Reconstructed Stem Cell Nanoghosts: A Natural Tumor TargetingPlatformNaama E. Toledano Furman, Yael Lupu-Haber, Tomer Bronshtein, Limor Kaneti, Nitzan Letko,Eyal Weinstein, Limor Baruch, and Marcelle Machluf*,†

Faculty of Biotechnology and Food Engineering, Technion − Israel Institute of Technology, Haifa 32000, Israel

*S Supporting Information

ABSTRACT: The ultimate goal in cancer therapy is achieving selective targeting of cancer cells.We report a novel delivery platform, based on nanoghosts (NGs) produced from the membranesof mesenchymal stem cells (MSCs). Encompassing MSC surface molecules, the MSC-NGsretained MSC-specific in vitro and in vivo tumor targeting capabilities and were cleared fromblood-filtering organs. MSC-NGs were found to be biocompatible. Systemic administration ofdrug loaded MSC-NGs demonstrated 80% inhibition of human prostate cancer.

KEYWORDS: Nanoghosts, drug delivery, mesenchymal stem cells, cancer targeted therapy

The ultimate goal in cancer therapy is a “magic bullet” thatallows selective targeting of cancer cells.1,2 Three main

considerations must be addressed when designing any suchdelivery system: biocompatibility, long circulation time, andselectivity.1 In cancer therapy, passively targeted drug-carryingparticles are still the predominantly used drug-delivery platform.1

On the basis of their nanosize and physical properties, suchsystems were shown to accumulate in the tumor surroundings,owing to the enhanced permeability and retention (EPR) effectof tumor vasculature and microenvironment. Passive targeting,however, is still limited due to varying degrees of tumorvascularization and permeability affected by the tumor type andstage.3 To overcome these limitations, active cancer targetingmoieties, such as antibodies, have been incorporated intopolymeric drug-carriers made from nanoparticles, micelles, orliposomes.4−6 However, the relatively short circulation times4

and the complexity of producing such actively targeted carriers7

hinder their clinical applications.Here we report on a novel targeted delivery platform, based on

nanoghosts (NGs) that are reconstructed from the whole cellmembrane of mesenchymal stem cells (MSCs). To ensuretargeting, the lineage integrity of the MSCs from which the NGswere produced was continuously validated using flow cytometryfor typical MSCmarkers (Supporting Information Figure S1). Incontrast to exosomes or other extracellular vesicles that are shedor bud from cells, MSC-NGs are manufactured in a reproducibleprocess by isolating intact MSC cell membranes (ghost cells),and homogenizing them into nanosized vesicles (nanoghosts)while entrapping a therapeutic of choice. This approach,presenting a new paradigm for active cancer-targeted drug-

delivery, is supported by our previous publication demonstratingthe in vitro targeting of HIV-infected cells by NGs expressing thereceptor for a viral ligand found on infected cells.8,9

The reasoning for choosing MSCs as a source to producecancer-targeting NGs lies in their hypo-immunogenicity andability to target many kinds of cancers at different developmentalstages.10,11 Such targeting was shown to involve both chemo-taxis12 and surface interactions.13 Nonetheless, isolated mem-brane fractions of tumor cells, and not their cytoplasmaticfractions, appear to contain the most potent MSC attrac-tants.14,15 The MSC targeting mechanism is also known to betumor-specific but not species-specific, allowing the targeting ofsusceptible tumors by MSCs isolated from different species.16,17

Moreover, MSCs expressing exogenous anticancerous proteins,suggested for cell-based cancer therapy due to their homingabilities and hypo-immunogenicity, demonstrate some benefitswhen administered as whole cells into animal models.18

Therefore, using MSC-derived NGs (MSC-NGs), a variety oftumors requiringMSC support10,19 may be targeted by their owninvitation, extended to these Trojan horses. Most importantly,this targeting system does not entail the elaborate production oftargeting molecules and their incorporation into passive vehicles,constituting a simpler and more clinically relevant approach thanexisting particulate drug-delivery vehicles. Unlike exosomes,shed-vesicles or cell-based delivery systems, which are predom-inantly intended for the delivery of products manufactured by the

Received: April 17, 2013Revised: June 6, 2013

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cells themselves, MSC-NGs can be made in different sizes andloaded with a variety of therapeutics, not only cell-made ones,through a reproducible and clinically relevant technologicalprocess.In the reported study, the efficacy of our MSC-derived NGs

against cancer was demonstrated using a prostate cancer modeland by encapsulating the biologic model drug sTRAIL, thesoluble form of TNF-related apoptosis-inducing ligand. sTRAIL,applied through cell-based therapy, was shown to haveconsiderable anticancerous impact when secreted into thetumor environment by transfected tumor cells20 or targetedMSCs.21 sTRAIL was selected due to its short biological half-lifeand hepatotoxicity, limiting its clinical use despite its apparentselectivity and potency. Moreover, sTRAIL administered in theform of controlled-release formulation showed no effect whenadministered without additional drugs, further emphasizing theshortcomings of previously reported delivery platforms.22

Physical Characterization of NGs. NGs were preparedfrom the cytoplasmatic membranes of human and rat MSCs(hMSC and rMSC, respectively), and human smooth musclecells (SMC, as a nonmesenchymal control). Briefly, the cells(Figure 1a) are harvested and hypotonically treated with tris-magnesium buffer followed by mild homogenization to allowcytosol removal without substantially disrupting cell membrane.Cells are centrifuged, precipitated, and washed several times toremove most nucleic matter, as apparent by staining with 4′,6-diamidino-2-phenylindole (DAPI, Figure 1b). The homogenized

cytoplasm-free cells (termed ghost cells or ghosts) are thenmildly sonicated and washed again (Figure 1c). As seen, somemembrane fractionation occurs in this step; nonetheless, theproduct is large enough to be observable by light microscopy andto be separated by low speed centrifugation, allowing furtherremoval of the cytosol and nuclei residues and resulting in muchless evidence for DAPI staining. The sonicated ghosts wereextruded into NGs in a medium containing sTRAIL andretrieved by ultracentrifugation. The NGs exhibited narrow sizedistributions with similar average diameters of ∼180 nm (Figure1d) and zeta potential of −12 mV (Figure 1e). sTRAILencapsulation efficiency was 30% (data not shown) and had noapparent effect on the size or unilamellar morphology of hMSC-NGs imaged with Cryo-TEM (Figure 1f). Out of the sTRAILreleased during five days in 37 °C (18 ± 4 μg out of 300 μg/sample), about 70% was released during the initial six-hour burst-release, followed by a linear sustained release profile (Figure 1g).All hMSC surface markers (>50%) were retained on the NGs, asshown by flow cytometric analysis of Dynabeads conjugated withhMSC-NGs and immunostained (Figure 1h). The overallmarker retention was slightly reduced compared to MSC-NGsthat were not PEGylated (Supporting Information Figure S2a).Substantial marker retention was also demonstrated by directflow cytometry of PEGylated MSC-NGs that were not attachedto Dynabeads (Supporting Information Figure S2b).

In vitro: Targeting, Cytotoxicity, and Immunogenicityof hMSC-NGs. Fluorescence-activated cell sorting (FACS)

Figure 1. Physical characterization of NGs. Flourscent micrscopy images of (a) human MSCs counterstained with DAPI before processing into (b)homogenized ghosts (circled by a white dashed line) and (c) sonicated ghosts. Images are representative of at least three independent samples. (d) Sizeand (e) zeta-potential of NGsmade from hMSC, rMSC, and human smooth muscle cells (SMC, n = 3). (f) Representative Cryo-TEM images (n > 3) ofhMSC-NGs. (g) Cumulative sTRAIL release from hMSC-NGs; 100% release refers to the total amount of protein released during the time of the assay,which equals 18± 4 μg out of 300 μg per sample (n = 6). (h) Representative (n = 3) FACS histograms ofMSCmarkers on the surface of NGs conjugatedwith Dynabeads to achieve a FACS detectable size (see Supporting Information Figure S2 for analyses of non-PEGylated or PEGylated Dynabeads-freeMSC-NGs). Data is represented as mean ± SD.

