Treatment of Invasive Brain Tumors Using aChain-like NanoparticlePubudu M. Peiris1,2,3, Aaron Abramowski3,4, James Mcginnity1,3, Elizabeth Doolittle1,2,3,Randall Toy1,2,3, Ramamurthy Gopalakrishnan2,3, Shruti Shah1,3, Lisa Bauer2,3,5,Ketan B. Ghaghada6,7, Christopher Hoimes8,9, Susann M. Brady-Kalnay9,10,James P. Basilion1,2,3,9, Mark A. Griswold2,3,9, and Efstathios Karathanasis1,2,3,9
Abstract
Glioblastoma multiforme is generally recalcitrant to currentsurgical and local radiotherapeutic approaches. Moreover,systemic chemotherapeutic approaches are impeded by theblood–tumor barrier. To circumvent limitations in the latterarea, we developed a multicomponent, chain-like nanoparticlethat can penetrate brain tumors, composed of three iron oxidenanospheres and one drug-loaded liposome linked chemicallyinto a linear chain-like assembly. Unlike traditional small-molecule drugs or spherical nanotherapeutics, this oblong-shaped, flexible nanochain particle possessed a unique abilityto gain access to and accumulate at glioma sites. Vasculartargeting of nanochains to the avb3 integrin receptor resultedin a 18.6-fold greater drug dose administered to brain tumors
than standard chemotherapy. By 2 hours after injection, whennanochains had exited the blood stream and docked at vas-cular beds in the brain, the application of an external low-power radiofrequency field was sufficient to remotely triggerrapid drug release. This effect was produced by mechanicallyinduced defects in the liposomal membrane caused by theoscillation of the iron oxide portion of the nanochain. In vivoefficacy studies conducted in two different mouse orthotopicmodels of glioblastoma illustrated how enhanced targeting bythe nanochain facilitates widespread site-specific drug delivery.Our findings offer preclinical proof-of-concept for a broadlyimproved method for glioblastoma treatment. Cancer Res; 75(7);1356–65. �2015 AACR.
IntroductionThe invasive forms of brain tumor, such as glioblastomamulti-
forme (GBM), are terminal upon diagnosis and no new protocolshave been developed in more than 30 years. Current approachesfor the treatment of glioma are limited in their effectiveness,because GBM tumors are characteristically diffuse, highly inva-sive, nonlocalized, and drug penetration across the blood–tumorbarrier (BTB) is poor for most chemotherapeutic agents (1, 2).Today, systemic chemotherapy is not the primary treatment ofchoice for brain tumors due to the presence of the BTB. In an effort
to avoid having to penetrate the BTB, implantable biodegradabledrug depots within a brain tumor are currently being used inclinical practice to localize a chemotherapeutic and allow forcontrolled drug delivery (3). However, this method relies on drugdiffusion froma central core. As a result, drug usually cannot reachthe tumor periphery where the most aggressive cells persist.Therefore, the ideal drug delivery system should be based onsystemic intravascular administration, which utilizes the tumor'sown blood supply for transport allowing for drug deliverythroughout the tumor and its invasive sites.
Notably, nanoparticles have shown promise, because theycan be designed not only to carry a range of cytotoxic drugs, butalso to "smuggle" the drug into intracranial tumors such asgliomas. For example, although the potent chemotherapeuticdrug doxorubicin (DOX) exhibits insignificant penetration ofthe BTB, it was demonstrated in patients with glioblastomasthat long circulating liposomal nanoparticles containing doxo-rubicin achieved a 13- to 19-fold higher accumulation ofdoxorubicin in brain cancerous lesions compared with thenormal brain (4). However, even though the BTB compromisesthe impermeable nature of the blood-brain barrier (BBB),blood vessels are not nearly as leaky as the angiogenic vesselsobserved in other cancer types (5). Thus, nanoparticles exhibitrelatively low penetration into gliomas with a patchy, near-perivascular distribution, resulting in failure to deliver drugs tothe difficult-to-reach invasive sites of brain tumors (6).
