Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles

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International Journal of Pharmaceutics 450 (2013) 241– 249

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International Journal of Pharmaceutics

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Pharmaceutical nanotechnology

Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles:New vehicles for magnetically targeted drug delivery

Liron Philosof-Mazora,1, George R. Dakwarb,1, Mary Popovb, Sofiya Kolushevaa,Alexander Shamesc, Charles Linderd, Sarina Greenbergc, Eliahu Heldmanb,David Stepenskyb,∗∗, Raz Jelineka,∗

a Department of Chemistry and Ilse Katz Institute of Nanotechnology, Faculty of Natural Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israelb Department of Clinical Biochemistry and Pharmacology, The Faculty of Health Sciences, Ben-Gurion University of the Negev, Israelc Department of Chemistry, Faculty of Natural Sciences, Ben-Gurion University of the Negev, Israeld Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Israel

a r t i c l e i n f o

Article history:Received 6 March 2013Received in revised form 9 April 2013Accepted 11 April 2013Available online 24 April 2013

Keywords:BolaamphiphilesIron oxide nanoparticlesMagnetic nanoparticlesNano-drug delivery systemsTargeted drug delivery

a b s t r a c t

Bolaamphiphiles – amphiphilic molecules consisting of two hydrophilic headgroups linked by ahydrophobic chain – form highly stable vesicles consisting of a monolayer membrane that can be usedas vehicles to deliver drugs across biological membranes, particularly the blood–brain barrier (BBB). Weprepared new vesicles comprising bolaamphiphiles (bolavesicles) that encapsulate iron oxide nanoparti-cles (IONPs) and investigated their suitability for targeted drug delivery. Bolavesicles displaying differentheadgroups were studied, and the effect of IONP encapsulation upon membrane interactions and celluptake were examined. Experiments revealed more pronounced membrane interactions of the bolavesi-cles assembled with IONPs. Furthermore, enhanced internalization and stability of the IONP–bolavesicleswere observed in b.End3 brain microvessel endothelial cells – an in vitro model of the blood–brain barrier.Our findings indicate that embedded IONPs modulate bolavesicles’ physicochemical properties, endowhigher vesicle stability, and enhance their membrane permeability and cellular uptake. IONP–bolavesiclesthus constitute a promising drug delivery platform, potentially targeted to the desired location usingexternal magnetic field.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Iron oxide nanoparticles (IONPs) have been a topic of intenseresearch in recent years due to their unique characteristics, such assmall dimensions, magnetic properties, biocompatibility, and havebeen applied for various biomedical applications (Colombo et al.,2012; Gao et al., 2009). For instance, encapsulation of pharmaceu-tical substances into nano-formulations that contain IONPs can beused to target the drug to the desired organs or body regions bymeans of an external magnetic field (Arruebo et al., 2007; Veisehet al., 2010). Moreover, IONPs have been extensively investigatedas contrast agents for magnetic resonance imaging (MRI) (Kimet al., 2011; Lee and Hyeon, 2012; Wang et al., 2001). For example,Feridex I.V.TM and Resovist® iron oxide-based MRI contrast agentwere approved (by the FDA and EMA, respectively) for detection

∗ Corresponding author. Tel.: +972 526839384.∗∗ Corresponding author. Tel.: +972 8 6477381; fax: +972 8 6479303.

E-mail addresses: [email protected] (D. Stepensky), [email protected] (R. Jelinek).1 These authors contributed equally to this work.

of liver lesions upon intravenous injection (Wang, 2011). Unfortu-nately, both these products were discontinued several years ago,in part due to the safety concerns. IONPs exhibit some attractiveproperties: they can be easily manufactured, spatially controlledwhile inside the human body by external (or internally implanted)magnetic fields that are considered physiologically safe, and theirlocalization can be detected using magnetic resonance imaging. Itshould be also noted that IONPs are generally considered biocom-patible and biodegradable (Reddy et al., 2012), since following itsrelease the free iron is integrated in the iron stores of the body, usedfor metabolic processes and eventually eliminated from the body.

Vesicular particles constitute potential vehicles with whichIONPs can be formulated and employed to deliver drugs to targettissues. Different techniques have been developed for synthesis ofIONPs-containing vesicles, usually core-shell structures in whicha magnetic iron oxide is coated by artificial lipid bilayers, thatare able to associate with living cells (Lesieur et al., 2011; Soenenet al., 2009). It has been suggested that IONPs-containing vesiclesare taken up (endocytosed) by different types of cells and accu-mulate in the lysosomes, and that this uptake can be enhancedby an external magnetic field, leading to targeted drug delivery

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242 L. Philosof-Mazor et al. / International Journal of Pharmaceutics 450 (2013) 241– 249

(Chorny et al., 2011). Furthermore, IONPs-containing liposomes orelectrospun fibers can be heated by an alternating magnetic fieldto trigger drug release or to produce local hyperthermia/ablation(Huang et al., 2012; Yallapu et al., 2011).

