PEGylated enhanced cell penetrating peptide nanoparticles for lung gene therapy

Abstract

The lung remains an attractive target for the gene therapy of monogenetic diseases such as cystic fibrosis (CF). Despite over 27 clinical trials, there are still very few gene therapy vectors that have shown any improvement in lung function; highlighting the need to develop formulations with improved gene transfer potency and the desirable physiochemical characteristics for efficacious therapy. Herein, we introduce a novel cell penetrating peptide (CPP)-based non-viral vector that utilises glycosaminoglycan (GAG)-binding enhanced transduction (GET) for highly efficient gene transfer. GET peptides couple directly with DNA through electrostatic interactions to form nanoparticles (NPs). In order to adapt the GET peptide for efficient in vivo delivery, we engineered PEGylated versions of the peptide and employed a strategy to form DNA NPs with different densities of PEG coatings. We were able to identify candidate formulations (PEGylation rates ≥40%) that shielded the positively charged surface of particles, maintained colloidal stability in bronchoalveolar lavage fluid (BALF) and retained gene transfer activity in human bronchial epithelial cell lines and precision cut lung slices (PCLS) in vitro. Using multiple particle tracking (MPT) technology, we demonstrated that PEG-GET complexes were able to navigate the mucus mesh and diffuse rapidly through patient CF sputum samples ex vivo. When tested in mouse lung models in vivo, PEGylated particles demonstrated superior biodistribution, improved safety profiles and efficient gene transfer of a reporter luciferase plasmid compared to non-PEGylated complexes. Furthermore, gene expression was significantly enhanced in comparison to polyethylenimine (PEI), a non-viral gene carrier that has been widely tested in pre-clinical settings. This work describes an innovative approach that combines novel GET peptides for enhanced transfection with a tuneable PEG coating for efficacious lung gene therapy.

Keywords: Cell-penetrating peptide (CPP); Gene therapy; Glycosaminoglycan-binding enhanced transduction (GET); Lung; Plasmid DNA (pDNA); Transfection.

Fig. 1.

DNA NP formation and characterisation. (A) FLR is a multi-domain peptide made up of a HS GAG binding domain (red), amphiphilic region (blue) and CPP (purple). When mixed with DNA, FLR peptides form electrostatic interactions with the negatively charged phosphate groups of the plasmid to form nanoparticles (NPs) through self-assembly. (B) The ability of GET peptides to bind DNA was assessed using a YO-PRO1 fluorescence-based assay. The graph shows a decrease in the percentage fluorescence as the peptide out competes the dye by complexing DNA at different tested (+/−) charge ratios (CRs). (C) Following the formation of DNA NPs, the physiochemical characteristics of complexes were investigated at different CRs. Hydrodynamic size and zeta-potential were measured in ultrapure water and 10 mM NaCl at pH 7.0, respectively. Error bars indicate SD, n = 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

The effect of PEGylation on the physiochemical properties of DNA NPs. (A) Schematic of non-PEGylated and PEGylated FLR peptides blended at different molar ratios to form DNA NPs with a tuneable coating of PEG on the outer surface. We hypothesized that by varying the PEG component we were able to form DNA NPs at different PEGylation rates. For example 20% PEGylation rate would represent a mixture of 20% PEGylated peptide and 80% non-PEGylated peptide. (B) To confirm the formation of DNA NPs we measured the diameter and charge of complexes at 0%, 10%, 20%, 40%, 60%, 80% and 100% PEGylation rates. The hydrodynamic size and zeta-potentials were measured in ultrapure water and 10 mM NaCl at pH 7.0, respectively. (C) We tested the ability of complexes to protect DNA from nucleases. Naked pDNA, 0% PEG DNA NPs and 100% PEG DNA NPs were incubated with DNase I for 30 min, digested using proteinase K and visualized using a gel shift assay. Degradation of DNA was evidenced by a shift, smear or loss of the DNA band. (D) Stability of PEGylated DNA complexes was investigated following 1 h incubation in ultrapure water or 10% (v/v) BALF in PBS. Hydrodynamic diameter of respective NPs was measured by DLS. Error bars indicate SD, n = 3. Two-tailed Student’s t-test, *P < .05, **P < .01.

Fig. 3.

In vitro uptake and transfection efficiency of DNA NPs at different PEGylation rates. (A) Human bronchial epithelial cells (BEAS2B-R1) cells were treated with rhodamine-labelled DNA complexes for 4 h, trypsinized to remove any extracellularly bound DNA and fluorescence intensity was measured using flow cytometry. (B-C) For the transgene expression study, BEAS2B-R1 cells were transfected for 24 h with 1 μg of a reporter gene expressing luciferase. (B) Transfection efficiency and (C) cell viability (PrestoBlue assay) were assessed at a 48 h time-point. Error bars indicate SD, n = 6. One-way ANOVA with Bonferroni’s post-test or one-way ANOVA with Dunnett’s test for the comparison of each treatment group with the control (for cell viability), *P < .05, **P < .01.

Fig. 4.

Multiple particle tracking of DNA NPs in ex vivo human CF sputum samples. The diffusion of DNA NPs at 0%, 40% and 60% PEGylation rates was investigated in freshly expectorated patient CF sputum samples. (A) Representative transmission electron micrographs of the respective NPs. Scale bar, 500 nm on original images and 250 nm on inserts. Following a 1 h incubation in sputum, movies of NP displacement were captured and analysed by MPT technology. (B) Representative trajectories of particles moving through CF sputum and (C) mean squared displacement (MSD) of particles. Dot plots of (D) ensemble-median MSD and (E) percentage mucus penetrating particles (MSD ≥ 1 μm²). Error bars indicate SD, n = 5–6 patient samples (> 100 DNA NPs tracked for each experiment). One-way ANOVA with Bonferroni’s post-tests; *P < .05, ** P < .01.

Fig. 5.

In vivo bio-distribution of PEGylated DNA complexes in healthy mouse lung models. Lung tissues were harvested 30 min following intratracheal administration of Cy5-labelled DNA NPs at 0% and 40% PEGylation rates. (A) Representative images of NP distribution in large airways and lung parenchyma following administration of the respective NPs (red). Scale bar, 0.2 mm. Cell nuclei were stained with DAPI (blue). Image-based quantification of (B) coverage of NPs in large airways and (C) distribution of NPs in the lung parenchyma. Error bars indicate SD, n = 3 mice/group (> 30 sections were analysed per mouse). Two-tailed Student’s t-test, *P < .05, **P < .01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

Transgene expression and safety profile of DNA NPs. (A) In vivo mouse lung transgene expression of luciferase activity following intratracheal administration of PEGylated DNA NPs. Acute toxicity of the complexes was assessed by (B) cell counts and (C) percentage of macrophages and neutrophils from BALF of treated lungs 24 h post administration. (D) Representative images of lung sections stained with haematoxylin and eosin. AW, airway, arrow indicates cellular infiltrate. Scale bar, 100 μm. (E) Histopathological scoring of lung inflammation. Error bars indicate SD, n = 8–9 mice/group for transgene expression and cell counts, n = 5 mice/group for histopathological scoring. One-way ANOVA with Bonferroni’s post-tests; *P < .05, **P < .01.