Protein transduction domains (PTDs) have been used for transduction of biologically active molecules (a.k.a. cargos) such as siRNA, drugs, and proteins across plasma membranes, with green or red fluorescent proteins (FP) being the venue to monitor the delivery. The advantages of PTDs include low toxicity to the cell and high efficiency of transport across cytoplasm membrane. Recently, researchers have started to explore the use of luminescent semiconductor nanocrystals (quantum dots, QDs) to deliver and to monitor the delivery of biomolecules. The high quantum yield and photostability of QDs allows long term monitoring, an advantage for many biomedical applications. The proposed mechanisms by which PTDs and QDs enter cells are different. Cargo- conjugated QDs have been reported to cross plasma membrane via classic clathin- mediated endocytosis, while internalization of cargo-conjugated PTD is thought to occur via actin-dependent macropinocytosis. To take advantage of unique properties of QD and PTD, we propose to develop a QD-PTD delivery and monitoring system. Because both QD and PTD are used, it is unclear what the uptake mechanism of the QD-PTD-cargo will be;consequently the cargo may end up in different subcellular localization. Further, it remains to be elucidated whether the cargo is dissociated from the QD-PTD due to intracellular physical or biochemical reactions. If so, the dissociation may lead to misidentification of subcellular localization of the cargo. The goals of this proposed study are 1) to create an efficient biomolecule delivery and monitoring system with a photostable long-term imaging molecule, 2) to investigate potential dissociation between the cargo and the delivery system, and 3) to reveal mechanisms of cellular uptake and subcellular localization. To achieve our goals, we propose to establish a two-color """"""""QD-PTD- surrogate cargo"""""""" delivery and monitoring system and use live cell imaging to monitor uptake and localization. We will synthesize green fluorescent QD, mercaptopropionic acid passivated ZnS@CdSe. The PTD to be used for our studies is nona-arginine. Surrogate cargoes are red or green FP to test dissociation and subcellular localization. QD will be attached to PTD via a covalent conjugation. Both covalent and noncovalent bonding between PTD and FP will be studied. PTD-FP denotes covalent bonding, while PTD/FP indicates nocovalent bonding. The QD-PTD-FP or QD-PTD/FP will be added to a human bronchoalveolar carcinoma-derived cell line, A549, for imaging studies. For the dissociation study, red FP will be selected to contrast with green QD. Potential dissociation has not been studied for PTD or QD delivered cargoes, but is critical for evaluation of a delivery system. Green FP will be used for the study of cellular uptake and subcellular localization in conjunction with red or blue cellular markers. Since both QD and PTD are used simultaneously, it is unclear what the uptake mechanism will be. We will determine whether cellular uptake of QD-PTD/FP is macropinocytosis or endocytosis using specific inhibitors. For either uptake, the intracellular QD-PTD/FP is likely to be transported by actin and/or microtubules. Inhibitors will be used to determine if intracellular localization is transport-dependent. The significance and the long term applications of this proposed study are in the biomedical field. The QD-PTD can be used as an efficient system to deliver and monitor molecules into the cell. Other versatile QDs and PTDs can be tested in the future. FP used in this study can be replaced by functional proteins, receptors, and siRNA for medical diagnosis, therapeutical agent delivery, receptor studies, and molecular mechanistic studies that are relevant to lung diseases and cancers.
The biomolecule delivery system established from this project can be a useful research tool for basic biological studies which lead to understanding of the nature of human diseases. This system can be further developed to carry functional therapeutical agents for medical treatments of various diseases. Thus, the objective of this proposal is much relevant to public health.
