Dendritic nanostructures can play a key role in drug delivery, due to the high density and variety of surface functional groups that can facilitate and modulate the delivery process. We have investigated the effect of dendrimer end-functionality on the activity of polyamido amine (PAMAM) dendrimer-methotrexate (MTX) conjugates in MTX-sensitive and MTX-resistant human acute lymphoblastoid leukemia (CCRF-CEM) and Chinese hamster ovary (CHO) cell lines. Two amide-bonded PAMAM dendrimer-MTX conjugates were prepared using a dicyclohexylcarbodiimide (DCC) coupling reaction: one between a carboxylic acid-terminated G2.5 dendrimer and the amine groups of the MTX (conjugate A) and another between an amine-terminated G3 dendrimer and the carboxylic acid group of the MTX (conjugate B). Our studies suggest that conjugate A showed an increased drug activity compared to an equimolar amount of free MTX toward both sensitive and resistant cell lines, whereas conjugate B did not show significant activity on any of the cell lines. Despite substantially impaired MTX transport by MTX-resistant CEM/MTX and RII cells, conjugate A showed sensitivity increases of approximately 8- and 24-fold (based on IC50 values), respectively, compared to free MTX. Co-incubation of the cells with adenosine and thymidine along with either conjugate A or MTX resulted in almost complete protection, suggesting that the conjugate achieves its effect on dihyrofolate reductase (DHFR) enzyme through the same mechanism as that of MTX. The differences in cytotoxicity of these amide-bonded conjugates may be indicative of differences in the intracellular drug release from the cationic dendrimer (conjugate B) versus the anionic dendrimer (conjugate A), perhaps due to the differences in lysosomal residence times dictated by the surface functionality. These findings demonstrate the feasibility of using dendrimers as drug delivery vehicles for achieving higher therapeutic effects in chemotherapy, especially in drug-resistant cells.

In this report, this region was further refined to TMDs 11-12 by digestion with 2-nitro-5-thiocyanatobenzoic acid. A transportcompetent cysteine-less hRFC was used as a template to prepare single cysteine-replacement mutant constructs in which each residue from Glu-394 to Asp-420 of TMD 11 and Tyr-435 to His-457 of TMD 12 was replaced individually by a cysteine. The mutant constructs were transfected into hRFC-null HeLa cells. Most of the 50 single cysteine-substituted constructs were expressed at high levels on Western blots. With the exception of G401C hRFC, all mutants were active for Mtx transport. Treatment with sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) had no effect on hRFC activity for all of the cysteine mutants within TMD 12 and for the majority of the cysteine mutants within TMD 11. However, MTSES inhibited Mtx uptake by the T404C, A407C, T408C, T412C, F416C, I417C, V418C, and S419C mutants by 25-65%. Losses of activity by MTSES treatment for T404C, A407C, T412C, and I417C hRFCs were appreciably reversed in the presence of excess leucovorin, a hRFC substrate. Our results strongly suggest that residues within TMD 11 are likely critical structural and/or functional components of the putative hRFC transmembrane channel for anionic folate and anti-folate substrates.

Reduced folates such as 5-methyl tetrahydrofolate and classical antifolates such as methotrexate are actively transported into mammalian cells by the reduced folate carrier (RFC). RFC is characterized by 12 stretches of mostly hydrophobic, ␣-helix-promoting amino acids, internally oriented N and C termini, and a large central linker connecting transmembrane domains (TMDs) 1-6 and 7-12. Previous studies showed that deletion of the majority of the central loop domain between TMDs 6 and 7 abolished transport, but this segment could be replaced with mostly non-homologous sequence from the SLC19A2 thiamine transporter to restore transport function. In this report, we expressed RFC from separate TMD1-6 and TMD7-12 RFC halfmolecule constructs, each with a unique epitope tag, in RFC-null K562 cells to restore transport activity. Restored transport exhibited characteristic transport kinetics for methotrexate, a capacity for trans-stimulation by pretreatment with leucovorin, and inhibition by Nhydroxysuccinimide methotrexate, a documented affinity inhibitor of RFC. The TMD1-6 half-molecule migrated on SDS gels as a 38 -58 kDa glycosylated species and was converted to 27 kDa by N-glycosidase F or tunicamycin treatments. The 40 kDa TMD7-12 half-molecule was unaffected by these treatments. Using transfected cells expressing both TMDs 1-6 and TMDs 7-12 as separate polypeptides, the TMD7-12 half-molecule was covalently radiolabeled with N-hydroxysuccinimide [ 3 H]methotrexate. No radioactivity was incorporated into the TMD1-6 half-molecule. Digestion with endoproteinase GluC decreased the size of the radiolabeled 40 kDa TMD7-12 polypeptide to ϳ20 kDa. Our results demonstrate that a functional RFC can be reconstituted with RFC half-molecules and localize a critical substrate binding domain to within TMDs 7-12.