Here we describe preparation and characterization new IONP-containing bolaamphiphilic lipid vesicles. Bolaamphiphiles are aunique class of compounds consisting of two hydrophilic head-groups connected to each end of a hydrophobic alkyl chain.Bolaamphiphiles can form vesicles that consist of a monolayermembrane surrounding an aqueous core (Fuhrhop and Wang,2004). Vesicles made from natural bolaamphiphiles, such asthose extracted from archaebacteria (archaesomes), are very sta-ble thermodynamically and, therefore, can be used for targeteddrug delivery (Grinberg et al., 2010). However, bolaamphiphilesfrom archaebacteria are heterogeneous and cannot be easilyextracted or chemically synthesized. Recently, custom-designedbolaamphiphiles were chemically synthesized in our laboratories(Grinberg et al., 2008; Popov et al., 2010) and novel unilamellarvesicles were prepared from these compounds. We have previouslyshown that these vesicles exhibit properties beneficial to controlledand targeted drug delivery, including the ability to deliver drugsacross the blood–brain barrier (BBB) to the brain (Dakwar et al.,2012; Popov et al., 2012).

Bolaamphiphilic vesicles (bolavesicles) may have certain advan-tages over conventional liposomes as potential vehicles for drugdelivery. Bolavesicles may have thinner membranes than compa-rable liposomal particles that are made of a bilayer membrane(Stern et al., 1992) and therefore, they possess larger inner vol-ume and hence, for small nanovesicles (diameter of less than100 nm), they can have a higher encapsulation capacity comparedto liposomes of the same diameter. Moreover, bolavesicles aremore stable than classical liposomes because of reduced lipidexchange due to the high energy needed to pull a hydrophilichead group via the hydrophobic domain within the monolayermembrane. Yet, bolavesicles can be readily destabilized by atriggering event that slightly changes the structure of the headgroups (e.g., by hydrolysis of the headgroups using a specificenzymatic reaction, such as acetylcholine headgroups cleavage byacetylcholinesterase, thus allowing controlled release of the encap-sulated material at the site of action (i.e., drug targeting)) (Popovet al., 2012).

In this study, we encapsulated IONPs in bolavesicles, charac-terized their biophysical properties, membrane interactions, andcell uptake. The results indicate that the IONPs significantly modu-late vesicle properties, giving rise to more pronounced membraneinteractions and higher vesicle stability. In particular, cell-uptakestudies using b.End.3 cells, murine brain microvessel endothe-lial cells which have been used as an in vitro model of theblood–brain barrier (Brown et al., 2007), indicate that associationof the IONPs with the bolavesicles enhance cell internalization andintracellular vesicle stability. This study points to potential use ofIONP/bolavesicle assemblies in drug delivery and targeting appli-cations. Specifically, encapsulation of IONPs in bolavesicles mightenable more efficient transport across biological barriers, as wellas control of vesicle cargo delivery and disposition inside the bodyusing external magnetic fields.

2. Materials and methods

2.1. Chemicals and materials

Iron(III) acetylacetonate (Fe(acac)3), diphenyl ether, 1,2-hexadecanediol, oleic acid, oleylamine, and carboxyfluorescein(CF) were purchased from Sigma Aldrich (Rehovot, Israel). Chlo-roform and ethanol were purchased from Bio-Lab Ltd. Jerusalem,

Israel. 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)(DMPG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine(DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),cholesterol (CHOL), cholesteryl hemisuccinate (CHEMS) were pur-chased from Avanti Lipids (Alabaster, AL, USA), The diacetylenicmonomer 10,12-tricosadiynoic acid was purchased from AlfaAesar (Karlsruhe, Germany), and purified by dissolving the powderin chloroform, filtering the resulting solution through a 0.45 �mnylon filter (Whatman Inc., Clifton, NJ, USA), and evaporationof the solvent. 1-(4 trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was purchased from MolecularProbes Inc. (Eugene, OR, USA). (4′, 6-diamidine-2′-phenylindole,dihydrochloride) (DAPI) was purchased from KPL Ltd., (MD, USA).

2.2. Bolaamphiphile synthesis

The bolaamphiphiles GLH19 and GLH20 were synthesized aspreviously described (Grinberg et al., 2008; Popov et al., 2010).Briefly, the carboxylic group of methyl vernolate or vernolic acidwas interacted with aliphatic diols to obtain bisvernolesters. Thenthe epoxy group of the vernolate moiety, located on C12 and C13of the aliphatic chain of vernolic acid, was used to introduce twoACh headgroups on the two vicinal carbons obtained after theopening of the oxirane ring. For GLH-20, the ACh head group wasattached to the vernolate skeleton through the nitrogen atom ofthe choline moiety. The bolaamphiphile was prepared in a two-stage synthesis: First, opening of the epoxy ring with a haloaceticacid and, second, quaternization with the N,N-dimethylamino ethylacetate. For GLH-19 that contains an ACh head group attachedto the vernolate skeleton through the acetyl group, the bolaam-phiphile was prepared in a three-stage synthesis, including openingof the epoxy ring with glutaric acid, then esterification of thefree carboxylic group with N,N-dimethyl amino ethanol and thefinal product was obtained by quaternization of the head group,using methyl iodide followed by exchange of the iodide ion bychloride using an ion exchange resin. Each bolaamphiphile wascharacterized by mass spectrometry, NMR and IR spectroscopy.The purity of the two bolaamphiphiles was >97% as determinedby HPLC.

