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

Therapeutics, Targets, and Chemical Biology

Multifunctional Telodendrimer NanocarriersRestore Synergy of Bortezomib and Doxorubicinin Ovarian Cancer TreatmentLili Wang1, Changying Shi1, Forrest A.Wright1, Dandan Guo1, Xu Wang1,Dongliang Wang2, Richard J.H.Wojcikiewicz1,3, and Juntao Luo1,3

Abstract

We have developed multifunctional nanoparticles for code-livery of bortezomib and doxorubicin to synchronize theirpharmacokinetic profiles and synergize their activities in solidtumor treatment, a need still unmet in the clinic. Micellarnanoparticles were formed by a spatially segregated, linear-dendritic telodendrimer containing three segments: a hydro-philic polyethylene glycol (PEG), a bortezomib-conjugatingintermediate, 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 drugdelivery. NP(BTZ-DOX) displayed excellent drug-loadingcapacity and stability, which minimized premature drug leak-age and synchronized drug release profiles. Bortezomib releasewas accelerated significantly by acidic pH, facilitating drug

availability in the acidic tumor microenvironment. Synergisticanticancer effects of combined bortezomib and doxorubicin wereobserved in vitro against both multiple myeloma and ovariancancer cells. NP(BTZ-DOX) prolonged payload circulation andtargeted tumors in vivo efficiently with superior signal ratios oftumor to normal organs. In vitro and in vivo proteasome inhibitionanalysis and biodistribution studies revealed decreased toxicityand efficient intratumoral bortezomib and doxorubicin deliveryby nanoformulation. NP(BTZ-DOX) exhibited significantlyimproved ovarian cancer treatment in SKOV-3 xenograft mousemodels in comparison with free drugs and their combinations,including bortezomib and Doxil. In summary, tumor-targetedand synchronized delivery system elicits enhanced anticancereffects and merits further development in the clinical setting.Cancer Res; 77(12); 3293–305. �2017 AACR.

IntroductionOvarian cancers remain ongoing challenges mainly due to the

development of drug resistance and the likely occurrence of cancermetastasis at the time of diagnosis (1). Relapse of disease iscommonly seen for ovarian cancers after the primary treatmentwith platinum-based therapeutics, which will be further treatedwith different chemodrugs (2). Monotherapy through a singlemechanism frequently shows limited efficacies due to intrinsic oracquired drug resistance in cancer treatment (3–6). Instead, drugcombinations with different mechanisms of action can kill cancercells synergistically andminimize the emergence of drug-resistantmutations (7). The development of novel and efficient drugcombinations may improve ovarian cancer treatment, as well as

for other solid tumor treatments. Proteasome inhibitors targetmany protein degradation pathways, providing rationale for theclinical use in combination therapy.

Bortezomib is a potent proteasome inhibitor that is approvedfor the treatment of multiple myeloma and other hematologicmalignancies (8, 9). Bortezomib binds to the threonine residuesin the active sites of the proteasome via boronic acid to block thedegradation of ubiquitinated proteins (10–12). Proteasome inhi-bition regulates protein levels, which may sensitize or antagonizeother drugs in cancer treatment. For example, bortezomib hasbeen shown to antagonize microtubule-interfering drugs (e.g.,paclitaxel) by inhibiting G2–Mtransition andMCL-1 degradationin both neuroblastoma (13) and ovarian cancer cells (14). Incontrast, bortezomib is able to sensitize DNA-damaging agents,for example, doxorubicin and cisplatin, by inhibiting NF-kBactivation (13).

Bortezomib-based combination chemotherapy has dominatedmultiple myeloma treatment in clinical practice (3). For example,bortezomib andDoxil combination yields significantly improvedefficacy when compared with single reagent in multiple myelomatreatments (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 clinicaltrials utilizing bortezomib as a single agent (8) or in combinationchemotherapy (16, 17) in treating solid tumors. This includes thetrial using bortezomib in combination withDoxil to treat ovariancancer (18). Although the reason is not clear for the differencebetween preclinical and clinical results, it is likely associatedwith the unfavorable and mismatched pharmacokinetic and

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

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

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

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

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

�2017 American Association for Cancer Research.

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

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

Previously, we have developed a well-defined linear-dendritictelodendrimer as a versatile delivery vehicle (35, 37–40). Themodular design and the precise telodendrimer synthesis enablerational nanocarrier engineering for either drug (41, 42) orprotein/peptide (43, 44) delivery. Recently, we have demonstrat-ed that drug binding affinity within a telodendrimer nanocarriercan be enhanced by decorating telodendrimers with drug-bindingmoieties identified via computational and experimental screen-ings (42). Bortezomib has a dipeptide structure lacking sufficienthydrophobicity for stable encapsulation in polymeric micelles.Notably, bortezomib can form a reversible boronate ester linkageefficiently for prodrug formation via coupling with cis-diols andbe released either under acidic condition or in the presence ofcompeting 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 drugrelease (48). In this study, we rationally modify three-layeredtelodendrimer with both bortezomib-conjugating moiety (BCM)and doxorubicin-binding moiety (DBM) to load bortezomib anddoxorubicin via reversible conjugation and affinitive encapsula-tion, respectively. Rhein (Rh)molecule asDBMwill be introducedto strengthen doxorubicin affinity within nanocarrier by takingadvantage of the pi-pi stacking. A collection of naturally occurringcis-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 doxorubicincoloaded nanocarrier is expected to enhance their accumulationsin solid tumors and release both drugs preferably in tumormicroenvironments to synergize their efficacy in ovarian cancertreatment.

Materials and MethodsSee 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 concentrationof 100 mmol/L. Bortezomib solution was mixed with equalvolumeof diol or catechol separately. The conjugationwas carriedout at 37�C for 18 hours. In situ proton nuclear magnetic reso-nance (1H-NMR) study for model compounds conjugationwas conducted by mixing bortezomib (20 mmol/L) with ChA(20 mmol/L) solutions in DMSO-d6 at the same volume ratio.

Telodendrimer synthesisThe telodendrimers were synthesized via solution-phase pep-

tide condensation reactions following the typical procedurereported in our previous publications (35, 40). The procedure isdetailed in Supplementary Information.

