Supporting Information

Photosensitive Pt(IV) prodrug-loaded nanoparticles exhibit controlled

drug release and enhanced efficacy in vivo.

Haihua Xiao, Gavin T. Noble, Jared F. Stefanick, Ruogu Qi, Tanyel Kiziltepe, Xiabin Jing* and Basar

Bilgicer*

CONTENTS

Materials and Methods 3

Scheme S1. Synthesis of Photosensitive Pt(IV)-Azide Complexes C1C4. 7

Figure S1. IR Spectra of Photosensitive Pt(IV)-Azide Complexes C1C4. 8

Figure S2. 1H NMR of Photosensitive Pt(IV)-Azide Complexes C1C4. 9

Figure S3. ESI-MS Photosensitive Pt(IV)-Azide Complexes C1C4. 10

Figure S4. Representative UVA Sensitivity and Stability in the Dark of C3 and NC3 11

Figure S5. Succinic acid release from C1 and C3 12

Figure S6. MALDI-MS Study of 5’-GMP Chelation by C3 13

Scheme S2. Depiction of polymer P1 self-assembly 14

Figure S7. UVA-Mediated Pt Release from NC1, NC2 and NC4 15

Figure S8. UV-Vis Spectra of Model Pt Complexes and NC2 Dialysates 16

Figure S9. Cell viability after prolonged UVA irradiation 17

Table S1. Physicochemical Properties of Micellar Nanoparticles NC1-NC4 18

References 18

2

MATERIALS AND METHODS

General Materials and Methods. The carrier polymer P1 was synthesized as per previously reported methods. [1] All other

commercially sourced chemicals and solvents were used without further purification. N-hydroxysuccinimide (NHS), 1-ethyl-

(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), 1R,2R-cyclohexanediamine (DACH) and succinic

anhydride were purchased from Sigma-Aldrich. Cisplatin (purity 99%) and K2PtCl4 (purity 99%) were bought from Shandong

Boyuan Chemical Company, China. 1H NMR spectra were measured using a Unity-300MHz NMR spectrometer (Bruker).

Fourier Transform Infrared (FT-IR) spectra were recorded on a Bruker Vertex 70 spectrometer. Electrospray mass spectrometry

(ESI-MS) measurements were performed on a Quattro Premier XE system (Waters) equipped with an electrospray interface

(ESI). An inductively coupled plasma optical emission spectrometer (ICP-OES, iCAP 6300, Thermoscientific, USA) was used

to measure platinum loading in the polymer-Pt(IV) conjugates and dialysis samples in drug release experiments. An

inductively coupled plasma mass spectrometer (ICP-MS, Xseries II, Thermoscientific, USA) was used for quantitative

determination of trace levels of platinum in the cell lysis liquid. Micelle size and polydispersity was measured using a DAWN

EOS DLS (Wyatt Technology, USA) equipped with a vertically polarized He-Ne laser. TEM images were taken using a JEOL

JEM-1011 electron microscope. Particle size and zeta potential measurements were conducted on a Malvern Zetasizer Nano

ZS. Kunming (KM) mice (68 weeks old) were purchased from Jilin University (Changchun, China). H22 cells (murine

hepatocarcinoma cell lines) were purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences,

Shanghai, China.

Synthesis and Characterization of Pt(IV)-Azide Complexes.

*Caution!* Although no problems were encountered during this work, heavy metal azides are known to be heat and shock-

sensitive detonators. Therefore, it is essential that any platinum azide compounds are handled with care. The chemical

synthesis of all novel compounds used in this study is outlined in Scheme S1. Novel compounds were characterized using IR

(Figure S1), 1H NMR (Figure S2) and ESI-MS (Figure S3).

Synthesis of c,c-[Pt(NH3)2(N3)2]. c,c-[Pt(NH3)2(N3)2] was prepared as previously reported,[2] and the procedure is outlined in

Scheme S1A; IR: cm-1 3277 (sh, NH2), 2050 (sh, N3).

Synthesis of c,c,t-[Pt(NH3)2(N3)2(OH)2]. c,c,t-[Pt(NH3)2(N3)2(OH)2] was prepared as previously reported,[2] and the

procedure is outlined in Scheme S1A; 1 H NMR: (300 MHz, d6-DMSO, 25 °C) : δH 4.81-5.18 (6H, m, NH3); IR: cm-1 3478

(sh, OH), 3265 (sh, NH2), 2050 (sh, N3)’ 550 (sh, Pt-OH).

