Materials and Measurements

Cisplatin was bought from Shandong Boyuan Chemical Company, China.1-(4-Aminophenyl)-1,2,2-triphenylethene, N-hydroxysuccinimide (NHS), pyridine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), methoxy polyethylene glycol (Mn = 8000 Da, mPEG8k), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Allyl glycidyl ether (AGE), 2,2'-Azobis(2-methylpropionitrile) (AIBN), succinic anhydride 1-(4-Aminophenyl)-1,2,2-triphenylethene (TPE) and 2-Aminoethanethiol hydrochloride were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). All other reagents were commercially available and used as received.

Measurements

Proton nuclear magnetic resonance (1HNMR) spectra were measured on a Bruker AVANCE DRX 400 spectrometer using deuterated dimethyl sulfoxide (DMSO d6) and deuterium oxide (D2O). The molecular weight and polydispersity index of the synthetic polymer were detected using a Tetrahydrofuran 515 gel permeation chromatograph (GPC). Diameter and polydispersity index were measured on Malvern ZETASIZER LAB. Transmission electron microscope (TEM) images were collected using a JEOL JEM-1011 transmission electron microscope with an acceleration voltage of 100 kV. An inductively coupled plasma mass spectrometer (ICP-MS, Xseries II, Thermo Fisher Scientific, USA) was used for the analysis content of Pt. Electrospray ionization mass spectrometry (ESI–MS) measurement was conducted on a Waters Quattro Premier XE system. The molecular weight and polydispersity index of the synthetic polymer were determined using a Tetrahydrofuran 515 gel permeation chromatograph (GPC). The molecular weight of PAGE-Pt-TPE was analyzed by using Autof ms800 (Autobio Diagnostics, Zhengzhou, China) systems.

Synthesis of Pt(IV) prodrug

Briefly, cisplatin (1 g) was suspended in H2O2 (20 mL, 30%) stirred for 24 h at room temperature. Finally, Pt(IV) was obtained by filtration and washing with water and ethanol.

Synthesis of Pt(IV)-COOH

The Pt(IV) prodrug (1 g, 2.9 mmol) and succinic anhydride (1.168 g, 11.6 mmol) were dissolved in N,N-Dimethylformamide (DMF, 20 ml). After reaction at 60 ℃ for 24 h, solution was poured into diethyl ether and filtering it for three time.

Synthesis of PAGE

Block copolymer PAGE was synthesized according to the literature with a slight change as shown in Scheme 1. Briefly, dried mPEG8k(2.0 mmol), 305 mg cesium hydroxide monohydrate (1.8 mmol, 0.9 equiv), and 40 mL dried toluene were added to a dry reaction bulb under argon/nitrogen atmosphere. In order to acquire partially deprotonated macroinitiator, it is necessary to evacuate at 90 ℃ for 3 h. After that, additional 100 mL of toluene was added and 6.84 g AGE (60.0 mmol, 15 equiv) was added. The polymerization reaction needs to be maintained for 48 h at 45 ℃ for 48 h under an inert atmosphere. To stop the polymerization was stopped, a trace of acetic acid–ethanol (50/50) solution was added.The reaction solution was poured into a mount of cold diethyl ether for 3 times and the polymers were collected and dried in vacuum for 8 h (85%).

Synthesis of PAGE-NH2

PAGE (5 g), 2-Aminoethanethiol hydrochloride (10 g, 62.5 mmol) and dried 20 mL of DMF were add to dry reaction bulb under inert atmosphere. After dissolution and addition of AIBN, the reaction maintained for 24 h at 65 ℃ under an inert atmosphere. DMF was removed at 50 ℃ under vacuum. Residue was redissovled in CH2Cl2 and precipitated in cold diethyl ether. After filtered and dried, mPEG-NH2 was acquired.