Bortezomib–telodendrimer formation and dual-drug loadingBortezomib was initially reacted with telodendrimer to form

bortezomib–telodendrimer conjugate. Dual-drug loaded nano-carriers with different ratios of bortezomib and doxorubicin wereformulated by varying the amount of bortezomib-conjugatedtelodendrimer anddoxorubicin. Adetailed procedure is describedin Supplementary Information.

In vitro drug releaseThe releases of bortezomib and doxorubicin from nanocarriers

were performed using a dialysis method. A 300 mL nanoformula-tion in PBS was placed in dialysis tubing (MWCO ¼ 3,500 Da)and immersed in a 40 mL release medium of PBS (pH 7.4) oracetate buffer (pH 5.5) at 37�C. Two microliters of aliquots fromdialysis tubing were sampled at predetermined intervals. Doxo-rubicin concentration was determined using fluorescence quali-fication at excitation/emission of 488/599 nm. The amount ofbortezomib was detected by fluorescence using alizarin as afluorescent reporter (49, 50). The quantitative bortezomib titra-tion by alizarin was validated for both bortezomib–mannitol andbortezomib–telodendrimer conjugates. Because of interference influorescence between doxorubicin and alizarin in bortezomibmeasurements, the release profile of bortezomib was conductedusing bortezomib-conjugated nanoformulations without doxo-rubicin loaded. Three microliters of bortezomib–telodendrimersampled were treated with 2 mL mannitol (10 mg/mL in DMSO)overnight followed by the addition of 25 mL alizarin solution(1 mg/mL in DMSO) for another 6 hours. The alizarin fluores-cence was examined at excitation/emission ¼ 485/625 nm.

Cell lines and animalsH929MMandSKOV-3ovarian cancer cell lineswere purchased

from ATCC (2009) without further authentication. The referencecancer cells were expanded and cryopreserved in liquid nitrogenuntil use. Both cell lines used were within 6–8 passages andsubcultured less than three passages. H929 cell was cultured inRPMI1640 medium and SKOV-3 cell was maintained in McCoy5A medium, supplemented with 10% FBS, 100 U/mL penicillinG, and 100 mg/mL streptomycin at 37�C using a humidified 5%CO2 incubator. Female athymic nude mice (Nu/Nu strain), 5–6weeks age, were purchased from The Jackson Laboratory. Allanimals were kept under pathogen-free conditions in accordancewith Association for Assessment and Accreditation of Laboratory

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Animal Care guidelines and allowed to acclimatize for at least4 days prior to any experiments. All animal experiments wereperformed in compliance with institutional guidelines followingprotocols approved by the Animal Use and Care AdministrativeAdvisory Committee. SKOV-3 cells (7� 106) in a 100-mLmixtureof PBS and Matrigel (1:1, v/v) without FBS were injected subcu-taneously into the flanks of nude mice to form nodules.

In vitro efficacy and synergistic effectsThe cytotoxicities of unloaded and drugs loaded nanoparticles

were studied using H929 and SKOV-3 cells and cell viability wastested via MTS assays. Cells were seeded in a 96-well plate at thecell densities of 8� 103 cells per well. After overnight incubation,the cells were treated with different formulations at serial con-centrations. After incubation, CellTiter 96 aqueous cell prolifer-ation reagent was added to each well according to the manufac-turer's instructions. Untreated cells served as negative controls.Results were obtained as the average cell viability of triplicateexperiments calculated by a formula of [(ODtreat � ODblank)/(ODcontrol � ODblank) � 100%].

Themedian-effect analysis proposed by Chou and Talalay (51)were used to evaluate synergistic drug combinations in vitro. Thismethod assesses the drug–drug interaction in terms of combina-tion index (CI), which is based on the relationship betweenconcentration and response. The CI was used to evaluate synergybetween doxorubicin and bortezomib against H929 and SKOV-3cells in vitro. CI analysis was performed by CompuSyn software.Values ofCI<1,CI¼1, andCI>1 indicate synergy, additivity, andantagonism, respectively.

In vitro proteasome inhibition studies20S ProteasomeActivity Assay Kit (EMDMillipore) was used to

measure proteasome activity. A total of 6 � 105 cells/well (H929and SKOV-3) were placed in a 6-well dish. Free bortezomib anddoxorubicin formulation (BTZþDOX) and BTZ-DOX coloadedPEG5kChA4-Rh4 nanoformulation [NP(BTZ-DOX)] were addedat 10 ng/mL bortezomib and 100 ng/mL doxorubicin equivalentconcentrations to their respective wells and incubated at 37�C for1 and 18 hours, respectively. Negative control groups were incu-bated with PBS. After incubation, cells were washed twice withcold PBS and lysed in 60 mL lysis buffer (50 mmol/L HEPES,5mmol/L EDTA, 150mmol/LNaCl, 1%Triton X-100, pH7.5) onice for 30 minutes. The lysate was centrifuged at 20,000 � g for15 minutes at 4�C. Proteasome activity assays were carried out bythe addition of 10 mL of the lysate, 10 mL of proteasome substrate(Suc-LLVY-AMC, 0.5 mmol/L), and 80 mL of the assay buffer[25 mmol/L HEPES, pH 7.5, 0.5 mmol/L EDTA, 0.05% NP-40,and 0.001% SDS (w/v)] into a 96-well plate with incubation at37�C for 1 hour. Proteasome activity was assessed viafluorescencespectroscopy at excitation/emission ¼ 380/460 nm (BioTek Syn-ergy H1).

Optical animal imagingNude mice bearing human SKOV-3 ovarian cancer xenografts

(approximately 500 mm3) were randomized into two groups(3 mice per group). DiD [a hydrophobic near-infrared (NIR)cyanine dye] was encapsulated in the nanocarrier together withdoxorubicin and bortezomib [DiD-NP(BTZ-DOX)] at a massratio of 4:1:0.1 (DiD/DOX/BTZ). One-hundred microliters ofDiD-NP(BTZ-DOX) solution was filtered with a 0.22-mm filter

to sterilize the solution before injection. An equal amount of DiDin ethanol solution was diluted with PBS, mixed with BTZþDOXand intravenously injected. Mice were anesthetized with isoflur-ane and optically imaged at designed time points using an IVIS 200(PerkinElmer) with the excitation/emission at 625/700 nm. After72 hours, the animals were sacrificed and all the major organs andtumors excised for ex vivo imaging to determine the in vivo biodis-tribution of nanoparticles. The associated fluorescence intensitieswere determined by Living Image software (Caliper Life Sciences)using operator-defined regions of interest (ROI) measurements.

