When utilized as a macromolecular drug targeting ligand, folic acid (Pte-Glu) has traditionally been coupled to peptides, proteins and lipids via one of its two carboxylate groups fortuitously located within a distal glutamyl moiety. It has been assumed in the literature that the gamma-glutamyl carboxylate of Pte-Glu is the preferred conjugation site for macromolecules enduring endocytosis via the folate-binding protein receptor. However, it is also possible that the steric placement of the attached macromolecule around the vitamin's pteridine moiety may be the more influential parameter controlling this delivery mechanism. Using solid-phase chemistries, we have synthesized dipeptide derivatives of pteroic acid for the purpose of identifying the preferred site onto which a macromolecule can be chemically attached without compromising its endocytosis potential. Thus, using fluorescent and radiolabeled conjugates, we have determined that macromolecules attached to Pte-Glu by either an alpha- or gamma-glutamyl linkage could associate with receptor-bearing cells at virtually identical levels. We further discovered that removal of the remaining un-conjugated glutamyl carboxylate had no inhibitory effect on cell uptake; and, the cytotoxicity of related momordin toxin conjugates were comparable among the various pteroate derivatives tested. From these observations we suggest that the preparation of endocytosis-competent pteroate-macromolecule conjugates is strongly influenced by the steric environment around the ligand's para-aminobenzoic acid moiety, and that no selective isomeric (i.e. alphaGlu versus gammaGlu) conjugation requirement necessarily exists.

Poly(ethylene glycol) of various sizes was used as a molecular spacer to separate the cell-targeting ligand, folate, from the surface of poly-L-lysine. The resulting ternary macromolecule (pLys-PEG-folate) was investigated in various formulations for its ability to transfect reporter plasmids into receptor-bearing HeLa and IGROV cell lines. Formulations were optimized with respect to DNA content, +/- charge ratio, and the size and amount of PEG substitution off the pLys backbone. Transfection activity was highest 48 h after sample introduction, and PEG 3400 was determined to be the most favorable spacer size tested. pLys-PEG-folate:DNA transfection was also found to be both concentration dependent and saturable; plus, it was blocked by the addition of excess-free folate, indicative of a specific mechanism of uptake. Transfection activity was virtually identical for complexes formed in 10% serum-supplemented media, deionized water, or Hepes buffer. And, cell viability remained greater than 85% at the highest concentrations of pLys-PEG-folate:DNA complexes tested (4.8 microg/mL pLys 331 000; 12 microg/mL DNA). Taken together, these observations provide evidence that pLys-PEG-folate:DNA complexes are taken up specifically by the folate endocytosis pathway, and that the intramolecular spatial distance of the ligand from the pLys backbone dramatically influences transfection.