Enhancing cellular uptake of activable cell-penetrating peptide–doxorubicin conjugate by enzymatic cleavage

Figures & data

Figure 1 (A) Proposed schematic diagram of ACPP-DOX conjugate for antitumor drug delivery. ACPP-DOX conjugate is inactivated initially and circulates freely in vivo. When the ACPP-DOX conjugate accesses the tumor tissue, it is triggered and activated by overexpressed extracellular MMPs (MMP-2/9). The CPP-DOX releases and translocates into cells. The ACPP-DOX conjugate includes a cargo of doxorubicin, polycational CPP, polyanional attenuating peptides, and MMP-specific substrates. (B) Synthesis scheme of ACPP-DOX conjugates: (a) doxorubicin (DOX), (b) N-succinimidyl-3-maleimidopropionate (SMP), (c) DOX-SMP, (d) ACPP, an activatable cell-penetrating peptide. (e) ACPP-DOX, a derivative of DOX linked with the ACPP. (C) ¹H-NMR spectrum of DOX-SMP. (D) FT-IR spectra of SMP, DOX, and DOX-SMP.

Abbreviations: ACPP-DOX, activable cell-penetrating peptide–doxorubicin; CPP, cell-penetrating peptide; MMP, matrix metalloproteinase.

Figure 2 Enzymatic-triggered cleavage of ACPP-DOX conjugate by MMPs in different ratios.

Note: (MMPs)/(ACPP-DOX) (w/w) of (A) 0.463:1 (B) 0.288:1 (C) 0.173:1 (D) 0.114:1, and (E) absence of MMP treatment.

Abbreviations: ACPP-DOX, activable cell-penetrating peptide–doxorubicin; MMP, matrix metalloproteinase.

Figure 3 (A) The uptake intensity of MCF-7 cells after treatment with ACPP-DOX conjugate in the absence (−) or presence of extra collagenase IV at concentrations of 20 μg/mL and 100 μg/mL. (B) The uptake intensity of MCF-7 cells after treatment with ACPP-DOX conjugate in the absence (−) or presence of extra MMP-2 at concentrations of 20 ng/mL and 200 ng/mL. (C) Cellular uptake intensity–time curve of DOX and ACPP-DOX in MCF-7 and HT-1080 cells at different incubation times. (D) Confocal microscopy images of ACPP-DOX or DOX incubated with HT-1080 and MCF-7 cells at 37°C for 0 hour–8 hours.

Notes: Red represents fluorescence of DOX. Blue represents fluorescence of Hoechst 33258 for nuclei staining. Scale bars: 100 μm. *P < 0.001; ^#P < 0.05; ns = no significance.

Abbreviations: DOX, doxorubicin; ACPP-DOX, activable cell-penetrating peptide–doxorubicin; MMP-2, matrix metalloproteinase-2.

Figure 4 Antiproliferative activity of DOX, ACPP-DOX, and ACPP against MCF-7 and HT-1080 cells.

Notes: Antiproliferative activity was determined using the MTT method after incubation for 24 hours in vitro. Data are represented as the mean ± standard deviation (n = 6).

Abbreviations: DOX, doxorubicin; ACPP-DOX, activable cell-penetrating peptide–doxorubicin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.