Molecular basis for in vivo anticancer effectsSKOV-3 bearing mice were distributed into three groups of

3 mice and were treated intravenously with PBS, BTZþDOX, andNP(BTZ-DOX) formulation, respectively on days 0 and 4 at a doseof 0.5 mg/kg bortezomib and 5 mg/kg doxorubicin equivalents.Themice were sacrificed on day 5, and the tumors were excised forthe proteasome activity measurement and apoptosis analysis,respectively. Tumors were homogenized in 200-mL lysis buffer(100 mmol/L Tris, pH 7.8, 150 mmol/L NaCl, 1% Triton-X100)per 50 mg tissue for 30 minutes on ice. The homogenate wascentrifuged at 20,000� g for 15 minutes at 4�C. The supernatantwas removed and centrifuged again to ensure complete removalof any precipitate. The proteasome activity was measured follow-ing the same protocol described above for the in vitro proteasomeinhibition study. The same tumor lysates were analyzed byWestern blot analysis to compare the protein ubiquitinationlevels in different groups. Gel loading buffer was added to theclarified lysates and incubated at 37�C for 30 minutes, subjectedto SDS-PAGE and transferred to nitrocellulose for Western blot-ting and probed with anti-ubiquitin (a gift fromDr. Martin Obin,Tufts University, Medford, MA) and anti-Grp94 (Stressgen #SPA-850) following the protocol described previously (52).

Intratumoral distribution and tumor pathology studiesTumors were collected from euthanized mice, freshly embed-

ded in Tissue-TekO.C.T. compound, and stored at�80�C. Tumortissue was sectioned to a thickness of 16 mm on a microtome-cryostat for fluorescence imaging of DiD biodistribution in tumorsites. For bioactivity studies, tumor samples were cut into 10-mmsections and fixed with 4% paraformaldehyde and stained withhematoxylin and eosin (H&E) for pathology analysis. Levels ofapoptosis in tumors treated with different formulations weredetected via TUNEL staining using an In SituCell Death DetectionKit, POD (Roche) following the manufacturer's instructions.Nuclei were counterstained with DAPI and tumor apoptosissignals were detected under a laser scanning fluorescence micro-scope (Leica).

In vivo anticancer efficacyWe implanted 6 nude mice per group with human ovarian

SKOV-3 cancer cells with the consideration of possible failure intumor growth. Nude mice bearing SKOV-3 xenograft tumors(approximately 100–150 mm3 in volume) were randomly divid-ed into six groups (n ¼ 5–6 per group), including control (PBS),bortezomib (0.5 mg/kg), BTZþDOX (0.5 mg/kg bortezomiband 5 mg/kg doxorubicin equivalents), BTZþDoxil (0.5 mg/kgbortezomib and 5 mg/kg doxorubicin equivalents), NP(BTZ-DOX) (0.5 mg/kg bortezomib and 5 mg/kg doxorubicin equiva-lents), and NP(BTZ-DOX) (0.8 mg/kg bortezomib and 8 mg/kg

Synergized BTZ/DOX Combination Codelivered by Nanocarriers

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doxorubicin equivalents). Treatments were intravenously admin-istered via tail vein injection on days 0, 4, and 8 for a total of threetreatments. Animal body weight and tumor volume were mon-itored over time. Seven days after the last treatment, approxi-mately 100 mL of blood was collected via tail bleeding for bloodcounts. The tumor sizes were measured with electronic calipers,and volumes calculated using the following formula: V ¼ (short-est diameter2 � longest diameter)/2. Animals were sacrificedwhen tumor volume exceeded 1,500 mm3, the tumor becamenecrotic, or body weight loss > 20% was observed.

Statistical analysisData are presented as means � SD, unless otherwise specified.

All statistical analyses were performed using Student t test forcomparison of two groups, one-way ANOVA for multiple groups.The level of statistical significancewas set withP < 0.05 consideredsignificant. IC50 valueswere calculated fromcell viability curves byfitting a dose–response model using sigmoidal functionwith variable Hillslope. Pharmacokinetic data were fitted to a

two-compartment model. Sample size estimation for in vivoefficacy study was based on our previous studies (45, 46). Wetargeted an effect sizeof two in the treatment groups, resulted froman anticipated 30% reduction in tumor volume and an estimatedSD of 15% of tumor inhibition at the end of experiment. Usingtwo-sample t test, a sample size of 5 per group will give an 80%power to detect the targeted effect size of 2 at a significance level of0.05, in both tumor growth inhibition and survival analysis,which reported the time of tumor volume exceeding 1,500 mm3.Animal survival data were analyzed descriptively using theKaplan–Meier method and the survival differences between twogroups were assessed using the Student t-test.

ResultsTo codeliver bortezomib and doxorubicin with the ultimate

goal of synergizing their anticancer effects by synchronizing drugaccumulations in tumor sites, we designed a series of multifunc-tional and well-defined three-layered telodendrimers (Fig. 1A),

Figure 1.

A, Chemical design of codelivery telodendrimer platform. B, Model reaction study of bortezomib (BTZ) conjugation by MALDI-TOF MS spectra showingbortezomib conjugates with CaA (top), chlorogenic acid (ChA, middle), and gluconic acid (GA, bottom). [MþNa]þ 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 PEG5k-NH2, PEG

5kCaA4-Rh4, PEG5kGA4-Rh4,

and PEG5kChA4-Rh4.

Wang et al.





consisting of a linear polyethylene glycol (PEG) block, a cis-diol–containing intermediate layer for bortezomib conjugation and adoxorubicin binding interior layer.

