Tumor-specific targeting and intracellular delivery mediated by the folate endocytosis pathway

Susan Wang, Purdue University

Abstract

Folate-conjugates have been previously shown to enter living cells via receptor-mediated endocytosis. In this dissertation, it is further demonstrated that smaller molecules such as deferoxamine and DTPA and large drug carriers such as liposome can also be delivered via the same route. Radiolabeled folate-conjugates are used as tumor-selective imaging agents in animal models. Folate-targeted liposomes are capable of delivering anticancer drugs and oligonucleotides into receptor-bearing cell cultures. Folate-conjugated metal chelator deferoxamine (DF) is synthesized as a candidate for in vivo tumor imaging. The $\sp{67}$Ga complex of DF-folate is rapidly and specifically taken up by cultured KB cells with a t$\sb{1/2}$ of $\sim$3 min. Biodistribution study in athymic mice bearing tumor xenografts tumor implants results in a tumor-to-blood ratio of $\sim$400 4 h after administration. Folate-conjugated macromolecules or liposomes are internalized into acidified compartments by the cell via receptor-mediated endocytosis. The compartmental pH following folate-mediated endocytosis is determined by ratioing confocal images of cells dual-labeled with folate-DM-NERF-dextran, a pH indicator, and folate-Texas Red-dextran, a pH-insensitive reference. The compartments have an average pH value of 5.0. The low pH environment in these compartments may facilitate the cytoplasmic release of some liposome-encapsulated drugs such as doxorubicin. Folate-PEG-liposome-encapsulated antisense oligonucleotides are non-destructively delivered into cultured tumor cells overexpressing the folate receptor. The greatly enhanced growth inhibitory effects can be blocked by excess amount of free folic acid as the competing agent in the culture medium. Novel pH-sensitive liposome formulations are constructed by encapsulating the amphipathic peptide EALA into the liposomes or attaching the peptide on the liposome headgroups. A fluorometric assay using propidium iodide is developed to monitor the cytoplasmic release of the liposomal contents following endocytosis.

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Biochemistry|Pharmacology|Oncology|Pharmaceuticals

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