A smart nanoplatform for enhanced photo-ferrotherapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Emerging therapies, such as ferroptosis mediated cancer therapy and phototherapy, offer new opportunities for HCC treatment. The combination of multiple treatments is often more effective than monotherapy, but many of the current treatments are prone to serious side effects, resulting in a serious decline in patients’ quality of life. Therefore, the combination therapy of tumor in situ controllable activation will improve the efficacy and reduce side effects for precise treatment of tumor. Herein, we synthesized a GSH-activatable nanomedicine to synergize photothermal therapy (PTT) and ferrotherapy. We utilized a near-infrared dye SQ890 as both an iron-chelating and a photothermal converter agent, which was encapsulated with a GSH-sensitive polymer (PLGA-SS-mPEG), to attain the biocompatible SQ890@Fe nanoparticles (NPs). In the tumor microenvironment (TME), SQ890@Fe NPs showed a GSH-activated photothermal effect that could increase the Fenton reaction rate. Meanwhile, the depletion of GSH could further increase ferroptosis effect. In turn, the increasing radical generated by ferrotherapy could impair the formation of heat shock proteins (HSPs) which could amplify PTT effects by limiting the self-protection mechanism. Overall, the intelligent nanomedicine SQ890@Fe NPs combines ferrotherapy and PTT to enhance the efficacy and safety of cancer treatment through the mutual promotion of the two treatment mechanisms, providing a new dimension for tumor combination therapy.


Preparation of SQ890@Fe NPs
The SQ890@Fe NPs was prepared through a nanoprecipitation method by the self-assembling of SQ890-Fe 3+ and the GSH sensitive polymer PLGA-SS-mPEG. SQ890-Fe (3 mg) and PLGA-SS-mPEG (100 mg) was fully dissolved in CH2Cl2. After the solvent was removed by thin-film dispersion method, ultrapure water (3 mL) was slowly added and ultrasonic dispersion was performed. SQ890@Fe NPs was obtained after dialysis purification and stored at 4 ℃ for further use. All chemicals were purchased from Alfa Aesar, Thermo Fischer Scientific, or Sigma-Aldrich and used without further purification. Ultrapure water was prepared using a Milli-Q Plus System.

Photothermal measurement
Various concentrations of SQ890@Fe NPs solution (1 mL), with GSH (10 mM), were illuminated with 808 nm light at various power densities to evaluate the photothermal properties, and the corresponding thermographic images were recorded using an IR camera.
For the photothermal conversion efficiency (η) evaluating, 1 mL SQ890@Fe NPs and 1 mL water were illuminated with 808 light for 500 s until reaching the maximum stable temperature and then spontaneous cooling to room temperature. The temperature variations were recorded and calculated.
For photostability tests, 1 mL SQ890@Fe NPs was irradiated for 5 min and cooled for 5 min, repeated these cycles 5 times, and recorded the temperature variation.

Reagents and Cell line and Measurement of cellular uptake in vitro
Rho B was assembled into NPs following the above mentioned methods. HepG2 cells (2.5 × 10 5 ) were seeded in 6-well plates for 12 h. After being cultivated with DMEM medium containing Rho B NPs (10 μM) for 2 h, 4 h, and 6h, the cells were washed with PBS and fixed with 4 % (W/V) paraformaldehyde . Then the cells were washed PBS and stained with Hoechest 33342 (2 μg/mL, blue) for 5 min. And the cellular uptake was visually determined using CLSM.

Cell viability assay in vitro
HepG2 cells (5 × 10 3 ) were seeded in 96-well plates and cultured 12 h for attachment. Thereafter, cells were treated with various concentrations of SQ890 NPs and SQ890@Fe NPs and illuminated with/without 808 nm light (1 W/cm 2 ) for 5 min at 4 h post-administration. The cells were then maintained for another 20 h. The cell viability was measured by MTT assay. All cell lines were purchased from American Type Lifestyle Collection (ATCC).

In Vitro Fluorescent imaging
HepG2 cells were incubated in a DMEM medium containing SQ890 NPs (20.0 μM) and SQ890@Fe NPs (20.0 μM) for 4h, the cells were then illuminatied with/without 808 nm light (1 W/cm 2 ) for 5 min. Thereafter, Calcein AM/PI buffer was added after washing the cells and cultivated for 30 min in dark. Then using an inverted fluorescent microscope to record the cells fluorescent images immediately.

Measurement of intracellular LPO level
Intracellular Lipid Peroxidation. HepG-2 cells were seeded into 6-well plate (1×106 cells/ well) and treated with PBS, SQ890@Fe NPs (25 μM), and SQ890@Fe NPs (60 μM) cultivated for 24 h. Next, after digested, resuspended, and fragmented, they were then centrifuged at 10000 rpm for 10 min and the supernatants collected. A commercial assay kit (Lipid peroxidation MDA assay kit) was used to evaluate intracellular LPO levels by UV-vis spectroscopy.
Animal models. The animal experiments were performed following a protocol approved by the Animal Care and Use Committee of Guangdong Medical University. The 5-6 weeks female BALB/c nude mice and Kunming mice were supplied by Guangdong Sijiajingda Biotechnology Co., Ltd. 5-6-week-old female BALB/c nude mice were used to establish the PDX HCC . Blocks of tumor tissues (about 3×3×3 mm 3 / block) were subcutaneously inoculated in the right hind leg of BALB/c nude mice.

In vivo PTT
The tumor-bearing mice were randomly divided into 5 groups (n=5). When the tumor volume reached about 80-100 mm 3 , mice were administrated with PBS, SQ890 NPs (5 mg/kg) and SQ890@Fe NPs (5 mg/kg) and illuminatied with/without 808nm light (1 W/cm 2 ) for 5 min at 8 h post-administration. During irradiating, thermographic pictures of mice were collected using an IR camera. Recording the tumor size and bodyweight every two days during the 10day treatment period. At the termination of treatment, mice from each group were sacrificed to gathered the tumors and major organs for TUNEL and H&E staining, and blood samples for the hematology analysis.

Blood biochemistry analysis
Blood was collected from the heart of tumor bearing mouse models in different treatment groups, and centrifuged for 15 min at 1,500g, 4 °C. Serum was isolated and collected in the fresh tubes. BUN, CREA, ALT, and AST in the serum were quantified to indicate the renal and hepatic function

Statistical analysis
Experimental data were analyzed using GraphPad Prism 9 or SPSS software. All data are expressed as the mean ± SEM. Statistical differences between groups were analyzed using independent two-sample t-tests. Differences between groups were considered significant at P < 0.05.   Figure S5. H&E staining of mice organs. H&E-stained slices obtained from the heart, liver, spleen, lung, and kidney of mice after various treatments. Scale bar: 25 μm.