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analyses were used to determine the selectivity of hMSC-NGsbinding to specific targets (PC3 and MCF7) in comparison totheir binding to two nonspecific targets: baby hamster kidney(BHK) cells and human smooth muscle cells and quantified aslog odds ratio (LOR). The hMSC-NGs exhibited time-dependent selectivity toward PC3 and MCF7 cells whencompared to both nonspecific targets (Figure 2a). PC3 cellswere selected for the continuation of our study because the NGsdemonstrate lesser selectivity toward them; that is, they present abigger challenge than MCF7 cells. hMSC-NGs were found toaccumulate inside the cytoplasm and nucleolus of PC3 cells overtime, as shown by confocal microscopy (Figure 2b). Whenincubated with NGs for more than 12 h, PC3 cells becamesurrounded by large clusters of NGs, also apparent inside thecells and fused with the cell membrane (Figure 2c). Free sTRAILand to a larger extent (p < 0.01) sTRAIL-encapsulating hMSC-NGs (NG-TRAIL) exhibited time-dependent cytotoxicity (p <0.001) toward PC3 cells compared to empty hMSC-NGs, whichdisplayed no cytotoxicity (Figure 2d). hMSC-NGs did not evokenitric oxide production or TNF-α expression by mouse leukemicmonocyte macrophage cells (RAW) when compared tolipopolysaccharides (LPS), which served as a positive control(p < 0.0001, Figure 2e). The nitric oxide levels secreted by cellsexposed to NGs were similar to those measured for the negativePLGA control (p > 0.05), while the TNF-α mRNA levels were

even lower than the negative control (p < 0.05) and similar to thebasal level, affirming the NGs’ lack of immunogenicity.

In vivo: Tumor Targeting, Biodistribution, and Hep-atotoxicity of hMSC-NGs. PC3 tumors were chosen forbiodistribution studies as they do not express CD90, a typicalhighly expressedMSC surface marker, making it easier to identifythe hMSC-NGs traces using antihuman-CD90. The validity ofCD90 staining for assessing the biodistribution was confirmed,given that no difference emerged between the CD90 staining ofpermeablized or intact cells incubated with MSC-NGs for 3 h(Supporting Information Figure S3). Mice were sacrificed 24 hand one week post NG administration. Tumors and bloodfiltering organs were harvested, dissociated into single-cellsuspensions, and compared to those of untreated mice usingFACS to detect human CD90 (Figure 3a). Significant NGaccumulation was found in the tumors and liver (p < 0.001) 24 hafter intraperitoneal (IP) or intravenous (IV) administrationwith no significant accumulation in other organs. One week postadministration, NGs were found only in the tumors (>50%) andonly in IP-treated mice. No expression of human CD90 on eitherthe tumor or blood filtering organs was found in untreated mice.Histological analysis of liver samples, harvested up to 4 weeksafter NG administration, revealed no hepatotoxicity orhistological abnormalities in immunocompetent C57BL mice(Figure 3b). Alanine transaminase (ALT) activity in the plasma,

Figure 2. In vitro targeting, cytotoxicity, and immunogenicity of hMSC-NGs. (a) Selectivity of NGs binding to PC3 and MCF7 cells. (b) Binding ofNGs (white arrows) to PC3 cells (NGs, red (DiI); cell, green (GFP); nucleolus, blue (DAPI)) evaluated using confocal microscopy over short (3 h) and(c) long (12 h) incubation times (NGs, red (DiI); cell, green (Di0)). (d) PC3 viability post incubation with empty NGs, free sTRAIL, and sTRAIL-containing NGs. Measurements were normalized to the viability of the untreated cells (100%). (e) Immunogenic potential of NGs evaluated using theGriess method and TNFα expression. Data are represented as mean ± SD. All micrographs are representative of at least three independent samplesimaged through three regions of interest (ROIs). Abbreviations: DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Di0, 3,3′-dioctadecyloxacarbocyanine perchlorate.

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as an indicator of liver functions, was evaluated 1, 2, and 4 weeksafter administration (Supporting Information Figure S4) and wasfound to be within the normal reference interval.23

In vivo Immunogenicity of hMSC-NGs. No changes werefound in the lymph node mRNA levels of IL-1β (Figure 4a) and

TNF-α (Figure 4b) of both treated and untreated C57BLmice 1,2, and 4 weeks post IP administration of hMSC-NGs (p > 0.05).The plasma levels of IL-1β, TNF-α, and IL-10 were undetectablein both treated and untreated mice (data not shown).Antitumor Efficacy of sTRAIL-Encapsulating NGs. NGs

made from human or rat MSCs, and human smooth muscle cells(as a nonmesenchymal control), encapsulating sTRAIL, wereinjected into tumor-bearing athymic nude mice (300 μgsTRAIL/mouse) via the IP route, which was previously foundto yield more targeted biodistribution than the IV route. A singledose of sTRAIL-containing human MSC-derived NGs (NG-TRAIL) was sufficient to induce significant inhibition of tumorgrowth compared to untreated mice (p < 0.001), mice treatedwith empty NGs (p < 0.05) or free sTRAIL (p < 0.01, Figure 5a).

This therapeutic effect is also demonstrated in SupportingInformation Figure S5 showing much smaller tumors, harvestedfrom mice treated with NG-TRAIL, compared to all othercontrols. Similar tumor inhibition (p < 0.01) was alsodemonstrated using sTRAIL-containing NGs derived from ratMSCs. No significant difference (p > 0.05) was found betweenhuman and rat MSC-derived NGs (Figure 5b, RAT-NG-TRAIL), although the human MSC-derived NGs seemed togenerate a slightly better effect during the last two time points.The mesenchymal source of the NGs, however, had a substantialeffect on their efficacy. sTRAIL-containing NGs derived fromhuman smooth muscle cells, having the same size and charge asMSC-NGs, did not inhibit tumor growth. Synthetic liposomescontaining sTRAIL (synLIP-TRAIL) also had no effect on tumorgrowth (Figure 5c). Two weeks after treatment, tumors wereharvested, dissociated into single-cell suspensions, and analyzedby flow cytometry for Annexin-V, a marker of cell apoptosis anddeath (Figure 5d). The percentage of apoptotic cells found intumors harvested from mice treated with NG-TRAIL (89%) wasgreater (p < 0.001) than that found in tumors of untreated mice(1%) and mice treated with empty NGs (11%) or free sTRAIL(53%). Substantial NG accumulation was detected in immuno-fluorescent tumor sections from mice treated with human or ratMSC-NGs (Figure 5e), compared with mice treated with NGsderived from human smooth muscle cells. Image analysis of theabove tumor sections (n = 3 per group) revealed a slightly higher,yet not statistically significant (p > 0.05), CD90-to-DAPI ratiofor the human MSC-derived NGs (NG-TRAIL, 2.4 ± 0.2) thanfor the rat NGs (RAT-NG-TRAIL, 1.9 ± 0.1).

Immunohistochemical Analysis of Treated Mice Tu-mors. Tumors from animals treated with NG-TRAIL werefound to express less CD31 and Ki-67 and more Caspase-3,indicating a reduction in the vascularization and proliferation andan increase in apoptosis compared to untreated mice or micetreated with empty hMSC-NG or free sTRAIL (Figure 6a).

