Microfluidic Nano-Assembly of Red-Blood-Cell (RBC) Lipids and Components for Engineering Extracellular Vesicles

Area of Science:

• Nanomedicine
• Biotechnology
• Materials Science

Background:

• Scalable and precise engineering of nanocarriers is a challenge in nanomedicine.
• Native extracellular vesicles (EVs) like red blood cell-derived EVs (RBCEVs) have limitations in yield, scalability, and cargo loading control.
• Current nanocarriers often lack tunable properties and immune compatibility.

Purpose of the Study:

• To develop a bottom-up strategy for creating engineered red blood cell extracellular vesicles (eRBCEVs).
• To achieve scalable synthesis of customizable eRBCEVs with controlled properties and broad cargo encapsulation.
• To establish eRBCEVs as a next-generation therapeutic platform for programmable delivery.

Main Methods:

• Utilized microfluidic diffusional mixing of purified red blood cell (RBC) lipids and components.
• Employed multi-physics simulation for parametric optimization of flow rates, lipid concentration, and channel geometry.
• Validated cargo encapsulation using cryo-electron microscopy (cryo-EM), chemical mapping, and high-resolution total internal reflection fluorescence microscopy (TIRFM).

Main Results:

• eRBCEVs demonstrated efficient encapsulation of diverse cargos including oligonucleotides, gold nanoparticles, hemoglobin, erythrocruorin, and adeno-associated virus (AAV) particles.
• Functionalized eRBCEVs with α-PD-L1 antibodies showed uptake in tumor organoids.
• In vivo studies in mice revealed sustained circulation and minimal immunogenicity compared to controls.

Conclusions:

• The developed microfluidic platform enables scalable synthesis of customizable eRBCEVs.
• eRBCEVs preserve RBC lipid features, offer cargo flexibility, and exhibit low immunogenicity.
• This platform represents a significant advancement for therapeutic delivery in gene therapy, immunomodulation, and nanomedicine.