Delivery of nucleic acids to ex vivo porcine airways using electrospray

Aim of the Study: Nucleic acid-based therapies have the potential to provide clinically meaningful benefit across a wide spectrum of lung disease. However, in vivo delivery remains a challenge. Here we examined the feasibility of using electrospray to deliver nucleic acids to both porcine tracheal tissue sections and whole lung ex vivo. Materials and Methods: The effect of electrospray solution, emitter gauge, flow rate and voltage on plasmid DNA integrity was examined by analyzing supercoiled:open circle structure ratio by gel electrophoresis. Optimal parameters were used to deliver luciferase DNA and mRNA and siRNA-FITC to tracheal tissue sections. Luciferase mRNA was delivered to whole porcine lungs ex vivo using a catheter and bronchoscope system. Luciferase activity and fluorescence were analyzed by luminometry and microscopy respectively. Results: The incidence of DNA plasmid nicking was greatest in a low salt solution without ethanol compared with 1% and 20% ethanol with salt. From a range of emitters tested, a 32 gauge emitter produced the best supercoiled:open circle structure ratio, likely because less voltage was required to produce a stable electrospray with this emitter. Lower flow rates also showed a trend towards reduced DNA nicking. GFP DNA electrosprayed at 5 kV and 6 kV resulted in lower levels of GFP expression in A549 lung cells following lipofection compared with 3 kV and 4 kV. Optimised parameters of 20% ethanol solution, 32 gauge emitter, low flow rates and voltages of 3–5 kV, nucleic acid molecules were successful for delivery of luciferase DNA and mRNA as well as siRNA-FITC to porcine tracheal tissue sections and for delivery of luciferase mRNA to whole porcine lungs via bronchoscope. Conclusions: We report ex vivo delivery of nucleic acids to porcine lung tissue via electrospray and bronchoscopic electrospray delivery of nucleic acid to an ex vivo porcine lung model

Anatomy and Bronchoscopy of the Porcine Lung. A Model for Translational Respiratory Medicine

The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome. As a result, the role of this model is likely to become even more important. For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. An increasing number of lung conditions are being studied and modeled in the pig. Genetically modified porcine models of cystic fibrosis have been generated that, unlike mouse models, develop lung disease similar to human cystic fibrosis. However, the scientific literature relating specifically to porcine lung anatomy and airway histology is limited and is largely restricted to veterinary literature and textbooks. Furthermore, methods for in vivo lung procedures in the pig are rarely described. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model

Delivery and viability compared with electroporation.

<p>Comparison of delivery efficiency (using 10kDa dextran-Alexa488) and cell viability and survival (using propidium iodide exclusion) for (A) the reversible permeabilization method and (B) electroporation. (C) The transfection score defined as (transfected cells/ total cells)x(viable cells/ total cells) for the two methods). <i>n</i> = 3, data are depicted as the mean ± standard deviation.</p

Cell-independent delivery.

<p>(A) Delivery of 3 μM 10 kDa dextran-Alexa488 to primary human fibroblasts and primary human MSC. (B) Efficiency of delivery was quantified by flow cytometry at 2 hr post-delivery. (C) Delivery of BSA-FITC to U266 and Jurkat suspension cells. (D) Efficiency of delivery was quantified by flow cytometry at 2 hr post-delivery. All photomicrographs are 10x magnification. <i>n</i> = 3, data are depicted as the mean ± the standard deviation. (MSC = mesenchymal stem cells; BSA-FITC = bovine serum albumin-fluorescein isothiocyanate).</p

Testing mechanisms of cargo uptake and subsequent membrane resealing.

<p>(A) Time course of uptake of 10 kDa dextran-Alexa488 into A549 cells analyzed by fluorescence microscopy consistent with simple diffusion post-delivery (10x mag.). (B) In A549 cells the uptake of EGFP mRNA was not inhibited either by pretreatment with Dynasore or chloropromazine to inhibit clathrin-mediated endocytosis or Nystatin or EIPA to inhibit caveolar-mediated endocytosis and micropinocytosis. (C) Lipofectamine 2000 was used as a positive control for endocytosis-mediated delivery. EGFP expression was reduced in lipofected cells treated with Dynasore. (D) PI uptake was analyzed by flow cytometry and the data indicate that the cells remain permeable to PI for up to 6 min post-treatment but then reseal and prevent uptake thereafter. <i>n</i> = 3, data are depicted as the mean ± standard deviation. (EIPA = 5-(N-Ethyl-N-isopropyl)amiloride; EGFP = enhanced green fluorescent protein; PI = propidium iodide; PBS = phosphate buffered saline).</p

Cell functionality and intracellular targeting.

