| Abstract | Mast cells (MCs), are hematopoetically-derived secretory immune cells that release preformed as well as de novo synthesized infammatory mediators in response to activation by several stimuli. Based on their role in infammatory responses, particularly in the lung and skin, MCs provide an efective target for anti-infammatory therapeutic strategies. Drug-delivery of lipophilic payloads to MCs can be challenging due to their functionally distinct intracellular structures. In the present study, pH-sensitive cationic lipid-based nanoparticles (LNPs) composed of DODMA, DODAP or DOTAP lipids that encapsulated a GFP or eGFP plasmid were constructed using non-turbulent microfuidic mixing. This approach achieved up to 75–92% encapsulation efciency. Dynamic light scattering revealed a uniformly sized and homogeneous dispersion of LNPs. To promote cellular internalization, LNPs were complexed with apolipoproteins, amphipathic proteins capable of binding lipids and facilitating their transport into cells. Cryo-TEM analysis showed that LNP structure was diferentially modifed when associated with diferent types of apolipoproteins. LNP preparations made up of DODMA or DODMA, DODAP and DOTAP lipids were coated with seven apolipoproteins (Apo A1, B, C3, D, E2, E4 and H). Diferentiated bone-marrow derived mouse mast cells (BMMCs) were exposed to apolipoprotein-LNP and internalization was measured using fow cytometry. Out of all the apolipoproteins tested, ApoC3 most efciently facilitated cellular internalization of the LNP into BMMCs as determined by GFP fuorescence using fow cytometry. These efects were confrmed in a less diferentiated but also interleukin-3-dependent model of mouse mast cells, MC/9. ApoC3-LNP enhanced internalization by BMMC in a concentration-dependent manner and this was signifcantly increased when BMMC were pre-treated with inhibitors of actin polymerization, suggesting a dependence on intracellular shuttling. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) decreased ApoC3-LNP internalization and reduced the expression of apolipoprotein E receptor 2 (ApoER2), suggesting that ApoC3-LNP binding to ApoER2 may be responsible for its enhanced internalization. Furthermore, ApoC3 fails to facilitate internalization of LNPs in Lrp8−/− KO BMMC that do not express ApoER2 on their cell surface. Altogether, our studies reveal an important role of ApoC3 in facilitating internalization of cationic LNPs into MCs. |
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