Mechanism of internalization of an ICAM-1-derived peptide by human leukemic cell line HL-60: influence of physicochemical properties on targeted drug delivery.

Peptide-mediated targeted delivery offers an attractive strategy for selective delivery of cytotoxic drugs to cancer cells. In this work, we have investigated the mechanism of internalization of cIBR peptide [cyclo(1,12)PenPRGGSVLVTGC] that is conjugated with fluorescein isothiocyanate (FITC) and doxorubicin (DOX) to give FITC-cIBR and DOX-cIBR conjugates, respectively. Internalization mechanisms of FITC-cIBR and DOX-cIBR were studied in LFA-1-expressing cells (HL-60) and LFA-1-deficient cells (HUVEC) under the following conditions: (a) at two different temperatures (4 and 37 degrees C), (b) in the presence of ATP-depleting agents (sodium azide and 2-deoxy- d-glucose), and (c) in the presence of a microtubule-disrupting agent (nocodazole). At 37 degrees C, FITC-cIBR was internalized by HL-60 cells and located in the endosomes; however, it was not internalized by LFA-1-deficient HUVEC. Incubation of FITC-cIBR at 4 degrees C or in the presence of nocodazole inhibited its endocytosis in HL-60 cells. The ATP inhibitors inhibited the internalization of FITC-cIBR but maintained its binding to cell surface receptors. In contrast, DOX-cIBR was diffusely distributed in the cytoplasm of LFA-1-expressing HL-60 cells following incubation at 37 degrees C. No inhibitory processes could block the entry or change the distribution pattern of DOX-cIBR into HL-60 cells, suggesting that DOX-cIBR uptake was not mediated by receptors such as LFA-1. DOX-cIBR was still found inside HUVEC, but with a distribution pattern somewhat different from that in HL-60 cells. The major entry mechanism of DOX-cIBR could be via passive diffusion because DOX-cIBR has an octanol/water distribution coefficient (Log D) of 1.15. Thus, DOX-cIBR is more lipophilic than FITC-cIBR with a Log D of 0.57. Therefore, the change in the hydrophobicity of the conjugate may alter the mechanism of entry of DOX-cIBR compared to that of FITC-cIBR. This study suggests that alteration of the physicochemical properties of drug-peptide conjugates can change the mode of uptake from receptor-mediated uptake to passive diffusion.