Controlled release of free doxorubicin from peptide–drug conjugates by drug loading

Covalent modification of a drug with a peptide moiety has been extensively used as an effective strategy to improve the drug's therapeutic outcome. One important consideration in the design of such a prodrug is the release of the free drug from the covalently bound form in a desired fashion. In most cases, the free drug release rate is controlled by the use of various chemical linkers that bridge the drug to the auxiliary segment. We report here that the degree of drug conjugation per peptide could also regulate the drug release in addition to its apparent effect on drug loading of the resulting conjugates. In this work, we synthesized three peptide-drug conjugates (NTD, d-NTD and q-NTD) in which the cell penetrating peptide Tat is covalently connected to one, two, or four doxorubicin, respectively, through a cathepsin B degradable tetrapeptide linker (-Gly-Phe-Leu-Gly-). We found that the number of doxorubicin within the conjugate impacts the release of doxorubicin in a significant way, with q-NTD showing the slowest release rate while NTD showing the fastest release rate. Our cellular uptake experiments reveal that q-NTD accumulated most effectively within cancer cells while NTD shows the lowest intracellular accumulation concentration. Interestingly, our cell viability assessment using a SRB assay reveals that d-NTD is the most potent conjugate against HepG2 human liver cancer cells. These results suggest that intracellular accumulation efficiency and the free drug release rate are two important factors that determine the in vitro efficacy of drug conjugates. To further validate this conclusion, we conjugated a short hydrocarbon onto the NTD to improve its cellular uptake, and found that the resulting conjugate, C16NTD, exhibited comparable intracellular accumulation as the q-NTD conjugate but superior anticancer activity due to its more effective release of free doxorubicin.