Synthesis of C1. To a solution of c,c,t-[Pt(NH3)2(N3)2(OH)2] (0.11 g) in anhydrous DMSO (3 ml) was added succinic

anhydride (32 mg) (Scheme S1A) and the reaction mixture stirred at room temperature for 24 hours in the dark. Cooled diethyl

ether (100 ml) was added to precipitate the product to obtain a bright yellow solid, which was washed several times with

diethyl ether, and dried. The product was isolated in 60% yield (85 mg). The product was characterized using IR (Figure S1A.

iv), 1H NMR (Figure S2B) and ESI-MS (Figure S3A); 1 H NMR: (300 MHz, d6-DMSO, 25 °C) : δH 2.33-2.39 (4H, m, -

3

OCH2CH2C-), 5.12-5.71 (6H, m, NH3); IR: cm-1 3469 (br, OH), 2051 (sh, N3), 1700 (sh, CO-OH), 1638 (sh, -PtOOC-C-), 557

(sh, Pt-OH). ESI-MS: (negative mode) m/z 446.2 [M-H]- (100%).

Synthesis of C2. To a solution of c,c,t-[Pt(NH3)2(N3)2(OH)2] (0.11 g) in anhydrous DMSO (10 ml) was added succinic

anhydride (158 mg) (Scheme S1A) and the reaction mixture was stirred at 50 °C for 24 hours in the dark. Cooled diethyl ether

(100 ml) was added to precipitate to obtain a bright yellow solid, which was redissolved in methanol and precipitated by

acetone and washed several times with acetone, and dried. Complex C2 was isolated in 50% yield (87 mg). The product was

characterized using IR (Figure S1A. v), 1H NMR (Figure S2C) and ESI-MS (Figure S3B); 1 H NMR: (300 MHz, d6-DMSO, 25

°C) : δH 2.31-2.59 (8H, m, -OCH2CH2C-), 5.41-6.10 (6H, m, NH3); (300 MHz, D2O, 25 °C) : δH 2.53 (8H, m, -OCH2CH2C-);

IR: cm-1 2065 (sh, N3), 1738 (sh, -CO-OH), 1650 (sh, -PtOOCC-); ESI-MS: (negative mode) m/z 546.3 [M-H]- (100%).

Synthesis of c,c-[Pt(DACH)Cl2]. c,c-[Pt(DACH)Cl2] was prepared as previously described,[3] (0.7 g, 77% yield).

Synthesis of c,c-[Pt(DACH)(N3)2]. c,c-[Pt(DACH)(N3)2] was prepared using a similar procedure as reported for c,c,t-

[Pt(NH3)2(N3)2]: c,c-[Pt(DACH)Cl2] (0.381g) was suspended in water (50 mL), to which AgNO3 (0.339 g) was added. The

reaction mixture was then stirred in the dark for 24 hours, after which the white AgCl precipitate formed was removed by

filtration. NaN3 (0.13g) was added to the clear solution and sharp yellow precipitates of the target compound formed. The c,c-

[Pt(DACH)(N3)2] precipitates (0.2 g, 51% yield) were filtered and collected. IR: cm-1 3270 (sh, NH2), 2051 (sh, N3).

Synthesis of c,c,t-[Pt(DACH)(N3)2(OH)2]. c,c-[Pt(DACH)(N3)2] (0.15 g) was suspended in water (10 ml), to which 30%

H2O2 (3 ml) was added. The reaction mixture was stirred in the dark for 24 hours. Thereafter, it was concentrated under

reduced pressure. Methanol (2 ml) was added to dissolve the concentrated products and then diethyl ether (100 ml) was added

to precipitate the product, c,c,t-[Pt(DACH)(N3)2(OH)2] (100 mg, 61%). The product was characterized using IR (Figure S1B.

iii) and 1H NMR (Figure S2D); 1 H NMR: (300 MHz, d6-DMSO, 25 °C) : δH 6.05-6.30 (2H, m, NH2), 6.73-6.97 (2H, m, NH2),

0.9-2.2 (10H, m, cyclohexane CH, CH2); IR: cm-1 3454 (br, -OH), 2068 (sh, N3), 540 (sh, -Pt-OH).