To circumvent the limitations of today's drugs in treatinginvasive brain tumors, a multicomponent, flexible chain-likenanoparticle was developed. The nanoparticle, termed nano-chain, is composed of three iron oxide nanospheres and one
1Department of Biomedical Engineering, Case Western Reserve Uni-versity, Cleveland, Ohio. 2Department of Radiology, Case WesternReserve University, Cleveland, Ohio. 3Case Center for ImagingResearch, CaseWestern ReserveUniversity,Cleveland,Ohio. 4Depart-ment of Biochemistry, Case Western Reserve University, Cleveland,Ohio. 5Department of Physics, Case Western Reserve University, Cle-veland, Ohio. 6Edward B. Singleton Department of Pediatric Radiol-ogy, Texas Children's Hospital, Houston, Texas. 7Department of Radi-ology, Baylor College of Medicine, Houston, Texas. 8University Hospi-tals Case Medical Center, Cleveland,Ohio. 9Case Comprehensive Can-cer Center, Case Western Reserve University, Cleveland, Ohio.10Department of Molecular Biology and Microbiology, Case WesternReserve University, Cleveland, Ohio.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Author: Efstathios Karathanasis, Case Western Reserve Uni-versity, 2071 Martin Luther King Jr. Drive, Wickenden Building, Cleveland, OH44106. Phone: 216-844-5281; Fax: 216-844-4987; E-mail: [email protected]
drug-loaded liposome chemically linked into a linear, chain-likeassembly (Fig. 1A and B; refs. 7, 8). The multicomponent natureand shape of the particle result in two unique features thatfacilitate effective treatment of difficult-to-treat brain tumorsusing a low dose of cytotoxic drugs as illustrated in Fig. 1C(7, 9, 10). Contrary to traditional small-molecule drugs ormore contemporary nanotherapeutics, the nanochain particlepossesses a unique ability to gain rapid access to and be deposit-ed at brain tumor sites. The nanochains are capable of trans-porting their drug cargo to brain tumors via highly specificvascular targeting of the vascular bed associated with the primarytumor mass and its invasive sites. Numerous studies have shownthat avb3 integrin is highly overexpressed on brain tumors'vascular bed, which has led to clinical trials testing integrinantagonists as antiangiogenic agents for patients with GBM(11–16). Furthermore, avb3 integrin is minimally expressed onnormal resting blood vessels (17, 18). Notably, RGD-targetednanoparticles are rapidly internalized by endothelial cells via theavb3 integrin receptor (13, 14, 19, 20). Hence, the nanochainutilizes a cyclic RGD peptide as a ligand to target the avb3 integrinreceptor on the endothelium of angiogenic blood vessels ofbrain tumors. The size, shape, and flexibility of the nanochainssignificantly increase the margination of the particles toward theblood vessel walls in microcirculation (i.e., continuous scaveng-ing of vascular walls), and targeting avidity of nanoparticles (i.e.,latching on vascular target) due to geometrically enhanced mul-tivalent attachment on the vascular target (9).
However, even after successful targeting to brain tumors, thedrug molecules must spread to all the cancer cells, especially thehard-to-reach ones, resulting in widespread anticancer activitythroughout the entire volume of tumors. Although nanoparti-cles typically release their content slowly, drug release fromnanochains can be remotely triggered due to mechanically in-duced defects of the liposomal membrane caused by the oscill-ation of the iron oxide portion of the nanochain in the presenceof an radio frequency (RF) field (7). Two hours later, afternanochains slip from the blood stream and dock on thevascular bed of brain tumors, a low-power RF field (10-kHzfrequency; 5 mT amplitude) is applied outside the body. Thefield causes the nanochain to vibrate, breaking open the drug-loaded liposome and spreading cytotoxic drugs to the entirevolume of glioma sites (7, 21).
In contrast with delivery of cancer drugs via passive intratu-moral accumulation, our strategy utilizes the overexpressed avb3integrin receptor as a docking site to establish well-distributeddrug reservoirs on the brain tumor vasculature, which can sub-sequently spread free drug in the tumor interstitium using an RFfield as an external trigger. In this study, we show that the synergyof nanochain's enhanced targeting and widespread drug deliverycapabilities facilitates enhanced treatment of brain tumor sites,which are otherwise inaccessible by conventional therapies.
Materials and MethodsMaterials
Theprimary antibody for the specific endothelial antigenCD31was purchased from BD Biosciences Pharmingen. Secondaryantibodies and cell culture media were obtained from Invitrogen.Cross-Linked Ethoxylate Acrylate Resin (CLEAR) resin, reactionvessels, other accessories for solid-phase chemistry, and the cyclo(Arg-Gly-Asp-D-Phe-Cys) or c(RGDfC) peptide were purchasedfrom Peptides International Inc. The cross-linkers 3,30-Dithiobis(sulfosuccinimidylpropionate; DTSSP) and sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC),and the cleaving agent Tris[2-carboxyethyl] phosphine (TCEP)wereobtained from Thermo Fisher Scientific. Polyethylene glycol(PEG) conjugateswerepurchased fromLaysanBio.General solventsand chemicals were obtained from Thermo Fisher Scientific. Doxo-rubicin was obtained from Sigma.
Synthesis and characterization of nanoparticlesThe nanochains were fabricated using a two-step solid-phase
chemistry based on a previously published method (7, 8). Moredetails on the synthesis of the nanochain particles can be foundin previous publications (7, 8, 21).