2.3. Synthesis of magnetite nanoparticles

(IONPs): Fe(acac)3(2 mmol) was mixed in phenyl ether (20 mL)with 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), and ole-ylamine (6 mmol) under argon and was heated to reflux for 30 min.After cooling to room temperature, the dark-brown mixture wastreated with ethanol under air, and a dark-brown material wasprecipitated from the solution. The product was dissolved in chlo-roform in the presence of oleic acid (2 mmol) and oleylamine(2 mmol) and re-precipitated with ethanol to obtain 4-nm Fe3O4nanoparticles (nanoparticle size has been measured by TEM, seeFig. 1A).

2.4. Bolavesicle preparation and characterization

Bolaamphiphiles, cholesterol, and CHMES (2:1:1 mole ratio)were dissolved in chloroform for GLH-20 or a mixture of chloroformand ethanol for GLH-19. For the IONPs-containing formulations,0.5 mg magnetite nanoparticles dispersed in chloroform wereadded to the mix. The solvents were evaporated under vacuum andthe resultant thin films were hydrated in 0.2 mg/mL CF solutionin PBS and probe-sonicated (Vibra-Cell VCX130 sonicator, Sonicsand Materials Inc., Newtown, CT, USA) with amplitude 20%, pulseon: 15 s, pulse off: 10 s to achieve homogenous vesicle dispersions.


L. Philosof-Mazor et al. / International Journal of Pharmaceutics 450 (2013) 241– 249 243

Fig. 1. IONPs-containing bolavesicles characterization. (A) Cryo-TEM image of the prepared IONPs. Scale bar 20 nm; (B) Cryo-TEM images of bolavesicles. Left: empty vesicleswithout IONPs; right: vesicles with encapsulated IONPs. Scale bar 50 nm; (C) Electron paramagnetic resonance (EPR) spectra of free IONPs (not associated with bolavesicles;dotted lines), and IONPs associated with bolavesicles (solid lines). Left: GLH-19, right: GLH-20. The insets show the magnified peak areas.

Vesicle size and zeta potential were determined using a ZetasizerNano ZS (Malvern Instruments, UK).

2.5. Electron paramagnetic resonance (EPR)

EPR spectra of IONPs re-suspended in chloroform (in presenceof oleic acid and oleylamine; i.e., the same form of IONPs that wasused for generation of the IONPs-containing formulations) or of theIONPs-embedded bolavesicles resuspended in PBS were obtainedusing a Bruker EMX-220 X-band (� ∼ 9.4 GHz) EPR spectrometerequipped with an Oxford Instruments ESR 900 temperature acces-sories and an Agilent 53150A frequency counter. Spectra wererecorded at room temperature with the non-saturating incidentmicrowave power 20 mW and the 100 KHz magnetic field modula-tion of 0.2 mT amplitude. Processing of EPR spectra, determinationof spectral parameters were done using Bruker WIN-EPR software.

2.6. Cryogenic transmission electron microscopy (cryo-TEM)

Specimens studied by cryo-TEM were prepared. Sample solu-tions (4 �L) were deposited on a glow discharged, 300 mesh, laceycarbon copper grids (Ted Pella, Redding, CA, USA). The excess liq-uid was blotted and the specimen was vitrified in a Leica EM GPvitrification system in which the temperature and relative humid-ity are controlled. The samples were examined at −180 ◦C using aFEI Tecnai 12 G2 TWIN TEM equipped with a Gatan 626 cold stage,and the images were recorded (Gatan model 794 charge-coupleddevice camera) at 120 kV in low-dose mode.

2.7. Lipid/polydiacetylene (PDA) assay

Lipid/polydiacetylene (PDA) vesicles (DMPC/PDA, 2:3 moleratio) were prepared by dissolving the lipid components inchloroform/ethanol and drying together in vacuo. Vesicles weresubsequently prepared in double distilled water (DDW) by probe-sonication of the aqueous mixture at 70 ◦C for 3 min. The vesiclesamples were then cooled at room temperature for an hour andkept at 4 ◦C overnight. The vesicles were then polymerized usingirradiation at 254 nm for 10–20 s, with the resulting emulsionsexhibiting an intense blue appearance. PDA fluorescence wasmeasured in 96-well microplates (Greiner Bio-One GmbH, Fricken-hausen, Germany) on a Fluoroscan Ascent fluorescence plate reader(Thermo Vantaa, Finland). All measurements were performed atroom temperature at 485 nm excitation and 555 nm emission usingLP filters with normal slits. Acquisition of data was automaticallyperformed every 5 min for 60 min. Samples comprised 30 �L ofDMPC/PDA vesicles and 5 �L bolaamphiphilic vesicles assembledwith IONPs, followed by addition of 30 �L 50 mM Tris-base buffer(pH 8.0). A quantitative value for the increasing of the fluorescenceintensity within the PC/PDA-labeled vesicles is given by the fluo-rescence chromatic response (%FCR), which is defined as follows(Raifman et al., 2010):

%FCR = FI − F0

F100× 100 (1)

where FI is the fluorescence emission of the lipid/PDA vesiclesafter addition of the tested membrane-active compounds, F0 is the



fluorescence of the control sample (without addition of the com-pounds), and F100 is the fluorescence of a sample heated to producethe highest fluorescence emission of the red PDA phase minus thefluorescence of the control sample.

