Low Molecular Weight Alginate Oligosaccharides as Alternatives to PEG for Enhancement of the Diffusion of Cationic Nanoparticles Through Cystic Fibrosis Mucus

Abstract

Airway mucus is a major barrier to the delivery of lipid-based nanoparticles in chronic airway diseases such as cystic fibrosis (CF). Receptor-Targeted Nanocomplexes (RTN), comprise mixtures of cationic lipids and bifunctional peptides with receptor-targeting and nucleic acid packaging properties. The aim of this study is to improve the mucus-penetrating properties of cationic siRNA and mRNA RTNs by combining them with low molecular weight alginate oligosaccharides, OligoG and OligoM. Cationic RTNs formulated with either alginate become strongly anionic, while PEGylated messenger RNA (mRNA) and short interfering RNA (siRNA) RTNs remain cationic. Both alginates enhance mucus diffusion rates of cationic siRNA and mRNA RTNs in a static mucus barrier diffusion model, with OligoG particularly effective. PEGylation also enhance mucus diffusion rates of siRNA RTNs but not mRNA RTNs. Electron microscopy shows that RTNs remained intact after mucosal transit. The transfection efficiency of OligoM-coated mRNA RTNs is better than those coated with OligoG or PEG, and similar to cationic RTNs. In siRNA RTN transfections, OligoM is better than OligoG although 1% PEG is slightly better than both. The combination of cationic RTNs and alginate oligosaccharides represents a promising alternative to PEGylation for epithelial delivery of genetic therapies across the mucus barrier while retaining transfection efficiency.

Keywords: cystic fibrosis; mRNA; mucus penetration; nanoparticles; siRNA.

Figure 1

Alginate Structures showing charge distribution.

Figure 2

Transwell static mucus diffusion assay.

Figure 3

Experimental design RTN formulations were prepared with or without PEG and alginates, size and charge were measured, then RTN formulations were added to the mucus layer. In the mucus modification study, formulations OligoG or Oligo M were added directly to the mucus. RTN samples were collected pre and post mucus transition for TEM analysis while size and charge of RTN formulations were analyzed prior to addition to mucus.

Figure 4

Schematic illustration of RTN, RTN/OligoM and RTN/OligoG. Anionic molecules OligoM or OligoG bind to the surface of cationic RTN due to electrostatic attraction. The surface charge of the formulations (RTN/OligoM and RTN/OligoG) become anionic.

Figure 5

Impedance Dw/Dm of mRNA RTNs or siRNA RTNs with or without OligoG or OligoM. Bar chart of the impedance (Dw/Dm) of RTNs containing, a) mRNA, and, b) siRNA. The diffusion rate in the CF mucus‐membrane system was calculated as described in Methods. The data represent means ±SE, n ≥ 3. The non‐parametric Kruskal–Wallis test with post‐hoc Dunn's test was performed to assess significance (*p<0.05).

Figure 6

TEM images of mRNA RTNs and siRNA RTNs before or after transition. mRNA RTN, a) before, and, b) after mucus‐membrane transition. mRNA RTN premixed with OligoG, c) before, and, d) after transition of mucus‐membrane barrier. mRNA RTN premixed with OligoM, e) before, and, f) after mucus‐membrane transition. siRNA RTN g) before, and, h) after transition. siRNA RTN premixed with OligoG, i) before, and, j) after mucus‐membrane transition. siRNA RTN premixed with OligoM, k) before, and, l) after transition of the mucus‐membrane barrier. The scale bars are shown on each image.

Figure 7

Luciferase mRNA transfection efficiency and cell viability of CFBE and NHBE BMI‐1 cells transfected with OligoG or OligoM. a) CFBE BMI‐1, and, b) NHBE BMI‐1 cells, transfected with C18DOPE containing peptide E and Luciferase mRNA with OligoG or OligoM. The luciferase activity was assessed after 24 h (n = 6). One‐way ANOVA with post‐hoc Dunnett's test was performed to assess significance relative to mRNA RTN. Resazurin cell viability assay of, c) CFBE BMI‐1, and, d) NHBE BMI‐1 cells, 24 h after transfection. One‐way ANOVA with post‐hoc Dunnett's test was performed to assess significance to untransfected cells (UT), n = 6. All the data represent means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 8

siRNA transfection efficiency of CFBE BMI‐1 cells transfected with OligoG or OligoM. CFBE BMI‐1 cells were transfected with 100 nm GAPDH siRNA RTN, with or without OligoG or OligoM. 48 h after transfections, the cells were harvested, and the silencing efficiency was assessed by qRT‐PCR. The silencing efficiency of GAPDH siRNA was normalized to irrelevant (non‐targeting, negative control) siRNA under the same conditions. Student's t test was performed to test significance, as indicated by *p < 0.05, **p < 0.01, ***p < 0.001. All the data represent means ± SE, n = 3.