Cytosolic protein delivery using pH-responsive, charge-reversible lipid nanoparticles

Yusuke Hirai,Hisaaki Hirose,Shiroh Futaki,Tomohiro Asai,Miki Imanishi

doi:10.1038/s41598-021-99180-5

Yusuke Hirai, Hisaaki Hirose + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-99180-5

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Abstract

Although proteins have attractive features as biopharmaceuticals, the difficulty in delivering them into the cell interior limits their applicability. Lipid nanoparticles (LNPs) are a promising class of delivery vehicles. When designing a protein delivery system based on LNPs, the major challenges include: (i) formulation of LNPs with defined particle sizes and dispersity, (ii) efficient encapsulation of cargo proteins into LNPs, and (iii) effective cellular uptake and endosomal release into the cytosol. Dioleoylglycerophosphate-diethylenediamine (DOP-DEDA) is a pH-responsive, charge-reversible lipid. The aim of this study was to evaluate the applicability of DOP-DEDA-based LNPs for intracellular protein delivery. Considering the importance of electrostatic interactions in protein encapsulation into LNPs, a negatively charged green fluorescent protein (GFP) analog was successfully encapsulated into DOP-DEDA-based LNPs to yield diameters and polydispersity index of < 200 nm and < 0.2, respectively. Moreover, ~ 80% of the cargo proteins was encapsulated into the LNPs. Cytosolic distribution of fluorescent signals of the protein was observed for up to ~ 90% cells treated with the LNPs, indicating the facilitated endocytic uptake and endosomal escape of the cargo attained using the LNP system.

Highlights

Proteins are among the essential molecules involved in maintaining cellular h omeostasis[1]
Few reports have been published on the intracellular delivery of proteins, especially those including precise confocal laser scanning microscopy (CLSM) analyses of the cellular fates of cargo proteins
We established an approach in producing Lipid nanoparticles (LNPs) based on the pH-sensitive, charge-reversible lipid DOP-DEDA, attaining efficient protein delivery into cells

Summary

Introduction

Proteins are among the essential molecules involved in maintaining cellular h omeostasis[1]. Three major issues have to be cleared in formulation of LNPs: (i) formulation of LNPs with defined particle sizes and dispersity, (ii) efficient encapsulation of cargo proteins into LNPs, and (iii) effective cellular uptake and endosomal release into the cytosol to obtain the expected activity. These are especially important when delivery of precious proteins including antibodies are intended. Effective siRNA delivery was achieved in human cancer cells using this LNP system, yielding a marked inhibition of cancer cell growth by inducing the knockdown of pololike kinase-126 This DOP-DEDA-based LNPs may have a potential applicability to protein delivery, no previous study has been made to evaluate this.

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