Figure 3. In vivo prostate tumor targeting, biodistribution, and hepatotoxicity of hMSC-NGs. Harvested tumors were dissociated into single cells andanalyzed by flow cytometry for human CD90 as an indicator of NG fusion. Positive expression is calculated in the designated markers normalized to theuntreated control group (black curves) based on the test events following (a) IP (blue curves) or IV (purple curves) administration (see SupportingInformation Figure S3 for CD90 expression on permeablized or intact PC3 cells incubated with NGs). (b) Histological analysis of liver samples post IPadministration of NGs (see Supporting Information Figure S4 for ALT activity in the mouse plasma). Representative micrographs of at least threeindependent samples analyzed in each group are presented. Micrograph legend: * centrilobular veins;▶ bile ductile, identifying the centrilobular andthe periportal regions of the liver, respectively.

Figure 4. Lymph node proinflammatory cytokine levels. The levels ofproinflammatory cytokines (a) IL-1β and (b) TNF-α determined usingreal-time RT-PCR normalized to GAPDH and relative to the untreatedcontrols. Data are represented as mean ± SD.

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Quantitative analysis of immunohistochemistry (IHC) micro-

graphs (using the Mann−Whitney U test) statistically validated

these results (p < 0.05, Figure 6b−d).

Here we propose a new approach for utilizing MSC-derivedNGs as a natural and highly specific drug-delivery platform thatcan be easily produced and targeted to a variety of pathologiesincluding cancers. This unique platform encompasses the surface

Figure 5. Antitumor efficacy of sTRAIL-encapsulating NGs. (a) IP injection of free sTRAIL, NG-TRAIL, and empty NGs (n = 16) made from hMSC(see Supporting Information Figure S5 depicting the harvested tumor). (b) IP injection of NG-TRAIL, prepared from rMSC (RAT-NG-TRAIL) andhuman smooth muscle cells (SMC-NG-TRAIL) compared with untreated mice and mice treated with human MSC-derived NGs (NG-TRAIL). (c) IPinjection of synthetic liposomes containing sTRAIL (synLIP-TRAIL) compared to untreated mice or mice treated with NG-TRAIL. Tumors weredissociated into single-cell suspensions, analyzed by flow cytometry for Annexin-V, and compared with tumors harvested from untreated mice or micetreated with empty NG or free sTRAIL. (d) Dot-plots (n ≥ 3) for each experimental group presented as forward-scatter (FSC) vs Annexin-V labeling.(e) Representative images of tumor sections taken from mice treated with NG-TRAIL, RAT-NG-TRAIL and SMC-NG-TRAIL immunostained withantibodies (red) against hMSC, rMSC and human smooth muscle cells, respectively, and counterstained with DAPI. Data are represented as mean ±SEM ** = p < 0.01; *** = p < 0.001.

Figure 6. Tumor immunohistochemical analysis. (a) Tumors subjected to histopathological and immunohistochemical analyses for morphology(H&E), vascularization (CD31), proliferation (Ki-67), and apoptosis (Caspase-3). Representative micrographs of tumor sections (n ≥ 3). (b)Microvessel density, (c) proliferation, and (d) apoptosis indices as determined using the Mann−Whitney U test compared to untreated mice. Data arerepresented as mean ± SD. Statistical significance indicators: * = p < 0.05; ** = p < 0.01.

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molecules of MSC cytoplasmatic membranes, exploiting theirnatural, specific, and unique tumor targeting capabilities.10,11,24

The NGs we describe are fundamentally different from exosomesand shed-vesicles in their production and possible applications.Furthermore, NGs made from mammalian cell membranes havebeen studied previously only as explanatory models for thecytoplasmatic membrane and as possible vaccine adjuvants.25,26

To the best of our knowledge, this is the first comprehensivereport demonstrating that intrinsically targeted NGs can beutilized as drug-carriers.The NGs we prepared from human MSCs, rat MSCs, and

human smooth muscle cells were shown to resemble commonnanoliposomal drug-delivery systems. The simple technologicalprocess, used for the production of NGs, yielded a reproducibleproduct devoid of nucleic residues. The cell origin, whethermesenchymal or not, human or rat, had no effect on the NG size,charge andmorphology. The loading capacity of NGs and releaseprofiles of sTRAIL were found to be similar to those previouslyreported for synthetic liposomes, exhibiting a short burst-releasefollowed by a linear sustained release profile. These similarities tosynthetic liposomes hint at the NGs’ potential to serve asversatile drug carriers, which is at least comparable if not betterthan conventional liposomes. PEGylation was implemented(using activated PEG5000) to reduce possible NG opsonin-dependent uptake and increase circulation time in a waypreviously shown to improve the in vivo transfection efficiencyof lentiviral vectors and to facilitate liposomal escape from thereticuloendothelial system.27,28 When compared to un-PEGylated NGs (Supporting Information Figure S2a), thePEGylation seemed to partially mask two of the four tested MSCsurface markers. Nonetheless, the remaining markers were stillfound in substantial quantities (>50%) and native confirmation(compared to intact cells, see Supporting Information FigureS1), which was evidently sufficient to support active targeting.The expression of correctly oriented MSC markers on the NGs′surface also confirms the preservation of membrane asymmetry,which is crucial for stability and targeting.29

The in vitro binding to prostate and breast cancer cells wasfound to be specific and time dependent compared to thenonspecific targets, BHK, and human smooth muscle cells.Human CD90, which is widely expressed on MSC-NGs but noton the tumor cells, served as a marker to follow up the NGs invitro and later in vivo. The two nonspecific targets were selectedas representatives of either nonhuman transformed cells orhuman primary benign cells, emphasizing that MSC-NGselectivity is tumor-specific and not species-specific, providingfurther evidence for active targeting. MSC-derived NGs wereseen fused with, on the surface of, and within the cytoplasm oftarget cells. This wide yet selective MSC-NG localization mayalso explain the potent time-dependent cytotoxicity demon-strated toward PC3 cells. Overall, these findings suggest thatNGs may be used as selective and potent drug-carriers to treatvarious MSC-associated cancers.Once established in vitro, the biodistribution and antitumor

efficacy of MSC-NGs were investigated in a nude mice modelinoculated with human prostate cancer, again using humanCD90, which is widely expressed on MSC-NGs but not on thetumor cells. Our data reveal that NGs accumulate in the tumorsharvested frommice one day after IV or IP administration. Sevendays post administration, substantial NG accumulation wasnoted only in the tumors from IP-administered mice. The NGsfrom the tumors of IV-administered mice, and all blood filteringorgans of both IP- or IV-administered mice, were clear. This

more targeted biodistribution via the IP in comparison to IVroute was previously reported for various nanovesicles and wasattributed to their prolonged retention in the peritoneum,resulting in sustained release to the blood.30 These studies alsoshowed that IP-administered negatively charged PEGylatednanovesicles, such as our own, achieved high blood levels despitethe first-pass effect.MSC-NGs administered into immunocompetent mice ex-

hibited no immunogenicity, hepatotoxicity or side-effects, asexpected from the safe nature ofMSC cell-therapy applications.31

A previous study investigating the effect of the secondaryimmune response on the efficacy of allogeneic MSCs (allo-MSCs) have shown that multiple infusions of high allo-MSCs didnot change the overall health or immune status of recipientnonhuman primates.32 In this study, Beggs et al. also showed thatmultiply transplanted allo-MSCs were detected for at least onemonth in muscle biopsies of half of the animals. Accordingly, webelieve that NGs made from allo-MSCs can be safelyadministered in multiple doses despite concerns about asecondary immune response. However, our system lacks thecytoplasmatic immunosuppressive mechanisms of intact MSCand therefore merits further immunological investigation, despitethe PEGylation which may reduce the immunogenicity of theNGs. Nonetheless, we believe that in light of the many promisingclinical trials with allo-MSC, the clinical applicability of multipleadministrations of allo-MSCs, or their derived products, is stillviable.33,34