Model reactions of bortezomib conjugation with BCMsThe reactivities of BCMs with bortezomib in the formation of

boronate were evaluated initially before their conjugation ontotelodendrimers to demonstrate the feasibility for telodendrimermodification. The explicit formations of bortezomib conjugateswith CaA, ChA, and GA were confirmed by MALDI-TOF MS anddisplayed as sodium adducts of molecular ion peaks (Fig. 1B). Abivalent conjugate of bortezomib with ChA was detected at m/z1073.674, which is correlated with the chemical structure of ChA(Fig. 1A) containing both cis-diol and catechol functionalities. 1H-NMR analysis provides further spectrometric evidence for theconjugation between bortezomib and diols (Supplementary Fig.S1). The disappearance of characteristic phenol resonances indi-cates the success complexation of catechol with boronic acid intoChA–BTZ conjugate. The association constant of diols for borte-zomib chelation was measured to be 370, 290, and 34 M�1 forChA, GA, and CaA, respectively (Supplementary Fig. S2; refs. 50,53). The results show that ChA has the highest affinity forbortezomib, which is in agreement with literature reports thatthe catechols have greater reactivity than cis-diols in reacting withboronic acid (30, 53). Consequently, these biocompatible com-pounds were chosen as BCMs to functionalize telodendrimers tovalidate the methodology for nanocarrier design and optimizebortezomib conjugation.

Codelivery telodendrimer synthesisThe teloendrimers were constructed using solution peptide

chemistry with the structural scaffold shown in Fig. 1A. Theoligolysine-based scaffold is spatially segregated into two den-dritic domains for dual-drug loading. A flexible oligo-ethyleneglycol spacer is placed between the two domains to reduce sterichindrance. Telodendrimers were synthesized from PEG5k-NH2

(�5 kDa); the stepwise synthetic pathway is detailed in Supple-mentary Fig. S3. The oligolysine components were synthesized bythe coupling of the orthogonally protected lysine via Boc- orFmoc-protecting chemistries. The efficient amide bond formationallows the construction of telodendrimers with ease and efficien-cy. The chemical structures of all the intermediates in teloden-drimer synthesis have been confirmed by 1H-NMR with the clearsignal assignments and accurate peak integration (SupplementaryFigs. S4–S10). Molecular weight determination by MALDI-TOFMS reveals the maintained narrow polydispersity and accuratemass increase according to the intermediate chemical structures

throughout the telodendrimer synthesis (SupplementaryFig. S11). The functionalizations of telodendrimers with BCMswere carried out at the final step throughDIC/NHS chemistry. Thecodelivery telodendrimers bearing DBM (Rh) and BCMs (GA,CaA, and ChA) are denoted as PEG5kGA4-Rh4, PEG

5kCaA4-Rh4,and PEG5kChA4-Rh4 (chemical structures are shown in Fig. 1A).

The structural integrity and molecular weight distribution ofthe resultant codelivery telodendrimers were characterized byMALDI-TOFMS. Similar to the PEG-NH2 precursor, monomodaland narrowly dispersed molecular weight distributions weredetected with molecular peaks centered at m/z of 7,780, 7,640,and 8,210 for PEG5kGA4-Rh4, PEG

5kCaA4-Rh4, and PEG5kChA4-Rh4 telodendrimers, respectively (Fig. 1C). As shown in Table 1,the observed molecular weights are close to the theoretical valuescalculated based upon the PEG 5000 precursor. The slight dis-crepancies in molecular weight detected by MALDI-TOF MS canbe attributed to the entanglement of high-molecular weightpolymers, which are likely to hinder the desorption of ionizedspecies. The chemical composition of codelivery telodendrimerswere further confirmed using 1H-NMR spectroscopy. A represen-tative 1H-NMR spectrum for the telodendrimer PEG5kChA4-Rh4 isshown in Supplementary Fig. S12. Well-resolved resonances ofthe olefin protons in chlorogenicmoieties at 6.21–6.26 and 7.46–7.49 ppm, aromatic protons at 6.77, 6.98, and 7.04 ppm, andphenolic protons at 9.12 and 9.54 ppm were observed. Theintegration values of these signature peaks match closely to thepredicted values relative to the characteristic methoxy proton ofPEG at 3.25 ppm, highlighting the well-defined structure of thetelodendrimer. In addition, the apparent peak broadening in thetelodendrimer compared with the small molecule (Supplemen-tary Fig. S12) is indicative of reduced chain mobility after thecovalent attachment to the telodendrimer. 1H-NMR spectra forPEG5kGA4-Rh4 and PEG5kCaA4-Rh4 are shown in SupplementaryFigs. S13 and S14.

BTZ–telodendrimer conjugationThe presence of cis-diols/catechol on the designed telodendri-

mers provides an efficient anchor for bortezomib conjugation viareversible boronate bond. In this study, the drug-loading contents(DLC) for bortezomib were set at 10% in nanotherapeuticsproduction. For BTZ–telodendrimer prodrug formation, a 10%DLC gives a molar ratio of telodendrimer to drug of approxi-mately 1:2. The progress of the coupling reaction was monitoredby alizarin dye assay and UV-Vis absorbance of unboundedbortezomib at 270 nm. The results revealed that the bortezomibcoupling reaction completed after 8-hour incubation at 37�Cwith an initial bortezomib concentration of 20 mg/mL

Table 1. Characterization and drug loading properties of codelivery telodendrimers

Mn (Da) CMCc Nanoparticle sized (nm) DLE%e DLE%f

Telodendrimers Theo.a Obs.b (mg/mL) NP NP(BTZ) NP(BTZ-DOX) (DOX) (BTZ)

PEG5kGA4-Rh4 7,910 7,780 5.3 31 � 12 41 � 15 34 � 13 93 � 2 89 � 2PEG5kCaA4-Rh4 7,840 7,640 4.1 28 � 10 29 � 12 28 � 9 96 � 2 80 � 5PEG5kChA4-Rh4 8,540 8,210 4.2 25 � 8 23 � 9 20 � 8 97 � 1 94 � 4

Abbreviations: BTZ, bortezomib; CMC, critical micelle concentration; DOX, doxorubicin.aTheoretical molecular weight.bObserved molecular weight was determined by MALDI-TOF MS analysis.cThe CMC was determined by dye micellization method.dNanoparticle size was measured by DLS and expressed as mean � half-peak width.eDLE of doxorubicin was determined by fluorescence analysis of drug-loaded micelle solutions before and after centrifugation.fDLE of bortezomib on telodendrimer was quantified by 1H-NMR analysis.