2.8. Fluorescence anisotropy

Giant vesicles (GUV’s) were prepared through the rapid evap-oration method (Moscho et al., 1996). Briefly, GUVs comprisingDMPE and DMPG (1:1 mole ratio) were prepared through dis-solving the lipid constituents in chloroform/ethanol (1:1, v/v),subsequently adding to round-bottom flask (250 mL) containingchloroform (1 mL). The aqueous phase (5 mL of PBS buffer, pH 7.4)was then carefully added along the flask walls. The organic solventwas removed in a rotary evaporator under reduced pressure (finalpressure 40 mbar) at room temperature and 40 rpm rotation speed.After evaporation for 4–5 min, an opalescent fluid was obtainedwith a volume of approximately 5 mL. The fluorescence probe TMA-DPH was incorporated into the DMPE/DMPG vesicles by adding thedye dissolved in tetrahydrofuran (1 mM) to the vesicle solution tothe final concentration of 0.22% (molar ratio) and incubating for30 min at room temperature. 30 �L of bolavesicles (10 mg/mL) wereadded to 30 �L of the TMA-DPH/DMPE/DMPG vesicles followedby addition of low ionic strength PBS buffer (NaCl/10, pH = 7.4) toa total volume of 1.0 mL. TMA-DPH fluorescence anisotropy wasmeasured at 428 nm (excitation 360 nm) on an FL920 spectro-fluorimeter (Edinburgh Instruments, UK). Anisotropy values wereautomatically calculated by the spectrofluorimeter software usingconventional methodology.

2.9. Cell culture

b.End3 immortalized mouse brain capillary endothelium cellswere kindly provided by Prof. Philip Lazarovici (Institute for DrugResearch, School of Pharmacy, The Hebrew University of Jerusalem,Israel).The b.End3 cells were cultured in DMEM medium supple-mented with 10% fetal bovine serum, 2 mM l-glutamine, 100 IU/mLpenicillin and 100 �g/mL streptomycin (Biological Industries Ltd.,Beit Haemek, Israel). The cells were maintained in an incubator at37 ◦C in a humidified atmosphere with 5% CO2.

2.10. Internalization of CF by the cells in vitro

b.End3 cells were grown on 24-well plates or on coverslips(for fluorescence-activated cell sorting (FACS) and fluorescencemicroscopy analysis, respectively). The medium was replaced withculture medium without serum and CF solution, or tested bolavesi-cles (equivalent to 0.5 �g/mL CF), or equivalent volume of themedium were added to the cells and incubated for 5 h at 4 ◦C or at37 ◦C. At the end of the incubation, cells were extensively washedwith complete medium and with PBS, and were either detachedfrom the plates using trypsin-EDTA solution (Biological IndustriesLtd., BeitHaemek, Israel) and analyzed by FACS (FACSCalibur FlowCytometer, BD Biosciences, USA), or fixed with 2.5% formalde-hyde in PBS, washed twice with PBS, mounted on slides usingMowiol-based mounting solution and analyzed using an Olym-pus FV1000-IX81 confocal microscope (Olympus, Tokyo, Japan)equipped with 60x oil objective. All the images were acquired usingthe same imaging settings and were not corrected or modified.

2.11. Live confocal imaging

b.End3 cells were grown on 24-well plates, after 24 h, themedium was replaced with culture medium without serum andCF solution, or studied bolavesicles (equivalent to 0.5 �g/mL CF),or equivalent volume of the medium were added to the cells and

incubated for 5 h in an incubator at 37 ◦C in a humidified atmo-sphere with 5% CO2. At the end of the incubation period, the cellswere washed with growth medium and with PBS. The nucleuswas stained with DAPI (100 �g/mL in PBS). Subsequently, the cellswere detached from the plates using Trypsin-EDTA solution andwashed again with PBS. Live imaging was performed using a ZeissLSM 510-NLO system with an Axiovert 200 M inverted microscope(Carl Zeiss Inc., Germany) tuned to 405 nm and 63 × 1.4 NA ZeissPlan-Apochromat oil immersion objective. Videos were recordedwithout a magnet, and with a magnet placed on different sides ofthe well.

2.12. Statistical analysis

The fluorescence anisotropy data are presented as mean andstandard deviations (SD) or standard errors of mean (SEM). Statis-tical differences between the control and the studied formulationswere analyzed using ANOVA followed by Dunnett post-test usingInStat 3.0 software (GraphPad Software Inc., La Jolla, CA, USA). Pvalues of less than 0.05 were defined as statistically significant.

3. Results and discussion

3.1. IONP/bolavesicle characterization

In this study we employed two synthetic bolaamphiphiles thatwere designed and characterized in our laboratories (Scheme 1).Both compounds have cationic headgroups derived from acetyl-choline (ACh). GLH-20 can be hydrolyzed by cholinesterases (ChE),and GLH-19 that is not digested by ChE (Scheme 1). These twobolaamphiphiles were shown to form spherical vesicles that coulddeliver encapsulated materials across biological barriers such ascell membranes and the blood–brain barrier.

