Nanoemulsions and nanocapsules as carriers for the development of intranasal mRNA vaccines

Abstract

The global emergency of coronavirus disease 2019 (COVID-19) has spurred extensive worldwide efforts to develop vaccines for protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our contribution to this global endeavor involved the development of a diverse library of nanocarriers, as alternatives to lipid nanoparticles (LNPs), including nanoemulsions (NEs) and nanocapsules (NCs), with the aim of protecting and delivering messenger ribonucleic acid (mRNA) for nasal vaccination purposes. A wide range of prototypes underwent rigorous screening through a series of in vitro and in vivo experiments, encompassing assessments of cellular transfection, cytotoxicity, and intramuscular administration of a model mRNA for protein translation. As a result, two promising candidates were identified for nasal administration. One of them was a NE incorporating a combination of an ionizable lipid (C12-200) and cationic lipid (DOTAP), both intended to condense mRNA, along with DOPE, which is known to facilitate endosomal escape. This NE exhibited a size of 120 nm and a highly positive surface charge (+ 50 mV). Another candidate was an NC formulation comprising the same components and endowed with a dextran sulfate shell. This formulation showed a size of 130 nm and a moderate negative surface charge (-16 mV). Upon intranasal administration of mRNA encoding for ovalbumin (mOVA) associated with optimized versions of the said NE and NCs, a robust antigen-specific CD8 + T cell response was observed. These findings underscore the potential of NEs and polymeric NCs in advancing mRNA vaccine development for combating infectious diseases.

Keywords: Intranasal vaccination; Nanoparticles; Polymeric nanocapsule; SARS-CoV-2; mRNA vaccine.

Fig. 1

Citotoxicity of NE-mGFP (top) and NC-mGFP (bottom), at different mGFP concentrations, in HeLa cells at 24-hours post transfection Abbreviations: CS: chitosan. DX: dextran sulphate. mGFP: mRNA encoding for GFP. NE: nanoemulsion. NC: nanocapsule. PC: positive control, lipofectamine. PR: protamine sulphate EP. Values represent the mean ± standard deviation (n ≥ 3)

Fig. 2

GFP transfection efficiency of NE-mGFP (top) and NC-mGFP (bottom) nanocarriers, at different mGFP concentrations. Transfection efficiency is expressed in percentage of GFP positive cells (bars, left axis) and mean fluorescence intensity (symbols, right axis) in HeLa cells, 24 h post transfection Abbreviations: CS: chitosan. DX: dextran sulphate. mGFP: mRNA encoding for GFP. NE: nanoemulsion. NC: nanocapsule. PC: positive control, lipofectamine. PR: protamine sulphate EP. A significant comparison was performed using two-way ANOVA followed by Turkey’s multiple comparison tests between the highest concentration and lower concentration (top) and between the highest concentration of each group (bottom). p-values < 0.05 were considered statistically significant (*). Also, (**) if p-value < 0.01, and (****) if p-value < 0.0001. Values represent the mean ± standard deviation (n ≥ 3)

Fig. 3

Quantification of whole-body luciferase signal of NE-mLuc and NC-mLuc formulations, after intramuscular administration. Each animal was injected in both legs and imaged by IVIS at different time points (6, 24, and 48 h) Abbreviations: DX: dextran sulphate. mLuc: mRNA encoding for luciferase. NE: nanoemulsion. NC: nanocapsule. PP: PGA-PEG or PEG (5 kDa)-b-PGA (10) (Na). A significant comparison was performed using two-way ANOVA followed by Turkey’s multiple comparison tests between the groups, at 6 h. p-values < 0.05 were considered statistically significant (*). Also, (***) if p-value < 0.001, and (****) if p-value < 0.0001. Values represent the mean ± standard deviation (n ≥ 3)

Fig. 4

Percentage of OVA-specific CD8⁺ T cells upon intranasal administration con NE-13-mOVA and NC-4-DX-mOVA, obtained by flow cytometer, in blood (collected on day 7 post-administration, left) and splenocytes (collected on day 10 post-administration, right). The percentage of antigen-specific CD8⁺ T cells was gated as CD45⁺/CD8⁺/Dextramer⁺ Abbreviations: DX: dextran sulphate. mOVA: mRNA encoding for ovalbumin. NE: nanoemulsion. NC: nanocapsule. PBMCs: peripheral blood mononuclear cells. A significant comparison was performed using one-way ANOVA followed by Turkey’s multiple comparison tests between the groups. p-values < 0.05 were considered statistically significant. Values represent the mean ± standard deviation (n ≥ 3)

Fig. 5

IFN-γ-producing spot-forming units (SFU) from splenocytes, by ELISpot assay (collected on day 10) after intranasal administration of NE-13-mOVA and NC-4-DX-mOVA. Study was performed in unstimulated (left) and OVA-stimulated splenocytes Abbreviations: DX: dextran sulphate. mOVA: mRNA encoding for ovalbumin. NE: nanoemulsion. NC: nanocapsule. A significant comparison was performed with mixed-effects analysis followed by Dunnett’s multiple comparison tests between the groups. All comparisons were not significant (n ≥ 3)