Tumor modelAll animal procedures were conducted under a protocol
approved by the Institutional Animal Care and Use Committeeof Case Western Reserve University (Cleveland, OH). The CNS-1rodent glioma tumor model was used for these studies. Five- to8-week-old NIH athymic nude mice (20–25 g each) were housedin the Athymic Animal Core Facility at Case Western ReserveUniversity according to institutional policies. CNS-1 cells were
Figure 1.Illustration of the nanochain particleand its therapeutic effect on braintumors. A, schematic of a linearnanochain particle composed of threeiron oxide nanospheres and one drug-loaded liposome. B, TEM image ofnanochain particles. C, illustration ofthe successful delivery of nanochain-baseddrug to invasive brain cancer viavascular targeting and RF-triggereddrug release.
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infected with GFP encoding lentivirus, harvested for intracranialimplantation by trypsinization, and concentrated to 1� 105 cells/mL in PBS. Mice were anesthetized by intraperitoneal administra-tion of 50 mg/kg ketamine/xylazine and fitted into a stereotaxicrodent frame. Cells were implanted at AP¼þ0.5 and ML¼ – 2.0from bregma at a rate of 1 mL/minute in the right striatum at adepth of �3 mm from dura. A total of 200,000 cells wereimplanted per mouse. Similar procedures were used for the 9Lglioma model.
Histologic evaluationOnce the appropriate tumor sizes were established (�8 days
after tumor inoculation), the animals were injected intravenouslywith the nanochain particles at a dose of 0.5 mg/kg doxorubicin.At 2 hours after injection, the animals were exposed to the RF fieldfor 60minutes. Animals were euthanized 24 hours after injection,and organswere extracted and analyzedhistologically for locationof brain tumor (CNS-1-GFP cells). Serial tissue sections of 12-mmthickness were stained for the specific endothelial antigen CD31and with the nuclear stain DAPI. The tissue sections were imagedat�5,�10, and�20magnification on the Zeiss AxioObserver Z1motorized FL inverted microscope. To evaluate the spread ofdrugs in relation to the location of nanochains, we used Prussianblue to stain iron. Direct fluorescence (red) imaging was used fordoxorubicin. Histologic sections were imaged on a Zeiss AxioObserver Z1 motorized FL inverted microscope. To obtain animage of the entire section, a montage of each section was madeusing the automated tiling function of the microscope.
Survival studyOnce the tumors were established (�5 days; histologically
confirmed), the animals were injected intravenously with thenanochain at a dose of 0.5 mg/kg doxorubicin. At 2 hours afterinjection, a 60-minute application of the RF field (amplitude`B ¼ 2 mT, frequency f ¼ 10 kHz, RF power ¼ 3–5 Watts) wasemployed using a custom-made solenoid (N ¼ 105 turns, innerdiameter ¼ 2.8 cm; ref. 7). During this procedure, the animalswere anesthetized through the administration of inhalant iso-flurane. Two subsequent treatments were applied at time intervalsof 2 days at the samedoxorubicin dose followed by exposure toRFfollowing identical protocol to the first cycle (if applicable).Following the same schedule for the treatments and RF applica-tion, control groups included animals treated with nanochains(no RF), liposomal doxorubicin with RF, free doxorubicin, andsaline. After tumor inoculation, mice were monitored daily forany abnormal symptoms. The well-being of the animals tookpriority in decisions about euthanasia or other interventions.When animals showed a 10% loss of body weight, they wereeuthanized in a CO2 chamber. Although the 10%weight loss wasthe primary endpoint criterion for the vast majority of the ani-mals, two animals in the CNS-1 gliomamodel, one animal in thedoxorubicin-treated group, and one animal in the nanochain-treated group (þRF), had to be euthanized because the animalssuffered from inactivity and lethargy. At the terminal point, thetumormass at the primary site had grown significantlywith afinalsize of about 1.8 mm. Although the final size of the tumor at theprimary site does not indicate the degree of tumor invasiveness,we histologically observed a considerable number of distant siteswith dispersive brain tumor cells in the case of the CNS-1 gliomamodel. Time of death was determined to be the following day.
Statistical analysisMeans were determined for each variable in this study and
the resulting values from each experiment were subjectedto one-way ANOVA with post hoc Bonferroni test (SPSS 15).A P value of less than 0.05 was used to confirm significantdifferences. Normality of each dataset was confirmed using theAnderson–Darling test.
ResultsSynthesis and characterization of nanochains
To fabricate the multicomponent nanochain particles, a step-wise solid-phase chemistry approach was developed. In the firststep (Fig. 2A), amine-PEG functionalized iron oxide nanospheres(hydrodynamic diameter of 27 nm)were conjugated onto amine-functionalized CLEAR resin via a homo-bifunctional cross-linkerreactive toward primary amines containing a disulfide bridge. Theiron oxide nanospheres were allowed to bind to the solid supportand then cleaved off using a reducing agent. The thiolytic cleavageliberated the iron oxide nanosphere from the solid supportconverting the amines to a different chemical functionality (thiolgroup) on the portion of the nanosphere's surface that was linkedto the resin. The second step (Fig. 2B) used the same type of resinand the modified nanospheres were introduced in a step-by-stepmanner using a hetero-bifunctional cross-linker for conjugationbetween primary amine and sulfhydryl groups. As a final com-ponent, an amine functionalized doxorubicin-loaded liposomewith a hydrodynamic diameter of 35 nm was added. It should benoted that each step included multiple washing cycles to removeany unbound nanospheres and excess reagents from the nano-particle–resin complex. In the end, the thiol of the cysteine residueof the c(RGDfC) peptide was used to conjugate the targetingligand to the remaining amine-terminated PEG on the surface ofnanochains. Finally, the avb3 integrin-targeting nanochains werecleaved off the resin and recovered.