Finally, a single IP administration of sTRAIL-containingMSC-NGs was sufficient to inhibit tumor growth by more than 70%.NGs prepared from human or rat MSCs significantly inhibitedtumor growth for up to two weeks. Although, there was nostatistically significant difference between the potent tumorinhibition achieved by human or rat MSC-NGs (two-wayANOVA), human MSC-derived NGs did seem to produce abetter effect during the last two time points (days 11 and 13).This therapeutic outcome also corresponds with our resultsshowing a higher CD90-to-DAPI ratio in tumors harvested frommice treated with human-derived MSC-NGs, implying theexistence of higher NGs accumulation compared to rat-derivedNGs. Free sTRAIL and empty NGs exhibited much less, but stillsignificant, antitumor effects. Evidently, the tumor progressionafter administration of free sTRAIL or empty NGs was quitesimilar despite substantial difference in the ratio of apoptotic cells(53 and 11%, respectively). This finding may be attributed tovarious and different mechanisms that lead to similar tumorinhibition regardless of apoptosis. In case of free sTRAIL, theentire effect is probably due to apoptosis,20 whereas the emptyNGs may act both by disrupting the target cell membranes,leading to some apoptosis, and by interacting with the tumorenvironment. Accordingly, the binding and fusion of the NGswith the tumor cells may disrupt the cell plasma membrane andcytoskeleton leading to a cytotoxic effect. A similar effect was alsodemonstrated in our previous publication, showing that emptytargeted NGs, made from CCR5-expressing nonhuman cell-lines, selectively decreased the viability of target cells expressingthe CCR5 ligand (gp120).8 This effect was also demonstratedwith conventional liposomes and was shown to be mediumdependent. The anticancer effect of empty NGs may be alsoattributed to their interactions with different components of thetumor microenvironment such as angiogenic blood vessels,inflammatory cells, and MSC transition to tumor-associatedfibroblasts.35,36 Moreover, the NGs may compete with

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endogenous MSCs and interfere with the tumor support theyprovide.In contrast to MSC-derived NGs, smooth muscle cell-derived

NGs, which have similar size and physical properties as theMSC-NGs, failed to target the tumor, lending further support to thebenefit of active targeting over passive targeting based on theEPR effect.5 The lack of targeting by these nonmesenchymal cell-derived NGs, along with the specific targeting of a human tumorby NGs derived from both rat and human MSCs, proves that thetargeting is tumor-specific and not species-specific. Moreover, wecompared our system to synthetic liposomes that are of similarsize and structure as our MSC-NGs, serving as the closestsynthetic control to our NGs. These synthetic liposomes, whichare widely investigated as drug-carriers for various cancers,5,37

had no effect when administered under similar conditions,emphasizing that passive targeting alone is not sufficient tomediate an effective therapeutic outcome, which is in contrast toour highly efficient actively targeted MSC-NGs. Our in vivoefficacy studies were further supported by flow cytometry andhistological analyses demonstrating the scope of the therapeuticeffect, revealing increased apoptosis with reduced proliferationand blood supply to the tumor. Comparing the results of theAnnexin-V FACS analyses with those of the immunohistochem-ical staining for Caspase-3 reveals that more cells were shown toprogress into later apoptotic stages, when harvested from NG-TRAIL treated mice, than mice treated with free sTRAIL. Tumorcells from NG-TRAIL treated mice may also undergo Caspase-independent apoptosis,38 which may lead to higher Annexin-Vstaining, despite similar Caspase-3 levels found in the tumor cellsfrom mice treated with NG-TRAIL or free sTRAIL.A similar therapeutic effect to what we have achieved using

NG-TRAIL was previously demonstrated for prostate cancer,using monoclonal antibodies against N-cadherin, which is highlyexpressed in castration-resistant prostate cancer; however, itrequires more frequent and higher dosing.39 Our therapeuticoutcome is comparable to that demonstrated by DeMarra et al.40

who used no less than three administrations per week ofliposomes encapsulating Zoledronic acid and exceeded the effectachieved by a weekly administration of an imatinib-mitoxantroneliposomal formulation.41 The efficiency of our delivery system iseven more compelling in light of the results reported bySrivastava et al.,42 which demonstrated no inhibition of tumorgrowth after two weeks and as many as four IV administrations ofsimilar quantities of free sTRAIL. The efficacy of our system alsoexceeded that of previously reported liposomal formulationscontaining sTRAIL tested on glioblastoma and lung cancer.37,40

Although tested on tumors other than prostate cancer, theseformulations exhibited a similar in vitro effect on these tumors aswas exhibited on prostate cancer by our own system. Never-theless and in contrast to our results, the reported liposomalsystems were shown to achieve substantial tumor inhibition onlywhen sTRAIL was administered along with liposomal DOX.37,40

The clinical advantage of our delivery system is even moredefinitive when compared with previously reported sTRAIL-PLGA systems that had to be locally administered at higher andmore frequent doses, achieving no effect unless formulated withadditional drugs.22

In conclusion, we believe that our work holds huge medicinalpotential, opening a new avenue for drug-delivery for treatmentof yet incurable cancer and other diseases. Although furtherresearch is required into the exact targeting mechanism, hereinwe demonstrate a proof-of-concept for a model platformentrapping therapeutics and achieving specific tumor targeting

and growth inhibition. This platform exhibited an unprecedentedtherapeutic effect compared with synthetic liposomes orpreviously reported targeted drugs in similar model systems.The mechanism governing MSC surface interactions with cancercells is probably that underlying the ability of MSC-derived NGsto achieve specific targeting and tumor inhibition whenformulated with a drug-of-choice. It is also possible that thevarious integrins found on the surface of the MSC-NGs (seeSupporting Information Table S1), which are known to take partin other MSC interactions (wound healing, angiogenesis, and theimmune response), are associated with cancer as well. Therefore,further to their therapeutic potential, MSC-NGs can be used asan efficient tool to investigate the mechanisms underlying MSCinvolvement in cancer, possibly revealing additional targets forinterfering with cancer progression. Moreover, based on MSChoming abilities such a platform can be readily extended to targetdifferent cancers and other pathologies (e.g., genetic, viral,autoimmune, etc.), which are manifested in the expression ofunique surface markers to which endogenous or exogenoustargeting molecules may be found on MSCs or other cells.

■ ASSOCIATED CONTENT

*S Supporting InformationFigure S1: Flow cytometric analyses of human bone marrowMSC using typical positive and negative MSC surface markers.This data is presented to demonstrate the lineage integrity of theMSCs fromwhich theNGs are prepared. The expression levels ofthe intact cells can also be compared with the expression of MSCmarkers on MSC-NGs depicted in Figures 1e and S2. Figure S2:A flow cytometric analysis of typical positive MSC markers on(a) non-PEGylated MSC-NGs, and (b) PEGylated MSC-NGsusing direct analysis without utilizing Dynabeads conjugation (asdepicted in figure 1h). Figure S3: CD90 expression onpermeablized and intact PC3 cells following incubation withMSC-NGs. These results are presented to support the validity ofCD90 staining for assessing the NG biodistribution as presentedin Figure 3a. Figure S4: ALT activity in the plasma of mice treatedwith MSC-NGs. These results are related to histologicalhepatotoxicity analysis depicted in Figure 3b. Figure S5:Representative images of tumors harvested 2 weeks post IPadministration of sTRAIL-containing NGs. These resultssupport the tumor size progression depicted in Figure 5a.Table S1: The results of a proteomic analysis on MSC-NGsreaffirming the preservation of ECM-associated integrins that isrelated to our discussion of the cancer targeting mechanism.Supplemental experimental procedures: A detailed description ofthe experimental protocols. This material is available free ofcharge via the Internet at http://pubs.acs.org..

