Harnessing Ferroptosis and Photothermal Effects: A New Paradigm in Aggressive Lymphoma Therapy

Background: Methotrexate (MTX) is a cornerstone drug in chemotherapy regimens for aggressive lymphomas. Recent studies have indicated that MTX promotes ferroptosis by inhibiting dihydrofolate reductase, thereby reducing the conversion of dihydrofolate to tetrahydrofolate (BH4) and disrupting the GCH1/BH4 pathway. MTX also inhibits various reductases, reduing reductive substances, such as glutathione (GSH) and making ferroptosis irreversible. However, application of MTX faces several challenges, including poor solubility, insufficient efficacy and sever toxicities. Moreover, in highly aggressive lymphomas, tumor cells aggregate to form a localized mass., creating an obstacle for drug penetration. Previously, we developed MTX-loaded micelles (MLMs) using copolymer monomethyl polyethylene glycol-polycaprolactone. MLMs enhanced therapeutic efficacy while mitigated adverse actions. However, MLMs alone was insufficient to treat Burkitt lymphoma, necessitating combination therapies. Photothermal therapy (PTT) is an innovative therapy utilizing conversion of light into heat through photothermal effect. PTT induces apoptosis and necrosis in tumor cells while minimizing damage to normal tissues due to their differential thermal tolerance. Prussian blue (PB), (Fe₃[Fe(CN)₆]₂), exhibits strong absorption in the near-infrared region, demonstrating excellent photothermal conversion capabilities. We aim to develop a multifunctional system combining PB nanoparticles (PBNPs) with MLMs. The MLMs reduce BH4 and other reductive substances, together with Fe²⁺ from PBNPs, induce ferroptosis. PTT induces apoptosis and necrosis. And the elevation of local temperature facilitates targeted release of MTX from MLMs at the tumor site, thus reducing cytotoxicity to normal tissues.

Methods: MLMs were prepared as previouly reported. PBNPs were synthesizedvia a one-pot method, followed by surface modification with hyaluronic acid. The NPs were characterized for materialological parameters. The experimental subjects received following treatments: saline, MLMs, PBNPs, PBNPs+laser, MLMs+PBNPs and MLMs+PBNPs+laser. In vitro studies were conducted using Raji cells, where the therapeutic efficacy was assessed through CCK-8 and flow cytometry. We established Burkitt lymphoma models with standard/high tumor burden to investigate the therapeutic efficacy and toxicity. We monitored tumor volume and body weight, collected blood samples for biochemical and blood routine analyses and harvest the major organs and tumors for pathological examination (HE, Ki-67 and TUNEL staining). To elucidate the possible mechanisms, we observed the treated cells by TEM and conducted transcriptome sequencing to analyze gene expression alterations. ROS, lipid peroxides (LPO), BH4, and GSSG/GSH ration were measured using respective assy kits. FerroOrange staining was conducted to evaluate intracellular iron.

Results: We successfully synthesized two types of NPs. PBNPs had a spherical morphology, a moderate particle size (80-100 nm), and a uniform dispersion. Photothermal conversion experiments demonstrated that under 808 nm laser irradiation, the temperature of the PBNPs significantly increased, reaching 50-65°C. Both In vitro and in vivo experiments showed that PBNPs+MLMs achieved efficient inhibition of lymphoma, which was enhanced when PBNPs were irradiated with laser. And the combination treatment resulted in tumor eradication in 2/6 mice in standard group. Toxicity evaluation indicated that PBNPs alone or PBNPs+MLMs +laser exhibited minimal systemic toxicity. Transcriptome sequencing revealed that upregulated genes were primarily enriched in pathways related to oxidative stress, autophagy, and ferroptosis, whereas downregulated genes were enriched in pathways associated with cell proliferation and metabolism. TEM found the treated cells exhibited mitochondrial shrinkage and reduction in mitochondrial cristae. FerroOrange staining indicated increased intracellular Fe²⁺. Decreased levels of BH4 and GSSG/GSH, along with increased levels of ROS and LPO were detected, further supporting ferroptosis and oxidative stress.

Conclusion:We developed a multifunctional paradigm integrating PTT and chemotherapy to enhance therapeutic efficacy while reducing toxicities, which represents a novel therapeutic strategy with promising translational potential.