(Supplementary Fig. S15). Bortezomib conversions are estimatedto be 86%, 70%, and 94% for GA-, CaA-, and ChA-containingtelodendrimers, respectively. Conjugation efficiency was thenquantified using 1H-NMR by comparing the signal integrationof methoxy proton on the PEG chain at 3.25 ppm with dimethylproton on bortezomib ranging from 0.74 to 0.93 ppm (Supple-mentary Figs. S16–S18). A summary of loading efficiencies ispresented in Table 1. Bortezomib drug loading efficiency (DLE)reflected by NMR study is determined to be 89% � 2%, 80% �5%, and 94% � 4% for PEG5kGA4-Rh4, PEG

5kCaA4-Rh4, andPEG5kChA4-Rh4, respectively. Alizarin dye assay further con-firmed that approximately 10% wt bortezomib content was pre-sented in the final telodendrimer prodrug (Supplementary Fig.S19). The overall conjugation efficiency is correlated with thedensity and reactivity of the cis-diol/catechol groups as confirmedby molecular bindings study (Supplementary Fig. S2). In eachmolecule of ChA and GA, there are two pairs of cis-diol/catecholmoieties available for bortezomib attachment, which ultimatelyleads to more effective conjugation. CaA with only one reactivesite shows lower coupling efficiency in prodrug formation.

Codelivery telodendrimer nanocarrier characterizationThe self-assembly behavior of telodendrimers was character-

ized by critical micelle concentration and dynamic light scattering(DLS) measurements (Supplementary Fig. S20 and Table 1). Thehigh encapsulation capacity of doxorubicin by codelivery telo-dendrimers is attainable due to the strong interactions betweendoxorubicin and rhein through pi-pi stacking and hydrophobicinteractions (42). TheDLE of doxorubicin at 10wt% feeding ratiowere determined to be 93%� 2%, 96%� 2%, and 97%� 1% forPEG5kGA4-Rh4, PEG

5kCaA4-Rh4, and PEG5kChA4-Rh4, respective-

ly (Table 1). Bortezomib-conjugated telodendrimers self-assem-bled with doxorubicin into monodispersed micelles with smallsizes ranging from20 to 40 nm for all telodendrimer platforms, asevident by bothDLS and TEM studies (Fig. 2A–D). RepresentativeTEM images showed that the empty micelle formed by PEG5k-

ChA4-Rh4 are elongated micelles in shape (Fig. 2C), which iscorrelated with pi-pi stacking of Rh moieties observed in ourprevious studies (42). After dual-drug loading, a morphologytransformation from a rod-like shape to a spherical structure wasobserved, suggesting the disruption of long-range molecularstacking (Fig. 2D).

In vitro drug releaseThe stabilized doxorubicin encapsulation at the core region of

micelles is further confirmed by the sustained doxorubicin releasefrom micelles as compared with free doxorubicin (Fig. 2E). Over24-hour dialysis, 50% to 70% of doxorubicin was released fromtelodendrimer nanoformulations, DOX-PEG5kGA4-Rh4, DOX-PEG5kCaA4-Rh4, and DOX-PEG5kChA4-Rh4, which were signifi-cantly slower than free doxorubicin (100% release at 8 hours). Incontrast, less than 20%of doxorubicin can be released fromDoxilwithin 24 hours and no significant drug release was observedafterwards, which may limit the drug availability in cancer treat-ment. The difference in release rates from three telodendrimernanoformulations is likely attributed to the varying hydropho-bicity of diol/catechol functionalities at the intermediate layerbetween the doxorubicin-affinitive core and the PEG shieldingcorona. Relativelymore hydrophilic GA shows lower capability tosustain doxorubicin, leading to slightly faster release; while more

hydrophobic CaA and ChA interfaces create additional barrier toprevent the doxorubicin diffusion. A modest increase in doxoru-bicin release rate from DOX-PEG5kChA4-Rh4 nanoformulationwas observed at pH 5.5, due to the increased solubility ofdoxorubicin (Fig. 2E).

The release profiles in Supplementary Fig. S21 indicate thatbortezomib release from telodendrimer conjugates are signifi-cantly slower compared with the bortezomib–mannitol (freedrug formulation) in PBS (pH 7.4) at 37�C. The boronate esteris sensitive to both acidic pH and the cis-diol containing sugar, forexample, glucose. The bortezomib releases from telodendrimerconjugates were found to be dual stimuli-responsive as designed.About 39% of bortezomib was released within 24-hour incuba-tion at pH7.4,whichwas slightly increased to48% in the presenceof an elevated glucose concentration (50 mmol/L; Fig. 2F).Bortezomib release was significantly accelerated under acidiccondition (pH 5.5), showing 75% of bortezomib release after24 hours. The pH-triggered bortezomib release was furtherenhancedby thepresence of glucose,which indicates thepreferredbortezomib release from nanoformulations on-demand at tumormicroenvironments and in the acidic lysosome after tumor celluptake.

Biocompatibility of nanocarriersThe in vitro toxicities of the designed telodendrimers were

evaluated in terms of hemolytic property and cytotoxicity. Allthree telodendrimers show negligible in vitro hemolytic activitytoward red blood cells after 24-hour incubation at concentrationsranging from 10 to 1,000 mg/mL (Supplementary Fig. S22A). Invitro cell culture studies revealed noncytotoxicity of telodendri-mers with concentrations ranging from 0.32 to 625 mg/mL asdetermined by MTS assay (Supplementary Fig. S22B). We furtherexamined the potency of bortezomib–telodendrimer conjugatesinH929MMcells. The results demonstrated that the bortezomib–telodendrimer conjugates maintained drug potency, having IC50

values of 0.49, 3.49, and1.74ng/mL for BTZ-PEG5kGA4-Rh4, BTZ-PEG5kCaA4-Rh4, and BTZ-PEG5kChA4-Rh4, respectively. Theslightly elevated IC50 values as compared with that of BTZ–mannitol (0.23 ng/mL) could be attributed to the sustainedbortezomib release from the nanoparticles. PEG5kChA4-Rh4 exhi-bits superior properties in drug loading, release profile and in vitroefficacy, and therefore was selected for further studies.