■ AUTHOR INFORMATION

Corresponding Author*E-mail: machlufm@tx.technion.ac.il. Tel: 972-4-829-4916; 972-4-829-3079. Fax: 972-77-887-1951.

Present Address†(M.M.) The Laboratory for Cancer Drug Delivery & Cell BasedTechnologies, Faculty of Biotechnology & Food Engineering,Technion−Israel Institute of Technology (IIT). Haifa, Israel,32000.

Author ContributionsN.E.T.F., Y.L.-H., T.B., and L.K. have equally contributed to thiswork.

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The manuscript was written with the contributions of allauthors. All authors have given approval to the final version of themanuscript.NotesThe authors declare the following competing financialinterest(s):Two of the authors, Marcelle Machluf and TomerBronshtein, share ownership over the patent entitled liposomalcompositions and uses of same which depict some of theconcepts presented herein and are currently pending at nationalphase (US publication number: US2012/0164214A1; Europeanpatent publication number: EP2470164 A2).

■ ACKNOWLEDGMENTSMCF7 cells and bFGF were kindly donated by Professor GeraNeufeld from the Department of Anatomy and Cell Biology,Faculty of Medicine, IIT. RAW cells were kindly provided byProfessor Ester Meyron-Holtz, IIT. PGEX-2TK plasmid waskindly donated by Professor Stanley Lipkowitz from theUniformed Services University of Health Science in Bethesda,MD. Pathological analysis was provided by Ori Brenner − B.VSc(Diplomate, ACVP), The Weizmann Institute of Science, Israel.We further wish to thank Meital Levy-Mishali for her technicalassistance. The research was supported by The Israeli ScienceFoundation (ISF) under F.I.R.S.T. Grant 2015103. The partialsupport of the Russell Berrie Nanotechnology Institute,Technion − IIT, is thankfully acknowledged. The financialsupport and contribution of the Ed Sattel and Bert RichardsonFoundations is greatly appreciated.

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Nano Letters Letter

dx.doi.org/10.1021/nl401376w | Nano Lett. XXXX, XXX, XXX−XXXH

Reconstructed Stem Cell Nanoghosts: A Natural Tumor Targeting

Platform

SUPPLEMENTAL INFORMATION

Naama E. Toledano Furman†, Yael Lupu-Haber†, Tomer Bronshtein†, Limor

Kaneti†, Nitzan Letko, Eyal Weinstein, Limor Baruch, and Marcelle Machluf*

Faculty of Biotechnology and Food Engineering, Technion – Israel Institute of

Technology, Haifa, Israel 32000

† These authors have equally contributed to this work.

*Corresponding author

Marcelle Machluf, Ph.D.

The Laboratory for Cancer Drug Delivery & Cell Based Technologies

Faculty of Biotechnology & Food Engineering

Technion-Israel Institute of Technology

Haifa, 32000

Israel

Tel: 972-4-829-4916, 972-4-829-3079

Fax: 972-77-887-1951

Email: machlufm@tx.technion.ac.il

SUPPLEMENTAL DATA

Validating the lineage integrity of mesenchymal stem cells

Human bone marrow

mesenchymal stem cells (MSC,

Lonza™, Basel, Switzerland)

were used throughout this work

as a source for the production

of cell-derived nano-ghosts

(NGs). To validate their lineage

integrity, each MSC batch was

analyzed by flow cytometry for

typical MSC positive markers

(CD44, CD90 and CD105) and

the negative marker CD31.

Briefly, 1:100 mouse anti-

human CD90, CD105, CD44

and CD31 and 1:200 FITC-

conjugated goat anti mouse

IgG/IgM (BD PharmingenTM,

San Diego, CA) were used for

FACS analysis against isotype controls according to the manufacturer’s protocols. As

can be seen in figure S1, all positive MSC markers were widely expressed with no

expression of the negative marker - CD31.

Figure S1: Lineage integrity of MSCs used for NG production. Human bone marrow derived MSCs were analyzed by flow cytometry for typical MSC positive markers (CD44, CD90 and CD105) and the negative marker CD31. Representative histograms are presented (n≥3). Percent of positive expression is calculated based on the test events (blue curves) in the designated markers normalized to the isotype control (black curves).

Validating the preservation and orientation of

membranal proteins on the NGs

Un-PEGylated NGs were conjugated with

Dynabeads™, labeled with fluorescent primary

antibodies against CD90, CD105, CD44 and

CD29, and analyzed by FACS to compare the

retention of MSC surface markers to that of

PEGylated MSC-NGs (Fig 1h). As can be seen

in figure S2a, the un-PEGylated NGs retained a

substantial expression of all tested markers

(>50%). CD105 and CD44 levels on the un-

PEGylated NGs were similar to those measured

on the PEGylated ones, whereas the CD90 and

CD29 levels were 20% and 35% higher on the

un-PEGylated NGs, respectively. These results

and the ones presented in figure 1h, demonstrate

that the majority of the tested surface markers

are retained by the NGs’ and that the PEGylation

does not hinder their ability to bind specific

antibodies. To validate the retention of correct

membrane asymmetry, PEGylated MSC-NGs

were also analyzed without conjugation to

Dynabeads™. The MSC-NGs were

immunostained with fluorescent anti CD29 and

CD90 and analyzed by FACS using no primary

Figure S2: Conformation and orientation of MSC markers on the surface of NGs. (a) Non-PEGylated MSC-NGs were conjugated with Dynabeads™, labeled with fluorescent primary antibodies against typical MSC surface markers and analyzed by FACS. (b) PEGylated MSC-NGs without Dynabeads™ were labeled with fluorescent primary antibodies against CD90 and CD29 and analyzed by FACS using no threshold on the forward scatter to detect the nano-particles. Representative histograms are presented (n≥3). Percent of positive expression is calculated based on labeled NGs (blue curves) in the designated markers normalized to unlabeled NGs (black curves).

threshold. The primary FACS threshold, usually set along the forward scatter (FSC),

was discarded to allow detection of the small sized NGs. Two more samples were

analyzed apart from the labeled and unlabeled NGs, namely clear buffer and buffer

with only antibodies. Both control samples revealed no FACS-detectable events,

reaffirming the FACS’ ability to detect and distinguish NGs. Similar to what was

shown with Dynabeads™-conjugated NGs (Figs. S2a and 1h), the conformation and

orientation of CD29 and CD90 were substantially retained (>50%) on the NG

membranes (Fig. S2b).

CD90 expression on permeablized and intact PC3 cells following incubation with

MSC-NGs

Monoclonal antibodies against human CD90 were used in our biodistribution studies

(presented in figure 3a) to detect the remains of MSC-derived NGs that may have

underwent membrane fusion with their target (PC3) or other cells. To assure the

validity of CD90 staining for assessing the

biodistribution, and since staining of intact

non-permeablized cells might miss particles

that may have been taken through endocytosis,

we compared the staining of intact and

permeablized cells. PC3 cells were incubated

with human MSC-derived NGs for 3 hrs, left

intact or permeablized using Tween-20,

stained with monoclonal antibodies against

human CD90, and analyzed by FACS. As can

be seen in figure S3, no significant change was

Figure S3: CD90 expression on permeablized and intact cells following incubation with NGs. PC3 cells were incubated with human MSC-NGs for 3 hrs, washed, harvested, left intact or permeablized, stained with anti human CD90 monoclonal antibodies, and analyzed by FACS. Analysis is representative of at least three similar results.

found in the CD90 expression between permeablized (55%) and intact cells (57%).

Accordingly, we believe that despite cellular degradation of NGs that may have been

up taken by endocytosis, these results imply that the majority of the particles undergo

membrane fusion, at least when the tumor is the subject of their targeting—thereby,

validating our method.