Synthesis of PAGE-Pt

In general, large excess of Pt(IV)-COOH prodrug (245 mg, 0.46 mmol), EDC (265 mg, 1.38 mmol), and NHS (159 mg, 1.38 mmol) were added into reaction bulb and 8 mL of DMF was injected. The solution was activated for 4 h in ice bath. After that, poured PEGA-NH2 (184 mg, 0.023 mmol) into reaction solution and maintained the reaction at 25 ℃ for 3 days. Finally, the solution was poured into Spectra/Por Regenerated Cellulose membrane (MWCO = 3500), which was dialyzed with purified water for 2 days. After freeze-dried, PAGE-Pt was obtained.

Synthesis of PAGE-Pt-TPE

In general, PAGE-Pt polymeric prodrug (100 mg, 0.01 mmol), EDC (46 mg, 0.24 mmol), and NHS (28 mg, 0.24 mmol) were added into reaction bulb and 8 mL of DMF was injected. The solution was activated for 4 h in ice bath. After that, poured TPE (69.4 mg, 0.2 mmol) into reaction solution and maintained the reaction at 25 ℃ for 3 days. Finally, the solution was poured into Spectra/Por Regenerated Cellulose membrane (MWCO = 3500), which was dialyzed with purified water for 2 days. After freeze-dried, PAGE-Pt-TPE was obtained.

Reductive drug release of PGAE-Pt-TPE

To simulate the release process of PAGE-Pt-TPE in vivo. PAGE-Pt-TPE (2 mg) was dissolved in different phosphate buffer, inluding 1 mL of PBS (0.01 M, pH 7.4), PBS (0.01 M, pH 7.4, 1 mg/ml SA), PBS (0.01 M, pH 5.0) or PBS (0.01 M, pH 5.0, 1 mg/ml SA). The solution was poured into a dialysis bag (MWCO = 500) and 19 mL of the corresponding PBS solutions was added. Drug release was conducted at 37 ℃ in an air bath shaker.

Cell culture

Human cervical carcinoma HeLa cells were cultured with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS). The U14 cells were cultured in the ascites of mice. These and murine tumor cells U14 were acquired from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.

Cellular uptake of TPE

For TPE release in cells, The germfree coverslips were added into 6-well plates. HeLa cells were cultured on it (2 × 10 5 cells per well) for 24 h. PAGE-Pt-TPE was dissolved in DMEM (Pt: 54 µM) and the initial medium was substituted with PAGE-Pt-TPE solution for 2 h and 4 h, respectively. After cultured for corresponding time, HeLa cells were washed with cold PBS. For CLSM observation, the HeLa cells on the coverslips were fixed using formaldehyde(4%) for 15 min 25 ℃. We observed fluorescence images using a CLSM.

Cell viability

Cells were seeded in 96-well plates (5 × 103 cells per well). After incubated in DMEM for 24 h, the medium was added with cisplatin, TPE, Pt(IV), PAGE-Pt and PAGE-Pt-TPE from 3.375 to 216 µM (final Pt concentration from or equal molar amount Pt). After incubating for 48 h, MTT was added, and 96-well plates was at incubator for another 4 h. Afer removal of MTT solution, 150 mL of DMSO was added to each well to dissolve the formazan crystals formed in the cells. After mixed for 5 min, the absorbance was acquired at 490 nm using a microplate reader.

Animals used

Kunming (KM) mice were bought from Jilin University. The experimental mice bearing U14 xenograft tumor model were acquired by subcutaneously injecting U14 cells (1 × 106, 0.1 mL PBS) into right legs of mice. All the in vivo study protocols were approved by the local institution review board and performed according to the Ethics Committee of the Beijing Tiantan Hospital of Capital Medical University, China. After volume of subcutaneous U14 tumor model reach to 500 mm3, the animals were anaesthetized with pentobarbital sodium and the tumor was collected from sacrificed mice. It was cut into pieces and treated with homogenizer and pressure cell disruptor. After that, supernatant was collected by centrifugation.