Impact of nebulizers on nanoparticles-based gene delivery efficiency: in vitro and in vivo comparison of jet and mesh nebulizers using branched-polyethyleneimine

Abstract

Nanoparticles-based gene delivery has emerged as a promising approach for the treatment of genetic diseases based on efficient delivery systems for therapeutic nucleic acids (NAs) into the target cells. For pulmonary diseases such as cystic fibrosis (CF), chronic obstructive pulmonary diseases (COPD), infectious disease or lung cancer, aerosol delivery is the best choice to locally deliver NAs into the lungs. It is, therefore, important to investigate the effects of nebulization conditions on the efficiency of delivery. To this purpose, the non-viral vector branched polyethyleneimine (b-PEI, 25 kDa) was investigated for plasmid delivery by aerosol. Two types of nebulizers, jet nebulizer and mesh nebulizer, were compared regarding the properties of the nanoparticles (NPs) formed, the efficiency of NAs delivery in vitro and in vivo models and the pulmonary deposition. The results indicate that the mesh nebulizer has a better gene delivery performance than the jet nebulizer in this application. This superiority was demonstrated in terms of size, concentration, distribution of NPs and efficiency of NAs delivery. However, pulmonary deposition appears to be similar regardless of the nebulizer used, and the difference between the two systems lies in the inhalable dose. These results underline the crucial role of nebulization techniques in optimizing aerosol-mediated gene delivery by b-PEI and highlight the potential of mesh nebulizers as promising tools to improved gene therapy. Therefore, the comparison must be performed for each gene therapy formulation to determine the most suitable nebulizer.

Keywords: Gene delivery; aerosol; branched-polyethyleneimine; cystic fibrosis; jet nebulizer; lung; mesh nebulizer; nebulization.

Figure 1.

NPs Characterization before and after nebulization; a) Z-average (d.nm) and polydispersity index (PDI) of NPs (250 µg pDNA, N/P 40) before and after nebulization using jet or mesh nebulizer. Three different measurements of three different samples were performed and results are represented as mean ± SD; b) Zeta potential (mV) of NPs (250 µg pDNA, N/P 40) before and after nebulization using jet or mesh nebulizer. Three different measurements of three different samples were performed and results are represented as mean ± SD; c) Electrophorese of b-PEI complex before and after nebulization using jet or mesh nebulizer. To release pGM144, experimentations were performed in presence (+ds) or absence (−ds) of dextran sulfate. For all graphical representations, a Student t-test was performed, **** corresponding to a p value < 0.0001, *** corresponding to a p value < 0.001, ** corresponding to a p value < 0.01 and ns corresponding to non-significant.

Figure 2.

NPs concentration before and after nebulization; a) concentration (particles/mL) as a function of NPs size (nm) before and after nebulization using jet nebulizer. Five different measurements of three different samples were performed and results are represented as mean ± SD; b) Concentration (particles/mL) as a function of NPs size (nm) before and after nebulization using mesh nebulizer. Five different measurements of three different samples were performed and results are represented as mean ± SD; c) Percentage of NPs between 100 to 180 nm before and after mesh nebulization; d) concentration of NPs before and after nebulization using jet or mesh nebulizer. Results are expressed in particles/mL. For all graphical representations, a Student t-test was performed, **** corresponding to a p value < 0.0001 and ns corresponding to non-significant.

Figure 3.

Exposure box validation; a) heat map of the min-max normalized mist repartition inside the exposure box. A549 cells were used to determine this repartition based on luciferase reported gene. Five different nebulization have been performed with b-PEI/pGM144 (250 µg pDNA, N/P 40) using jet or mesh nebulizer to this purpose. The heatmap visualizes the spatial distribution of values across the grid, highlighting patterns of clustering or gradients. The spatial grid was organized as an 8x12 matrix, where each cell corresponds to a physical location on the grid. Values were scaled using a color gradient to enhance interpretability. Brighter colors (yellow) correspond to higher values, while darker colors (blue) correspond to lower values.; b) Boxplot of the distribution of values across three predefined spatial zones: Center, Middle, and Periphery.

Figure 4.

Transfection efficiency and cell viability; a) Luciferase expression and cell viability 24h after transfection on four different submerged cell lines (A549, 16HBE14o-, CFBE41o-, Calu-3) after nebulization using jet or mesh nebulizer of a b-PEI complex solution. Luciferase activity is expressed in relative light unit per milligram of totals proteins (RLU/mg of proteins) and cell viabilities are expressed as a percentage of viability reported to non-transfected cells. Three different nebulization per cells line have been performed and results are represented as mean ± SD. A Student t-test was performed to compare the both nebulizers, **** corresponding to a p value < 0.0001 and ** corresponding to a p value < 0.01; b) Luciferase expression and cell viability 24 h after transfection on 16HBE14o- cell cultivated under air/liquid interface after nebulization using jet or mesh nebulizer of a b-PEI complex solution. Three different nebulization have been performed and 4 transwells have been used by aerosol. Luciferase activity is expressed in relative light unit per milligram of totals proteins (RLU/mg of proteins) and cell viabilities are expressed as a percentage of viability reported to non-transfected cells. Results are represented as mean ± SD. A Mann-Witney test was performed to compare both nebulizers, * corresponding to a p value < 0.05.

Figure 5.

Luciferase expression of lung crushed of mice previously exposed or not to an aerosol of b-PEI complex solution using jet or mesh nebulizer. Results are expressed in relative light unit per milligram of totals proteins (RLU/mg of protein). Five different mice per condition have been used and results are represented as mean ± SD. A Mann-Witney test was performed to compare both nebulizer ** corresponding to a p value < 0.01.

Figure 6.

a) Normalized mass distribution as a function of aerodynamic diameter generated using jet or mesh nebulizer. Three nebulizations were performed per nebulizer; b) Median mass aerodynamic diameter (MMAD, µm) and c) geometric standard deviation (GSD) calculated based on the normalized mass distribution; d) comparison of inhalable and non-inhalable dose between jet and mesh nebulizer. The result is expressed in the percentage of introduced pDNA dose. Three nebulizations were performed per nebulizer and a Student t-test was performed to compare both nebulizer, **** corresponding to a p value < 0.0001, ns corresponding to non-significant.