Polymer-Coated Extracellular Vesicles for Selective Codelivery of Chemotherapeutics and siRNA to Cancer Cells.

Combination therapies involving small-interfering RNA (siRNA)-mediated gene silencing and small-molecule drugs are of high interest for cancer treatment. Among the current gene delivery carriers, cell-derived extracellular vesicles (EVs) are particularly promising candidates due to their high biocompatibility, low immunogenicity, in vivo stability, and inherent targeting ability. Here, we developed a multifunctional EV platform capable of selective codelivery of siRNA and doxorubicin (DOX) to cancer cells. siRNA was first loaded into engineered lipid-hybridized EVs (eEVs) to serve as a core. Subsequently, DOX was incorporated into a polyelectrolyte shell surrounding eEVs, which was deposited by layer-by-layer (LbL) assembly. This approach resulted in the production of a stable EV-polymer complex (LbL-eEV) with a diameter of 140.2 ± 9.0 nm and zeta potential of +22.1 ± 0.5 mV. Experiments were performed to assess cellular uptake, cytotoxicity, and gene silencing efficacy in lung adenocarcinoma cells (A549), with noncancerous fibroblast cells (CCL-210) used as a control. The results demonstrated that the LbL-eEV complex can traffic through cells and release siRNA in the cytoplasm, while delivered DOX enters nuclei to induce programmed cell death. Moreover, the inherent selectivity of the particles for cancer cells resulted in effective gene silencing and cancer killing efficiency with reduced cytotoxicity to normal cells. Synchronous delivery of siRNA and DOX was also verified by flow cytometry analysis of single cells. In summary, these data provide a proof of concept for engineering EVs to deliver multiple therapeutics and suggest that LbL-eEVs are a promising drug delivery platform for targeting cancer.

Engineered extracellular vesicles with synthetic lipids via membrane fusion to establish efficient gene delivery.

The low yield of extracellular vesicle (EV) secretion is a major obstacle for mass production and limits their potential for clinical applications as a drug delivery platform. Here, we mass produced engineered extracellular vesicles (eEVs) by fusing the surface composition of EVs with lipid-based materials via a membrane extrusion technique. A library of lipids (DOTAP, POPC, DPPC and POPG) was fused with EVs to form a hybrid-lipid membrane structure. Uniform lamellar vesicles with a controlled size around 100 nm were obtained in this study. Particle number characterization revealed this extrusion method allowed a 6- to 43-fold increase in numbers of vesicles post- isolation. Further, exogenous siRNA was successfully loaded into engineered vesicles with ~ 15% - 20% encapsulation efficiency using electroporation technique. These engineered extracellular vesicles sustained a 14-fold higher cellular uptake to lung cancer cells (A549) and achieved an effective gene silencing effect comparable to commercial Lipofectamine RNAiMax. Our results demonstrate the surface composition and functionality of EVs can be tuned by extrusion with lipids and suggest the engineered vesicles can be a potential substitute as gene delivery carriers while being able to be mass produced to a greater degree with retained targeting capabilities of EVs.