In vitro cellular uptakeH929 MM cells were incubated with dual-drug loaded PEG5k-

ChA4-Rh4, NP(BTZ-DOX), as well as the free drug combination(BTZþDOX), and imaged using confocal microscopy to investi-gate the overall cellular internalization. Free doxorubicin rapidlytranslocated into the nucleus with little fluorescence detected inthe cytoplasm, even with brief incubation for 30 minutes (Sup-plementary Fig. S23A). In sharp contrast, doxorubicin in nano-formulation showed exclusive distribution in cytoplasm within30minutes (Supplementary Fig. S23A) and gradual translocationinto the nucleus after 2-hour incubation (Fig. 2G). In addition,cells treated with the NP(BTZ-DOX) had stronger colocalizationof doxorubicin within the lysosomal compartments (stained bylysotracker green) compared with a low level of colocalization forBTZþDOX treatment, suggesting the endocytotic pathway fornanocarrier uptake. In addition, the cellular uptake of NP(BTZ-DOX) was founded to be hindered at 4�C, indicating the energy

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dependent process for nanoparticle uptake. As for BTZþDOX, celluptake at 4�Cwasnot affected (Supplementary Fig. S23A). Furthercell lysis and drug extraction analysis quantitatively reveals thatthe cell uptake profile of the nanoformulation is temperature andconcentration dependent (Supplementary Fig. S23B–S23E).

In vitro synergistic effect in cancer treatmentThe systemic toxicity and in vivo efficacy largely relies on the

optimal therapeutic ratios loaded and the amount of drugsdelivered to tumor sites. As such, we first assessed the in vitrosynergistic effect of BTZ-DOX coloaded by our nanoparticles inkilling cancer cells. In agreement with other studies (54), resultsshow that bortezomib couldmarkedly enhance theH929MMcellkilling at subtoxic concentrations of doxorubicin. Cell viabilityanalysis demonstrated that the IC50 value for doxorubicin inH929 cells was 229.3 ng/mL in the absence of bortezomib or7.33 ng/mL in combination with bortezomib at a 1:4 molar ratio(Fig. 3A and B). Similarly, NP(BTZ-DOX) at different BTZ/DOXdrug ratioswere tested in SKOV-3 ovarian cancer cells (Fig. 3C and

D). We assessed the synergistic effect of bortezomib and doxo-rubicin using awhole cell killing panel and analysis via theChou–Talalay method (51). The CIs were observed to be generally lessthan 1 at different BTZ/DOX ratio, indicating synergistic effects ofthe two drugs. Only at high percentage of cell killing range, lowBTZ/DOX ratio, for example, 1:4 and 1:10, showed antagonism inboth cell lines, which however showed significant synergism atlower concentrations (Fig. 3E and F). The CI at 50% cancer cellinhibition (CI50) were detected to be significantly smaller than 1for a wide range of BTZ/DOX ratios from 1:1 to 1:10, indicatingtheir strong synergism in killing both cancermodels (Fig. 3E and Fand Supplementary Table S1).

Biodistribution and pharmacokinetic profileNear-infrared fluorescence (NIRF) imaging was utilized as a

noninvasive method to monitor real-time tissue distribution andtumor accumulation of nanocarriers in vivo. DiD, a NIRF dye, wascoloaded into NP(BTZ-DOX) micelles to probe in vivo biodistri-bution of nanoparticles. The in vivo whole-body fluorescent

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 PEG5kChA4-Rh4 micelles (A and C) and BTZ-DOX coloaded nanocarriers (B and D). E,In vitro cumulative doxorubicin (DOX) release from free doxorubicin, Doxil, and doxorubicin encapsulated nanocarriers in PBS (pH 7.4) or acetate buffer (pH 5.5).F, In vitro cumulative bortezomib (BTZ) release from BTZ–mannitol (Velcade mimicking formulation) and BTZ–PEG5kChA4–Rh4 conjugate under indicatedconditions. G, Cellular uptake behavior of BTZþDOX and NP(BTZ-DOX) by H929 cancer cells via confocal microscopy at 37�C for 2 hours. Scale bar, 30 mm.






imaging showed that DiD-labeled NP(BTZ-DOX) micelles grad-ually accumulated at the SKOV-3 tumor xenografts throughoutthe 72 hours period after tail vein injection. In contrast, weaktumor fluorescence was observed in mice in the control groupinjected with free drugs (DiD–BTZþDOX; Fig. 4A). At 72-hourpostinjection, tumors and other major organs were harvested forex vivo NIRF imaging to compare the tissue distribution of for-mulations. As shown in Fig. 4B, DiD–NP(BTZ-DOX) micelleswere mainly accumulated in tumors with more than four-foldhigher intensity than that in the vital organs, for example, liver,

lung, spleen, and kidney. Mice treated with DiD–BTZþDOXshowed the majority accumulations in spleen and lung withlower accumulation in the tumor (Fig. 4C). During in vivo imag-ing, blood sampleswere collected andDiDfluorescent signalweremeasured to compare the pharmacokinetic profiles. As shownin Fig. 4D, nanoformulation exhibits significantly prolongedcirculation times with the 7.5-fold increase of AUC and eight-fold increase in half-life. Following ex vivo imaging, the distribu-tion of DiD signals in tumor slices were observed under fluores-cent microscope. DiD signal was observed throughout tumor

Figure 3.

In vitro cell viability analysis of H929 MM (A and B) and SKOV-3 ovarian (C and D) cancer cells for 72-hour incubation with BTZ–mannitol, bortezomib (BTZ)-conjugated, and BTZ-DOX coloaded nanocarriers with different mass ratios. Cell viabilities were plotted against bortezomib concentration (A and C) anddoxorubicin concentration (B and D), respectively. The CI plot (Chou–Talalay method) showing synergism of NP(BTZ-DOX) with varying mass ratios ofBTZ/DOX in H929 (E) and SKOV-3 (F) cells. CI < 1, synergy; CI ¼ 1, additivity; CI > 1, antagonism.

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tissue slices treated with DiD–NP(BTZ-DOX), whereas signal washardly detectable in tumor treatedwithDiD–BTZþDOX(Fig. 4E).

