Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery.

Erica Quagliarini,Giulio Caracciolo,Luca Digiacomo,Valentina Tassinari,Junbiao Wang,Lishan Cui,Barbara Sartori,Cristina Marchini,Laura Masuelli,Daniela Pozzi,Francesca Giulimondi,Heinz Amenitsch,Serena Renzi,Roberto Bei,Augusto Amici

doi:10.3390/pharmaceutics13081292

Abstract

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

Highlights

The results showed that lipid nanoparticles (LNPs) produced with a lower total flow rate (TFR) were generally associated with keratinocytes (HaCaT), epidermoid cervical cancer (CaSki) and immortalized keratinocytes higher transfection efficiency (TE) and lower cytotoxicity, especially once compared to LipofectamineTM 3000, the
While TE of PEGylated LNP2 increased with increasing oleoyl phosphatidylethanol-amine (DOPE), Cholesterol and PEG-lipid 1,2-dioleoyl-snof plasmid DNA (pDNA)/well, the opposite trend was found for PEGylated LNP8
The development of LNPs encapsulating RNA types has proved that formulations and manufacturing processes need to be adapted to each type of cargo and are not interchangeable

Summary

Introduction

To exert their function properly, nucleic acids (NAs) such as messenger RNA (mRNA), short interfering RNA (siRNA), and plasmid DNA (pDNA) need to reach their target tissue without any alterations of their complex structures and, subsequently, interact with cytosol and/or nucleus of target cells [1]. Free NAs are highly susceptible to rapid degradation in biological media and clearance from the circulation. As demonstrated in a seminal paper by the Nobel Prize M.R. Capecchi, even if directly injected in the cell cytoplasm naked NAs do not work at all [2]. Even if directly injected in the cell cytoplasm naked NAs do not work at all [2]

Methods

Discussion

Conclusion