Multifunctional Telodendrimer Nanocarriers Restore Synergy of Bortezomib and Doxorubicin in Ovarian Cancer Treatment

Abstract

We have developed multifunctional nanoparticles for codelivery of bortezomib and doxorubicin to synchronize their pharmacokinetic profiles and synergize their activities in solid tumor treatment, a need still unmet in the clinic. Micellar nanoparticles were formed by a spatially segregated, linear-dendritic telodendrimer containing three segments: a hydrophilic polyethylene glycol (PEG), a bortezomib-conjugating intermediate, and a dendritic doxorubicin-affinitive interior. Bortezomib-conjugated telodendrimers, together with doxorubicin, self-assembled into monodispersed micelles [NP(BTZ-DOX)] with small particle sizes (20-30 nm) for dual drug delivery. NP(BTZ-DOX) displayed excellent drug-loading capacity and stability, which minimized premature drug leakage and synchronized drug release profiles. Bortezomib release was accelerated significantly by acidic pH, facilitating drug availability in the acidic tumor microenvironment. Synergistic anticancer effects of combined bortezomib and doxorubicin were observed in vitro against both multiple myeloma and ovarian cancer cells. NP(BTZ-DOX) prolonged payload circulation and targeted tumors in vivo efficiently with superior signal ratios of tumor to normal organs. In vitro and in vivo proteasome inhibition analysis and biodistribution studies revealed decreased toxicity and efficient intratumoral bortezomib and doxorubicin delivery by nanoformulation. NP(BTZ-DOX) exhibited significantly improved ovarian cancer treatment in SKOV-3 xenograft mouse models in comparison with free drugs and their combinations, including bortezomib and Doxil. In summary, tumor-targeted and synchronized delivery system elicits enhanced anticancer effects and merits further development in the clinical setting. Cancer Res; 77(12); 3293-305. ©2017 AACR.

Figure 1

A, Chemical design of co-delivery telodendrimer platform. B, Model reaction study of BTZ conjugation by MALDI-TOF MS spectra showing BTZ conjugates with caffeic acid (CaA, top), chlorogenic acid (ChA, middle) and gluconic acid (GA, bottom). [M+Na]⁺ were found at m/z 551.785 (CaA-BTZ), 725.358 (ChA-BTZ), 1073.674 (ChA-2BTZ), 567.724 (GA-BTZ) and 916.259 (GA-2BTZ). C, MALDI-TOF MS spectra of PEG^5k-NH_2, PEG^5kCaA₄-Rh₄, PEG^5kGA₄-Rh₄, and PEG^5kChA₄-Rh_4.

Figure 2

In vitro nanocarrier characterizations. A–D, Characterization of particle sizes and morphologies. Hydrodynamic sizes of nanoparticles measured by DLS (A and B) and TEM images with negative staining (C and D) for PEG^5kChA₄-Rh₄ micelles (A and C) and BTZ-DOX co-loaded nanocarriers (B and D). E, In vitro cumulative DOX release from free DOX, Doxil and DOX encapsulated nanocarriers in PBS (pH 7.4) or acetate buffer (pH 5.5). F, In vitro cumulative BTZ release from BTZ-mannitol (Velcade mimicking formulation) and BTZ-PEG^5kChA₄-Rh₄ conjugate under indicated conditions. G, Cellular uptake behavior of BTZ+DOX and NP(BTZ-DOX) by H929 cancer cells via confocal microscopy at 37 °C for 2 h. Scale bar, 30 μm.

Figure 3

In vitro cell viability analysis of H929 MM (A and B) and SKOV-3 ovarian (C and D) cancer cells for 72 h incubation with BTZ-mannitol, BTZ-conjugated and BTZ-DOX co-loaded nanocarriers with different mass ratios. Cell viabilities were plotted against BTZ concentration (A and D) and DOX concentration (B and E), respectively. The combination index (CI) plot (Chou–Talalay method) showing synergism of NP(BTZ-DOX) with varying mass ratios of BTZ/DOX in H929 (C) and SKOV-3 (F) cells. CI < 1 indicates synergy; CI = 1 indicates additivity; CI > 1 indicates antagonism.

Figure 4

In vivo (A) and ex vivo (B) NIRF optical images of SKOV-3 bearing mice injected intravenously with free DiD-BTZ+DOX and DiD-NP(BTZ-DOX) formulations. C, The ex vivo tumor and organ uptake profiles of DiD. D, Blood concentrations of DiD were monitored at different time points after tail vein injection to compare the pharmacokinetics. E, Representative ex vivo fluorescence imaging of SKOV-3 tumor tissue sections 72 h post-injection of DiD and DiD-NP(BTZ-DOX). Blue = DAPI, red = NIR DiD. Scale bar, 50 μm.

Figure 5

In vitro and ex vivo proteasome inhibition and apoptosis analysis. A, In vitro proteasome activity of cultured H929 and SKOV-3 cells following exposure to PBS control, free BTZ+DOX and NP(BTZ-DOX) for 1 or 18 h at 37 °C (n = 3; **, P < 0.01, ***, P < 0.001). B–D, Ex vivo proteasome inhibition and apoptosis studies of tumor homogenate from in vivo treatment on SKOV-3 bearing mice of PBS control, free BTZ+DOX, and NP(BTZ-DOX) groups. B, Ex vivo proteasome activity assay (n = 3; *, P < 0.05 compared to PBS and BTZ+DOX groups). C, Western blot analysis of tumor homogenates from PBS control, free BTZ+DOX, and NP(BTZ-DOX) groups, showing BTZ-induced accumulation of ubiquitinated species (~125–400 kDa). Grp94 served as a loading control. D, Representative H&E (upper row) and TUNEL staining (lower row) of ex vivo SKOV-3 tumor tissue sections.

Figure 6

In vivo anticancer efficacy studies. In vivo body weight changes (A), tumor growth curves (B) and Kaplan–Meier survival curves (C) of SKOV-3 ovarian cancer xenograft bearing mice after intravenous treatment with different BTZ and DOX formulations (three injections on days 0, 4, and 8). Data are displayed as mean ± SD (n = 5–6; ***, P < 0.001).