Synthesis of C3. To a solution of c,c,t-[Pt(DACH)(N3)2(OH)2] (0.1 g) in anhydrous DMSO (3 ml) was added succinic

anhydride (23.4 mg) (Scheme S1B) and the reaction mixture stirred at room temperature for 24 hours in the dark. Cooled

diethyl ether (100 ml) was added to precipitate a light yellow solid, which was collected, washed several times with diethyl

ether and dried. The product complex C3 was isolated in 60% yield (74 mg). The product was characterized using IR (Figure

S1B. iv), 1H NMR (Figure S2E) and ESI-MS (Figure S3C). 1 H NMR: (300 MHz, d6-DMSO, 25 °C) : δH 1.03-2.2 (10H, m,

cyclohexane CH, CH2), 2.39 (4H, m, succinic -OCCH2CH2CO-), 8.11-6.35 (4H, m, NH2); IR: cm-1 2065 (sh, N3), 1724 (sh, -

CO-OH), 1628 (sh, -OOC-C-); ESI-MS: (negative mode) m/z 526.3 [M-H]- (100%).

Synthesis of C4. To a solution of c,c,t-[Pt(DACH)(N3)2(OH)2] (0.1 g) in anhydrous DMSO (3 ml) was added succinic

anhydride (117 mg) (Scheme S1B) and the reaction mixture stirred at room temperature for 24 hours in the dark. Cooled

diethyl ether (100 ml) was added to precipitate a light yellow solid, which was re-dissolved in methanol and precipitated by

acetone and washed several times with acetone, collected and dried. The product complex C4 was isolated in 60% yield (88

mg). The product was characterized using IR (Figure S1B. v), 1H NMR (Figure S2F) and ESI-MS (Figure S3D); 1 H NMR:

4

(300 MHz, d6-DMSO, 25 °C) : δH 0.91-2.12 (10H, m, cyclohexane CH, CH2), δH 2.37 (4H, m, - succinic OCCH2CH2CO-),

8.11-5.7 (4H, m, NH2); IR: cm-1 2060 (sh, N3), 1716 (sh, -CO-OH), 1628 (sh, -OOC-C-); ESI-MS: (negative mode) m/z 626.4

[M-H]- (100%).

Preparation of Micellar Nanoparticles of Pt(IV)-azide Complexes. Pt(IV)-azide complexes C1-C4 were dissolved in

water (5% w/v), added to an aqueous solution of EDC and NHS (1.5 eq. per carboxyl group each) and stirred for ten minutes.

The polymer P1 (1 amine eq. per Pt(IV)-azide complex carboxyl group) was then added and the solution stirred at in the dark

for 24 h. P1 has 114 ethylene glycol, 20 polycaprolactone (PCL) and 10 PLL repeat units. The polymer conjugates of C1-C4

were then purified by dialysis (1,000 MWCO) for 12 h and lyophilized. To form NC1-NC4, polymer conjugates of C1-C4 (50

mg) were dissolved in DMF (10 ml) and then water (50 ml) was added dropwise with stirring. The micellar nanoparticles were

then dialyzed (1,000 MWCO) against water to remove the DMF and lyophilized for storage.

Measurement of UV-Vis Spectra of Model Pt Complexes. In order to determine the major Pt(II) species produced during

the UVA irradiation procedure, aqueous solutions of model compounds were prepared by dissolving the Pt drugs into water at a

concentration of cisplatin (0.24 mg/ml), c,c-[Pt(NH3)2(N3)2] (0.06 mg/ml), c,c,t-[Pt(NH3)2(N3)2(OH)2] (0.17 mg/ml), c,c-

[Pt(DACH)Cl2] (0.1 mg/ml), c,c-[Pt(DACH)(N3)2] (0.075 mg/ml) and c,c,t-[Pt(DACH)(N3)2(OH)2] (0.15 mg/ml). Species c,c-

[Pt(NH3)2(H2O)2]2+ and c,c-[Pt(DACH)(H2O)2]2+ were prepared by reacting cisplatin (0.1 mg/ml) and c,c-[Pt(DACH)Cl2] (0.05

mg/ml) with AgNO3.3 UV-vis spectra were taken of each solution and are shown in Figure S6A, B.

Determination of Pt Species Released from Micellar Nanoparticles by Dialysis. In order to confirm the identity of

released Pt and to give insight into the drug release mechanism, UV-vis spectra of NC2 and NC3 dialysates were taken.

Mechanistic information was inferred by comparing the shape of the UV-vis curve present in the dialysate to the model

compounds shown in Figure S6A, B. As shown in Figure S6C, D, in the dark the N3→Pt peak at 258 nm is clear in the UV-vis

spectra of NC2 dialysates, implying that the N3→Pt remained intact in the dark and Pt(IV) was released from the micellar

nanoparticles. However, the peak at 258 nm was significantly reduced in dialysates of NC2 taken after UVA irradiation (Figure

S6E, F) and although initially some N3→Pt remained intact, little N3→Pt was observed after 6 h and was completely

diminished after 10 h. This implied a combined mechanism of Pt release where hydrolysis of C1-C4 from the micellar

nanoparticles and UVA activation of Pt(IV) could occur simultaneously.