<p>(A) Alexa Fluor^® 488-labelled tyramide substrate demonstrated activity and localization of HRP in CHO cells following delivery of HRP. (B) Increasing production of fluorescent DCF product with increasing dose of HRP delivered into CHO cells compared with cells where HRP was delivered by pipette. (C) GFP expression following delivery of GFP mRNA. (D) Cell viability remained above 75% up to 168hr post-delivery. (E) Up to 3 doses of GFP mRNA (4 μg) were delivered. GFP expression was analyzed 24 hr after the final dose. (F) Confocal microscopy image illustrates co-delivery to A549 cells: DAPI (300 nM), Mitotracker Red (50 μM) and Phalloidin-Alexa488 (0.33 μM) correspond to blue nuclei, red mitochondria and green actin filaments, respectively. <i>n</i> = 3, data are depicted as the mean ± the standard deviation. (HRP = horseradish peroxidase; DCF = dichlorofluorescein; GFP = green fluorescent protein; DAPI = 4',6-diamidino-2-phenylindole).</p

Delivery solution.

<p>(A) 150 μM PI or 3 μM 10 kDa Dextran-Alexa488 in 200 μl delivery solution was delivered to A549 cells using a micropipette. Immediately after delivery, uptake of PI was visible throughout the cell population but no uptake of dextran was apparent. With 20 μl delivery solution, PI uptake was apparent where the solution first landed in the well (drop zone) but not in other areas. Low level uptake of 10 kDa Dextran-Alexa488 was also observed in the drop zone. (B) LDH release measured at 24 hr post-delivery indicated that 37.2±4.8%, 44.6±1.9% and 51.4±4.7% cells were damaged when 200 μl delivery solution alone, delivery solution containing PI or delivery solution containing 10 kDa Dextran-Alexa488 respectively was applied. In contrast, LDH release was 5.1±6.0, 10.5±1.3% and 5.6±3.1% respectively for these solutions when a 20 μl volume was applied. All photomicrographs are 10x magnification. <i>n</i> = 3, data are depicted as the mean ± standard deviation. (DS = delivery solution only; PI = propidium iodide; LDH = lactate dehydrogenase).</p

Examples of delivery of diverse cargoes to CHO cells.

<p>(A) Proteins of varying sizes were labelled with FITC and 4 μg protein per well was delivered to CHO cells and analyzed by fluorescence microscopy at 2 hr post-delivery, 10x magnification. (B) Increased efficiency of delivery of beta-lactoglobulin was demonstrated with increasing concentration of protein delivered. (C) Full length anti-rabbit-Alexa488 secondary antibody was successfully delivered. (D) GFP mRNA (5 μg) was delivered twice (10 μg/well in total) into cells using the permeabilization method and expression of GFP protein was observed by fluorescence microscopy at 24 hr post-delivery. (E) Luciferase mRNA (5 μg) was delivered twice (10 μg/well total) into cells using the permeabilization method and expression of luciferase was quantified by luminometry at 24 hr post-delivery. For lipofection, luciferase mRNA (5 μg) was delivered per well. (F) pGFP (5 μg) was delivered twice (10 μg/well total) into cells using the permeabilization method and expression of GFP protein was observed by fluorescence microscopy at 24 hr post-delivery. (G) pGLuc (10 μg) was delivered into cells using the permeabilization method and expression of luciferase was quantified by luminometry at 24 hr post-delivery. For lipofection, 0.5 μg pGLuc was delivered per well. <i>n</i> = 3, data are depicted as the mean ± standard deviation. (GFP = green fluorescent protein; pGFP = plasmid encoding GFP; pGLuc = plasmid encoding <i>Guassia</i> luciferase).</p