The search for smart, selective drug delivery systems is reshaping cancer therapy. Among emerging nanotechnologies, extracellular vesicle-mimetic systems derived from red blood cells (Nano-erythrosomes, or NanoEs) unique advantages: high biocompatibility, scalability, and the ability to pass biological barriers.

This work introduces a modular two-step engineering strategy that transforms red blood cell-derived nanovesicles into tumor-targeted carriers:

i) surface functionalization via bio-orthogonal click chemistry using a fluorescent peptide targeting the oncofetal Extra Domain-B (EDB) of fibronectin, a tumor-specific matrix protein absent in healthy tissues;

ii) therapeutic loading with the chemotherapeutic drug Paclitaxel (PTX) through a mild sonication protocol.

Click-based functionalization proved to be efficient, yielding over 60% of NanoEs displaying the targeting peptide. To assess the targeting specificity and therapeutic potential of the engineered NanoEs, we employed the millifluidic MIVO cancer-on-chip model that mimics the tumor microenvironment, including relevant matrix components and cell-cell interactions. This platform enabled real-time, high-resolution monitoring of nanoparticle-cell interactions under dynamic flow conditions. Thanks to this approach we demonstrated that anti-EDB-NanoEs exhibit significantly enhanced uptake in EDB-positive cancer cells compared to unmodified vesicles. The peptide retained its binding specificity when conjugated, enabling precise recognition of the tumor microenvironment. Despite a moderate drug loading efficiency (~1.5% by HPLC-MS), PTX-loaded NanoEs elicited a strong cytotoxic response in tumor cells, confirming that the low-dose delivery via targeted nanocarriers can achieve significant therapeutic effects.

This work introduces a modular and reproducible platform to generate red blood cell-derived nanocarriers with selective tumor-homing capability. By combining click chemistry with physical drug encapsulation, we propose a next-generation strategy for precision oncology.