bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: new vehicles for magnetically...
TRANSCRIPT
![Page 1: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/1.jpg)
G
I
P
Bv
LQ1
ADa
b
c
d
e
8fQ2
ARRAA
KBIMNT
1
rsb2tumeae
Q3
0h
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
ARTICLE IN PRESS Model
JP 13259 1–9
International Journal of Pharmaceutics xxx (2013) xxx– xxx
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
j o ur nal ho me page: www.elsev ier .com/ locate / i jpharm
harmaceutical nanotechnology
olaamphiphilic vesicles encapsulating iron oxide nanoparticles: Newehicles for magnetically targeted drug delivery
iron Philosof-Mazora,1, George R. Dakwarb,1, Mary Popovc, Sofiya Kolushevaa,lexander Shamesf, Charles Linderd, Sarina Greenberga, Eliahu Heldmanc,avid Stepenskye,∗∗, Raz Jelineka,∗
Department of Chemistry and Ilse Katz Institute of Nanotechnology, Faculty of Natural Sciences, Ben-Gurion University of the Negev, Beer Sheva, IsraelDepartment of Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, IsraelDepartment of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, IsraelDepartment of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, IsraelDepartment of Clinical Biochemistry and Pharmacology, The Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva4105, IsraelDepartment of Chemistry, Ben-Gurion University of the Negev, Israel
a r t i c l e i n f o
rticle history:eceived 6 March 2013eceived in revised form 9 April 2013ccepted 11 April 2013vailable online xxx
eywords:olaamphiphiles
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-
ron oxide nanoparticlesagnetic nanoparticlesano-drug delivery systemsargeted drug delivery
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.
40
41
42
43
44
45
46
47
48
49
. Introduction
Iron oxide nanoparticles (IONPs) have been a topic of intenseesearch in recent years due to their unique characteristics, such asmall dimensions, magnetic properties, biocompatibility, and haveeen applied for various biomedical applications (Colombo et al.,012; Gao et al., 2009). For instance, encapsulation of pharmaceu-ical substances into nano-formulations that contain IONPs can besed to target the drug to the desired organs or body regions byeans of an external magnetic field (Arruebo et al., 2007; Veiseh
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
t al., 2010). Moreover, IONPs have been extensively investigateds contrast agents for magnetic resonance imaging (MRI) (Kimt al., 2011; Lee and Hyeon, 2012; Wang et al., 2001). For example,
∗ Corresponding author. Tel.: +972 526839384.∗∗ Corresponding author.
E-mail addresses: [email protected] (D. Stepensky), [email protected] (R. Jelinek).1 1These authors contributed equally to this work.
378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2013.04.017
50
51
52
53
54
© 2013 Elsevier B.V. All rights reserved.
Feridex I.V.TM and Resovist® iron oxide-based MRI contrast agentwere approved (by the FDA and EMA, respectively) for detectionof 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 target
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
tissues. 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. It has been suggested that
55
56
57
58
![Page 2: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/2.jpg)
ING Model
I
2 ourna
Ieutcah
cugBm2tbdfeb(vsaa2
tdr(atstttbsQ4yaao
tclislboiIceau
2
2
h
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
ARTICLEJP 13259 1–9
L. Philosof-Mazor et al. / International J
ONPs-containing vesicles are taken up (endocytosed) by differ-nt types of cells and accumulate in the lysosomes, and that thisptake can be enhanced by an external magnetic field, leading toargeted drug delivery (Chorny et al., 2011). Furthermore, IONPs-ontaining liposomes or electrospun fibers can be heated by anlternating magnetic field to trigger drug release or to produce localyperthermia/ablation (Huang et al., 2012; Yallapu et al., 2011).
