Porous Pt Nanospheres Incorporated with GOx to Enable Synergistic Oxygen‐Inductive Starvation/Electrodynamic Tumor Therapy

Abstract Glucose‐oxidase (GOx)‐mediated starvation by consuming intracellular glucose has aroused extensive exploration as an advanced approach for tumor treatment. However, this reaction of catalytic oxidation by GOx is highly dependent on the on‐site oxygen content, and thus starvation therapy often suffers unexpected anticancer outcomes due to the intrinsic tumorous hypoxia. Herein, porous platinum nanospheres (pPts), incorporated with GOx molecules (PtGs), are synthesized to enable synergistic cancer therapy. In this system, GOx can effectively catalyze the oxidation of glucose to generate H2O2, while pPt triggers the decomposition of both endogenous and exogenous H2O2 to produce considerable content of O2 to facilitate the glucose consumption by GOx. Meanwhile, pPt induces remarkable content of intracellular reactive oxygen species (ROS) under an alternating electric field, leading to cellular oxidative stress injury and promotes apoptosis following the mechanism of electrodynamic therapy (EDT). In consequence, the PtG nanocomposite exhibits significant anticancer effect both in vitro and in vivo. This study has therefore demonstrated a fascinating therapeutic platform enabling oxygen‐inductive starvation/EDT synergistic strategy for effective tumor treatment.


Characterization
The sample morphology was examined using a field-emission scanning electron microscopy (FESEM,. Transmission electron microscope (TEM) imaging and elemental mapping were acquired using FEI Tecnai F20 TEM. The crystalline phase identification was determined by an X-ray diffraction instrument (RIGAKU D/MAX 2550/PC). The zeta potential and dynamic light scattering (DLS) size distribution of samples were measured by a Malvern zetasizer (Nano-ZS). The nitrogen adsorption-desorption isotherms were measured by an automatic surface area and porosity analyzer (Tristar 3020, Micromeritics). Inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer NexION300X) was adopted for quantifying the Pt concentration. The absorbance spectra were measured by a spectrophotometer (Shimadzu, UV-2700). The fluorescent images were observed under a fluorescence inversion microscope (Nexcope, NIB900). The cell apoptosis and necrosis was determined by a flow cytometry (Beckman Coulter, CytoFLEX LX). The images of H&E and Ki-67 staining was obtained with a digital slide scanner (Hamamatsu, NanoZoomer 2.0-RS), and the TUNEL images were acquired by a digital slide scanner (Olympus, VS120).

Synthesis of porous Pt nanosphere incorporated with GOx (PtG)
Porous platinum nanospheres (pPts) were synthesized following a one-pot method.
Typically, 25 mg Pluronic F-127 was added to 2.5 mL aqueous solution of 0.84 mM KBr. 1 mL 40 mM H2PtCl6 aqueous solution and 2.5 mL 0.14 M ascorbic acid solution were added into the mixture dropwise. The solution was stirred in a 70℃ oil bath for 12 h. Subsequently, the mixture was cooled down to ambient temperature and centrifuged at 12000 rpm for 10 min. In order to remove the residual Pluronic F-127, the collected sample was washed repeatedly with ethanol and water. Then, methoxy PEG sulfhydryl (mPEG-SH, Mw = 5000) was used surface modification of pPts. Briefly, 1 mg pPts and 5 mg mPEG-SH were mixed in 5 mL ultra-pure water under ultrasonication for five minutes. After 48 h PEGylation process, the PEGylated pPts were collected by centrifugation and washed with water for three times.
Finally, to prepare the GOx functionalized nanoplatform, GOx and the sample were dispersed in water and stirred overnight. Finally, the final sample was obtained by centrifugation.

Evaluation of electrodynamic activity
The experimental instruments were arranged following the previous literature. A function signal generator was employed as the power source to provide electric output. The electrocatalytic activity was assessed by measuring the degradation of methylene blue. Briefly, different amounts of pPts were added to 2 mL PBS containing 15 μM MB. The function signal generator was connected, and a square-wave electrical signal with certain voltage and frequency was applied. The current was monitored and adjusted with the aid of a multimeter.
After powering on for different period (0, 5, 10, 20, 30 and 40 min), 50μL of the mixture was collected and diluted with PBS to 200 μL, and the UV-vis absorbance spectra of MB were collected.

