Targeting Triple Negative Breast Cancer Cells with Novel Cytotoxic Peptide–Doxorubicin Conjugates

Abstract

In this study, we have designed and synthesized two novel peptide-drug conjugates (PDCs) where the drug, doxorubicin (Dox), is linked to the peptide via a succinimidyl thioether bond or a hydrazone linker. A highly specific and proteolytically stable breast cancer cell targeting peptide (WxEAAYQrFL) is conjugated to Dox to synthesize peptide-Dox thioether (1) or hydrazone (2) conjugate. The evaluation of the stability in water, media, and human serum showed that the conjugate 1 with the succinimidyl thioether linkage is more stable compared to the acid-sensitive hydrazone containing conjugate 2. The cytotoxicity studies showed that the two PDCs were as toxic as free Dox toward the triple negative breast cancer (TNBC) cells and were 7-30 times less toxic (IC50 1.2-4.7 μM for TNBC cells versus 15-39 μM for non-cancerous cells) toward the noncancerous breast cells compared to the free doxorubicin (IC50 0.35-1.5 μM for TNBC cells versus 0.24 μM for non-cancerous cells). The results from the comparative study of the two PDCs suggest that both may have translational potential for TNBC treatment.

Evaluation of a Keratin 1 Targeting Peptide-Doxorubicin Conjugate in a Mouse Model of Triple-Negative Breast Cancer

“…The hydrazone group in the conjugate facilitates the release of the drug under mildly acidic conditions; however, it also makes it less stable. Previously, we found that when incubated with 25% human serum, the conjugate showed a half-life of ≈6 h, mainly cleaving at the hydrazone bond, giving unmodified Dox and the peptide with the linker portion ( Figure 1 ) [ 29 ]. After intravenous administration in mice, the PDC showed a half-life of around 1.7 h in blood ( Figure 2 c), which incidentally is much longer than the half-life of Dox (in min) [ 35 ].…”

“…We used a linear 18-4 peptide that was engineered to bind breast cancer cells via the cell-surface K1 receptor [23,28] The cellular uptake of peptide 18-4 and its PDC takes place via receptor-mediated endocytosis in breast cancer cells [25,48]. A PDC (Figure 1) using linear 18-4 peptide was synthesized to evaluate the specificity of the conjugate toward TNBC cells [29]. The conjugate showed specific toxicity toward the TNBC cells (IC 50 1.2-2.2 µM) and was 9-fold less toxic to normal breast tissue-derived MCF-10A cells (IC 50 15.1 µM).…”

“…Peptide-Dox conjugate (PDC, Figure 1 ) was synthesized and purified, as described previously [ 29 ]. Doxorubicin hydrochloride salt (Dox.HCl) was purchased from LC Laboratories (Woburn, MA, USA), while daunorubicin hydrochloride (Dau) and aldoxorubicin (Aldox) were from MedChem Express (Monmouth Junction, NJ, USA).…”

“…We engineered peptides, such as linear 18-4 and cyclic analogues, for specific uptake by breast cancer cells (MCF-7 and MDA-MB-231) via cell surface K1 mediated endocytosis [ 25 , 27 ]. Further, K1 targeting linear peptide 18-4 was used to synthesize four peptide-doxorubicin conjugates with different linker chemistries, such as ester, amide, succinimidyl thioether, and hydrazone [ 28 , 29 ]. We showed specific uptake of the targeted PDCs via receptor mediated endocytosis in MCF-7 and MDA-MB-435-MDR cancer cells [ 28 ].…”

“…We showed specific uptake of the targeted PDCs via receptor mediated endocytosis in MCF-7 and MDA-MB-435-MDR cancer cells [28]. The PDCs with K1 targeting peptide 18-4 were more cytotoxic to TNBC cells (IC 50 1.2-4.7 µM) compared to non-cancerous human mammary epithelial MCF-10A cells (IC 50 15.1-38.6 µM), while free drug (doxorubicin) was equally cytotoxic to both cancer and non-cancerous cells (IC 50 0.24-1.5 µM) [29]. To explore the in vivo efficacy and evaluate the potential of K1 targeting PDC for TNBC treatment, we report here the antitumor activity of one of these peptide-doxorubicin conjugates (Figure 1), where the peptide (18-4) is conjugated to Dox via an acid-sensitive N-acyl hydrazone linker in a mouse model for TNBC.…”