To assemble IONP loaded bolavesicles we first synthesizedmonodisperse Fe3O4 IONPs (Fig. 1A) coated with a hydrophobiclayer of a surfactant (oleic acid) to prevent aggregation. The IONPswere then dispersed in an organic solvent containing the bolaam-phiphiles GLH-19 or GLH-20 together with membrane stabilizers(cholesterol and cholesteryl hemisuccinate) and the organic sol-vent was dried under vacuum. The thin film that was formed washydrated in buffer and probe-sonicated to form IONPs-containingvesicles (see experimental section for more details). The bolavesi-cles were highly stable in aqueous solutions and could be keptfor long time periods (weeks) without undergoing disintegrationor aggregation. Fig. 1B, C and Table 1 present characterization ofthe IONPs-containing bolavesicles. In particular, the experimentswere designed to determine whether the IONPs were encapsu-lated within the bolaamphiphile vesicles, and to what degree theirco-assembly with the bolaamphiphiles altered the properties (size,morphology, surface charge) of the bolavesicles.

Table 1 depicts bolavesicle size distributions (with and withoutencapsulated IONPs) determined by dynamic light scattering (DLS),and the corresponding zeta potential values of the vesicles. Table 1demonstrates that the IONPs did not significantly modify the vesiclesize. However, in both types of bolavesicles (comprising of GLH-19 and GLH-20 bolaamphiphiles, respectively) inclusion of IONPsslightly reduced the zeta-potential, suggesting that association ofthe IONPs reduced the exposure of the positive surface charge,likely due to some reorganization of the lipids/bolaamphiphile con-stituents and interactions between the head-groups and IONPs.

Cryogenic-transmission electron microscopy (cryo-TEM) exper-iments further highlight the structural properties of the IONPs-containing bolavesicles (Fig. 1B). In particular, the representativecryo-TEM images in Fig. 1B reveal distinct patterns of IONPslocalization inside and outside the vesicles, depending on the



GLH-19

GLH-20

OO

O(CH2)10 O

O

HO

NO

O O

O

OH

NO

OOCl Cl

O(CH2)10 O

O

HO

O

ONO

O

ClO

OH

O

ON O

O

Cl

Scheme 1. Bolaamphiphiles used in this study.

bolaamphiphile composition. Specifically, in case of GLH-19bolavesicles, the IONPs appear to localize in vicinity to the vesi-cle membrane in a dispersed form, with part of the IONPs presentinside the bolavesicles, while the other part is outside the bolavesi-cles (Fig. 1B). In contrast, when GLH-20 was used for vesicleformation, the IONPs appear as clusters inside the bolavesicles,not in close vicinity to the membrane. These distinct forms ofIONP/bolavesicle association most likely reflect the different chem-ical structures of the bolaamphiphiles. Specifically, the positivelycharged choline moiety head group in GLH-19 is located at the endof the alkyl side-chain (see Scheme 1). The repulsion between thepositive groups at the vesicle interface might allow the hydropho-bic IONPs to penetrate and reside in vicinity to the bolaamphiphilelayer, as depicted in Fig. 1B. In the case of GLH-20, the choline islocated further down in the bolaamphiphile alkyl chain (Scheme 1),resulting in a more condensed bolaaphiphile layer (or a strongerrepulsion between the headgroups and the IONPs within the vesi-cle membrane). Consequently, the IONPs appear to be localizedinside the bolavesicle core rather than close to the bolaamphiphilemonolayer membrane.

The electron paramagnetic resonance (EPR) data shown inFig. 1C confirm that the IONPs are exposed to different molecularenvironments in the GLH-19 and GLH-20 bolavesicle formula-tions. EPR spectra of aqueous solutions containing control IONPsnot associated with bolavesicles (Fig. 1C, dotted-line traces) con-sist of an intense, slightly asymmetric signal characteristic ofsuper-paramagnetic single-domain NPs (Köseoglu et al., 2004).Association of the IONPs with the bolavesicles resulted in sig-nificant modulation of the EPR spectra (Fig. 1C, solid traces).Specifically, the EPR spectra of the IONPs/bolavesicles are notice-ably broadened, ascribed to inter-particle distance which is notkinetically averaged, due to interaction of the IONPs with thebolavesicles. Importantly, the spectral changes were clearly corre-lated to the type of bolaamphiphile; the broad EPR component wasmuch more dominant in GLH-20 vs. GLH-19 bolavesicles (Fig. 1C,right vs. left panels). This result corroborates the cryo-TEM datashown in Fig. 1B, pointing to more condensed association of theIONPs inside the GLH-20 bolavesicles, resulting, most likely, in lessnanoparticle mobility (and hence broadened EPR signal).

3.2. Membrane interactions of IONPs-containing bolavesicles

To investigate the interactions of the new IONPs-containingbolavesicles with biological membranes, we applied fluorescencespectroscopy in conjunction with lipid bilayer model systems(Fig. 2). Fig. 2A depicts a kinetic experiment in which theIONPs-containing bolavesicles were incubated with biomimetic

lipid/polydiacetylene (PDA) vesicles (Jelinek and Kolusheva, 2001).The lipid/PDA vesicle platform was shown to mimic lipid bilayersystems, providing spectroscopic means for monitoring bilayerinteractions of membrane-active species through recording thechromatic/fluorescent transformations of PDA (Kolusheva et al.,2000a).

