Strategies for Improved pDNA Loading and Protection Using Cationic and Neutral LNPs with Industrial Scalability Potential Using Microfluidic Technology

Abstract

Purpose

In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity.

Methods

The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical “post-formulation” DNA addition vs “pre” addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake.

Results

Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the “post” method, while maintaining their characteristics up to 100 µg/mL of pDNA with the “pre” addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes.

Conclusion

In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.

Keywords:

• gene therapy
• lipid nanoparticles
• microfluidics
• lipoplexes
• DNA delivery

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This research was funded by Fondazione Telethon grant number GGP19113, “Pigment Epithelium-derived Factor (PEDF) peptide as therapeutic agents for inherited retinal degeneration”; PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – M4 C2 I1.4, Potenziamento strutture di ricerca e creazione di “campioni nazionali di R&S” su alcune Key Enabling Technologies, finanziato dall’Unione europea – NextGenerationEU - CN00000041 National Center for Gene Therapy and Drugs based on RNA Technology, ‘CN3’ - Spoke 1; PNRR M4 C2-I1.3 Project PE_00000019 “HEAL ITALIA” CUP E93C22001860006 of University of Modena and Reggio Emilia. The views and opinions expressed are those of the authors only and do not necessarily reflect those of the European Union or the European Commission. Neither the European Union nor the European Commission can be held responsible for them.