In vivo studies. Kunming (KM) mice (68 weeks old) were purchased from Jilin University (Changchun, China). The use of

mice for this study was approved by the Animal Ethics Committee of Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences. H22 cells were purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of

Sciences, Shanghai, China cultured as described before.[4,5] Tumor bearing mice were induced by subcutaneously injecting

1×106 H22 cells into the back of the mice.

Blood Clearance. Clearance was measured by injecting platinum-based drugs, once, via the tail vein into mice at 5 mg

Pt/kg. Mice were then sacrificed at various time intervals. Blood was obtained via cardiac puncture mixed with 65% v/v nitric

acid (1 mL) and the Pt concentration measured using ICP-MS. 3 mice were used per group.

5

Tumor Growth Inhibition. Mice were randomly divided into 5 groups (10 mice per group) when tumor volumes reached

~100200 mm3. Mice in each group were treated with either oxaliplatin (5 mg or 10 mg Pt/kg body weight), C3 (10 mg Pt/kg

body weight), NC3 (5 mg Pt/kg body weight) or PBS. All drugs were injected intratumorally on days 0 and 4 and the mice

were kept in the dark. For UVA activation, mice were UVA treated for 1 h on days 1 and 5, 24 h after drug injection. After each

UVA treatment, mice were returned to the dark. The body weight and tumor volume were measured every two days and tumor

volumes calculated.[4,5]

Statistical analysis. The data were expressed as mean ± standard deviation (SD). Student’s t-test was used to determine the

statistical difference between various experimental and control groups. Differences were considered statistically significant at a

level of p < 0.05.

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SUPPLEMENTARY FIGURES

Scheme S1. Synthesis of novel photosensitive Pt(IV)-azide complexes C1C4.

7

Figure S1. IR spectra of (A) cisplatin (i), c,c-[Pt(NH3)2(N3)2] (ii), c,c,t-[Pt(NH3)2(N3)2(OH)2] (iii), complex C1 (iv), and C2 (v); (B) c,c-[Pt(DACH)Cl2] (i), c,c-[Pt(DACH)(N3)2] (ii), c,c,t-[Pt(DACH)(N3)2(OH)2] (iii), complex C3 (iv), and C4 (v). The appearance of a peak at ~2050 cm1 confirmed the presence of N3. After the formation of C1 and C3, the 3480 cm1 OH band is weakened and broadened and two C=O peaks characteristic of the coordinated carboxyl group (1635 cm1) and free carboxyl group (1700 cm1) appeared, confirming the addition of succinic acid. After the formation of C2 and C4 similar changes occurred and the Pt-OH stretch at 544 cm1 disappeared.

8

Figure S2. 1H NMR spectra of (A) c,c,t-[Pt(NH3)2(N3)2(OH)2] (d6-DMSO), (B) C1 (d6-DMSO), (C) C2 (d6-DMSO (upper) and D2O (lower)), (D) c,c,t-[Pt(DACH)(N3)2(OH)2] (d6-DMSO), (E) C3 (d6-DMSO), and (F) C4 (d6-DMSO).

9

Figure S3. Theoretical isotope pattern (insets) and experimental results of complex C1 (A), C2 (B), C3 (C) and C4 (D) measured by ESI-MS (negative mode). Complexes C1-C4 displayed major molecular ions at m/z = 446.2, 546.3, 526.3 and 626.4 respectively, in good agreement with their theoretical isotope patterns (inset).

10

Figure S4. UVA sensitivity and dark stability of photosensitive Pt(IV) prodrugs C1-C4. (a) UV-vis spectra of C3 upon UVA irradiation from 0 to 90 min. The maximum peak at λ = 258 nm dropped dramatically, demonstrating the breakdown of the N3→Pt bond upon UVA irradiation. (b) Normalized UV absorbance (258 nm) of aqueous solutions of C1-C4 versus irradiation time. (c) Stability of C1-C4 in the dark. Normalized UV absorbance (258 nm) of aqueous solutions was unchanged for all samples, which indicated high stability in the dark.

