Intranasal mRNA therapeutic design represents a compelling strategy by offering a minimally invasive delivery route that concentrates mRNA activity at the nasal mucosa, promotes localized biological responses, and limits systemic distribution and first-pass metabolism. However, biological barriers such as the mucus layer remain significant obstacles to effective delivery. In this study, we developed an intranasal mRNA delivery platform based on non-PEGylated cationic liposomes composed of SPC, DPPC, cholesterol, and DDAB, fabricated using a microfluidic approach. Among the tested formulations, B4 lipoplexes (SPC, DPPC, cholesterol, and DDAB at a 2:3:1:0.5 M ratio) demonstrated desired properties, including a positive surface charge of +21.0 mV, a particle size of 140 nm, a low polydispersity (0.28), and as high as 84% mRNA encapsulation. The lipoplexes were systematically evaluated for physicochemical characteristics, mucoadhesiveness, mucus penetration, in vitro cell viability and uptake, and transfection efficiency both in vitro and in vivo. The formulation demonstrated strong mucoadhesive behavior while maintaining efficient mucus penetration. Fluorescence imaging confirmed robust cellular uptake and successful cytosolic mRNA delivery, resulting in high transfection efficiency. Notably, the lipoplexes encapsulating GFP and SARS-CoV-2 Omicron spike protein mRNA achieved efficient in vitro transfection with minimal cytotoxicity in A549 and HEK293T cells, as confirmed by MTT assay. Importantly, the lipoplexes containing 0.25-2 µg mRNA did not trigger significant pro-inflammatory cytokine responses (IL-1β, IL-6, TNF-α, and MCP-1) in THP-1 cells. Following intranasal administration in C57BL/6 mice, firefly luciferase mRNA-loaded B4 lipoplexes exhibited efficient adhesion to and penetration through nasal mucus, facilitated transfection of cells lining the nasal cavity, and produced localized reporter protein expression without detectable off-target expression. Collectively, these findings highlighted the strong potential of the non-PEGylated cationic lipoplexes as a safe and effective intranasal mRNA delivery platform, offering a noninvasive targeting strategy that broadens the applicability of lipid-based mRNA therapeutics.
