[S2] H. Wang, Y. Chen, X. Y. Li and Y. Liu, Mol. Pharmaceutics, 2007, 4, 189.

[S3] K. Han, Q. Lei, H. Z. Jia, S. B. Wang, W. N. Yin, W. H. Chen, S. X. Cheng and

X. Z. Zhang. Adv. Funct. Mater., 2015, 25, 1248.

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[S4] Y. Y. Yuan, J. Liu and B. Liu, Angew. Chem., 2014, 126, 7291.

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Scheme S1. (A) Chemical structure of Ac-PEG8-K(PpIX)DPLGVRD3K(Ad)-CONH2.

(B) Synthetic process and chemical stucture of Ad-PEG-TK-DOX.

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Figure S1 (A) ESI-MS of lipoic acid modified β-CD (β-CD-SS). (B) TGA analysis of

Au-β-CD NPs.

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Figure S2. ESI-MS of Ac-PEG8-K(PpIX)DPLGVRD3-K(Ad)-CONH2.

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Figure S3. ESI-MS of (A) Ad-PEG, (B) Ad-PEG-TK and (C) Ad-PEG-TK-DOX.