Alanine Transaminase (ALT) activity in the plasma of mice treated with MSC-NGs

To determine hepatotoxicity, ALT activity was

analyzed in the plasma of immunocompetent

C57BL mice harvested 1, 2 and 4 weeks after

IP administration of MSC-NGs. As can be

seen in figure S4, ALT activity was within the

normal reference values (Boehm et al. 2007,

Biological chemistry 388(5):547-54),

indicating no liver damage. These results

further support the lack of hepatotoxicity also

demonstrated in the main text using

histological analysis of liver sections taken

from mice 1, 2 and 4 weeks after IP

administration of MSC-NGs (Fig. 3b).

Tumor size analysis

Tumors were harvested from NUDE mice 2 weeks after IP administration of empty

NGs, free sTRAIL, and sTRAIL containing NGs (TRAIL-NG) and compared with the

size of tumors from untreated (control) mice. As can be seen in figure S5, tumor sizes

Figure S4: ALT activity in mice treated with MSC-NGs. NGs (5•107 NG-cell-eq./mice) were IP injected into male C57BL mice (n=6 mice/group). 1, 2 and 4 weeks post administration the ALT activity in the mice plasma was analyzed using ALT activity kit.

correspond to their final sizes, as depicted in

figure 5, revealing substantial growth

inhibition after a single administration of

sTRAIL containing NGs and also showing

some effect of empty NGs and free sTRAIL.

Analysis of MSC-NGs surface proteome

To further analyze their protein composition,

membrane proteins were isolated from human

MSC-derived NGs using a Plasma Membrane

Protein Extraction Kit (Abcam®, Cambridge,

UK). The composition of the extracted protein

samples were analyzed at the proteomics

center of the Technion – Israel Institute of Technology. Briefly, the samples were

digested by Trypsin and the resulting peptides were analyzed by LC-MS/MS. The

peptide mix was fractionated by HPLC and electro-sprayed onto an ion-trap mass

spectrometer (Thermo-Fisher Scientific Inc., Waltham, MA). Mass spectrometry was

performed in order to analyze the peptides’ mass-to-charge ratio spectra and to

determine the proteins’ mass. For additional analysis and identification, the peptides

were further fragmented by collision induced dissociation and analyzed again. The

peptides were identified by Sequest 3.31 software (J. Eng and J. Yates, San Jose, CA)

against the human part of the uniprot database. All protein results are given as Uniport

Accession Numbers. Using this analysis, more than 500 proteins were identified on

the NG surface, among them many integrins involved in cellular interactions and

interactions between MSCs and the extracellular matrix (Tab. S1). The identified

Figure S5: Tumors harvested from treated mice. Tumors from untreated mice (control) and mice treated with empty NGs, free sTRAIL, and sTRAIL-containing MSC-NGs were harvested 2 weeks after IP administration. Representative images are presented out of at least 16 animals per group. Each blue square is 0.5 x 0.5 cm.

proteins are presented along with their accession numbers and Ppro, indicating the

probability of finding a match as good as or better than the observed match by chance.

Table S1. Extracellular matrix-associated integrins on NGs’ surface – proteomics analysis.

Remarks ppro Accession No. Reference

Fibronectin receptor. Also known as Integrin β-1. 10-10 P05556 CD29

Also known as Thy-1. 10-6 P04216 CD90

Also known as gp90 lymphocyte homing/adhesion receptor and as extracellular-matrix-III receptor.

10-7 P16070 CD44

Part of the TGF-β receptor. Also known as Endoglin. 10-7 P17813 CD105

Vitronectin receptor. Also known as CD51. 10-8 P06756 Integrin α-V

Creates a fibronectin receptor with the beta-1 subunit. Also known as CD49e.

10-6 P08648 Integrin α-5

Collagen receptor. Also known as CD49b. 10-8 P17301 Integrin α-2

Potent inhibitor of the complement membrane attack complex, inhibiting cell lysis.

10-8 P13987 CD59

Involved in cell adhesion. 10-5 P18084 Integrin β-5

Joins integrin β1 to bind ECM. 10-9 Q9UKX5 Integrin α-11

Joins integrin β1 to bind ECM. Also known as CD49c.

10-4 P26006 Integrin α-3

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Cells and media

Human bone marrow derived mesenchymal stem cells (MSCs, Lonza™, Basel,

Switzerland) were cultured in alpha-MEM (Sigma-Aldrich™, St. Louis, MO)

supplemented with 5 ng/ml basic fibroblast growth factor (bFGF). Rat bone marrow

MSCs (Lonza™) were cultured in low-glucose DMEM (Sigma-Aldrich™). Human

prostate cancer cell-line PC3 (ATCC: #CRL-1435™) was grown on HAM/F-12

nutrient-mixture (Sigma-Aldrich™). Human breast cancer cell-line MCF7, human

smooth muscle cells (SMC, Lonza™), baby hamster kidney cells  (BHK, ATCC:

#CCL-10™), and mouse leukaemic monocyte macrophage cells (RAW 264.7, ATCC

#TIB-71™) were grown in high-glucose DMEM (Biological Industries, Beit-

Ha’Emek, Israel). Media were supplemented with 10% Gibco® FBS, 1% Pen-Strep®,

and 0.4% Fungizone® – all purchased from Invitrogen™, Carlsbad, CA. Cultures

were maintained at 37°C in a humidified incubator with 5% CO2.

Production of human recombinant sTRAIL

PGEX-2TK plasmid expressing human GST-sTRAIL (soluble TNF-α Related

Apoptosis Inducing Ligand) was kindly donated by Prof. Stanley Lipkowitz from the

Uniformed University of Health Science in Bethesda, MD. Recombinant GST-

sTRAIL was expressed in IPTG induced Escherichia coli BL-21/DE3 (Stratagene,

Santa Clara, CA) and purified using GSH beads (GE Healthcare, Uppsala, Sweden)

and Amicon Ultra 15101K centrifugal filter units (Millipore, Billerica, MA). SDS-

PAGE was used to confirm sTRAIL production. The concentration of sTRAIL was

measured using the Bradford assay (Bio-Rad, Hercules, CA) and Peprotech™

sTRAIL detection ELISA kit (PeproTech™, Rehovot, Israel) according to the

manufacturer’s protocol.

Preparation of Nano-Ghosts (NGs)

NGs were prepared from the cytoplasmatic membranes of three source cells: human

and rat MSCs, and from smooth muscle cells – as a non-mesenchymal control. Source

cells (107 cells) were harvested, washed with PBS and re-suspended in cold Tris-

Magnesium buffer (TM-buffer), pH 7.4, comprising 0.01 M Tris and 0.001 M MgCl2

(Sigma-Aldrich™). Cells were homogenized at 22,000 rpm for 1 min (IKA®,

Taquara, RJ, Brazil) and a sucrose (Sigma-Aldrich™) solution in TM-buffer (60%

w/v) was added immediately after homogenization to a final concentration of 0.25 M.

The homogenized cells were centrifuged at 6,000 g for 15 min at 4°C and the

resulting pellet was then washed twice with 0.25 M sucrose in TM-buffer (pH 7.4).

The re-suspended pellet was sonicated for 5 seconds at 27% amplitude using

VibraCell VCX750 (Sonics & Materials Inc., Newtown, CT) and centrifuged at 6,000

g for 15 min at 4°C. The pellet was washed twice with 0.25 M sucrose in TM-buffer

(pH 8.6) as before. The re-suspended pellet was extruded 11 times through 0.4 µm

polycarbonate membranes (Osmonics Inc., Minnetonka, MN) using LIPEX®

(Northern Lipids, Burnaby, Canada) and ultra-centrifuged for 45 min at 150,000 g at

4°C. The resulting NG pellet was re-suspended in TM-buffer (pH 8.6). For the

production of sTRAIL-encapsulating NGs (NG-TRAIL), sTRAIL was added to the

sonicated cell sample (5•107 cells) just before extrusion, to a final concentration of 0.1

mg/ml. sTRAIL-containing NGs were then prepared by extrusion and retrieved by

ultracentrifugation as above. The efficiency of sTRAIL encapsulation was evaluated

using the Bradford protein assay comparing permeablized (using 0.01% TritonX100

in PBS) empty and sTRAIL-containing NGs.

