Synergistic Anticancer Therapy by Ovalbumin Encapsulation‐Enabled Tandem Reactive Oxygen Species Generation

Abstract The anticancer efficacy of photodynamic therapy (PDT) is limited due to the hypoxic features of solid tumors. We report synergistic PDT/chemotherapy with integrated tandem Fenton reactions mediated by ovalbumin encapsulation for improved in vivo anticancer therapy via an enhanced reactive oxygen species (ROS) generation mechanism. O2 .− produced by the PDT is converted to H2O2 by superoxide dismutase, followed by the transformation of H2O2 to the highly toxic .OH via Fenton reactions by Fe2+ originating from the dissolution of co‐loaded Fe3O4 nanoparticles. The PDT process further facilitates the endosomal/lysosomal escape of the active agents and enhances their intracellular delivery to the nucleus—even for drug‐resistant cells. Cisplatin generates O2 .− in the presence of nicotinamide adenine dinucleotide phosphate oxidase and thereby improves the treatment efficiency by serving as an additional O2 .− source for production of .OH radicals. Improved anticancer efficiency is achieved under both hypoxic and normoxic conditions.


Animal Experimental Ethical Inspection Form
28% ammonia solution was added dropwise under mechanical stirring at 700 rpm and oleic acid (4.00 g, 14 mmol) was subsequently added. The reaction mixture was heated to 70 °C for 1 h under stirring at 500 rpm and afterwards at 110 °C for 2 h. After precipitation of Fe3O4 NPs capped with oleic acid, the precipitate was rinsed five times with deionized water and dried in an oven at 65 °C overnight. The Fe3O4 NPs were transferred into water solutions for encapsulation in nanocapsules according to our previous work. [4]

Synthesis of Ovalbumin Nanocapsules
First, 50 mg ovalbumin and 7.2 mg NaCl were dissolved in 500 µL water at a stirring rate of 200 rpm. For the encapsulation of therapeutic agents, calculated amounts of Fe3O4 NPs, cisplatin, and/or NBS were added in the aqueous phase at this step. Next, 35.8 mg surfactant P((E/B)-b-EO) was dissolved in 7.5 g cyclohexane. The mixture was poured to the aqueous phase under stirring at 500 rpm. The pre-emulsion was homogenized by ultrasonication for 180 s (30 s ultrasonication, 10 s pause) with ice cooling at 70% amplitude using a Branson 450W sonifier and a 1/2' tip. Separately, 10.7 mg P((E/B)-b-EO) was dissolved in 5 g cyclohexane and 2 mg TDI was added to the solution. This mixture was added dropwise to the obtained miniemulsion for 5 min and the reaction was allowed to proceed for 24 h at 25 °C. Afterwards, excessive S4 surfactant was removed from the dispersion by repetitive centrifugation and replacement of the supernatant with fresh cyclohexane.
Next, the obtained nanocapsules (in cyclohexane) were transferred to an aqueous medium. 600 µL dispersion from cyclohexane was added dropwise to 5 mL 0.1 wt% SDS aqueous solution placed in an ultrasound bath. Subsequently, the sample was stirred with an open cap overnight to evaporate the cyclohexane. Excess SDS was removed via four centrifugation steps by replacing the supernatant with water.
For loading the nanocapsules with therapeutic agents, 250 µL Fe3O4 NPs dispersion (30 mg mL -1 in water) and 250 µL DMSO solution containing 20 mg cisplatin or/and 20 mg NBS were used and the amount of water was reduced accordingly to maintain 500 µL as the total volume for the water phase.

Biodegradability of Nanocapsules and Release of Payloads
Degradation of ovalbumin nanocapsules was studied by incubating the nanocapsule dispersions with serine protease trypsin. Specifically, 1 mL of dispersions with a solid content of 2wt% were incubated with 20 mg trypsin (gibco) at 37 °C. The mixture solutions were placed in dialysis tubes with molecular weight cut-off of 14 kDa. The dialysis tubes were then immersed in 20 mL milli-Q water and incubated at 37 °C in a shaking culture incubator. During the release experiment, 1 mL of dialysis medium was taken at given intervals and equal volume of water was added to keep the volume constant. Nanocapsules was treated in the same way in the absence of trypsin as control groups. The release of NBS in dialysis medium was quantified by measuring its absorbance at λ = 660 nm by using an Infinite M1000 plate reader (Tecan, Austria). A calibration curve of absorption against concentration of NBS is provided in the Supporting Information.
The details for hypoxic experiments: fresh medium was bubbled with nitrogen for 30 min to obtain hypoxic medium. Then all the cells for hypoxic experiments will incubate in this medium. Before the cells are used for experiments, the incubators are kept in incubator chamber (MIC-101, Billups-Rothenberg) at 37 °C in a humidified, 2% O2, 5% CO2, and 93% N2 atmosphere for more than 8 h. Meanwhile, an oxygen detector (Nuvair, O2 Qucikstick) was used to real time monitor the oxygen content ( 2% O2) in the chamber.

Cell Imaging Experiments
MCF-7 cells were planted onto 35 mm confocal dishes and incubated at 37 °C under 5% CO2 for 24 h. After obtained 80% cell density, the cells were washed by PBS for S6 three times, then nanocapsule dispersion (2 μg mL -1 ) was added. The images were collected at specific times. The red fluorescence was excited at 635 nm, and collected at 650-700 nm. The fluorescence of Hoechst 33342 was excited at 405 nm and collected on 420-480 nm. h. After that, the cells were collected into tubes. Nuclear extracts were obtained using a Nuclear/Cytosol Fractionation Kit (BioVision Research). The nuclear extraction is a standard method that is widely used. [5][6][7] Finally, the Pt contents were determined by ICP-MS (PerkinElmer NexION 2000 ICP-MS).

Cytotoxicity Test
Cell viability was assessed on formazan crystals by MTT For viability test, 10 μL MTT (5 mg mL -1 ) prepared in PBS was added to each well, and the plate was incubated at 37 °C for 4 h in a 5% CO2 humidified incubator. The medium was then carefully removed and the purple crystals were dissolved in 150 μL of DMSO. The absorbance at 570 nm was measured on a microplate reader (Thermo Fisher Scientific). Cell viability = (ODexperimental group -ODblank control) / (ODnegative control -ODblank control) × 100%, wherein the negative and blank controls were the unmedicated group and the blank medium group, respectively.  FePtNBS@OVA+DFOA+Light.

Intracellular Hydroxyl Radical Detection
The detection for hydroxyl radical was performed by using the similar procedure in Intracellular superoxide anion free radical detection. The concentration for HPF is 10 μM. The fluorescence of HPF was excited at 488 nm and collected on 500-550 nm. nm, and the emission wavelength of the red channel was 600-700 nm.

In Vivo Fluorescence Imaging
All animal operations were preformed according to the institutional animal use and care regulations approved by Ethics Committee, Dalian University of Technology In addition, mice treated with the same administration were dissected after 6 h, and tumor tissues and main organs (i.e., heart, liver, spleen, lung, and kidney) were imaged ex vivo. During the imaging process, the excitation wavelength was 635 nm and the collected emission wavelength was 680-720 nm.