Because of the simplified purification procedure and easyhandling of multiple reaction vessels, the solid-phase–basedsynthesis enables us to manufacture large amounts of nano-chains that exhibited a high degree of uniformity. On the basisof analysis of multiple TEM images (minimum count was200 particles), the majority of the particles (>85%) is com-posed of nanochains with three iron oxide spheres and oneliposome (7, 21). As shown in Fig. 2C, the hydrodynamic sizeof the nanochain particle and each constituent nanosphere, asmeasured by dynamic light scattering (DLS), verified the TEMfindings. Because of the high intraliposomal space available fordrug encapsulation and the efficient remote loading technique(22), the entrapment efficiency was very high (drug cargo: 6.8�10�5 ng doxorubicin per particle). To determine the contribu-tion of blood plasma to leakage of doxorubicin, Fig. 2D showsan in vitro stability test. The dialysis curve plateaued afterapproximately 3 hours, and the nanochain exhibited a leakageof approximately 6.5% of the total encapsulated doxorubicinafter 24 hours, which was comparable with the leakage of atypical 100-nm liposome.
Vascular targeting of brain tumorsTo evaluate the nanochain's ability at targeting brain
tumors, we compared the avb3 integrin-targeting nanochainswith avb3 integrin-targeting 30-nm liposomes, their nontar-geting variant and free doxorubicin. For this study, we selected
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the orthotopic CNS-1 glioma model. Recent reviews havecompared the most commonly used rodent glioma models(e.g., C6, 9L) in terms of pathologic and genetic similaritieswith the human disease (23, 24). The CNS-1 model is one ofthe few models that recapitulate the microenvironment of thehuman disease and display several histologic features anddiffuse growth and invasive pattern similar to human GBM.In addition to their ability to express several glioma markers, athree-dimensional cryo-imaging technique showed that therodent CNS-1 glioma cells is a valid system to study the highlydispersive nature of glioma tumor cells along blood vesselsand white matter tracts in vivo (25).
The animals were injected with the various doxorubicin for-mulations via tail vein at a dose of 0.5 mg/kg doxorubicin 8 daysafter orthotopic tumor inoculation. The animals were transcar-dially perfused with heparinized PBS 24 hours after intravenousadministration, and brains were retrieved and analyzed for doxo-rubicin content following an established protocol (26). At thistime point, doxorubicin was undetectable in blood circulation.We predominantly collected the tumor mass at the primary sitebecause it was very challenging to identify the margins of theinvasive sites of the tumor. Vascular targeting of nanochainsresulted in a 4.7% of the administered dose being localized inbrain tumor (Fig. 3A), which was 18.6-fold higher than freedoxorubicin. Free doxorubicin and nontargeted liposomes exhib-ited very low accumulation (<1%of injected dose) at brain cancersites. The targeted liposomes and nontargeted nanochains dis-played an accumulation of 1.45%and 1.75%of the injected dose,
respectively, in brain tumors. As expected, the levels of doxoru-bicin in normal brain were undetectable in the case of all theformulations.
After evaluating the significant targeting enhancement of doxo-rubicin to brain tumors using nanochains, we used noninvasiveimaging to assess the therapeutic efficacy of the nanoparticle withor without the RF application. By infecting the CNS-1 cells withGFP encoding lentivirus, the expression of GFP of the CNS-1 cellsenabled in vivo imaging using a Maestro fluorescence imagingsystem (Supplementary Fig. S1A). As a metric of the response ofbrain tumors to various treatments (n ¼ 5 in each group),quantification of fluorescence signal was used (Fig. 3B). Becauseof the emission of GFP in the green zone of the visible spectrum,the depth-dependent attenuation of the fluorescence signal pri-marily by the skull (and the brain tissues) does not allow accuratequantification of the number of CNS-1 glioma cells in the case ofin vivo imaging (Supplementary Fig. S1B). However, the relativedifferences of fluorescence signal among the various treatmentscan be used as a comparison in a semiquantitative manner. At 8days after orthotopic tumor inoculation, the animals were intra-venously injected with nanochains at a dose of 0.5 mg doxoru-bicin per kg of body weight. A 60-minute application of the RFfield (amplitude B¼ 5mT, frequency f¼ 10 kHz) was used at thepoint of maximum accumulation of the nanochain in braintumors (2 hours after injection; ref. 7). Previous in vitro studiesidentified that a 60-min application of the RF field at the selectedoperating conditions guarantees approximately 100% release ofnanochain's drug cargo (7). Maestro imaging was performed
Figure 2.Reaction scheme of the controlled assembly of nanochains using solid-phase chemistry. A, in the first step, chemical bifunctionality on the surfaceof parent iron oxide nanospheres is topologically controlled, resulting in nanospheres with two faces, one displaying only amines and the other only thiols.B, in the second step, the two unique faces on the parent nanosphere serve as fittings to chemically assemble them into nanochains. C, sizedistribution of nanochain particles and their parent nanospheres obtained by DLS (data presented as mean � SD). IO, iron oxide. D, comparison of in vitroblood plasma stability of nanochains to 30-nm and 100-nm liposomal doxorubicin. In a typical leakage procedure, 1 mL of formulation was placed indialysis tubing with 100 k MWCO and dialyzed against blood plasma at 37�C.
