Photodynamic‐Chemodynamic Cascade Reactions for Efficient Drug Delivery and Enhanced Combination Therapy

Abstract Nanomedicines with photodynamic therapy and reactive oxygen species (ROS)‐triggered drug release capabilities are promising for cancer therapy. However, most of the nanomedicines based on ROS‐responsive nanocarriers still suffer from serious ROS consumption during the triggered drug release process. Herein, a photodynamic‐chemodynamic cascade strategy for the design of drug delivery nanosystem is proposed. A doxorubicin hydrochloride‐loaded ROS‐responsive polymersome (DOX‐RPS) is prepared via the self‐assembly of amphiphilic poly(ethylene glycol)‐poly(linoleic acid) and poly(ethylene glycol)‐(2‐(1‐hexyloxyethyl)‐2‐devinyl pyropheophorbide‐α)‐iron chelate (PEG‐HPPH‐Fe). The RPS can effectively deliver a drug to tumor site through passive targeting effect. Upon laser irradiation, the photosensitizer HPPH can efficiently generate ROS, which further causes in situ oxidation of linoleic acid chain and subsequent RPS structural destruction, permitting triggered drug release. Intriguingly, catalyzed by HPPH‐Fe, ROS will be regenerated from linoleic acid peroxide through a chemodynamic process. Therefore, ROS‐triggered drug release can be achieved without ROS over‐consumption. The in vitro and in vivo results confirmed ROS generation, triggered drug release behavior, and potent antitumor effect of the DOX‐RPS. This photodynamic‐chemodynamic cascade strategy provides a promising approach for enhanced combination therapy.

LA solution for activation. Subsequently, PEG-PAMA (40 mg) and TEA (30 μL) were dissolved in THF (4 mL) and added into the mixture. The reaction was carried out at room temperature for 24 h. The product was dialyzed in DMSO and pure water and then lyophilized to generate PEG-PLA. The PEG-PSA was synthesized under the same experimental conditions except that the LA was replaced with SA.

Synthesis of PEG-HPPH-Fe
The PEG-HPPH was first synthesized ( Figure S2). HPPH (15 mg), EDC (10 mg), DMAP (0.5 mg) were dissloved in dichloromethane (10 mL). PEG-OH (90 mg) was added into the mixture for reaction overnight at room temperature. Then the dichloromethane was removed by reduced pressure distillation. The product was dialyzed in pure water and then lyophilized to generate PEG-HPPH. The PEG-HPPH-Fe was synthesized according to a previous report. [1] PEG-HPPH was incubated with 10 fold excess free iron(II) acetate in methanol for 1 hour at room temperature under argon. Free metal was removed by dialysis. The PEG-HPPH-Fe was then lyophilized. 1 H NMR spectra were recorded on a JEOLGX 400 D spectrometer (400 MHz) with CDCl 3 as the solvent and tetramethyl silane as an internal standard. The degree of polymerization of PEG-PBOCAMA determined by NMR is ~24.

Preparation and characterizations of DOX-RPS and DOX-NRPS
The DOX-RPS was prepared as follows: PEG-PLA and PEG-HPPH-Fe were dissolved in 4 mL of dichloromethane at room temperature to obtain the organic phase. DOX hydrochloride was dissolved in 0.5 mL of pure water and mixed with the organic phase under sonication.
The obtained emulsion was added to 8 mL of pure water and sonicated for another 120 s. The organic solvents were evaporated on a rotary evaporator to form DOX-RPS suspension. The DOX-NRPS was prepared under the same experimental conditions except that the PEG-PLA was replaced with PEG-PSA. RPS and NRPS without drug loading were also prepared. The morphology of the RPS and NRPS was observed by Tecnai TF30 transmission electron microscope (TEM) (FEI, Hillsboro, OR). The effective particle diameters and zeta potential of the samples were determined by a SZ-100 nano particle analyzer (HORIBA Scientific, USA) at room temperature. UV-vis absorption spectra of the samples were measured by Genesys 10S UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA).

ROS generation
In order to study the ROS generation through photodynamic and chemodynamic reactions, polymersomes without Fe (denoted as RPS1 and NRPS1) were prepared under the abovementioned experimental conditions except that the PEG-HPPH-Fe was replaced with PEG-HPPH. The singlet oxygen generation of RPS1 under laser irradiation was evaluated by a fluorescence singlet oxygen sensor green (SOSG) method. Briefly, SOSG was mixed with RPS1 suspension, the final SOSG oncentration was adjusted to 2 μM. The fluorescence spectra of the mixture solution before and after laser irradiation (671 nm) were measured under excitation at 498 nm. The generation of ROS through Fenton-like reaction was determined by a TMB assay. The RPS1 suspensions without or with 671 nm laser preirradiation (100 mW cm -2 , 5 min) were mixed with TMB solution. Absorption spectra were measured in the presence or absence of catalytic Fe 2+ ions.

In vitro drug release
The in vitro DOX release behaviors of the samples in different conditions were evaluated at 37 °C (n = 3). The samples without or with 671 nm laser irradiation were dispered in media (2 mL) and added to dialysis bags (MWCO: 3500 Da) and placed in environmental media (20 mL). At appropriate time points, 2 mL of the medium was taken out and replaced with the same amount of fresh medium. The amount of the released DOX was measured by UV-Vis spectrophotometer at the wavelength of 480 nm.

In vitro cell experiments
The U87MG, A549 and 293T cell lines were purchased from American type culture collection (ATCC). For cytotoxicity study, cells were seeded into 96-well plates at a density of 3 × 10 3 cells per well (n = 5) and incubated with different concentrations of RPS for 48 h.
The relative cell viabilities were measured by MTT assay.
To assess the intracellular ROS generation, U87MG cells were seeded into 8-well plates and incubated with DCFH-DA (15 μM) and different samples (RPS1, NRPS1, laser pre-irradiated RPS1 and laser pre-irradiated NRPS1) for 2 h. Then the intracellular ROS level was determined by flow cytometry (FCM) analyse.
To assess the cellular uptake of samples, U87MG cells were seeded into 8-well plates.

In vivo PET imaging
Deferoxamine (DFO) conjugated polymer was synthesized by reaction between SCN-DFO and excessive amino groups of PEG-PAMA. Then the DFO-PEG-PLA was synthesized by the above-mentioned method. DFO-modified DOX-RPS was prepared to chelate with the radionuclide zirconium-89 ( 89 Zr). [2] The 89 Zr-DOX-RPS solution (100 μL, 200 μCi) was intravenously injected into U87MG tumor-bearing mice. An Inveon small-animal PET scanner (Siemens, Erlangen, Germany) was used for the scanning at indicated time points after injection. At 72 h post-injection, the mice were sacrificed and the major organs were collected and assayed for radioactivity using a gamma counter. The percent of injected dose/gram of tissue (%ID/g) was then calculated (n = 3).