Empty NGs and sTRAIL-containing NGs were PEGylated according to the method

previously used for the PEGylation of lentiviral vectors, as published by Croyle et al.

2004 (J. Virol. 78:912-921). Briefly, succinimidyl-succinate activated monomethoxy-

PEG5000 (Sigma-Aldrich™) was added to the NG sample at a polymer-to-

membrane-protein ratio of 10:1 (w/w), as determined by the Bradford protein assay.

PEG conjugation was performed with gentle agitation for 2 hours, at room

temperature and stopped by adding 10X l-lysine (Sigma-Aldrich™) with respect to

the amount of added PEG. Un-reacted PEG, excess lysine, and reaction by-products

were eliminated by buffer exchange over a Micro-Bio Spin P-30 column (Bio-Rad)

equilibrated with TM-buffer (pH 8.6).

Physical characterization of NGs: Size, Zeta-potential and morphology

The size and Zeta-potential of NGs (2•107 cell eq./ml) in TM-buffer (pH 8.6) were

analyzed by number and intensity correlation of dynamic light scattering (DLS), and

Zeta-potential analysis using Zetasizer® Nano-Series® (Malvern Instruments,

Malvern-Worcestershire, UK). Samples were also imaged by Cryo-TEM (Philips

CM120, 120 kV) using an Oxford CT-3500 Cryo-holder and digitally recorded as

previously published by us in Bronshtein et al. 2011 (J. Control Release. 151(2):139-

48).

The release kinetics of sTRAIL

To determine sTRAIL’s release kinetics, sTRAIL-containing NGs (107 NG-cell-

eq./ml) were incubated in 0.5 ml PBS at 37oC on a rocking platform (200 RPM) for

up to 5 days. NG samples were removed after 2, 4, 6, 24, 48, and 120 hrs of

incubation and precipitated by 45 min ultra-centrifugation at 150,000 g and 4°C. The

supernatant, containing the released sTRAIL, was removed and analyzed using

Peprotech™ sTRAIL detection ELISA kit against an appropriate standard curve. The

remaining NG pellet was resuspended in the same medium and volume as above, and

returned for further incubation.

Conformation and orientation of membranal proteins

NGs were covalently adsorbed (2 NG-cell-eq./bead) to the surface of Tosyl-activated

M-280 Dynabeads™ (Invitrogen™) as previously published by us in Bronshtein et al.

2011, and according to the Dynabeads™ manufacturer’s protocol, replacing the

conjugated proteins with NGs. NG-conjugated beads were analyzed by flow

cytometry (FACS Calibur, BD™, San Diego, CA) for typical MSC markers against

isotype controls and un-conjugated beads using 1:100 mouse anti-human CD90,

CD105, CD44 and CD29 and 1:200 FITC-conjugated goat anti-mouse IgG/IgM

(BD™), according to the manufacturer’s protocols.

In-vitro targeting of prostate and breast cancer

Fluorescently-labeled NGs were prepared from human MSCs treated with 0.01%

(w/v) fluorescent membranal tracker 1,1′-dioctadecyl-3,3,3′,3′-

tetramethylindocarbocyanine perchlorate (DiI, Invitrogen™) according to the

manufacturer’s protocol. NGs were then incubated with two specific target cells: PC3

and MCF7 and two non-specific target cells: BHK and SMC (9 NG-cell-eq./cell), in 1

ml medium for 15 min to 3 hrs. After incubation, cells were washed with PBS,

harvested with Trypsin-EDTA (Sigma-Aldrich™) and analyzed by flow cytometry

for DiI labeling. The selectivity of PC3 and MCF7 binding was calculated as the LOR

of the DiI-positive populations between the specific target and the non-specific

control cells (BHK and SMC).

In-vitro NG targeting of PC3 cells: Confocal imaging

DiI-labeled NGs, made from human MSCs, were incubated with GFP-expressing PC3

cells (9 NG-cell-eq./cell) in 1 ml medium for up to 3 hours. PC3 cells were then

thoroughly washed, counterstained with 4',6-diamidino-2-phenylindole Fluoromount-

G (DAPI-Fluoromount-G, SouthernBiotech, Birmingham, Alabama) and imaged

using a Zeiss LSM510-META confocal microscope (Manheim, Germany). In another

experiment, PC3 cells, previously labeled with 3,3′-dioctadecyloxacarbocyanine

perchlorate (Di0, Invitrogen™), were incubated with the DiI-labeled NGs for 12 hrs.

Evaluating the in vitro cytotoxicity of NG-TRAIL

PC3 cells were seeded (1.5•104 cells/well) in a 96-well plate (NunclonTM, Roskilde,

Denmark) and allowed to recover overnight. Free sTRAIL (25 ng/well), human MSC-

derived NGs (1.25•106 NG-cell-eq./well), and sTRAIL-containing NGs (1.25·106 cell

eq./well) were added to the cultures. Cell viability was followed-up using the

AlamarBlue™ assay (Invitrogen™) against an appropriate calibration curve and

according to the manufacturer’s protocol. The relative cell viabilities were then

calculated in respect to the untreated control, set as 100%, and plotted against the

incubation time.

Evaluating the in vitro immunogenic potential of NGs

RAW 264.7 cells (3•105), kindly provided by Prof. Ester Meyron-Holtz from the

Technion, were seeded in 6-well plates (NunclonTM), cultured in 2 ml of complete

medium (see: cells and mediums section) and allowed to recover overnight. The

medium was replenished and the cells were exposed to 100 µg NGs (107 NG-cell-eq.),

1 µg of lipopolysaccharides (Sigma-Aldrich™), and 1 mg PLGA particles prepared

according to a salt leaching protocol previously reported by Caspi et al. 2007 (Circ.

Res. 100:263). The in vitro immunogenic potential of the NGs was evaluated 18 hrs

later by assessing the levels of secreted nitric oxide, and the expression of TNFα

mRNA. Nitric oxide levels were measured and determined against a standard

calibration curve (0 to 100 µM sodium nitrite) as the free stable nitrite form (NO2-)

using the Griess reagent system (Promega™, Madison, WI) and according to the

manufacturer’s instructions. To determine TNFα mRNA levels, cells were harvested,

homogenized, and treated by tri-reagent (Sigma-Aldrich™) to extract the mRNA that

was then reverse-transcribed in a PTC-200 PCR cycler (MJ Research, Waltham, MA)

using a VersoTM cDNA kit (Thermo Scientific, MA, USA). TNFα levels (normalized

to GAPDH) were quantified by PerfeCTa® SYBR® Green FastMix®, ROXTM

(Quanta Biosciences, MD, USA) using Applied Biosystems 7300 qPCR (Life

technologies®, Carlsbad, CA) with the following primers:

TNF-α Forward: 5'-GCCTCCCTCTCATCAGTTCT

Reverse: 5'-TGGTGGTTTGCTACGACGTG

In vivo studies

All animal experiments were performed in compliance with the Council of Animal

Experiments, Israeli Ministry of Health’s guidelines for the care and use of laboratory

animals. Animal Ethics Committee approval No.: IL-009-01-2011.

