International Journal of Nanomedicine (May 2024)
Strategies for Improved pDNA Loading and Protection Using Cationic and Neutral LNPs with Industrial Scalability Potential Using Microfluidic Technology
Abstract
Ilaria Ottonelli,1,* Elisa Adani,2,* Andrea Bighinati,2 Sabrina Cuoghi,1 Giovanni Tosi,1,3 Maria Angela Vandelli,1 Barbara Ruozi,1 Valeria Marigo,2,3 Jason Thomas Duskey1 1Nanotech Lab, Te.Far.T.I., Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Center for Neuroscience and Neurotechnology, Modena, Italy*These authors contributed equally to this workCorrespondence: Jason Thomas Duskey, Nanotech Lab, Te.Far.T.I., Department of Life Sciences, University of Modena and Reggio Emilia, Via Giuseppe Campi 103, Modena, MO, Italy, 41125, Tel +390592058573, Email [email protected] Valeria Marigo, Department of Life Sciences, University of Modena and Reggio Emilia, Via Giuseppe Campi 287, Modena, MO, 41125, Italy, Tel +390592055392, Email [email protected]: 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
