Design and characterization of lipid-enveloped PBAE particles. (A) Schematic of structure and composition of lipid-coated PBAE particles and mRNA cargo association. (B, C) Size histograms of particles prepared via nanoprecipitation (B) or emulsion/solvent evaporation (C) methods. (D) Measurement of absorbance of particle suspensions as a function of solution pH for both lipid and PVA stabilized particles. (E) Representative cryoEM image of lipid-enveloped PBAE particles, showing the presence of the lipid coating at the particle surfaces (scale bar = 50 nm).

pH-responsive lipid-enveloped PBAE particles chaperone the delivery of the membrane-impermeable dye molecule calcein into the cytosol of dendritic cells. DC2.4 cells were incubated for 1 h with calcein alone or calcein and lipid-coated poly-1 or PLGA particles, washed to remove unbound particles, then imaged live by confocal microscopy (A–D) or analyzed by flow cytometry (E–G). Confocal images of DC2.4 cells incubated with calcein alone (A) or calcein and lipid-coated PBAE particles (B) (LHS = fluorescence overlays: red, nanoparticles; green, calcein. RHS = bright-field images). (C, D) Cells co-incubated with calcein and either lipid-coated PBAE particles (C) or lipid-coated PLGA particles (D) (LHS = Green, calcein. RHS = bright-field images). Representative images of PEGylated particles are shown here, but a similar trend was observed with non-PEGylated particles. Flow cytometry scatter plots of particle fluorescence vs. calcein fluorescence (E) and histograms (F) for cells treated with calcein or calcein + PBAE particles compared with untreated control cells or cells incubated with particles alone. (G) Average percentage of cells exhibiting cytosolic/nuclear calcein distribution after 1 h incubation with lipid-coated PBAE particles or lipid-coated PLGA particles compared to calcein-only control. Shown are the mean ± SD from triplicate samples.

Assessment of nanoparticle cytotoxicity. DC2.4 cells were incubated with increasing doses of PEGylated or non-PEGylated lipid-coated poly-1 particles, or PVA-stabilized poly-1 particles for 1 h or 12 h at 37 °C, washed, detached and then stained with DAPI and annexin V to detect apoptotic and necrotic cells by flow cytometry. Shown are the percentages of live (DAPI⁻ annexin V⁻) cells relative to untreated controls. Error bars represent standard deviation of triplicate samples.

RNA adsorption to PEGylated or non-PEGylated lipid-coated PBAE particles in water. (A) Kinetics of poly I:C adsorption to lipid-coated PBAE particles for 300 μg particles incubated with 4 μg RNA in 300 μl of water. (B) Binding of Poly I:C at varying concentrations to 300 μg particles in 300 μl of water for 2 h. (C, D) Binding efficiency (C) and RNA loading (D) of mRNA or poly I:C assessed for RNA (4 μg) added to 300 μg particles for 2 h in 300 μl of water. (E) Size histograms of particles before and after mRNA adsorption at optimized loading conditions (4 μg mRNA added to 300 μg particles in 300 μl of water). (F) Release of poly I:C from nanoparticles incubated in RPMI medium containing 10% FBS at 37 °C. (G) 1 μg mRNA (either free mRNA (Naked) or bound to PEGylated PBAE nanoparticles (NP)) was incubated with 2 or 10% FBS for 5 min or 1 h at 37 °C (or left untreated, Cntl) and analyzed by electrophoresis on a 1% agarose gel. Upper panel shows mRNA detected in the loading wells when mRNA was loaded onto particles and lower panel shows intact free mRNA (Cntl) and degraded mRNA fragments obtained when samples were exposed to serum nucleases. Naked mRNA is completely destroyed by 1 h when exposed to 10% FBS.

Lipid-coated PBAE particles mediate cytosolic mRNA delivery and transfection in dendritic cells in vitro. (A, B) To observe cytosolic mRNA delivery, DC2.4 cells were incubated for 1h in the presence of LysoTracker green to stain endolysosomes (green) and either naked Cy3-labeled mRNA (red) in serum-free Opti-mem (A) or Cy3-labeled mRNA loaded on particles in complete medium (10% FBS) (B). To study transfection, DC2.4 cells were incubated for 12 h in complete medium (10% FBS) with either naked mRNA (1 μg/ml) or an equivalent dose of mRNA adsorbed to PEGylated or non-PEGylated lipid-coated PBAE particles (75 μg particles/ml), then washed and imaged live by confocal microscopy at 37 °C or analyzed by flow cytometry. (C) Confocal images of DiD-labeled particles (blue), Cy3-labeled mRNA (red), and GFP expression (green) with fluorescence/bright-field overlays in DC2.4 cells. (D–H) Flow cytometry analysis of DC2.4 cells showing mean particle uptake (D), representative histograms of fluorescent mRNA uptake (E) and mean frequency of mRNA⁺ cells (F), representative cytometry histograms of GFP expression (G), and mean frequency of GFP⁺ cells (H). Shown are means ± SD from duplicates in 2 independent experiments.

In vivo transfection in C5BL/6J mice following intranasal administration of firefly luciferase-encoding mRNA either in soluble form or adsorbed on fluorescent lipid-enveloped PBAE particles. (A) Fluorescence image of mice immediately following intranasal particle administration. (B) Total fluorescence signal at the inoculation site at 0 and 6 h quantified from groups of particle-treated or control mice. (C) Bioluminescence images of mice 12 h following mRNA administration. (D) Bioluminescence signal at the inoculation site at 6, 12 and 24 h quantified from groups of treated and control mice. ***,p <0.001, **, p <0.01 and *, p <0.05. Data shown from one representative of two independent experiments (n = 4 mice/group).