Bio-distribution and specific in vivo tumor targeting by NGs

Six week-old male Athymic nude mice (Harlan labs, Jerusalem, Israel) were

subcutaneously inoculated in the right leg flank with 106 PC3 cells. Tumor volume

was evaluated using the following correlation:

23 [mm]width [mm]length 0.52][mm Volume . NGs (5•107 NG-cell-eq./mice)

suspended in 50 µl PBS were intravenously (IV) or intraperitoneally (IP)

administrated to randomized groups once tumor volumes reached an average of 100

mm3. Mice were sacrificed 24 hrs and 1 week post NG administration and their

tumors and blood filtering organs (spleen, kidneys, liver, lungs) were harvested and

dissociated into single cells as previously published by Daenen et al. 2009 (Mol.

Cancer Ther. 8(10):2872-81). Dissociated cells were washed twice and analyzed for

NG remainders by flow cytometry using PE-Cy5 anti-human CD90 (Biolegend™,

San Diego, CA).

Determining the in vivo hepatotoxicity of NGs

NGs (5•107 NG-cell-eq./mice) were IP injected into 5 week-old male C57BL mice

(n=6 mice group, Harlan labs, Jerusalem, Israel). The mice were sacrificed 1, 2 and 4

weeks after administration and their livers were harvested. Plasma levels, taken 1 and

2 weeks post treatment, were analyzed for alanine transaminase (ALT) activity using

Alanine Transaminase Activity Assay Kit (Cayman Chemical Company, Ann Arbor,

MI) according to the manufacturer’s manual. Liver samples were preserved in 10%

neutral buffered formalin (Sigma-Aldrich™) and sent to certified pathological

analysis by ACVP diplomate at The Weizmann Institute of Science, Rehovot, Israel.

Each liver sample was trimmed into 2-4 slices stained with hematoxylin and eosin

(H&E), analyzed, and reported.

In vivo immunogenicity

NGs (5•107 NG-cell-eq./mice) were IP injected into 5 week-old male C57BL mice

(n=6 mice group, Harlan labs, Jerusalem, Israel). The mice were sacrificed 1, 2 and 4

weeks after administration and their plasma and inguinal lymph nodes were harvested.

Plasma samples were pooled and analyzed for TNF-α, IL-10 and IL-1β levels using

MAX TM Deluxe Sets ELISA kits (Biolegend™) according to the manufacturer’s

instructions. Harvested lymph nodes were homogenized, mRNA was extracted using

tri-reagent, according to the manufacturer’s instructions (MRC, Cincinnati, OH), and

reverse-transcribed in a PTC-200 PCR cycler using a VersoTM cDNA kit (Thermo

Scientific, Billerica, MA). TNFα and IL-1β levels (normalized to GAPDH) were

quantified by PerfeCTa® SYBR® Green FastMix®, ROXTM (Quanta Biosciences,

Gaithersburg MD) using Applied Biosystems 7300 qPCR with the following primers:

TNF-α Forward: 5'-GCCTCCCTCTCATCAGTTCT-3’

Reverse: 5'-TGGTGGTTTGCTACGACGTG-3’

IL-1β Forward: 5'-AGGATGAGGACATGAGCACC-3’

Reverse: 5'-ATGGGAACGTCACACACCAG-3’

Anti-tumor efficacy of sTRAIL-encapsulating NGs

NGs encapsulating 0.6 sTRAIL/NG (w/w) were prepared from the cytoplasmatic

membranes of human MSCs (NG-TRAIL), rat MSCs (RAT-NG-TRAIL), and human

SMCs (SMC-NG-TRAIL) as a non-mesenchymal control. Synthetic liposomes,

encapsulating sTRAIL (synLIP-TRAIL), were prepared from egg-PC, cholesterol and

DSPE-PEG(2000) using weight ratios of 65:15:15 (Avanti polar lipids, Alabaster,

AL) by thin film hydration followed by extrusion through 0.2 µm polycarbonate

membranes using LIPEX® extruder (Northern Lipids Inc., Burnaby, Canada).

Eight week-old nude mice, previously inoculated with 106 PC3 cells, were

randomized and divided into seven groups (A to G, n=16 mice/group). Group A, the

control group, was not treated. Once tumors reached a volume of 100 mm3 the mice in

Groups B to E were IP administered with the following formulations: B) NG-TRAIL

(5•107 NG-cell-eq./mice) delivering 300 µg sTRAIL/mouse; C) RAT-NG-TRAIL

(same quantities as above); D) SMC-NG-TRAIL (same quantities as above); E)

synLIP-TRAIL (1.25 mg/mice); F) Empty, human MSC-derived NGs (5•107 NG-cell-

eq./mice); and G) free sTRAIL (300 µg sTRAIL/mouse); Tumor volumes were

measured for 15 days and compared between the test (B and C) and the control groups

(A and C – F).

Annexin-V labeling of dissociated tumor cells

At the end of the efficacy trials, tumors were harvested from untreated mice (Group

A), mice treated with NG-TRAIL (Group B), mice administered with NG (Group F),

and mice treated with free sTRAIL (Group G). The harvested tumors were dissociated

into single-cell suspensions as previously done for the biodistribution assays above,

and analyzed by flow cytometry for Annexin-V labeling using a MEBCYTO

apoptosis kit (Medical & Biological Ltd, Nagoya, Japan).

Immunofluorescent analysis of harvested tumors

To further investigate NG localization, tumors were harvested from untreated mice

(Group A), mice treated with NG-TRAIL (Group B), mice treated with RAT-NG-

TRAIL (Group C), and mice administered with SMC-NG-TRAIL (Group D).

Harvested tumors were embedded in OCT (Tissue-Tek, Sakura Fineteck Inc.,

Torrance, CA), frozen on dry ice, and sliced into 10 μm thick sections using a Leica®

Cryostat (Manheim, Germany). Tumor sections were stained with PE/Cy5-conjuagted

anti-human CD90, APC-conjugated anti-rat CD90 and PE-conjugated anti-human

CD97 (for SMC) antibodies (Biolegend™), and imaged using a Zeiss LSM510-

META confocal microscope.

Histological analysis of harvested tumors

Tumors harvested from untreated mice (Group A), mice treated with NG-TRAIL

(Group B), mice administered with empty NG (Group F), and mice treated with free

sTRAIL (Group G) were sliced into 10 μm thick sections. Tumor sections were

stained with hematoxylin-eosin (H&E) and subjected to immunohistochemistry (IHC)

analysis using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA),

with the following primary antibodies: CD31 (1:100; BD Biosciences, San Jose, CA)

for micro-vessel staining; cleaved caspase-3 (1:100; Cell Signaling, Beverly, MA) for

in situ detection of apoptosis; and Ki67 (1:100; Lab Vision, Kalamazoo, MI) for in

situ detection of proliferation. Detection was carried out using 3,3V-diaminobenzidine

chromogen (Sigma-Aldrich™), which results in a positive brown staining. Sections

were counterstained with DAPI (Sigma-Aldrich™), dehydrated in ethanol, and

mounted with glass cover-slips. Negative control slides were obtained by omitting the

primary antibody. Apoptosis and proliferation were quantified by determining the

percentage of positively stained cells (stained cells divided by total nuclei) in 10

randomly chosen fields per tissue section at 200x magnification. Microvessel density

was determined by NIS Elements image analysis software (NIKON Instruments,

Amstelveen, The Netherlands) using 10 randomly chosen fields per section at 200x

magnification. Significant differences in microvessel density, proliferation, and

apoptosis indices were determined using the Mann-Whitney U test. Values of p<0.05

were considered statistically significant.

Statistics

Results are presented as the mean ±SD (standard deviation) or mean ±SEM (standard

error of the mean) of at least triplicates. Statistical significance in the differences of

the means was evaluated by two-tailed t-test. Analysis of variance (ANOVA) was

used to test the significance of differences among groups using JMP 6 software

(SAS™, Cary, NC).