Proteasome inhibition and tumor apoptosisIn vitro proteasome inhibition assay was performed on H929

and SKOV-3 cells after incubation with bortezomib-based com-bination formulation at 10 ng/mL bortezomib and 100 ng/mLdoxorubicin equivalent concentrations (Fig. 5A). The proteasomeactivity was inhibited by the bortezomib treatments in bothBTZþDOX and NP(BTZ-DOX) formulations. After 1-hour incu-bation, the lysate from cells exposed to NP(BTZ-DOX) showedsignificantly higher proteasome activity compared withBTZþDOX counterpart. Additional incubation (18 hours, 37�C)with NP(BTZ-DOX) elicited more proteasome inhibition. The

kinetically slower proteasome inhibition by NP(BTZ-DOX) wasexpected due to sustained BTZ release. The reduced proteasomeinhibition for NP(BTZ-DOX) in vitro implies the potentiallyreduced systemic off-target toxicity of the nanotherapeutics,which is associated with the spike of the free drug concentrationin circulation. Therefore, it is promising to reduce toxicity andincrease the tolerable dosages of nanoformulations in cancertreatment. Accordingly, themaintained efficacy of NP(BTZ-DOX)in cell culture as shown in Fig. 3 indicated that sustained drugrelease at tumor sites would not compromise the anticancerefficacy of nanoformulations.

Wenext investigated the ability of bortezomib anddoxorubicincombination in inducing in vivo proteasome inhibition at tumorsites. Nude mice bearing SKOV-3 ovarian cancers were treated

Figure 4.

In vivo (A) and ex vivo (B) NIRF optical images of SKOV-3 bearing mice injected intravenously with free DiD–BTZþDOX 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 tocompare the pharmacokinetics. E, Representative ex vivo fluorescence imaging of SKOV-3 tumor tissue sections 72-hour postinjection of DiD and DiD–NP(BTZ-DOX). Blue, DAPI; red, NIR DiD. Scale bar, 50 mm.






with PBS, BTZþDOX, and NP(BTZ-DOX), respectively, on day 0and day 4. On day 5, mice were sacrificed and tumors wereharvested. Half of each tumor was homogenized and proteinswere extracted for measurement of proteasome activity andsubstrate ubiquitination. As shown in Fig. 5B, proteasome activitywas significantly reduced in the tumors treated with thenanoformulation compared with PBS control and BTZþDOXtreatment (P < 0.05). Western blot analysis (Fig. 5C) revealedthe accumulation of ubiquitinated species in both bortezomib-

treated groups, demonstrating directly that the processing ofubiquitinated substrates is inhibited by bortezomib. However,only slightly increased or comparable ubiquitination wasobserved for NP(BTZ-DOX) compared with BTZþDOX, despitethat tumor proteasome activity were not inhibited by free drugcombination (Fig. 5B). This may be because of relatively slowdegradation of ubiquitinated proteins after the restoration ofproteasome activity from bortezomib inhibition. Importantly,significant reduced tumor cell density and treatment-induced

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 toPBS control, free BTZþDOX, and NP(BTZ-DOX) for 1 or 18 hours at 37�C (n ¼ 3; �� , P < 0.01; �� , P < 0.001). B–D, Ex vivo proteasome inhibition and apoptosisstudies of tumor homogenate from in vivo treatment on SKOV-3–bearing mice of PBS control, free BTZþDOX, and NP(BTZ-DOX) groups. B, Ex vivoproteasome activity assay (n ¼ 3; � , P < 0.05 compared with PBS and BTZþDOX groups). C, Western blot analysis of tumor homogenates from PBS control,free BTZþDOX, and NP(BTZ-DOX) groups, showing bortezomib (BTZ)-induced accumulation of ubiquitinated species (�125–400 kDa). Grp94 served as aloading control. D, Representative H&E (top) and TUNEL staining (bottom) of ex vivo SKOV-3 tumor tissue sections.

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tumor necrosis were observed in tumors treated with NP(BTZ-DOX) in H&E staining (Fig. 5D, top row). Furthermore, theTUNEL staining demonstrated the enhanced tumor apoptosis inthe tumors treated with NP(BTZ-DOX) in comparison with bothPBS and BTZþDOX treatment (Fig. 5D, bottom row).

In vivo anticancer treatmentBased on the bortezomib dose in animal treatments reported

in literatures (26, 28), SKOV-3 ovarian cancer xenograft modelswere treated with different formulations intravenously at theequivalent bortezomib dose of 0.5 mg/kg and doxorubicin doseof 5mg/kg on days 0, 4, and 8. Having demonstrated the reducedproteasome inhibition in cell culture in vitro as shown in Fig. 5A,NP(BTZ-DOX) is expected to be more tolerable than free drugs inmice. Therefore, a 60% escalation in dose for NP(BTZ-DOX) wasinjected in BALB/c mice to test toxicity, in comparison withBTZþDOX, for three treatments on the same schedule (day 0,4, and 8). No significant body weight losses were found in micetreated with NP(BTZ-DOX) at 0.8/8 mg/kg. In contrast,BTZþDOX showed body weight loss near 20% at 0.8/8 mg/kgdose (Supplementary Fig. S24). Given the decreased in vivotoxicity by nanoformulation, an elevated dose at 0.8/8 mg/kgwere tested forNP(BTZ-DOX) inparallelwith other treatments. Asshown in Fig. 6A, body weights of mice in all treatment groupsexperiencedmodest bodyweight losses of <15%,whichwere fullyrecovered after day 20, indicating the tolerable toxicity associatedwith the treatments (Fig. 6A). Blood count analysis performed onday7 after the third administration revealed thenormal counts forall treatment groups (Supplementary Table S2). The tumorvolumes in the SKOV-3 tumor bearing mice were continuouslymeasured to monitor the tumor growth (Fig. 6B). Free drugbortezomib and BTZþDOX treatments delayed tumor growth incomparison with PBS control group. The combination of borte-zomib with a nanoformulation of Doxil exhibited improvedtumor inhibition, which is likely due to the sustained drug releaseof Doxil. Our combinational nanoformulation significantlyinhibited tumor growth at the identical dose level compared withother groups. With the increased dose level at 0.8/8 mg/kg, NP(BTZ-DOX) dramatically improved anticancer efficacy. The ani-mal survival data in Fig. 6C revealed that NP(BTZ-DOX) signif-icantly extended the mean survival time to 52 days at the samedose level in comparison to PBS (24 days, P < 0.001), bortezomib

(30 days, P ¼ 0.001), BTZþDOX (37 days, P ¼ 0.005), andBTZþDoxil (42 days, P ¼ 0.016). Noticeably, NP(BTZ-DOX)treatment showed a prolonged mean survival of 58 days at adose of 0.8/8mg/kg BTZ/DOX. The enhanced anticancer effects inthese SKOV-3 ovarian cancer xenograft models by the optimizedNP(BTZ-DOX)s are attributed to their improved tumor targeting,reduced systemic toxicity and the synchronized drug availabilityat tumor sites.

