# Ethanol-Guided Hybridization of Extracellular Vesicles with Liquid-Crystalline Lipid Nanoparticles

## TL;DR

This paper introduces a new method to combine extracellular vesicles with lipid nanoparticles using ethanol, preserving their structure and function for potential use in nanomedicine.

## Contribution

A novel ethanol-guided microfluidic method for hybridizing EVs with LCNPs while maintaining structural and functional integrity.

## Key findings

- Hybrid nanoparticles retain long-range cubic order with EV membrane proteins in nanodomains.
- Ethanol stabilizes a sponge-like intermediate that guides controlled fusion and structural stability.
- Hybrid LCNPs show enhanced uptake in HEK293t cells and preserved EV biofunctionality.

## Abstract

Hybrid nanosystems
that integrate biological and synthetic lipid
assemblies hold great promise for tailoring nanoscale interfaces with
programmable chemical and structural functionality. However, existing
approaches to hybridize extracellular vesicles (EVs) with lipid nanoparticles
(LNPs) compromise either the EV bioactivity or the native supramolecular
organization of synthetic LNPs, undermining structure-dependent functionality.
Here, we introduce an ethanol-mediated microfluidic assembly route
that enables the in situ formation and hybridization
of nonlamellar liquid-crystalline lipid nanoparticles (LCNPs) with
red-blood-cell-derived EVs (RBCEVs) in a single step. This process
exploits ethanol-induced interfacial reorganization to drive EV incorporation
without compromising the LCNP cubic architecture. Synchrotron small-angle
X-ray scattering (SAXS) and cryogenic electron microscopy reveal hybrid
nanoparticles that retain long-range cubic order, with RBCEV membrane
proteins localized within phase-segregated nanodomains. Single-particle
Raman analysis and enzymatic assays confirm molecular-level hybridization
and preserved EV biofunctionality. Hybrid LCNPs also exhibit enhanced
uptake in HEK293t cells. Mechanistic SAXS studies uncover that ethanol
transiently stabilizes a swollen sponge-like intermediate, which mediates
controlled fusion and acts as a structural template upon solvent removal,
imparting long-lasting structural stability. This study elucidates
the physicochemical mechanism of ethanol-guided hybridization between
biogenic systems and soft nanostructured colloids, establishing design
principles for structurally controlled nanohybrids with broad applicability
in nanomedicine.