A multifunctional nanoplatform for ferroptosis inducing/chemo/photothermal therapy  enhances therapeutic effect and prevents adverse gastrointestinal reaction of methotrexate

Abstract

Abstract Backgrounds: Methotrexate (MTX) is pivotal in treating aggressive lymphomas. Recent studies show MTX not only interferes with DNA synthesis but also perturbs the redox balance in cancer cells. MTX disrupts the GCH1/tetrahydrobiopterin (BH4) pathway by inhibiting dihydrofolate reductase to promote ferroptosis. It inhibits various reductases, reducing levels of reductive substances like glutathione (GSH) and promoting ferroptosis. However, clinical application of MTX is hindered by several factors, including poor drug permeation (particularly in lymphomas with bulky masses), limited efficacy, and significant toxicities. Among numerous adverse effects associated with high-dose MTX, gastrointestinal toxicity poses a particularly challenging issue. It may cause endogenous intra-abdominal infections due to the disruption of the intestinal barrier, which can be life-threatening for lymphoma patients with myelosuppression after chemotherapy. Previously, we developed a nanomicelle formulation using monomethyl polyethylene glycol-polycaprolactone (MPEG-PCL), which showed advantages over traditional MTX injections. Based on this research, we propose a novel multimodal therapeutic strategy combining MTX-loaded micelles (MLMs) and Prussian blue nanoparticles (PBNPs) for a multimodal therapy. Prussian blue, chemically known as ferric ferrocyanide, is an FDA approved antidote for thallium and cesium. PBNPs exhibit excellent near-infrared (NIR) absorption and photothermal conversion capabilities, also provide iron ions catalyze the Fenton reaction, generating reactive oxygen species (ROS) that oxidize cell membrane lipids. This combination leverages chemotherapy, photothermal therapy (PTT), and ferroptosis to achieve efficient tumor cell killing and enhance the release of micelle-encapsulated MTX at elevated temperatures within the tumor microenvironment. Methods: MLMs were prepared as previously reported, and PBNPs were synthesized and surface-modified with hyaluronic acid for stability and targeting. Nanoparticles were characterized for physicochemical and photothermal properties. In vitro efficacy was assessed using Raji cells treated with PBS, MLMs, PBNPs (with/without 808 nm laser irradiation), and MLMs combined with PBNPs (with/without laser) through CCK-8 assay and flow cytometry. Mechanisms were explored via TEM, transcriptome sequencing and measurements of key metabolites (ROS, lipid peroxidation, malondialdehyde, BH4, GSH) and iron concentration . In vivo studies were conducted using Burkitt lymphoma mouse models with standard (150 mm³) and high (800 mm³) tumor burdens. Mice received treatments consistent with in vitro studies, with drugs administered intravenously every other day for six sessions. Local tumor irradiation was performed using an 808 nm laser at a power density of 1.0 W/cm2 for 5 min. Tumor volume and body weight were monitored. Fecal samples, tumor and organ tissues were analyzed post-treatment to assess status of tumor suppression and gastrointestinal toxicity using histological and molecular techniques. Results: PBNPs exhibited strong NIR absorption and efficient photothermal conversion, significantly increasing temperatures under laser irradiation to effectively kill lymphoma cells. Combined MLMs and PBNPs (with/without laser) enhanced antitumor efficacy both in vitro and in vivo. Notably, in the standard group, the combination treatment of MLMs and PBNPs (with laser) achieved tumor eradication in 2 out of 6 mice without obvious toxicity. As for gastrointestinal toxicity, MLMS induced severe diarrhea, significant decrease of tight junction proteins (ZO-1, Occludin, and Claudin-1) as well as the diversity and abundance of the intestinal microbiota. This adverse effect was mitigated by PBNPs (with laser). RNA sequencing revealed that upregulated genes were primarily enriched in pathways related to oxidative stress, autophagy, and ferroptosis, whereas downregulated genes in pathways associated with cell proliferation and metabolism.TEM showed a characteristic morphology of mitochondrial shrinkage, membrane rupture, and a reduction in mitochondrial cristae. Elevated level of ROS, LPO and MDA further confirmed ferroptosis. Conclusion: A novel multifunctional nanoplatform combining ferroptosis induction, chemotherapy, and photothermal therapy demonstrated superior therapeutic efficacy and reduced toxicity in treating aggressive lymphoma. This strategy presents a promising avenue for clinical translation.