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Endosome is a major barrier to efficient gene transfection by synthetic vectors because if the vectors are trapped in the endosome, they will traffic to the lysosome where the DNA is enzymatically degraded. Our hypothesis which serves as the rationale for the design of ortho ester-based lipids and lipoplexes is that cationic lipids which can quickly hydrolyze into membrane-destabilizing fragments in response to a small drop of pH should improve the gene transfection efficiency by facilitating the endosome escape. We designed and synthesized five ortho ester-based acid-labile cationic lipids ( 1-5 ) and developed nine lipoplexes comprising the five lipids. HPLC and LC/MS studies revealed that the ortho ester linkage in lipids ( 1-5 ) hydrolyzed at mildly acidic endosomal pH 5.5. Dioleyl glycerol was identified to be the major hydrolysis product of lipids 1, 2, and 3 . Oleoyl alcohol and 1-oleyloxy-2-trimethylamionium-3-propanol were identified to be the major hydrolysis products of lipids 4 and 5 . Photon Correlation Spectrometry (PCS) studies revealed that acidic endosomal pHs triggered the aggregation of lipoplexes comprising 1, 2, and 3 . Lipoplexes comprising 4 and 5 retained their size over 50 hours at acidic pHs. The fluorescence assay indicated that the ortho ester-based lipoplexes comprising lipids 1, 2, and 3 quickly destabilized a model biomembrane in response to the acidic pH. Acidic pH did not cause the membrane destabilization by lipoplexes comprising 4 and 5 . These results demonstrate that ortho ester-based lipoplexes comprising lipids 1, 2, and 3 hydrolyze into membrane-destabilizing fragments in response to acidic pH. Luciferase gene transfection was conducted on CV-1cultured cells. The lipoplexes comprising ortho ester-based cationic lipids 1, 2, and 3 significantly enhanced the luciferase gene expression. Two lipoplexes 2 /DOPE/DNA and 3 /DOPE/DNA mediated 45-fold and 116-fold, respectively, higher luciferase expression in CV-1 cells compared to the pH-insensitive lipoplex DOTAP/DOPE/DNA. The gene transfection efficiency correlates well with the pH-triggered membrane-destabilization by the ortho ester-based lipoplexes.

The overall goal of this project was to develop, optimize, and test an ultrasound-responsive targeted nanobubble for delivering osteoporosis-related silencing genes such as Cathepsin K small interfering RNA (CTSK siRNA) for osteoporosis treatment. The nanobubbles were synthesized using an in situ sonochemical method. The nanobubble (NB) is composed of a gas core made from perfluorocarbon, stabilized with albumin, encapsulated with CTSK siRNA, and embedded with alendronate (AL) for bone targeting (CTSK siRNA-NB-AL). Following its development, the responsiveness of CTSK siRNA-NB-AL to a therapeutic ultrasound probe was examined. The results of biocompatibility tests with human bone marrow-derived mesenchymal stem cells proved no significant cell death (p > 0.05). When the CTSK siRNA-NB-AL was supplemented with human osteoclast precursors, they suppressed osteoclastogenesis. Thus, this project establishes the potential of nanotechnology and ultrasound to deliver genes into the osteoclasts. This research also presents a novel ultrasound responsive and targeted nanobubble platform that can be used as a gene, drug, and/or oxygen delivery system for various diseases including cancer, neurodegenerative diseases, or bone disorders.

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