The time-dependent fluorescence curves in Fig. 2A, correspond-ing to the PDA fluorescence induced by binding of the bolavesiclesto the lipid/PDA assemblies, point to significant differences inmembrane interactions between the two types of the bolavesicles(GLH-19 vs. GLH-20). Specifically, Fig. 2A demonstrates that GLH-19bolavesicles gave rise to significantly higher fluorescence emis-sion following incubation with DMPG/DMPC/PDA as comparedto the GLH-20 bolavesicles. This enhanced fluorescence emissionis due to more pronounced interactions of GLH-19 bolavesicleswith the membrane, most likely ascribed to the positive cholinemoieties displayed at the bolavesicle membrane surface that areconsequently attracted to the negatively-charged lipid/PDA vesi-cles (which effectively mimic the negative plasma membrane ofmammalian cells) (Kolusheva et al., 2000b).

The PDA fluorescence emission data in Fig. 2A also underscoredifferences in membrane interactions between the empty (IONPs-free) bolavesicles and bolavesicles entrapping IONPs. Specifically,in both bolavesicle formulations (GLH-19 and GLH-20), the pres-ence of the IONPs significantly enhanced bilayer interactions,reflected in the higher PDA fluorescence (dashed curves in Fig. 2A).This effect was particularly pronounced in the case of GLH-19 – forwhich the inclusion of IONPs induced significantly higher, rapidlyincreasing fluorescence intensity (top broken curve in Fig. 2A). Thisresult is consistent with the cryo-TEM data shown in Fig. 1B point-ing to accumulation of the IONPs in vicinity to the bolavesiclemembrane, which interacts with the lipid membrane during theLipid/PDA assay. In comparison, localization of the IONPs inside theGLH-20 bolavesicles, as seen in the cryo-TEM image in Fig. 1B, isexpected to result in a lesser disruption of the lipid/PDA membraneinterface, giving rise to lower fluorescence intensities (Fig. 2A, bot-tom curves).

To gain further information on the extent of bilayer inser-tion and lipid reorganization following IONP association with thebolavesicles, we carried out fluorescence anisotropy experimentsemploying giant unilamellar vesicles (GUVs) (Moscho et al., 1996)that contain DMPE/DMPG phospholipids and the fluorescencedye trimethylammonium-diphenylhexatriene (TMA-DPH, Fig. 2B).DPH-containing hydrophobic molecules have been widely used formonitoring fluidity in lipid bilayers; specifically, the fluorescenceanisotropy of bilayer-anchored DPH is a sensitive probe for changesin fluidity induced by membrane-active species (Lentz, 1989).

Table 1IONP/bolavesicle sizes and surface charges.

Bolavesicle composition Hydrodynamic diameter(nm) (mean ± SEM)

Poly dispersityindex (PDI)

Zeta potential, mV(mean ± SD)

GLH-19/cholesterol/CHEMS 127 ± 33 0.054 41.4 ± 4.4GLH-19/cholesterol/CHEMS + 0.5 mg/mL IONPs 114 ± 46 0.109 38.6 ± 1.1GLH-20/cholesterol/CHEMS 115 ± 46 0.109 32.4 ± 1.0GLH-20/cholesterol/CHEMS + 0.5 mg/mL IONPs 110 ± 60 0.169 27.0 ± 2.9



Fig. 2. IONP/bolavesicle interactions with model membranes. (A) Lipid/PDA assay. PDA fluorescence emission (excitation 485 nm, emission 540 nm) following incubation ofbolavesicles or IONP loaded bolavesicles with DMPC/PDA vesicles. (B) Fluorescence anisotropy of DPH-TMA/DMPE/DMPG GUVs with bolavesicles or IONP loaded bolavesicles(10 mg/mL). Values are means + SD of two experiments (n = 2). Significant differences between the control and the studied formulations were analyzed using ANOVA followedby a Dunnett post-test: *P < 0.05, **P < 0.001.

In similar fashion to the biomimetic lipid/PDA assay results(Fig. 2A), the fluorescence anisotropy data in Fig. 2B underscoredifferences both between GLH-19 and GLH-20 bolavesicles, aswell as between the IONPs-containing bolavesicles and IONPs-free bolavesicles. Specifically, Fig. 2B shows a marked decrease inanisotropy when the DPH-containing GUVs were incubated withGLH-19 bolavesicles as compared to the GLH-20 bolavesicles. Thelower fluorescence anisotropy is indicative of higher mobility ofthe DPH dye, which echoes the PDA assay data (Fig. 2A) pointing tosignificantly greater bilayer disruption by the GLH-19 bolavesiclesas compared to the GLH-20 bolavesicles.

The fluorescence anisotropy data in Fig. 2B also highlightthe significant impact on membrane interactions of IONPs incor-poration within the bolavesicles. Indeed, for both GLH-19 andGLH-20, the IONPs-containing bolavesicles gave rise to markedlylower fluorescence anisotropy of DPH following incubation withthe DPH-TMA/lipid GUVs, compared to the respective IONPs-freebolavesicles. This result reflects more pronounced lipid reorgani-zation induced by binding of the IONPs-containing bolavesiclesand again corroborates the interpretation of the PDA assay datain Fig. 2A.

