Guanidinium-rich cell-penetrating peptidomimetic-doxorubicin conjugates show linker-dependent, reduced acute toxicity to cardiomyocytes and normal breast epithelium while retaining activity against a HER2-positive breast cancer model (SKBR3 spheroids).

Abstract

Doxorubicin (DOX) is a cornerstone anthracycline in breast cancer therapy, but its use is constrained by cumulative cardiotoxicity. We asked whether conjugation to a guanidinium-rich cell-penetrating peptidomimetic widens the window between anticancer activity and cardiomyocyte toxicity, or merely rescales potency. DOX was linked by azide-alkyne click chemistry to an eight-guanidinium peptidomimetic, H-O2Oc-[Dap(GO₂)]₈-O2Oc-NH₂, through either a hexynoyl linker (CuAAC; DOX-ACA-HEX-G8) or a dibenzocyclooctyne linker (SPAAC; DOX-ACA-DBCO-G8), so that the pair differs in a single variable. Both conjugates and free DOX were tested in three-dimensional spheroids of HER2-positive SKBR3, triple-negative MDA-MB-231, non-tumorigenic mammary epithelium (HB2) and human AC16 cardiomyocytes (0.5-50 μM; 24, 48 and 72 h; CellTiter-Glo 3D), and in a three-read-out control panel (LDH, ATP, DNA). Free DOX was potent but non-selective, killing SKBR3, HB2 and AC16 with comparable 72 h IC₅₀ values (<0.5, 0.78 and <0.5 µM) and leaving no meaningful window. DOX-ACA-HEX-G8 matched DOX against SKBR3 at 48-72 h (IC₅₀ < 0.5 µM) while being 15-fold (HB2) and >18-fold (AC16) less toxic to the non-malignant lines. Because the SKBR3 IC₅₀ is censored for both DOX and DOX-ACA-HEX-G8, their selectivity indices cannot be compared; the window is therefore established directly at matched concentration, without extrapolation: at 0.5 µM/72 h free DOX left SKBR3 at 6% and AC16 at 14% of control (an 8-percentage-point separation), whereas DOX-ACA-HEX-G8 left SKBR3 at 32% and AC16 at 122% (90 percentage points). The linker-matched DOX-ACA-DBCO-G8 was less selective, showing the effect tracks with linker chemistry. In the triple-negative model, DOX-ACA-HEX-G8 gave no window at all, and DOX-ACA-DBCO-G8 only a marginal one (selectivity index 1.6 for AC16 and 2.3 for HB2, both versus MDA-MB-231), although both conjugates were internalized. Both produced a pronounced low-dose hormetic signal in the non-malignant lines; a cell-free spike-recovery control excluded an artifact of the luminescence read-out, and the biomass read-out excluded proliferation, so the effect is metabolic and its mechanism remains open. DOX-ACA-HEX-G8 is a lead for anthracycline delivery with reduced acute cardiomyocyte toxicity, contingent on mechanistic studies of uptake and drug release.