Gene and drug delivery by gelatin nanospheres for cystic fibrosis.

摘要

Research into new clinical therapies for cystic fibrosis has focused primarily on either pharmacological agents or gene delivery systems. This study examines the potential of gelatin nanospheres to simultaneously deliver drugs and genes to induce CFTR expression in targeted cells. Nanospheres with an average size of 230nm were formed by the complex coacervation of gelatin and DNA at appropriate conditions of salt, temperature, and mixing speed. Drugs such as chloroquine and sodium 4-phenylbutyrate (PBA) were easily coencapsulated by their inclusion in the reaction mixture. Sustained release of DNA and PBA was demonstrated in vitro by protease digestion of the nanospheres and could be controlled by varying the crosslinking density of the gelatin matrix. The optimal nanosphere formulation for gene transfection was determined by comparing luciferase expression in 293 cells and included: transferrin conjugation for cell targeting and uptake, chloroquine coencapsulation for endosomal escape, and surface adsorbed calcium. Gelatin nanospheres were shown to efficiently transfect a human tracheal epithelial cell line (9HTE) and deliver a human CFTR cDNA to rabbit airway epithelial cells. Rabbits treated with the nanospheres; showed airway staining that was localized to the apical membrane surface of epithelial cells, demonstrating that the expressed CFTR was correctly trafficked to its site of function. Nanosphere DNA persisted in the nucleus of epithelial cells for at least one month. Airway expression in rabbit lungs was confirmed using a green fluorescence protein (GFP) gene encapsulated in gelatin nanospheres. Bronchial epithelial brushings obtained from nanosphere-treated and control airways showed strong GFP fluorescence in a high percentage of cells. These results demonstrated the efficacy of gelatin nanospheres as an in vivo gene delivery vehicle for CF.; An attractive drug candidate for CF drug therapy is sodium 4-phenylbutyrate (PBA). The action of PBA has been shown to restore CFIR function on the plasma membrane of ΔF508-expressing bronchial epithelial cells in vitro . Nanospheres with encapsulated PBA were tested on the bronchial epithelial cell line IB3-1 (ΔF508/W1282X) and examined for CFIR induction. Induction of CFIR expression was detected by antibody staining in nanosphere and free PBA treated cells. However, the intensity of staining was greater in nanosphere-treated cells although the dose of PBA in nanospheres was about 2500 times less than the free drug dose. The role of PBA in upregulating certain gene promoters was tested with a CAT gene driven by the CMV promoter. Nanospheres including both the CAT construct and PBA transfected cells 2 to 10 times more efficiently than in the absence of the drug. These effects strongly indicate that nanospheres can deliver a high and sustained local dose of PBA inside cells, thus improving bioavailability of the drug. This report demonstrates the potential for a combined gene and drug delivery vehicle to more effectively correct CF chloride conductance than either gene or drug alone in patients with the ΔF508 mutation.