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before and 48 hours after injection to allow for the cytotoxiceffects of doxorubicin to occur. Figure 3B shows that the signal ofthe untreated animals doubled within 48 hours. Although freedoxorubicin decelerated the disease, the brain tumor continuedgrowing as indicated by the increase of fluorescence signal. Evenwith the nanochain's enhanced targeting efficacy, the nanochaintherapy (without RF) resulted in a similar anticancer effect to freedoxorubicin. However, animals treated with nanochain followedby application of the RF field exhibited a substantial decrease offluorescence signals, which indicates the additional therapeuticeffects of the RF-triggered release. Furthermore, no temperatureincrease of the brain tissue was observed due to the RF field(Supplementary Fig. S2), indicating that the triggered releasemechanism of drug from the nanochains is not based onhyperthermia.
Histologic evaluationAfter evaluating the targeting capabilities and anticancer effects
of the nanochain in a macroscopic manner, we assessed thelocalization of nanochain in invasive glioma and the degree andtopology of doxorubicin delivery with or without the applicationof the RF field. Histologic analysis was performed on separategroups of mice 24 hours after injection (n ¼ 3). A representativeimage of a brain section is shown in Fig. 4A (left) displaying thepresence of the primary tumor and clusters of invasive cancer cellsdispersed in the brain. Bright field microscopy of the samehistologica section stained with hematoxylin-eosin is shown inSupplementary Fig. S3. Most notably, application of the RF fieldresulted in widespread delivery of doxorubicin at both the pri-mary and invasive sites of brain tumor (of Fig. 4A, right). We then
focuses on an invasive site (shown by the yellow circle in Fig. 4A)to assess the ability of the nanochain to deliver drugs to dispersingglioma cells. Imaging at higher magnification of this invasive siteshowed that nanochains were selectively localized on the endo-thelium associated with cancerous sites (Fig. 4B, left). Notably,application of the RF field facilitated the spreading of doxorubicinat distant cells away from nanochain deposits (Fig. 4B, right).Although the RF-triggered release resulted in widespread deliveryof doxorubicin to the target site (Fig. 4C), no spread of doxoru-bicin was observed in the case of nanochain-treated animals thatwere not exposed to RF, since the fluorescence signal of intrali-posomal doxorubicin is quenched (Fig. 4D). The in vivo anticancereffects of the nanochain treatment followed by RF observed inhistology are consistent to cell cytotoxicity studies (Supplemen-tary Fig. S4). Although 30-nm liposomal doxorubicin, 100-nmliposomal doxorubicin, and nanochain without RF exhibitedmoderate cytotoxicity (less than 30% relative cytotoxicity),RF-triggered release of doxorubicin from nanochain had signif-icant cytotoxic effects (65% cytotoxicity), which was comparablewith the effect of free doxorubicin (75% cytotoxicity), indicatingthe release of free doxorubicin molecules from the nanochainparticles upon application of the RF field.
Survival studiesThe therapeutic effect of the nanochain treatment was deter-
mined in two orthotopic glioma models by measuring survivaltimes. Control treatments included free doxorubicin followed byRF and nanochain without RF. The treatments were administeredthree times (Fig. 5A), each at a dose of 0.5 mg/kg doxorubicin.Targeted nanochains exhibited a 2.6-, 3.2-, and 6-fold higher
Figure 3.Evaluation of the ability of nanochains to target invasive brain tumors in mice. A, CNS-1 cells (2 � 105) were implanted in the right striatum at a depth of 3 mmfrom dura. At 8 days after tumor inoculation, the animals were injected with doxorubicin, nontargeted liposomes, integrin-targeting liposomes, nontargetednanochains, and targeted nanochains. All formulations were administered at a dose 0.5 mg doxorubicin per kg of body weight (n ¼ 4 mice per group; � , P <0.01 by Student t test). At 24 hours after injection, animals were euthanized, brain tumors were excised, and their doxorubicin content was extracted andmeasured using an established method. B, as a metric of the response of brain tumors to various treatments (n ¼ 5 in each group), quantification offluorescence intensity (FI) was used. The stable expression of green fluorescence protein within the CNS-1 cells enabled in vivo imaging using a CRiMaestro fluorescence imaging system. All formulations were administered at day 8 after tumor inoculation at a dose of 0.5 mg doxorubicin per kg biweekly.In the case of treatments combined with the RF field, animals were exposed for 60 minutes to an RF field (amplitude B ¼ 5 mT, frequency f ¼ 10 kHz)using a custom-made solenoid coil. The y-axis represents the normalized difference of fluorescence signal between days 8 and 10 (calculation of normalizedvalue [(FIday8- FIday10)/FIday8]x100; n ¼ 5 in each group; �, P < 0.01 by Student t test). DOX, doxorubicin.
