A Tailored Multifunctional Anticancer Nanodelivery System for Ruthenium‐Based Photosensitizers: Tumor Microenvironment Adaption and Remodeling

Abstract Ruthenium complexes are promising photosensitizers (PSs), but their clinical applications have many limitations. Here, a multifunctional nano‐platform PDA‐Pt‐CD@RuFc formed by platinum‐decorated and cyclodextrin (CD)‐modified polydopamine (PDA) nanoparticles (NPs) loaded with a ferrocene‐appended ruthenium complex (RuFc) is reported. The NPs can successfully deliver RuFc to the tumor sites. The release of RuFc from the NPs can be triggered by low pH, photothermal heating, and H2O2. The combined photodynamic and photothermal therapy (PDT‐PTT) mediated by PDA‐Pt‐CD@RuFc NPs can overcome the hypoxic environment of tumors from several aspects. First, the platinum NPs can catalyze H2O2 to produce O2. Second, vasodilation caused by photothermal heating can sustain the oxygen supplement. Third, PDT exerted by RuFc can also occur through the non‐oxygen‐dependent Fenton reaction. Due to the presence of PDA, platinum NPs, and RuFc, the nanosystem can be used in multimodal imaging including photothermal, photoacoustic, and computed tomography imaging. The NPs can be excited by the near‐infrared two‐photon light source. Moreover, the combined treatment can improve the tumor microenvironments to obtain an optimized combined therapeutic effect. In summary, this study presents a tumor‐microenvironment‐adaptive strategy to optimize the potential of ruthenium complexes as PSs from multiple aspects.

In order to quantitatively detect the cytotoxicity of TPPDT-PTT mediated by PDA-Pt-CD@RuFc, the MCTSs were irradiated with one or two lasers (808 nm: 1 W cm −2 , 10 min; 810 nm (two-photon laser): 100 mW, 20 min, 80 MHz, 100 fs) and incubated for another 24 h, the adenosine triphosphate (ATP) concentration was measured using the Cell TiterGlo 3D Cell Viability kit (Promega) as we previously described. [2] RT-qPCR Measurement of the Expression of HIF-1α and MDR1 Genes: RT-qPCR was performed as we previously described [3] using a Roche LightCycler 480 Detection System (Roche, USA) and SYBR Green I Master (Roche, USA). 4T1 cells were incubated in hypoxia (1%) or normoxia (21%) atmosphere. Briefly, 4T1 cells were treated with PDA-Pt-CD@RuFc (25 or 50 μg mL −1 ) for 6 h. Total RNA is isolated using the Trizol reagent (Life technologies, USA). The synthesis of cDNA was done using the iScript cDNA synthesis kit (BIO-RAD, USA). GAPDH was used as the reference gene. The primer sequences are listed as below. Analysis of MMP: MMP was detected by JC-1 staining as we previously reported. [3] Briefly, 4T1 cells were treated with PDA-Pt-CD@RuFc (50 μg mL −1 ) for 24 h. The cells were then irradiated with a 450 nm laser (17 mW cm -2 , 1 min) and an 808 nm laser (1 W cm -2 , 10 min). Then the cells were stained with JC-1 and analyzed by confocal microscopy.
The cells were stained and analyzed immediately by confocal microscopy and flow cytometry.
after intravenous injection. Before imaging, the mice were injected with anesthetics intraperitoneally (4% chloral hydrate, 200 μL) for anesthetization. The CT images were captured by an Inveon Multimodality PET/CT-system for small animals (Preclinical Solutions, Siemens Healthcare Molecular Imaging, USA).
In Vivo Antitumor Activity Evaluation: BALB/c mice carrying 4T1 tumors were randomly divided into 9 groups: (i) saline control group; injection group. When tumor volume reaches 100-150 mm 3 , the mice were treated with PDA-Pt-CD@RuFc NPs (200 μL, 1 mg mL −1 ). The groups with i.t. injection were irradiated immediately after administration. The groups with i.v. injection were irradiated 4 h after administration. For the combined treatment groups, the treatments were carried out in the order of PDT followed by PTT. Irradiation conditions: 450 nm, 12 W cm -2 , 5 min; 808 nm, 1 W cm -2 , 3 min. The weight and tumor size of mice were recorded every two days.
H&E Staining: All groups of mice with in vivo antitumor activity evaluation were dissected. The tumor tissues and the main organs (heart, liver, spleen, lung and kidney) were fixed with paraffin and examined using H&E staining as previously described. [4] The pictures were taken by a digital microscope (Leica QWin).  At 12 h post-injection, mice in group (2) and (4)  Immunofluorescence Staining: The specimens were preparation as described above.

Enzyme-Linked
After incubation with the primary antibody overnight at 4 °C, the specimens were incubated with the fluorochrome-conjugated secondary antibody for 1 h at room temperature. The nuclei were stained with DAPI, and the images were observed and collected under a Nikon inverted fluorescence microscope.
Statistical analysis: All biological experiments were performed at least twice with triplicates in each experiment. Representative results were depicted in this report and data were presented as means ± standard deviations (SD).

Supporting Scheme, Figures and Tables
Scheme S1. The synthetic route of RuFc.                             [5] as the reference.  [6] as the reference.