A pH-responsive prodrug biomimetic nanoplatform based on cis-aconitic anhydride-modified methotrexate for targeting therapy of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral cavity. Conventional chemotherapy often shows limited effectiveness due to poor bioavailability, lack of specificity, and harmful effects on healthy tissues. Nanotechnology, however, offers promising strategies for cancer therapy. In particular, carrier-free nanoparticles (NPs) that incorporate anticancer drugs have emerged as a novel approach in nanomedicine, though it in vivo stability and drug release kinetics of these carriers require optimization. In this study, we developed a methotrexate (MTX)-based, pH-responsive prodrug biomimetic nanoplatform (T12-RBCM@CM NPs) specifically designed for targeted OSCC treatment. MTX was first structurally modified with cis-aconitinic anhydride (CAA) to form pH-responsive drug self-delivery systems (CAA-MTX (CM) NPs). Next, red-blood-cell membrane (RBCM) was modified with T12 peptide (a peptide targeting the transferrin receptor (TFR) on the tumor cells' surface), which was coated onto CM NPs to yield the T12-RBCM@CM NPs platform. This system leverages the specific interaction between the T12 peptide and the overexpressed TFR on tumor cells for active targeted delivery. Moreover, it extends circulation time in vivo through the CD47 protein “don't eat me” signal on the RBCM surface. In vitro studies showed that T12-RBCM@CM NPs enhanced tumor cell uptake efficiency, induced apoptosis, and suppressed cell proliferation. In vivo experiments revealed that T12-RBCM@CM NPs exhibited prolonged circulation and targeted tumor delivery, effectively inhibiting tumor growth with favorable biological safety. In conclusion, T12-RBCM@CM NPs constitute an efficient and targeted therapeutic platform, providing a novel approach for OSCC treatment. • pH-Responsive prodrug design: The cis-aconitinic anhydride (CAA)-modified methotrexate (MTX) (CM NPs) enables rapid responsive drug release in the acidic microenvironment of tumors (pH ~6.8) and intracellular lysosomes (pH 5.0~6.5), ensuring targeted therapy with reduced impact on healthy tissues. • Active targeting drug delivery system: The incorporation of the T12 peptide facilitates specific binding to the overexpressed transferrin receptor (TFR) on tumor cells, significantly enhancing the nanoparticle’s targeting capability and drug uptake efficiency for Oral squamous cell carcinoma (OSCC) treatment. • Prolonged circulation and immune evasion: The platform leverages the "don’t eat me" signal from the CD47 protein on red blood cells membrane (RBCM) to effectively evade immune clearance, markedly prolonging systemic circulation time and improving drug accumulation at the tumor site. • Anti-tumor efficacy and biocompatibility: In preclinical models, T12-RBCM@CM NPs demonstrated robust tumor growth inhibition both in vitro and in vivo , alongside excellent biocompatibility and non-toxicity to major organs, positioning it as a promising strategy for OSCC therapy.