TY - JOUR
T1 - Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery
AU - Quagliarini, Erica
AU - Renzi, Serena
AU - Digiacomo, Luca
AU - Giulimondi, Francesca
AU - Sartori, Barbara
AU - Amenitsch, Heinz
AU - Tassinari, Valentina
AU - Masuelli, Laura
AU - Bei, Roberto
AU - Cui, Lishan
AU - Wang, Junbiao
AU - Amici, Augusto
AU - Marchini, Cristina
AU - Pozzi, Daniela
AU - Caracciolo, Giulio
PY - 2021/8/19
Y1 - 2021/8/19
N2 - 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.
AB - 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.
KW - lipid nanoparticles
KW - microfluidics
KW - transfection efficiency
KW - lipofectamine
KW - Lipofectamine
KW - Transfection efficiency
KW - Lipid nanoparticles
KW - Microfluidics
UR - http://www.scopus.com/inward/record.url?scp=85113477946&partnerID=8YFLogxK
U2 - 10.3390/pharmaceutics13081292
DO - 10.3390/pharmaceutics13081292
M3 - Article
SN - 1999-4923
VL - 13
JO - Pharmaceutics
JF - Pharmaceutics
IS - 8
M1 - 1292
ER -