Catalytic abilities of PtGs
To evaluate the catalytic ability of PtG, the pH changes of PtG in PBS were initially examined, both in the presence or the absence of glucose. Glucose (1 mg/mL) was added to PBS solution containing PtG under stirring. The pH value was monitored by a pH meter.
In order to explore the role of pPt played in the enzymatic activity of GOx, PBS solutions containing 1 mg/mL glucose were treated with PtG or free GOx. At different time intervals, the solutions were collected to measure the glucose and H2O2 concentration. The dissolved oxygen content was monitored by an oxygen meter.
The glucose-consuming ability of PtG was evaluated through DNS method. Briefly, glucose (1 mg/mL) was dissolved into 10 mL phosphate-buffered saline, and then PtGs and GOx (1 μg/mL GOx) were added respectively. At particular time points, 0.5 mL solution was 4 extracted and mixed with 1.5 mL DNS solution. The mixture was heated in boiling water for 5 min, followed by quenching in room temperature water for another 20 min. The UV-vis absorbance spectra was detected and the absorbance of each sample at 600 nm was measured to quantify the concentration of glucose.
The H2O2 concentration was determined through a colorimetric method with horseradish peroxidase (HRP) and TMB. Briefly, glucose was added into PBS solution with PtG or free GOx, respectively. At predetermined time points, 200 μL of the solution was collected and mixed with 300 μL HRP (1 U/mL), 1.5 mL PBS (pH = 5.8) and 300 μL TMB (2 mM) ethanol solution. The peak UV-vis absorbance of ox-TMB was recorded to calculate the variation of H2O2 concentration.
The dissolved oxygen content was measured by a dissolved oxygen meter. The procedure was similar to that of the glucose consumption to compare PtG and free GOx. In addition, to verify the oxygen compensation ability of pPts in presence of GOx and glucose, the dissolved oxygen content was also measured after adding H2O2 to the pPt aqueous solution.

Cell culture
4T1 cancer cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin at 37℃ in a humidified 5% CO2 atmosphere. To create the hypoxic and normoxic culture condition, the oxygen content of incubator was set as 2% and 20%, respectively.

In vitro cytotoxicity assay
For cytotoxicity assay, 4T1 cells were seeded in 96-well plate with a density of 10 4 cells per well for 24 h. Subsequently, the medium was replaced with fresh RMPI-1640, followed by addition of pPt, PtG and free GOx with vaired concentrations. After 24 h incubation, the supernatant was removed, and fresh medium containing 10% CCK-8 was added to each well.
After further incubation for 1h, the absorbance of each well was measured on a microplate reader at 450 nm.

5
To evaluate the starving therapeutic effect of PtG, the cells were seeded, and the culture medium was replaced with fresh RPMI-1640 medium of different glucose content.
Subsequently, cells were co-cultured with gradient concentrations of PtG. After 24 hours, the relative cell viabilities were measured via standard CCK-8 assay.
For EDT treatment, 4T1 cells were seeded in 24-well plates for 24 h. Then, the same concentration of pPt and PtG (60 μg/mL) were added to the media and cultured for 4h. A function signal generator was connected with the cells in series. Using a multimeter, the electric current was set at 5 mA for 10 min. After 24 h, a standard CCK-8 assay was carried out to evaluate the relative cell viabilities.
To further investigate the self-boosting effect induced by pPt for GOx-mediated starvation, the cell viability test was assessed both in normoxic (20% O2) and hypoxic (2% O2) conditions. First, the 4T1 cells were seeded in a 96-well plate and incubated in a normoxic environment overnight. Afterwards, PtG and free GOx were added to the cells. In addition, to simulate the microenvironment, additional PtG with 100 μM H2O2 was added and culture for further 24 h. The cell viability was assessed by CCK-8 assay.

Colony formation assay
4T1 cells were seeded at ~2000 cells per well of a 6-well plate. After incubated for 24 h, the culture medium was replaced by RPMI-1640 medium containing pPt and PtG for 4 h, followed by EDT treatment. The untreated cells were set as the control. Then, the culture medium was removed and replaced with fresh RPMI-1640 medium. After 10~14 days, cells were washed with PBS twice, immobilized with 4% methanol for 10 min, and stained with crystal violet for 15 min at room temperature. Following rinsing with ultra-pure water, each well with formed colonies was photographed.
Live & Dead cell staining assay 4T1 cells were seeded in 6-well plates and cultured overnight, followed by treated with PBS, pPt and PtG, respectively. After 4h, the cells received EDT treatment and incubated 6 overnight. The untreated cells were set as the control. Then, all cells were stained by Calcein-AM and PI solutions and incubated at 37℃ for 30 min. The dye solutions were removed, and each well was washed with PBS for three times. Finally, the images were observed under fluorescence inversion microscope.