DiscussionThe application of multiple agents in vivo is complicated by

their independent pharmacokinetics, biodistribution, and off-target effects. The unsatisfying clinical observation for bortezomibcombination therapy in solid tumor treatment, including thebortezomib andDoxil combination, has been postulated to resultfromdiverse pharmacokinetics of payloads. A nanoparticle-basedcodelivery system is promising to deliver drug combinations totumor sites in a defined temporal and spatial manner at a ratio-metric dose. The integration of multiple drugs into one vehicle iscritical for yielding synergism in cancer treatment. The encapsu-lation of bortezomib and doxorubicin in a nanocarrier is chal-lenging due to their distinct chemical structures and physicalproperties. In our approach, bortezomib and doxorubicin wereloaded in functionally segregated telodendrimer nanocarriers viareversible conjugation and affinitive physical encapsulation,respectively, through the rational design of telodendrimers. Thestability of a nanoformulation determines the in vivo performanceand ultimate treatment efficacy. Polymer micelles with smallparticle sizes (<50 nm) are preferred for intratumoral drug deliv-ery (55). However, polymer micelles featuring dynamic assem-blies are generally associated with fast or even burst drug releasewhen administered systemically. Thus, a strategy to controlmicelle size and stability is to increase the affinity between thedrug and polymer within the core of the micelle. Rhein wasintroduced to the telodendrimer as a doxorubicin bindingmoietyvia pi-pi stacking, exhibiting strong affinity in doxorubicin loadingand stabilization. In an alternative approach, bortezomib wasfacilely conjugated onto telodendrimers through the reversibleboronate ester bond to synchronize drug release with doxorubi-cin. As a result, such nanoformulation sustained the drug releaseof both drugs in similar profiles (Fig. 2), which are otherwise

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 ovariancancer xenograft–bearing mice after intravenous treatment with different bortezomib (BTZ) and doxorubicin (DOX) formulations (three injections ondays 0, 4, and 8). Data are displayed as mean � SD (n ¼ 5–6; �� , P < 0.001).






dramatically mismatched for Doxil and bortezomib (Fig. 2E andF). It is promising to synchronize their pharmacokinetics anddrugdistributions in vivo to synergize their activities in cancer treat-ment. In addition, bothbortezomib anddoxorubicin can respondto acidic pH to release from the nanocarrier more efficiently,which enables on-demand drug release within the tumormicroenvironment.

Telodendrimers have a well-defined architecture and chemicalstructure via precise peptide chemistry as evidenced by NMR andMALDI-TOF MS analysis. In this study, the nanocarrier displayedattractive physiochemical characteristics as a bortezomib-basedanticancer drug codelivery vehicle, including accommodation fordifferent drug combination ratio, small particle size, high loadingcapacity, sustained drug release, and excellent stability and bio-compatibility. The optimized nanocarrier is able to synchronizedrug release profiles and increase tumor availability of bortezo-mib and Doxil for solid tumor treatment. The in vitro evaluationsdemonstrated that the combined delivery of bortezomib anddoxorubicin by our nanoformulation fosters their synergism intreating MM and ovarian cancer in cell culture.

Furthermore, in vivo studies revealed that NP(BTZ-DOX) pro-longed systemic circulation and delivered payloads to tumor sitesefficiently. The in vitro and in vivo proteasome inhibition, tumortargeting/distribution and tumor apoptosis analysis strongly cor-related with the improved anticancer effects and reduced sideeffects by the optimized combination nanotherapy. The molec-ular and pathological analysis evidenced the enhanced level ofapoptosis in tumors treated with combination therapeutics deliv-ered by nanoparticle, especially as it pertains to bortezomibactivity. Given the reduced systemic side effects, increased toler-ated dosage, targeted drug delivery, and synchronized drugresidency, NP(BTZ-DOX) exhibits significantly enhanced antican-cer effects in ovarian cancer treatment. Although the tumormicroenvironments in human patients are different fromthe xenografted tumors, the improved pharmacokinetics and

biodistribution of NP(BTZ-DOX) can be foreseen based on theiroptimized physiochemical properties, which is promising toreignite the activity of bortezomib in solid tumor treatments andmerit further development in the clinical setting.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Wang, D. Wang, J. LuoDevelopment of methodology: L. Wang, J. LuoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Wang, C. Shi, F.A. Wright, D. Guo, R.J.H.WojcikiewiczAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Wang, C. Shi, F.A. Wright, D. Wang, R.J.H.Wojcikiewicz, J. LuoWriting, review, and/or revision of the manuscript: L. Wang, X. Wang,D. Wang, R.J.H. Wojcikiewicz, J. LuoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Guo

AcknowledgmentsWe greatly appreciate Prof. StephanWilkens (SUNYUMU) for the assistance

in TEM analysis and Prof. GolamMohi (SUNY UMU) for the help in blood cellanalysis.

Grant SupportThis work was financially supported by the NIH grant NIBIB 1R21EB019607

to J. Luo, New York State Health Department Peter T. Rowley Breast Cancerproject DOH01-Rowley-2015-00067 to J. Luo, and Napi Family ResearchAwards to J. Luo.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 29, 2016; revised February 21, 2017; accepted April 4,2017; published OnlineFirst April 10, 2017.

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2017;77:3293-3305. Published OnlineFirst April 10, 2017.Cancer Res Lili Wang, Changying Shi, Forrest A. Wright, et al. Bortezomib and Doxorubicin in Ovarian Cancer TreatmentMultifunctional Telodendrimer Nanocarriers Restore Synergy of

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