Actin limits enhancement of nanoparticle diffusion through cystic fibrosis sputum by mucolytics.
BACKGROUND
The secretions in the cystic fibrosis (CF) airways contains high concentrations of polymers, including the respiratory mucins and varying amounts of DNA and actin, the debris of an aggressive neutrophilic inflammatory response to infection. Physical and chemical interactions between these polymers contribute to the viscoelastic nature of a material that is hard to clear without the use of mucolytics. Secretions retained in the CF airway not only restrict airflow and invite infection, but also act as a barrier to the delivery of inhaled drugs and gene therapy vectors to the underlying airway epithelium. The aim of this investigation was to develop a simple, sensitive, assay to measure the diffusion of nanospheres the size of liposomal gene therapy vectors through CF sputum, and to model the polymer interactions that limit diffusion and the diffusion-enhancing activity of mucolytics.


METHODS
The diffusion of 200 nm fluorescent carboxylated nanospheres through CF sputum was investigated using a diffusion assay based on the micro-Boyden chamber. Atomic force microscopy (AFM) was used to visualise and measure the pore diameter in CF sputum. The effect of the mucolytics deoxyribonuclease (DNase), N-acetylcysteine and gelsolin on the diffusion of nanospheres though synthetic biogels comprising mixtures of DNA, mucin and F-actin was also investigated.


RESULTS
CF sputum significantly retarded the diffusion of 200 nm nanospheres. Pore diameter in CF sputum was highly variable, with a mean greater than 200 nm. At concentrations found in the CF airway, DNA (1-10 mg/ml) and mucin (25-50 mg/ml) also significantly reduced the diffusion of nanospheres. The barrier effects of DNA and mucin were not additive, and the additional presence of F-actin (5 mg/ml) did not influence diffusion of the nanospheres. However, actin (5mg/ml) completely inhibited the ability of DNase (2.9 microg/ml) and N-acetylcysteine (5 mM) to enhance diffusion. The activity of the mucolytics, DNase and N-acetylcysteine, was not restored by the addition of the actin depolymerising agent gelsolin (250nM).


CONCLUSIONS
Actin does not contribute to the barrier properties of CF sputum, but is a key determinant of the ability of mucolytics to enhance drug diffusion through synthetic and biological mucus.
Broughton-Head
Victoria J
VJ
School of Pharmacy and Biomedical Sciences, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, St Michael's Building, White Swan Road, Portsmouth, PO1 2DT, UK.
Smith
James R
JR
Shur
Jagdeep
J
Shute
Janis K
JK
D000111:Acetylcysteine / Q000494:pharmacology; D000199:Actins / Q000378:metabolism*; D000293:Adolescent; D000328:Adult; D003550:Cystic Fibrosis / Q000503:physiopathology*; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000494:pharmacology; D004058:Diffusion; D004587:Electrophoresis, Agar Gel; D005100:Expectorants / Q000494:pharmacology*; D018260:Gelsolin / Q000494:pharmacology; D015316:Genetic Therapy; D006801:Humans; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force / Q000379:methods*; D009077:Mucins / Q000378:metabolism; D053758:Nanoparticles; D010316:Particle Size; D013183:Sputum / Q000378:metabolism*
D000199:Actins; D005100:Expectorants; D018260:Gelsolin; D009077:Mucins; D004247:DNA; D003851:Deoxyribonucleases; D000111:Acetylcysteine