3.3. Cell uptake of IONPs-containing bolavesicles

The biophysical experiments in Fig. 2 demonstrate more effi-cient membrane interactions of the IONPs-containing bolavesiclesas compared to their IONPs-free counterparts. We further inves-tigated whether this difference is still apparent in experimentsexamining the interaction of IONPs-containing and IONPs-freebolavesicles with brain capillary endothelial cells. To this end, weused murine b.End3 cells, which are among the most extensivelyused cell lines for brain uptake and permeability studies (Li et al.,2010). These cultured cells possess many features that are char-acteristic to the BBB (e.g., monolayer formation that expressesthe tight junction proteins ZO-1, ZO-2, occludin and claudin-5)(Brown et al., 2007). Previously, we used b.End3 cells to ana-lyze uptake and intracellular fate of bolavesicles encapsulatinga model fluorescently-labeled protein (BSA-FITC) (Dakwar et al.,2012) and a fluorescent marker (carboxyfluorescein, CF) (Popovet al., 2012).

b.End3 cells were used here to determine the extent of inter-nalization of the bolavesicles encapsulating CF as compared to freeCF by fluorescence activated cell sorting (FACS) at 4 ◦C and 37 ◦C,respectively (Fig. 3). The FACS data clearly show that the cells didnot internalize free CF at both temperatures (blue curves in Fig. 3).This outcome is expected since CF is negatively charged at physio-logical pH and does not interact with the negatively charged plasma

membrane of the cells. Incubation of the CF-loaded bolavesicles withthe cells at 4 ◦C resulted in little internalization of the dye, as can beseen from the shift of the FACS curves to the right (Fig. 3A,C). Thisshift was substantially higher at 37 ◦C indicating that the uptakeof the bolavesicles by the cells can be energy-dependent. The FACSdata also show that the uptake of GLH-19 bolavesicles appears tobe more efficient at 37 ◦C than that of GLH-20 bolavesicles (Fig. 3Band D), which is consistent with the more pronounced interac-tions of GLH-19 bolavesicles with membranes, as discussed above(Fig. 2). It also should be noted that association of IONPs with thebolavesicles appeared to enhance the uptake of the bolavesicles bythe cells, particularly in case of the GLH-20 bolavesicles (Fig. 3C andD), although to a small extent (see the slight shift of the green curvesto the right, as compared to the orange curves, and a small popu-lation of highly-fluorescent cells in the green curve on panel C ofFig. 3).

Confocal fluorescence microscopy analysis presented in Fig. 4provides further insight into the uptake, stability, and localizationof the IONPs-containing bolavesicles vs. IONPs-free bolavesicles.The microscopy data in Fig. 4 complements the FACS experi-ments, and provide visual depiction of CF internalization withinthe cells. Importantly, free IONPs [not encapsulated within bolavesi-cles] rapidly aggregate in solution and do not permeate intocells.

Several observations need to be emphasized based on thedata presented in Fig. 4. First, echoing the FACS experiments,CF was internalized by the bEnd.3 cells only when encapsulatedwithin the bolavesicles (IONPs-containing and IONPs-free alike).The confocal images also confirm that the GLH-19 bolavesicleswere endocytosed more efficiently than the GLH-20 bolavesicles,and that addition of IONPs to the formulation enhanced cellu-lar uptake efficiency. Notably, in the case of GLH-19 bolavesicles(IONPs-containing and IONPs-free), after 5 h incubation a sig-nificant amount of CF fluorescence was seen in the cytoplasmof the cells. By comparison, in the case of GLH-20 bolavesiclesafter 5 h, smaller amount of the fluorescent dye accumulatedinside the cells and a substantial number of (IONPs-containingand IONPs-free) bolavesicles were associated with the cell mem-branes (appearing as punctuated green fluorescence). This result isindicative of greater membrane permeation by GLH-19 bolavesi-cles, and consistent with the biophysical experiments discussedabove (Fig. 2).

Another important observation in Fig. 4 is the different dis-tribution patterns of the fluorescent CF marker inside the b.End3cells. In case of the GLH-19-based bolavesicles, diffuse green stain-ing is observed, indicating possible intracellular disruption of thebolavesicles following uptake by the cells. In a dramatic contrast,



Fig. 3. b.End3 cell uptake of IONP loaded bolavesicles analyzed by FACS. The cells were incubated with the vesicles or with control solutions for 5 h at 4 ◦C (left) or at 37 ◦C(right). At the end of the incubation the cells were extensively washed and analyzed by FACS.

Fig. 4. Bolavesicle-mediated uptake of the CF by the b.End3 cells. The cells wereincubated with the bolavesicles (IONPs-free or IONPs-containing) or with the con-trol solutions for 5 h at 37 ◦C. At the end of the incubation the cells were extensivelywashed, fixed with formaldehyde, stained with nuclear stain (DAPI) and analyzedusing confocal microscopy. Left column: DAPI fluorescence; Middle column: CFfluorescence; right column: merged images. The scale bar corresponds to 10 �m.

a significant number of the endocytosed GLH-20 IONP loadedbolavesicles were still intact inside the cells, as indicated from themixed (diffuse + punctuated) pattern of the green CF fluorescencein the cells.