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deposition in brain tumors than nontargeted nanochains,targeted liposomes, and nontargeted liposomes, respectively(Fig. 5B). Since the targeting efficiency of the nanochains to braintumors was found to be significantly higher than those formula-tions, we chose to only assess the effect of targeted nanochainswith or without the combination of the RF field on the survivalrate. Not surprisingly, the free doxorubicin treatment had negli-gible therapeutic benefits in the CNS-1model. On the other hand,even with their enhanced targeting capability, nanochains (with-out RF) exhibited amoderate effect. However, the survival time ofthe nanochain-treated animals followed by RF was significantlyincreased (25 � 3 days; mean � SD) when compared with thenanochain-treated group without RF (16� 1 days), doxorubicin-treated group (10� 3days), and the untreated group (9� 1 days).In comparison with standard chemotherapy (i.e., doxorubicin),the 2.5-fold increase in survival of the nanochain-treated animalsfollowed by RF is highly significant considering the highly inva-sive nature of the CNS-1 model.
To illustrate the therapeutic efficacyof thenanochain treatment,we also used the 9Lmodel, which is an aggressive but not invasive
glioma model. As expected, the therapeutic effect of the freedoxorubicin treatment was negligible as indicated by the survivaltime being similar to the untreated group. Although 100% ofthe untreated and doxorubicin -treated mice died within 28 days,40%of the nanochain-treated group followed by RFwas still aliveafter 77 days (Fig. 5C). At the terminal point, post portemmeasurements showed that the tumor at the primary site was1.8 � 0.17 mm (mean � SD) in size for both animal models.
The average weight progression for each group is shown inSupplementary Fig. S5. Because of the highly selective depositionof the nanochain particles at brain tumors and the subsequentefficient spreading of drug, this significant therapeutic outcomewas achieved at a very low dose (i.e., 0.5 mg/kg), which is 10- to20-fold lower than the typical clinical regimens of liposomaldoxorubicin.
DiscussionInvasive brain tumors are recognized as one of the deadliest
forms of cancer. This stems from the fact that radiation therapy
Figure 4.Histologic evaluation of the nanochaintreatment. A, histological evaluationof the anticancer effect of nanochainswas performed in the orthotopicCNS-1 model in mice [magnification,�5; green, CNS-1 glioma cells (GFP);blue, nuclear stain (DAPI); violet,doxorubicin]. Fluorescence imagingof an entire histologic section of thebrain shows the primary tumor and itsinvasive sites (left). Fluorescenceimaging of the same histologic sectionshows the widespread distribution ofdoxorubicin molecules after a 60-minapplication of RF (right). B, highermagnification imaging (�20) of aninvasive site shows the location ofnanochains (blue) with respect to thelocation of endothelial cells (green, CD31) and brain tumor cells (left), and theRF-triggered release of doxorubicin(right) in the same histologic section.Nanochains were visualized bystaining iron with Prussian blue. Thedistribution of doxorubicin moleculesis shown with (C) or without (D) RFwith respect to the location of cancercells. DOX, doxorubicin.
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and surgical resection are incapable of completely removingdeeply penetrating, diffuse brain tumors (1, 2). As a result,mediansurvival is merely prolonged to 12 months following the combi-nation of surgical resection and focal radiotherapy. At the sametime, even though potent cytotoxic drugs are available to oncol-ogists, systemic chemotherapy is not the primary treatment ofchoice formalignant brain tumors due to the presence of the BTB,which limits transport to lipophilic or low-molecular-weight,uncharged compounds. In an effort to prevent exposure ofhealthy organs to the toxic side effects of chemotherapeutics andavoid having to penetrate the BTB, implantable drug depotswithin a brain tumor are currently being used in clinical practice(3). For example, Gliadel, an intracranial implantable biodegrad-able polymer loaded with the lipophilic drug BCNU, extendsthe median survival of patients with grade IV glioma by 13.4
weeks compared with placebo (27). On the other hand, systemicadministration of nanoparticles has the potential to facilitateaccess to the entire brain tumor by utilizing the tumor's ownblood supply.
