Development of on-chip mixing and incubation platform for the loading of extracellular vesicles with an anti-cancer drug

Despite great progress achieved in the understanding of cancer biology, metastases are still synonymous of terminal illness. Therefore, developing targeted therapies for cancer is a clinical imperative. In recent years, extracellular vesicles (EVs) have been found to be involved in cancer development. EVs are nano-sized vesicles released from different cell types under both normal and pathological conditions that carry information leading to some important processes such as angiogenesis, metastasis, drug resistance and proliferation in cancer. For instance, EVs derived from Neuroblastoma (NB) - one of the most common solid tumors of childhood - are found to play a role in mediating its progression. From a therapeutic point of view, EVs represent an excellent drug delivery system: EVs intrinsically possess many attributes of drug delivery systems, since these particles have a long-circulating half-life, are well tolerated in the body, and are internalized by recipient cells. Although EVs have displayed some promising therapeutic applications, extensive multi-disciplinary efforts are still needed. Microfluidic technologies could be a valuable tool for the engineering of EVs, owing to the low amount of reagents and samples needed, high throughput and different phenomena that characterize this technology with respect to their large-scale counterparts. Considering such promises, the aim of the project is the design and development of a microfluidic device for generating drug-loaded EVs using microfluidic mixing and incubation. Mesenchymal Stem Cells (MSCs) are used as cell sources of EVs. EVs can be loaded with various therapeutic agents, including Verteporfin (VP). VP is a small hydrophobic porphyrin that recently provided positive results for its anti-cancer activity in different tumors, including NB. The platform is designed using AutoCAD® so that both a perfect mixing between MSCs-derived EVs and VP occurs within the mixing unit and the desired incubation time is achieved. The platform is then modeled, performing computational fluid dynamics simulations through COMSOL Multiphysics®. The master mold is fabricated with photolithography, and polydimethylsiloxane (PDMS) replicas of the chips were obtained with replica molding processes. Plasma treatment is used to form an irreversible hydraulic seal of the microfluidic platform to a glass support. The platform in this configuration is used to perform both fluid dynamic and biological validations. Finally, preliminary experiments are carried out to study the effect of VP-loaded EVs on NB target cells. Although the utilization of EVs for therapeutic drug delivery is still in its infancy, a more advanced understanding and systemic evaluation of their use will boost the development of EVs as a superior and effective drug delivery system that can bring breakthroughs to the field of cancer nanomedicine.