Highly compacted DNA nanoparticles with low MW PEG coatings: in vitro, ex vivo and in vivo evaluation

Abstract

Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-l-lysine and 10kDa polyethylene glycol (CK(30)PEG(10k)), mediate effective gene delivery to the brain, eyes and lungs in vivo. Nevertheless, we found that CK(30)PEG(10k) DNA nanoparticles are immobilized by mucoadhesive interactions in sputum that lines the lung airways of patients with cystic fibrosis (CF), which would presumably preclude the efficient delivery of cargo DNA to the underlying epithelium. We previously found that nanoparticles can rapidly penetrate human mucus secretions if they are densely coated with low MW PEG (2-5kDa), whereas nanoparticles with 10kDa PEG coatings were immobilized. We thus sought to reduce mucoadhesion of DNA nanoparticles by producing CK(30)PEG DNA nanoparticles with low MW PEG coatings. We examined the morphology, colloidal stability, nuclease resistance, diffusion in human sputum and in vivo gene transfer of CK(30)PEG DNA nanoparticles prepared using various PEG MWs. CK(30)PEG(10k) and CK(30)PEG(5k) formulations did not aggregate in saline, provided partial protection against DNase I digestion and exhibited the highest gene transfer to lung airways following inhalation in BALB/c mice. However, all DNA nanoparticle formulations were immobilized in freshly expectorated human CF sputum, likely due to inadequate PEG surface coverage.

Figure 1

Transmission electron microscopy of compacted DNA nanoparticles formulated with (A) CK₃₀PEG_10k (B) CK₃₀PEG_5k and (C) CK₃₀PEG_2k. Scale bar represents 200 nm.

Figure 2

Colloidal stability of rod-shaped DNA nanoparticles after storage at 4°C for 6 months. (A) Turbidity parameters, an indicator of colloidal stability, for various DNA nanoparticle formulations. A turbidity parameter less than −3.5 (above the dashed line) indicates DNA nanoparticles that are compacted and non-aggregated, whereas values greater than −3.5 (below the dashed line) indicate significant aggregation of DNA nanoparticles. (B) Non-aggregated fraction of compacted DNA nanoparticles as determined by sedimentation. Data represents the average of 3 independent experiments +/− the standard deviation. * denotes statistical significance (* P < 0.05, ** P < 0.01).

Figure 3

Stability against DNase I digestion over time of (A) free DNA, or DNA compacted with (B) CK₃₀PEG_10k, (C) CK₃₀PEG_5k, or (D) CK₃₀PEG_2k. Upper and lower bands represent nicked circular and supercoiled conformations of DNA.

Figure 4

In vivo gene expression following pulmonary administration. BALB/c mice (n = 8) received 50 µg compacted or naked pd1GL3-RL DNA in saline by oropharyngeal aspiration. Control mice (n=6) received saline alone. Luciferase activity in lung homogenates was measured 24 hrs after pulmonary administration. Data represents the average +/− the standard deviation. * denotes statistical significance (** P < 0.01).

Figure 5

Transport of rod-shaped DNA nanoparticles and PEG coated polystyrene (PS-PEG_2k) nanoparticles in fresh, undiluted CF sputum. (A) Ensemble-averaged geometric mean square displacement (<MSD>) of compacted DNA nanoparticles and PS-PEG_2k in CF sputum as a function of time scale (τ). The slope of m = 1 corresponds to unobstructed diffusive behavior. Data represents 3 independent experiments, with n ≥120 particles per experiment. Error bars represent standard error of the mean. (B) Representative trajectories of CK₃₀PEG_10k DNA nanoparticles and 210 nm PS-PEG_2k particles in CF sputum during 20 s movies. (C) The fraction of particles predicted to penetrate a 10 µm think CF sputum layer over time using Fick’s second law and diffusion coefficients obtained from tracking experiments.

Figure 6

Schematic illustration of PEG regimes. The mushroom regime exists when neighboring PEG chains do not overlap, [Γ/SA] < 1, where Γ is the total unconstrained PEG surface area coverage and [SA] is the total particle surface area [27]. The brush regime exists when neighboring PEG chains overlap, [Γ/SA] > 1, and stretch away from the surface.