Historically, attempts to improve nanoparticle homing to braintumors have relied on the EPR effect followed by targeting ofvarious receptors to direct the nanoparticle–drug complex intobrain tumor cells. However, even though the BTB slightly com-promises the nature of BBB, brain tumors consist of blood vesselsthat are not as leaky as the angiogenic vessels observed in othercancer types. This results in low penetration of nanoparticlesinto the brain tumor interstitium, resulting in failure to reachthemajority of the primary tumormass and especially its invasivesites (6). Furthermore, the EPR effect is typically noticeable atthe core regions of a brain tumor, while it is attenuated at theinvasive sites of brain tumors with dispersing cancer cells. This isdue to the fact that the BBB of invasive sites has a very highlikelihood to remain intact. Importantly, it is not uncommonto find dispersing brain tumor cells as far away as 4 cm from theprimary site (28). Along these lines, the nanochain-basedtherapy exploits the tumor vasculature as a docking site for thenanoparticles followed by RF-triggered drug release to spreadthe drug throughout the primary tumor and its invasive sites,which are nearly inaccessible by today's systemic therapeutics.
Although various receptors have been exploited to target nano-particles to cancer cells (e.g., folate, EGF receptors; refs. 29–34),our work suggests that avb3 integrin-mediated vascular targetingprovides highly selective targeting of brain tumors. In fact, thestructure and shape of the nanochains have been specificallydesigned to target the tumor vascular bed (9). The particle shapegoverns the navigation of circulating nanoparticles through dif-ferent biologic processes, including targeting of difficult-to-reachcancer sites (35).Oneof thepivotal steps dictating the transport offlowing nanoparticles is their margination toward the bloodvessel walls. Contrary to spherical nanoparticles, nanoparticleswith geometrical asymmetry (e.g., oblong shape) are subjected totorques, resulting in tumbling and rotation, which increase thelateral drift of nanoparticles toward the blood vessel walls inmicrocirculation (36–38). Furthermore, the particle shape alsogoverns the targeting avidity of nanoparticles using receptor-ligand systems. Compared with nanospheres, oblong-shapednanoparticles exhibit enhanced targeting avidity due to geomet-rically enhanced multivalent docking. Indeed, within 24 hoursafter injection, integrin-targeting of nanochains resulted in a 4.7%of the administered dose being localized in brain tumor, whichwas 3-fold higher than integrin-targeting liposomes. Regardingthe in vivo fate of the remaining nanochains, our previous pub-lication evaluated the organ distribution of nanochains, whichwas comparablewith the behavior of standard 100-nmPEGylatedliposomes (7). At 24 hours after injection, the nanochains weremostly found in the reticuloendothelial organs (liver and spleen).Even though such a significant portion of the injected doseaccumulated at the tumor site, the histologic studies show thatno spread of doxorubicin in the brain tumor was observed in theabsence of the external stimulus (i.e., RF field). Not surprisingly,even with the enhanced targeting, the nanochain treatment(without RF) provided only modest benefits in terms of pro-longed survival. This is primarily related to the drug release profilefrom nanoparticles. Although free drug in its molecular formquickly spreads within the tumor interstitium (39–41), nanopar-ticles, without any triggeringmechanism, release their content at a
Figure 5.Treatment of brain tumor using nanochains. A, the schedule of treatmentsand application of RF are shown with respect to tumor inoculation. Allformulations were administered at a dose of 0.5 mg doxorubicin per kgbiweekly. Each treatment was administered three times at day 5, 7, and 9after tumor inoculation. In the case of treatments combined with the RFfield, animals were exposed for 60 minutes to an RF field (amplitude B ¼ 5mT, frequency f ¼ 10 kHz) using a custom-made solenoid coil. B, thesurvival times of CNS-1 tumor-bearing animals are shown after treatmentwith saline (untreated group), doxorubicin followed by RF, nanochains (noRF), and nanochains followed by RF (n ¼ 5 mice in each group). C, thesurvival times of 9L tumor-bearing animals are shown after treatment withsaline (untreated group), doxorubicin followed by RF and nanochainsfollowed by RF (n ¼ 5 mice in each group). DOX, doxorubicin.
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relatively slow rate, once they deposit at the target site. This slowrelease generates a low temporal and spatial concentration gra-dient of the drug, resulting innoncytotoxic levels of the drug distalfrom the particle (41).