This finding is significant, since high stability of drug-encapsulating vesicles during endocytosis is desirable. It should benoted that the intracellular fate of the bolavesicles was assessedin this study following 5 h in vitro incubation. For the purpose oftargeted delivery, much shorter time periods would likely be suf-ficient and therefore the intracellular fate of the vesicles in vivomay be completely different. Indeed, we previously observedsubstantial brain accumulation of a fluorescent dye when deliv-ered encapsulated within GLH-20 bolavesicles at 30 min afterintravenous administration (Dakwar et al., 2012; Popov et al.,2012).

While the fluorescence confocal microscopy images in Fig. 4clearly show efficient uptake of encapsulated CF into b.End3 cells, itis important to verify that the IONPs did not leak out or dissociatedfrom the bolavesicles outside of the cells. To evaluate this issue, weperformed real time imaging of live b.End3 cells that endocytosedbolavesicles encapsulating both CF and IONPs, in the presenceand absence of an externally-placed magnet (Fig. 5; a video fileprovided in the Supporting Information). Fig. 5 demonstrates theremarkable effect of the magnet on the b.End3 cells incubatedwith IONPs-containing bolavesicles. Specifically, these cells rapidlymigrated toward an externally-placed magnet (Fig. 5A). In contrast,bolavesicles that contained only encapsulated CF, but not IONPs,were not affected by the magnet (Fig. 5B). This result indicatesthat the IONPs were indeed delivered by the bolavesicles into thecells (an alternative, and less likely scenario is that IONPs wereable to bind to the cell membrane, without being internalized). Itshould be emphasized that b.End3 cells did not endocytose freeIONPs (i.e., IONPs that are not associated with bolavesicles, dataon the lack of magnet effect on the cells incubated with free IONPsare not shown). This inefficient endocytosis of the free IONPsapparently stems from the low endocytosis rate of the b.End3 cells



Fig. 5. Cell mobility induced by an external magnetic field. Live confocal imagingof b.End3 cells following 5-h incubation with bolavesicles. Top row: Cells incubatedwith IONPs-containing bolavesicles (GLH-20). Rapid migration of the cells towardthe externally placed magnet was recorded; Bottom row: Cells incubated with con-ventional (IONPs-free) bolavesicles (GLH-20). No cell movement was observed.

and propensity of oleic-acid-coated IONPs to aggregate in aqueoussolutions, that may limit their accessibility to the cells.

4. Conclusions

We describe a novel formulation comprising iron oxidenanoparticles (IONPs) associated with bolaamphiphile vesicles.Characterization of the IONPs-containing bolavesicles using EPRand cryo-TEM (Fig. 1) confirmed that the IONPs were associatedwith the bolavesicles. Interestingly, the IONPs interacted differ-ently with GLH-19 and GLH-20 in the vesicular environments, mostlikely reflecting the distinct chemical structures of the two bolaam-phiphiles.

The incorporation of IONPs within the bolavesicles was shownto significantly enhance their interactions with membrane bilay-ers in model systems. Specifically, more pronounced binding tothe bilayer interface and higher bilayer fluidity were induced bythe membrane-interacting IONPs-containing bolavesicles as com-pared to the IONPs-free bolavesicles. This outcome possibly relatesto bolaamphiphile reorganization within the vesicular membranefollowing embedding of the IONPs, leading to higher exposure ofthe bolaamphiphiles’ positively charged moieties and consequentpronounced interactions with the cell plasma membranes (whichare usually negatively charged).

The studied bolavesicle-based formulations were efficientlyendocytosed by the b.End3 brain endothelial cells, even in theabsence of the magnetic field, leading to efficient accumulationof the encapsulated materials in these cells. These observationssuggest that IONPs-containing bolavesicles might be excellentcandidates for transport of different molecular cargoes through bio-logical barriers. Specifically, the outcomes of this study indicate thatinteraction with IONPs-containing bolavesicles leads to significantassociation/accumulation of the IONPs with the cells. As a result,these cells can be spatially manipulated using an external magneticfield.

Thus, the new IONPs/bolavesicle assembly might be used asa drug delivery and targeting vehicle. The encapsulated IONPsmay help to target drug-loaded bolavesicles to specific regionin vivo by an external magnetic field. Subsequently, it could bepossible to attain triggered bolavesicle decapsulation and drug

release applying a local alternating magnetic field. In futureexperiments we plan to determine in vivo tissue disposition ofIONPs-containing bolavesicles in live animals with and withoutapplication of external (constant and alternating) magnetic fields.

Conflict of interest statement

Eli Heldman, Sarina Grinberg and Charles Linder hold a patenton use of bolavesicle formulations for drug delivery. Eli Heldmanis employed by Lauren Sciences Ltd., New York, USA that developsbolavesicle-based technologies for treating brain diseases.

Acknowledgements

We thank Prof. Philip Lazarovici (Institute for Drug Research,School of Pharmacy, The Hebrew University of Jerusalem, Israel)for providing the b.End3 cells. This study was supported by theIsrael Science Foundation Grant No. 973/11 to David Stepensky, EliHeldman, Sarina Grinberg and Charles Linder.

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Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles

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