Cell apoptosis analysis
The cell apoptosis was assessed by flow cytometry. The cells were seeded in 24-well plates and incubated for 24 h. Then, cells were treated with PBS, pPt and PtG for 4 h, followed by EDT treatment. The untreated cells were set as the control. After 24 h, the cells were digested with trypsin and collected by centrifugation. After repeatedly washing, the cells were costained with Annexin V-FITC and PI for 20 min, followed by examination using a flow cytometry to determine the ratio of apoptotic and necrotic cells.

Synthesis of fluorescein isothiocyanate (FITC) labeled GOx (GOx-FITC)
The GOx-FITC was prepared following the known approach. Typically, 25 μL of FITC in DMSO solution (5 mg/mL) was mixed with 1 mL sodium carbonate-sodium bicarbonate buffer solution (pH 9.4) containing 10 mg/mL GOx, and stirred at 4 ℃ in the dark for 12h.
Subsequently, the solution was dialyzed in deionized water overnight in dark, followed by lyophilization.

Cellular uptake
To investigate the cellular internalization of the nanocomposites, 4T1 cells were seeded in 6-well plates and cultured for 24h, the medium was replaced with fresh medium containing pPt or PtGFITC. After four hours, the culture medium was removed, and the cells were washed with PBS before examination using fluorescence inversion microscope.

GOx release profile analysis
The GOx concentration in PtG and the release profile were determined with Bradford assay kit. Typically, 20 μL GOx containing supernatant was collected and mixed with 200 μL 7 Bradford solution. The absorbance of the solution was measured on a microplate reader at 560 nm.

Intracellular ROS detection
4T1 cells were seeded in 6-well plates and incubated for 24h, followed by the addition of pPt and PtG. After 4h, cells were stained with 20 μM DCFH-DA, and co-cultured for 30 min.
The cells were then treated with electric field at 5 mA for 10 min and incubated at 37℃ for 1h.
Cells incubated with PBS but without electric field were used as the control. After washing with PBS, the cells were fixed by 4% methanol for 10 min. The cell nuclei were stained with DAPI for 10 min at room temperature, followed by three-time washing with PBS. Finally, DCFH fluorescence was observed with fluorescence inversion microscope.

Intracellular pH and oxygen evaluation
4T1 cells were seeded in 6-well plates and incubated overnight. For intracellular pH detection, the cells were treated with PBS, pPt and PtG, followed by EDT treatment. The cells treated without samples and electric field were set as control. After 6~10 hours, the cells were stained with 5 μM BCECF-AM for 1 h, followed by observation using fluorescence inversion microscope.
For intracellular oxygen detection, the cells were seeded in 6-well plates. After incubation overnight, the culture medium was replaced by fresh RPMI-1640 medium containing 5 μM [Ru(dpp)3]Cl2. After 4h, the medium was removed, and the cells were rinsed with PBS for three times. Then, PBS, pPt and PtG were added and co-incubated under a normoxic condition for 4 h, followed by EDT treatment. After 6~10 hours, the cells were washed with PBS before fluorescence imaging. In addition, to verify the intracellular oxygen generation ability of pPt, the cells were incubated with pPt under hypoxic condition, while leaving the rest of the procedures remained unchanged.

In vivo antitumor efficacy
8 All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Zhejiang University and approved by the Animal Ethics Committee of Zhejiang University. To obtain the tumor-bearing mice, four-weeks-old female BALB/c mice were subcutaneously inoculated with 50 μL PBS suspension of 4T1 cells. When the tumor reached ~500 mm 3 in average size (14 days after inoculation), the mice were randomly divided into seven groups and treated respectively with: (1) PBS, (2) pPt, (3) PBS plus electric field, (4) free GOx, (5) PtG, (6) pPt plus electric field, (7) PtG plus electric field. The aqueous solutions (50 μL) of pPt, GOx and PtG were injected intratumorally, and the corresponding Pt concentration is 2 mg/mL. After 10 min, the tumor sites (Group 3, 6, 7) were exposed to 5 mA square wave electric field for 10 min. Since the day that the mice For histology analysis, tumors from each group were harvested 24 hours after the treatment.
Then, the tumor tissues were sliced and fixed in formalin and sectioned. Tissue slices were stained with H&E, Ki-67, and TUNEL before the examination using fluorescence microscopy.