Formulation of Nucleic Acid with PH-Responsive Amphipathic Peptides for Pulmonary Delivery

This dissertation, "Formulation of Nucleic Acid With PH-responsive Amphipathic Peptides for Pulmonary Delivery" by Wanling, Liang, 梁婉玲, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Nucleic acids could be used as therapeutic agents for the treatment of many different diseases, but poor delivery limits their clinical application. A series of pH-responsive amphipathic peptides containing histidine or 2,3-diaminopropionic acid (Dap) derivatives, LAH and LADap peptides, were investigated in this study as nucleic acid carriers for the treatment of respiratory infectious disease. LAH and LADap peptides are cationic, amphipathic pH-responsive peptides. The major attractive property of these peptides is their ability to promote endosomal escape, hence enhance transfection efficiency. Both LAH and LADap peptides were able to form complexes with DNA and small interfering RNA (siRNA) through electrostatic interaction and facilitated cellular uptake via endocytosis. In acidic environments (e.g. in endosomes and lysosomes), the peptides were released from the complexes and changed their conformational structures to display membrane-destabilizing activity. Subsequently, nucleic acids were released from the endosomal/lysosomal compartments into the cytoplasm with improved transfection efficiency. These peptides were effective in transferring DNA and siRNA in various cell lines including human lung epithelial cells. In addition, they were less toxic than the commercially available lipid-based transfection reagent LipofectamineTM 2000. LAH4-L1 and LADap(Me)6-L1 were the two most efficient peptides of the LAH and LADap peptide series respectively, and were selected for further formulation development for pulmonary delivery. To determine the optimal carrier for delivering nucleic acids to the lungs, bronchoalveolar lavage fluid (BALF) was used as a model of airway surface liquid in the in vitro transfection experiments. High transfection efficiency was maintained with LADap(Me)6-L1 peptide, making it a promising vector for pulmonary nucleic acid delivery. Two drying methods, spray drying and spray freeze drying, were investigated to prepare dry powder formulation containing pH-responsive peptides and nucleic acids for inhalation. Both methods could produce powders with desirable aerodynamic properties for inhalation. The physical integrity and biological activity of the nucleic acids were also successfully retained. Overall, SD powders performed better than SFD powders, suggesting that spray drying was a more suitable method to produce inhalable nucleic acid formulation. The clinical potential of the spray-dried powder formulation the peptide-based nucleic acid delivery system was further investigated for the treatment of lung diseases. siRNA has considerable therapeutic potential for treating respiratory infectious diseases including influenza. By introducing siRNA targeting the conserved region of viral genes encoding nucleocapsid protein (NP), viral replication can be inhibited in mammalian cells. Powder formulations containing antiviral siRNA against H1N1 influenza virus and pH-responsive peptides were produced by spray drying. The SD powders were characterized and found to be suitable for inhalation with good stability. The formulations mediated highly effective in vitro delivery of antiviral siRNA into mammalian lung epithelial cells, leading to significant inhibition of viral replication when the transfected cells were subsequently challenged with H1N1 influenza virus. SD siRNA powders containing pH-responsive peptides are promising inhalable f