Folate-mediated delivery of macromolecules into cultured animal cells

Christopher Paul Leamon, Purdue University

Abstract

Under physiological conditions, folic acid enters some cells via a membrane-bound folate-binding protein (FBP) receptor participating in endocytosis, while in other cells its entry is facilitated by a membrane transport protein. Like the free vitamin, folic acid-protein conjugates were found to bind and enter receptor-bearing cells via an endocytic process. Evidence for this mechanism of internalization was obtained from uptake studies using radiolabeled or fluorescent protein-folate constructs. Macromolecules entering cells via this pathway were also capable of reaching the cytosol in an intact functionally active form, as demonstrated by the specific killing of cells treated with folate conjugates of ribosome-inactivating proteins. Using electron microscopic localization techniques, folate-protein conjugates were found to be internalized by uncoated pits which matured into intracellular endosomes and multivesicular bodies. These conjugates were also found to escape from these internal compartments into the cytosol through either endosomal-Golgi interactions or as a consequence of endosomal fusion. Because over-expression of FBP receptors is a characteristic marker for cell transformation, it was postulated that receptor-bearing transformed cells may be selectively killed if treated with folate-toxin conjugates. To demonstrate this selectivity, non-transformed human cells were co-cultured with transformed FBP-positive human cells. The mixed-cell cultures were then treated with a variety of folate-toxin conjugates. The results showed that the malignant FBP-positive cells were selectively targeted and destroyed whereas the nontransformed cells were allowed to proliferate normally. Although many FBP-positive cell lines were selectively killed using this strategy, several cell lines were found to resist this treatment. Because it was believed that the folate-toxin conjugates within these cells could not escape from internal vesicle compartments, special endosomal translocation-competent conjugates were prepared and tested for cytotoxicity to these "resistant" cell types. The results of these studies showed that these "resistant" cells could in fact be selectively targeted and destroyed with the translocatable conjugates.

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Biochemistry|Chemistry|Oncology|Pharmacology

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