Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

Nambin Yim,Seung-Wook Ryu,Seunghee Lee,Won Do Heo,Jeongjin Kim,Woong Sun,Daesoo Kim,Kwang Ryeol Lee,Chulhee Choi,Kyungsun Choi,Mohammed R Shaker,Ji-Ho Park,Hojun Choi

doi:10.1038/ncomms12277

Abstract

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

Highlights

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases
We generated HEK293T cells that can produce EXPLORs loaded with mCherry as a target protein by introducing vectors for two fusion proteins: CIBN-conjugated enhanced green fluorescent protein (EGFP)-tagged CD9 (CIBN-EGFP-CD9) and mCherry-tagged cryptochrome 2 (CRY2)
Discussion protein transduction and lipid nanoparticle-mediated protein delivery methods have been proposed for direct protein delivery into target cells and tissues[6,7,27], many obstacles remain before these methods can be successfully used in vivo, including low purification efficiency, failure to separate from nanoparticles in recipient cells and induction of immune responses against host immune cells

Summary

Introduction

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. The absence of a separation mechanism between cargo proteins and lipid nanoparticles limits the efficiency of cytosolic delivery, preparation of these particles often involves complicated protein purification steps To address these limitations, we developed an optogenetically engineered exosome system EXPLORs (exosomes for protein loading via optically reversible protein–protein interactions) that can deliver soluble proteins into the cytosol via controlled, reversible protein–protein interactions (PPIs). We propose a novel protein-loading method in which cargo proteins can be actively loaded into exosomes through endogenous biogenesis processes, allowing for efficient delivery into the cytosol of target cells through controllable, reversible detachment from the exosomes. Once the cargo proteins are introduced into the exosomes via the process of endogenous biogenesis, they can be detached from CD9-conjugated CIBN by removal of the illumination source, resulting in their release into the intraluminal space of the exosomes and enabling efficient delivery to the cytosolic compartment of target cells

Methods

Results

Conclusion