Lipofection of plasmid DNA into human mast cell lines using lipid nanoparticles generated by microfluidic mixing

Abstract

Mast cells are important immune cells that have significant roles in mediating allergy and asthma. Therefore, studying the molecular mechanisms regulating these and other processes in mast cells is important to elucidate. Methods such as lipofection, transduction, and electroporation are often employed to dissect these mechanisms by disrupting gene expression in mast cell lines. However, as with other leukocytes, human mast cells (HMCs) are often refractory to the delivery of plasmids by lipofection. In this study, we investigated the utility of lipid nanoparticles (LNPs) containing the ionizable cationic lipids 1,2-dioleoyloxy-3-dimethylaminopropane, 1,2-dioleyloxy-3-dimethylaminopropane, or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane for the delivery of plasmid DNA into HMC lines. Herein, we demonstrate for the first time the use of LNPs to achieve significant and reproducible levels of plasmid DNA transfection in HMC-1.2 and laboratory of allergic diseases 2 (LAD2) cells. These levels reached 53.2% and 16.0% in HMC-1.2 and LAD2 cells, respectively; and outperformed Lipofectamine 3000 in both cases. Moreover, cell viability in the transfected cells remained above 65% for all LNP conditions tested. Together, these observations illustrate the efficacy of this technique for mast cell researchers and further support the use of LNPs for nucleic acid delivery into leukocytes.

The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their Lymph Node Transitivity and Distribution

“…LNPs prepared by microfluidic mixing were the first siRNA drug to be approved for use in 2018. Recent progress indicates that LNPs prepared by microfluidic mixing result in the formation of potent delivery systems for delivering macromolecules such as nucleic acids. − Moreover, microfluidic mixing technology has broken the size limitation of LNPs and allows LNPs with sizes of less than 30 nm to be prepared. There is no doubt that such LNPs will be actively used in the field of lymphatic delivery of macromolecules.…”

Potential mechanisms of anaphylaxis to COVID-19 mRNA vaccines

“…14,15 Because mast cells are poised to respond to pathogen danger signals, it is feasible that connective tissue mast cells in the muscle may degranulate in response to interaction with the LNP. A recent publication described efficient transfection of human mast cells using an LNP delivery system, 22 presumably via phagocytosis, suggesting that the mast cells may take up the COVID mRNA vaccines. After phagocytosis of the LNP, a dispersed component of the vaccine may directly stimulate mast cell degranulation.…”

Apolipoprotein C3 facilitates internalization of cationic lipid nanoparticles into terminally differentiated bone marrow-derived mouse mast cells

“…The LNP formulations were diluted with PBS (pH 7.4, without magnesium and calcium; Gibco) and subjected to three sequential dia ltration steps using Amicon Ultra centrifugal lters with a nominal molecular weight limit of 10 kDa (Millipore, Etobicoke, ON, Canada) [5]. The GFP-Hybrid-LNP and GFP-DODMA-LNP preparations were stable for at least 17 months and two years respectively when stored at 4 o C, with no detectable changes in size.…”

“…DNA encapsulation e ciencies were measured using a Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scienti c) [5]. Brie y, the amount of PicoGreen uorescence associated with free (unencapsulated) DNA (Ff) in the LNP solution was compared to the level of PicoGreen uorescence obtained following a 5 min lysis of the LNPs using 0.05% Triton X-100 (total uorescence, Ft).…”

“…Although cationic lipid nanoparticles are sometimes synthesized using the double emulsion-solvent evaporation method, it can result in heterogeneous populations of LNPs with differing sizes, compositions and incomplete encapsulation of payloads. We have previously used controlled micro uidic mixing to generate discrete populations of 50-150 nm LNPs with higher encapsulation e ciencies [5]. We have optimized several different formulations based on six speci c lipids: 1,2dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleoyl-3trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DMPE) and cholesterol.…”