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  • Therapeutics, Targets, and Chemical Biology

    Multifunctional Telodendrimer Nanocarriers Restore Synergy of Bortezomib and Doxorubicin in Ovarian Cancer Treatment Lili Wang1, Changying Shi1, Forrest A.Wright1, Dandan Guo1, Xu Wang1, Dongliang Wang2, Richard J.H.Wojcikiewicz1,3, and Juntao Luo1,3


    We have developed multifunctional nanoparticles for code- livery 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 hydro- philic polyethylene glycol (PEG), a bortezomib-conjugating intermediate, and a dendritic doxorubicin-affinitive interior. Bortezomib-conjugated telodendrimers, together with doxoru- bicin, 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 leak- age 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.

    Introduction Ovarian cancers remain ongoing challenges mainly due to the

    development of drug resistance and the likely occurrence of cancer metastasis at the time of diagnosis (1). Relapse of disease is commonly seen for ovarian cancers after the primary treatment with platinum-based therapeutics, which will be further treated with different chemodrugs (2). Monotherapy through a single mechanism frequently shows limited efficacies due to intrinsic or acquired drug resistance in cancer treatment (3–6). Instead, drug combinations with different mechanisms of action can kill cancer cells synergistically andminimize the emergence of drug-resistant mutations (7). The development of novel and efficient drug combinations may improve ovarian cancer treatment, as well as

    for other solid tumor treatments. Proteasome inhibitors target many protein degradation pathways, providing rationale for the clinical use in combination therapy.

    Bortezomib is a potent proteasome inhibitor that is approved for the treatment of multiple myeloma and other hematologic malignancies (8, 9). Bortezomib binds to the threonine residues in the active sites of the proteasome via boronic acid to block the degradation of ubiquitinated proteins (10–12). Proteasome inhi- bition regulates protein levels, which may sensitize or antagonize other drugs in cancer treatment. For example, bortezomib has been shown to antagonize microtubule-interfering drugs (e.g., paclitaxel) by inhibiting G2–Mtransition andMCL-1 degradation in both neuroblastoma (13) and ovarian cancer cells (14). In contrast, bortezomib is able to sensitize DNA-damaging agents, for example, doxorubicin and cisplatin, by inhibiting NF-kB activation (13).

    Bortezomib-based combination chemotherapy has dominated multiple myeloma treatment in clinical practice (3). For example, bortezomib andDoxil combination yields significantly improved efficacy when compared with single reagent in multiple myeloma treatments (15). Preclinical studies have shown that free borte- zomib is effective in several solid tumors, both in vitro and in vivo (16). However, a lack of therapeutic effect was observed in clinical trials utilizing bortezomib as a single agent (8) or in combination chemotherapy (16, 17) in treating solid tumors. This includes the trial using bortezomib in combination withDoxil to treat ovarian cancer (18). Although the reason is not clear for the difference between preclinical and clinical results, it is likely associated with the unfavorable and mismatched pharmacokinetic and

    1Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York. 2Department of Public Health and Preventive Medicine, State University of New York Upstate Medical University, Syracuse, New York. 3Upstate Cancer Center, State University of New York Upstate Medical University, Syracuse, New York.

    Note: Supplementary data for this article are available at Cancer Research Online (

    L. Wang and C. Shi are the co-first authors of this article.

    Corresponding Author: Juntao Luo, Department of Pharmacology, SUNY Upstate Cancer Center, State University of NewYorkUpstateMedical University, 750 East Adams Street, Syracuse, NY 13210. Phone: 315-464-7965; Fax: 315-464- 8014; E-mail:

    doi: 10.1158/0008-5472.CAN-16-3119

    �2017 American Association for Cancer Research.

    Cancer Research 3293

    on November 27, 2020. © 2017 American Association for Cancer Research. Downloaded from

    Published OnlineFirst April 10, 2017; DOI: 10.1158/0008-5472.CAN-16-3119

  • biodistributionprofiles of bortezomib andDoxil. Bortezomib can be taken up efficiently by red blood cells after intravenous administration (19), resulting in rapid clearance from plasma (�5 minutes; refs. 19–21). In contrast, Doxil has a half-life of approximately 55 hours in humans with very slow drug release and limited intratumoral diffusion due to the large particle size (�100 nm; ref. 22). Nanoparticle-based delivery systems could accommodate such variations in pharmacokinetics for tumor- targeted drug delivery, resulting in enhanced combination ther- apy (23–25).

    Nanoparticle-mediated bortezomibdelivery systemshavebeen developed that utilize either physical encapsulation or reversible conjugation, demonstrating improved anticancer efficacy (26– 32). Eliminating premature drug release from nanoparticles is critical to delivering a sufficient amount of drug to tumor cells via the enhanced permeability and retention (EPR) effect (33, 34). Both bortezomib and doxorubicin have relatively good solubility in aqueous solution. It poses challenges to control their release profile through physical encapsulation in nanoparticles, especial- ly for small-sized micelles (20–30 nm) with less physical barrier for drug diffusion, which is preferred for intratumoral drug delivery over large polymeric nanoparticles (>100 nm; refs. 35, 36). Therefore, specific nanocarrier design is needed for efficient codelivery of these two drugs to reboot the efficacy of bortezomib and foster their synergism in ovarian cancer treatment.

    Previously, we have developed a well-defined linear-dendritic telodendrimer as a versatile delivery vehicle (35, 37–40). The modular design and the precise telodendrimer synthesis enable rational nanocarrier engineering for either drug (41, 42) or protein/peptide (43, 44) delivery. Recently, we have demonstrat- ed that drug binding affinity within a telodendrimer nanocarrier can be enhanced by decorating telodendrimers with drug-binding moieties identified via computational and experimental screen- ings (42). Bortezomib has a dipeptide structure lacking sufficient hydrophobicity for stable encapsulation in polymeric micelles. Notably, bortezomib can form a reversible boronate ester linkage efficiently for prodrug formation via coupling with cis-diols and be released either under acidic condition or in the presence of competing diols (45, 46). The acidic extracellular tumor micro- environments (pH 6.2–6.8; ref. 47) and the more acidic subcel- lular compartments, for example, late endosome and lysosome (�pH 5.0), have been widely exploited in pH-triggered drug release (48). In this study, we rationally modify three-layered telodendrimer with both bortezomib-conjugating moiety (BCM) and doxorubicin-binding moiety (DBM) to load bortezomib and doxorubicin via reversible conjugation and affinitive encapsula- tion, respectively. Rhein (Rh)molecule asDBMwill be introduced to strengthen doxorubicin affinity within nanocarrier by taking advantage of the pi-pi stacking. A collection of naturally occurring cis-diol and/or catechol-containing biomolecules, for example, caffeic acid (CaA), chlorogenic acid (ChA), and gluconic acid (GA), will be selected as BCMs. The bortezomib and doxorubicin coloaded nanocarrier is expected to enhance their accumulations in solid tumors and release both drugs preferably in tumor microenvironments to synergize their efficacy in ovarian cancer treatment.

    Materials and Methods See Supplementary Information for detailed information on

    Materials and spectroscopic characterization.

    Model reactions of bortezomib conjugation with cis-diol/ catechol-containing compounds

    Bortezomib and cis-diol/catechol-containing compounds (CaA, ChA, and GA) were dissolved in DMSO at a concentration of 100 mmol/L. Bortezomib solution was mixed with equal vo