Although previous studies exploited vascular targeting for anti-angiogenic strategies (13, 42, 43), we coupled vascular targeting ofnanochains toaunique triggered releasemechanism to spreadhighamounts of drug into the hard-to-reach brain tumors. In a previousstudy (7), we assessed the relation of the nanochain's structure toRF-triggered drug release. Through their interaction with magneticfields at the selected frequency of 10 kHz, the iron oxidecomponent of the nanochain particle efficiently converts magne-tic energy to mechanical vibration, resulting in "mechanical"disruption of the liposomal membrane. Contrary to heat-induceddrug release achieved by other nanoparticle designs (e.g., thermo-sensitive nanoparticles incorporating iron oxide or gold nanopar-ticles; ref. 44), the releasemechanismofnanochains results in rapidand efficient drug release even from very low concentration ofnanoparticles. This ability stems from the structure of the nano-chains, in such fashion that the response of the nanoparticles to the10 kHz oscillatingmagnetic field is amechanical "vibration" of thechain, rather than true rotational motion or heat dissipation (7).For example, although application of RF induced rapid release ofdoxorubicin from nanochains at very low particle concentration,negligible doxorubicin release was observed from liposomesencapsulating doxorubicin and iron oxide nanospheres. Althougha nanochain containing more than 3 iron oxide nanospheres mayexhibit even faster triggered release profiles, a nanochain largerthan 150 nm in length (i.e., 4 or more iron oxide nanospheres)reduces significantly the extraction yield of nanochains from theresin during the solid-phase–based synthesis of the particles.Furthermore, the RF frequencies and power used in our systemare lower than those experienced in a conventional clinical MRI(e.g., 64 MHz at kilowatts of power). These RF fields are wellunderstood, and thus the design, cost, and clinical deployment ofsuch system present a low degree of difficulty.
In addition to the drug delivery barrier of gliomas, it is impor-tant to recognize that glioma cells tend to be particularly resistantto drugs. The underlying causes for the failure of therapies againstbrain tumors may be diverse but cellular hierarchies have beenidentified defined in glioblastomas with self-renewing tumor-initiating cells at the apex that frequently display resistance toconventional treatments and can migrate to cause tumor recur-rence (45–47). For our initial studies, we chose doxorubicin as adrug model candidate, because of its fluorescence properties andits ability to rapidly diffuse through cellularmembranes and reachnuclear DNA, which functions as a sink for doxorubicin (39–41).Although it is well established that free doxorubicin displays verypoor penetration into gliomas and is nearly inaccessible to braintumor cells (48–50), our data indicate the enormous potential ofthe nanochain therapeutic strategy to deliver doxorubicin toglioma cells. To address both challenges of drug delivery and
multidrug resistance, future studies will seek to integrate thenanochain technology with the appropriate combination of com-plementary drugs to enable effective treatment of the "generalpopulation" of glioma cells as well as the small fraction of gliomacells that are resistant.
In this study, we show that both the particle shape and themulticomponent nature of the nanochain particle played anessential role in its therapeutic efficacy against invasive gliomas.Coupling a vascular targeting strategy with the RF-triggered drugrelease of nanochains overcame the BTB issues, enabling highdrug concentrations and widespread delivery within the tumorand therefore provided an increased likelihood of highly effectivetreatment of invasive brain cancer using a low dose of cytotoxicdrugs.
Disclosure of Potential Conflicts of InterestM.A. Griswold reports receiving commercial research grant from Siemens
Healthcare (MRIBusinessUnit).Nopotential conflicts of interest were disclosedby the other authors.
Authors' ContributionsConception and design: P.M. Peiris, K.B. Ghaghada, S.M. Brady-Kalnay,J.P. Basilion, M.A. Griswold, E. KarathanasisDevelopment of methodology: P.M. Peiris, E. Doolittle, R. Toy, S. Shah,K.B. Ghaghada, C. Hoimes, S.M. Brady-Kalnay, M.A. Griswold, E. KarathanasisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.M. Peiris, A. Abramowski, J. Mcginnity, E. Doolittle,R. Toy, S. Shah, L. Bauer, E. KarathanasisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.M. Peiris, E. Doolittle, S. Shah, K.B. Ghaghada,C. Hoimes, E. KarathanasisWriting, review, and/or revision of the manuscript: P.M. Peiris, K.B. Ghaghada,C. Hoimes, S.M. Brady-Kalnay, J.P. Basilion, M.A. GriswoldAdministrative, technical, or material support (i.e., reporting or organizingdata, constructingdatabases): J.Mcginnity, R.Gopalakrishnan, K.B.Ghaghada,M.A. Griswold, E. KarathanasisStudy supervision: M.A. Griswold, E. Karathanasis
AcknowledgmentsThe authors thankDishenLin, Swetha Rao, and Samantha Tucci for helpwith
animal studies and histology.
Grant SupportThis work was supported by grants from the National Cancer Institute
(R01CA177716), the Clinical and Translational Science Collaborative of Cleve-land (UL1TR000439) from the National Center for Advancing TranslationalSciences component of the NIH, the Case Comprehensive Cancer Center(P30CA043703), and the Ohio Cancer Research Associates (E. Karathanasis).L. Bauer was supported by a fellowship from the National Cancer InstituteTraining Program in Cancer Pharmacology (R25CA148052).
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.
ReceivedMay28, 2014; revisedDecember 19, 2014; accepted January 4, 2015;published OnlineFirst January 27, 2015.
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