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Additional file 1: Figure S1. Physicochemical characterization for the storage stability of blank LPNs at 4 °C and room temperature (RT) tested over a time-course of 62 days post-preparation (A) hydrodynamic size, (B) PDI and (C) ζ-potential. Colloidal properties reveal stability of LPNs for all tested time-points and temperatures. N = 3, mean ± SD. Figure S2. (A1, A2) Physicochemical characteristics of blank LPNs and (B1, B2) blank CS-PLGA NPs tested under different physiological conditions using HBSS buffer, cell culture medium DMEM with and without 10% FCS following 2 h, 4 h and 24 h of incubation. While LPNs show only a significant change in colloidal properties after incubation in DMEM plus 10% FCS, CS-PLGA NPs elicit a significant difference in colloidal parameter for all tested buffers compared with untreated samples. However, the observed size changes are immediate but not increasing within the 24 h of observation. N = 3, mean ± SD. Figure S3. Representative dot plots and appropriate gating strategy for cytotoxicity assay in DC2.4 cells using blank LPNs and CS-PLGA NPs indicate a fluorescence shift and hence higher cytotoxicity for particles of higher concentrations (160 μg/mL). Figure S4. Summary of physicochemical properties for fluoresceinamine labeled blank and labeled mRNA complexed nanoparticles wit mRNA:NPs w/w ratio of 1:10, 1:20 and 1:30. (A) Indicates the hydrodynamic size, (B) PDI and (C) ζ-potential. N = 4, mean ± SD. Figure S5. Representative dot plots and corresponding gating strategy for the transfection studies in DC2.4 cells using mRNA:LPNs and mRNA:CS-PLGA NPs with JetPRIME® as the positive control, untreated and naked mRNA as negative control. Fluorescence shift reveals cells with mRNA-mCherry transgene expression. Figure S6. (A) Representative graphs obtained 24 h and 48 h post-transfection of A549 cells with mRNA:LPNs and mRNA:CS-PLGA NPs at different mRNA:NPs weight ratios using flow cytometry. (B) Representative confocal images of A549 cells 48 h post-transfection using mRNA:LPNs, JetPRIME® as positive control, naked mRNA as negative control. Red fluorescence reveals cells successfully transfected while their morphology remains consistent with non-transfected cells (green: cell membrane; blue: cell nucleus; scale bar 50 μm).