11

Figure S5. ESI-MS (negative mode) study of the released species from C1 (A) and C3 (B) in aqueous solution upon UV irradiation at 0 min, 2 h and 5 h respectively. C1 showed a major molecular ion peak at m/z 446.1 (de-protonated C1) and a dimer at 893.3 (0 min UVA irradiation). The relative abundance of C1 and its dimer decreased dramatically upon 2 h UVA irradiation. A molecular ion, corresponding to succinic acid appeared at m/z 116.8, denoting fast release of the axially coordinated succinic acid of C1 and reduction from Pt(IV) to Pt(II). After 5 hours UVA irradiation, the molecular ion peak of C1 and its dimer is no loger present and only succinic acid can be detected. Similar trends were observed with C3 upon UVA irradiation.

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Figure S6. MALDI-TOF MS study of the photo-reduction of complex C3 (1 mM) to active Pt(II) species after 1 h UVA irradiation and its subsequent chelation with 5’-GMP (5 mM) at 37 C (left for 12 h in the dark). (A) Postulated mechanism of the activation of C3 and chelation with 5’GMP. After UVA irradiation in the presence of 5’-GMP a major MS peak in the MALDI-TOF MS at m/z = 1002.2 ((DACH)Pt(5’-GMP) 2) was observed (B), indicating that C3 was converted to Pt(II) species in the presence of 5’-GMP and thereafter chelated with 5’-GMP. (C) Expanded views of the C3 spectrum from m/z = 600 to m/z = 840 showing 2 fragments of the ((DACH)Pt(5’-GMP)2) with the loss of 1 full GMP unit (m/z = 656.1) and loss of 1 ribose monophosphate (m/z = 806.1). The possible adducts and fragments in the MALDI-TOF-MS spectrum are shown in D and their theoretical isotopic patterns were used to identify the species observed.

13

Scheme S2. Depiction of the self-assembly of P1 in aqueous solution. The hydrophobic polycaprolactone unit forms the core of the micelle and the hydrophilic MPEG and PLL units are presented at the aqueous interface.

14

Figure S7. Pt drug release from NC1 (A), NC2 (B) and NC4 (C) at pH 5.0 (i, iii) and pH 7.4 (ii, iv) upon UVA irradiation (i, ii) and in the dark (iii, iv) as measured by ICP-OES. All micellar nanoparticles released Pt faster with UVA irradiation and release was further enhanced at pH 5 over pH 7.4.

15

Figure S8. UV spectroscopic analysis of dialysates of NC2 in the dark and after UVA irradiation. Spectra of the reference compounds (A) cisplatin (i), hydrated cisplatin (ii), c,c-[Pt(NH3)2(N3)2] (iii), c,c,t-[Pt(NH3)2(N3)2(OH)2] (iv) and (B) c,c-[Pt(DACH)Cl2] (i), c,c-[Pt(DACH)(N3)2] (ii), c,c-[Pt(DACH)(H2O)2]2+ (iii), and c,c,t-[Pt(DACH)(N3)2(OH)2] (iv). These spectra were used as a comparison to confirm the identity of the Pt released from the micellar nanoparticles NC1-NC4. UV-vis spectra of the dialysates of NC2 in the dark (C, D) and after UVA irradiation (E, F) at p 7.4 (C, E) and pH 5.0 (D, F) displaying the characteristic UV peak at 258 nm for the N3→Pt bond. C and D show the release of Pt(IV) from the micellar nanoparticle NC2 via hydrolysis from the P1 polymer chain in the dark. UVA irradiated samples (E, F) display diminished UV-vis peaks at 258 nm through destruction of the N3→Pt bond.

16

Figure S9. Cell viability after UVA irradiation. Cell viability of SKOV-3 cells UVA irradiated for 1 h, 2 h was determined using a standard MTT assay. The non-irradiated cells (0 min) were used as controls. After 1 h UVA irradiation, no morphology change was observed and cell viability was >98%.

17

Table S1. Physicochemical properties of Pt(IV) micellar nanoparticles (NC1-NC4) and non-Pt control P1.

Sample Mean diameter (nm) Zeta potential

(mV)

TDCa (w/w %) EDCb (w/w %)

TEM DLS

P1c 200 ± 8.4 220 ± 4.6 +49.3 ± 2.87 Not detected Not detected

NC1 96 ± 5.7 130 ± 3.4 +8.7 ± 1.42 15.3 12.0 ± 2.13

NC2 119 ± 6.5 145 ± 2.7 0 ± 0.61 14.0 9.8 ± 1.74

NC3 170 ± 4.6 205 ± 1.5 +6.4 ± 2.33 14.3 11.5 ± 0.85

NC4 137 ± 5.1 165 ± 0.93 +1.5 ± 0.95 13.4 10.0 ± 1.53

a: TDC – theoretical Pt drug content; b: EDC - experimental Pt drug content; c: previously published results.1

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