A multi-functional PEGylated gold(iii) compound: potent anti-cancer properties and self-assembly into nanostructures for drug co-delivery

Amphiphilic gold(iii) porphyrin–PEG conjugates can undergo self-assembly into nanostructures, resulting in promising anti-cancer properties with minimal systemic toxicity. The nanostructures can be employed as nanocarriers for drug co-delivery.

Flow cytometric analysis of co-culture model of HCT116 (pre-treated by CMF 2 HC dye) and NCM460 cells stained with CellEvent Caspase-3/7 Green Ready Detection Reagent after incubation with 1 and solvent control for 24 h Difference in in vitro cytotoxicity toward A2780 and drug-resistant A2780cis and A2780adr Table S2 Relative toxicity of 1-3 and Au1a toward cancer cells and NCM460 cells Table S3 Relative toxicity of 1-3 and Au1a toward cancer cells and MIHA cells Table S4 Relative toxicity of 1-3 and Au1a toward cancer cells and CCD-19Lu cells Table S5 In vitro cytotoxicity of 1, 3 and cisplatin toward non-tumorigenic liver L02 and gliocyte CHEM-5 cells Table S6 Relative toxicity of DOX toward cancer cells and MIHA cells through administration of DOX alone and administration by NC1 Table S7 Relative toxicity of DOX toward cancer cells and NCM460 cells through administration of DOX alone and administration by NC1 fetal bovine serum, ʟ-glutamine (2 mM) and penicillin/streptomycin (100 U/mL).

[Au(TPP-COO-PEG 5000 -OCH 3 )]Cl (1)
Complex 4 (22 mg, 0.026 mmol), HOBt (2.4 mg, 0.018 mmol) and PEG 5000 -OCH 3 (124 mg, 0.025 mmol) were dissolved in anhydrous dichloromethane. With stirring at 0 o C, DCC (5.1 mg, 0.025 mmol) in dichloromethane was added to the solution mixture, followed by the addition of triethylamine (7.2 µL, 0.052 mmol). The solution mixture was then stirred at 0 o C for 2 h, and allowed to warm to room temperature and reacted for 5 days. After evaporation of volatile solvent under reduced pressure, the crude product was dissolved in deionized water, and undissolved solid was filtered.
The aqueous solution was extracted with dichloromethane twice, and the combined organic layers were washed with dilute HCl (aq) solution twice followed by saturated NaCl (aq) solution. The organic layer was then dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was dissolved in minimum amount of dichloromethane, and precipitated as a red solid by dropwise addition of the dichloromethane solution to diethyl ether. The red solid obtained after precipitation for totally three times was further purified by column chromatography on neutral alumina, using dichloromethane and methanol (50:1, v/v) as eluent. The product was obtained as red solid. Yield = 115 mg (78%

[Au(TPP-CONH-PEG 5000 -OCH 3 )]Cl (2)
The procedure was similar to that for complex 1, except that H 3 CO-PEG 5000 -NH 2 •HCl (124 mg, 0.025 mmol) was used instead of HO-PEG 5000 -OCH 3 . The red solid obtained after precipitation was further purified by column chromatography on neutral alumina, using dichloromethane and methanol (50:1, v/v) as eluent. The product was obtained as red solid. Yield = 88 mg (60% Preparation of nanocomposites of 1 and DOX. The procedure was similar to that of the preparation of nanostructures of 1, except that both complex 1 (2.5 mg) and DOX were dissolved in the acetonitrile (0.5 mL). P.7 The gold content of the nanocomposites was determined by dissolving the nanocomposites in conc. HNO 3 / conc. HCl solution mixture (9:1, v/v; 30 µL), followed by ICP-MS analysis.

Analysis of induction of apoptosis in HCT116 and NCM460 cells after incubation with gold(III) complexes by CellEvent Caspase-3/7 Green Detection Reagent
staining and flow cytometry. The procedure is similar to that using FITC-Annexin V  with 1 vol% DMSO) instead of 1.

Determination of gold content in tissues of mice treated with complex 1, 3 and
Au1a at the end point of tumor growth inhibition study by ICP-MS. At the end P.14 point of tumor growth inhibition study, the mice treated with complex 1, 3 and Au1a, respectively, were sacrificed and tissues including liver, spleen, lung, kidney and heart were harvested. The tissues were digested with 10% HCl in concentrated HNO 3 (0.1 g of tissue/1 mL). The solution mixture was heated at 60 o C for 15 h, and then diluted 50 times by ultra-pure water for ICP-MS analysis.

Determination of in vivo biodistribution of complex 1, 3 and Au1a by ICP-MS.
Nude mice bearing HCT116 xenografts were treated with 1 (13 mg/kg), or equal molar amount of Au1a (2 mg/kg) or 3 (2 mg/kg) through intravenous injection. After 24 h of the injection, the mice were sacrificed and tissues from tumor, liver, spleen, lung, kidney and heart were harvested. The tissues were digested with 10% HCl in concentrated HNO 3 (0.1 g of tissue/1 mL). The solution mixture was heated at 60 o C for 15 h, and then diluted 50 times by ultra-pure water for ICP-MS analysis.

MS.
At the end point of tumor growth inhibition study, the mice treated with solvent control or complex 1 were sacrificed and the tumor tissues were harvested, followed by homogenization in 50% ice-cold acetonitrile (0.1 g/300 µL). The homogenate was             Gold content (g per g of tissue) 1 (4 mg/kg) Au1a (2 mg/kg) 3 (2 mg/kg)

Fig. S23
Biodistribution of gold complexes in nude mice bearing HCT116 xenografts after 14 days of treatment with 1 (4 mg/kg), Au1a and 3 (2 mg/kg), respectively, through intravenous injection. The gold content in homogenized tissues was quantified by ICP-MS. n = 5 (for Au1a and 3) and 6 (for 1). Data are shown as mean ± SD. P.39