The cytosolic space of cells is an important target for drug delivery systems and intracellular pathogens. Many drugs are membrane-impermeant because of their large molecular size and hydrophilic characteristics; therefore the need for their specialized, efficient cytosolic delivery strategy is greater than ever. Listeria monocytogenes enters endocytic compartments and utilizes the specialized mechanism of the pore-forming protein, Listeriolysin O (LLO), to breach the endosomal membrane to escape into the cytosol. LLO has been utilized in drug delivery systems and has demonstrated its powerful utility as an endosomolytic agent in generating non-viral/nonbacterial delivery systems that mimic the Listeria invasion to deliver exogenous macromolecules into the cytosol. The molecular mechanism of the necessary and sufficient agent, LLO, is still not clearly understood. Specifically, its pH-dependent activity and its molecular regulators, such as the required reduction of the unique single cysteine, are not clear at this point. The goal of the current grant proposal is to understand the key elements of LLO activity and its regulatory mechanisms, likely dependent on cell types and cellular differentiation and metabolic states, and to use this knowledge to better design cytosolic delivery and targeting strategies in the future. Key questions to be addressed are: (i) LLO interaction with cholesterol-containing membranes, its membrane-binding domain, pH sensitive elements, on/off rates of binding, oligomerization rate, and how and which domains of LLO regulate these processes, (ii) how the unique cysteine of LLO is reduced in the endocytic pathway to activate the LLO activity and how that is regulated, and (iii) how delivery strategies are affected by various regulatory factors of LLO. As the reduction of disulfide bonds in the endocytic compartment is essential in many cases of pathogenesis and drug delivery systems as much as in the regulation of LLO activity, a significant part of the proposal is dedicated to designing and characterizing probes to monitor the reduction in endosomes in general and to investigate cellular factors modulating the reduction processes. The results and information obtained from the proposed research will be extremely important and critical not only for the rational design of LLO-mediated macromolecule delivery and for the long-term strategy of efficient cytosolic delivery but also for any drug delivery strategy utilizing reversible disulfide bonds, as well as for the elucidation of the Listeria invasion mechanism. ? ?
| Sun, Xun; Provoda, Chester; Lee, Kyung-Dall (2010) Enhanced in vivo gene expression mediated by listeriolysin O incorporated anionic LPDII: Its utility in cytotoxic T lymphocyte-inducing DNA vaccine. J Control Release 148:219-25 |
| Choi, Suna; Lee, Kyung-Dall (2008) Enhanced gene delivery using disulfide-crosslinked low molecular weight polyethylenimine with listeriolysin o-polyethylenimine disulfide conjugate. J Control Release 131:70-6 |
| Stier, Ethan M; Mandal, Manas; Lee, Kyung-Dall (2005) Differential cytosolic delivery and presentation of antigen by listeriolysin O-liposomes to macrophages and dendritic cells. Mol Pharm 2:74-82 |