Here we describe preparation and characterization new IONP-ontaining bolaamphiphilic lipid vesicles. Bolaamphiphiles are anique class of compounds consisting of two hydrophilic head-roups connected to each end of a hydrophobic alkyl chain.olaamphiphiles can form vesicles that consist of a monolayerembrane surrounding an aqueous core (Fuhrhop and Wang,
004). Vesicles made from natural bolaamphiphiles, such ashose extracted from archaebacteria (archaesomes), are very sta-le thermodynamically and, therefore, can be used for targetedrug delivery (Grinberg et al., 2010). However, bolaamphiphilesrom archaebacteria are heterogeneous and cannot be easilyxtracted or chemically synthesized. Recently, custom-designedolaamphiphiles were chemically synthesized in our laboratoriesGrinberg et al., 2008; Popov et al., 2010) and novel unilamellaresicles were prepared from these compounds. We have previouslyhown that these vesicles exhibit properties beneficial to controllednd targeted drug delivery, including the ability to deliver drugscross the blood–brain barrier (BBB) to the brain (Dakwar et al.,012; Popov et al., 2012).
Bolaamphiphilic vesicles (bolavesicles) may have certain advan-ages over conventional liposomes as potential vehicles for drugelivery. Bolavesicles may have thinner membranes than compa-able liposomal particles that are made of a bilayer membraneStern et al., 1992) and therefore, they possess larger inner volumend hence, for small nanovesicles (diameter of less than 100 nm),hey can have a higher encapsulation capacity compared to lipo-omes of the same diameter. Moreover, bolavesicles are more stablehan classical liposomes because of reduced lipid exchange dueo the high energy needed to pull a hydrophilic head group viahe hydrophobic domain within the monolayer membrane. Yet,olavesicles can be readily destabilized by a triggering event thatlightly changes the structure of the head groups (e.g., by hydrol-sis of the headgroups using a specific enzymatic reaction, suchs acetylcholine headgroups cleavage by acetylcholinesterase, thusllowing controlled release of the encapsulated material at the sitef action (i.e., drug targeting) (Popov et al., 2012).
In this study, we encapsulated IONPs in bolavesicles, charac-erized their biophysical properties, membrane interactions, andell uptake. The results indicate that the IONPs significantly modu-ate vesicle properties, giving rise to more pronounced membranenteractions and higher vesicle stability. In particular, cell-uptaketudies using b.End.3 cells, murine brain microvessel endothe-ial cells which have been used as an in vitro model of thelood–brain barrier (Brown et al., 2007), indicate that associationf the IONPs with the bolavesicles enhance cell internalization andntracellular vesicle stability. This study points to potential use ofONP/bolavesicle assemblies in drug delivery and targeting appli-ations. Specifically, encapsulation of IONPs in bolavesicles mightnable more efficient transport across biological barriers, as wells control of vesicle cargo delivery and disposition inside the bodysing external magnetic fields.
. Materials and methods
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
.1. Chemicals and materials
Iron(III) acetylacetonate (Fe(acac)3), diphenyl ether, 1,2-exadecanediol, oleic acid, oleylamine, and carboxyfluorescein
PRESSl of Pharmaceutics xxx (2013) xxx– xxx
(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 C13 ofthe aliphatic chain of vernolic acid, was used to introduce two AChheadgroups on the two vicinal carbons obtained after the openingof the oxirane ring. For GLH-20, the ACh head group was attachedto the vernolate skeleton through the nitrogen atom of the cholinemoiety. The bolaamphiphile was prepared in a two-stage synthesis:First, opening of the epoxy ring with a haloacetic acid and, sec-ond, quaternization with the N,N-dimethylamino ethyl acetate. ForGLH-19 that contains an ACh head group attached to the vernolateskeleton through the acetyl group, the bolaamphiphile was pre-pared in a three-stage synthesis, including opening of the epoxyring with glutaric acid, then esterification of the free carboxylicgroup with N,N-dimethyl amino ethanol and the final product wasobtained by quaternization of the head group, using methyl iodidefollowed by exchange of the iodide ion by chloride using an ionexchange resin. Each bolaamphiphile was characterized by massspectrometry, NMR and IR spectroscopy. The purity of the twobolaamphiphiles was >97% as determined by 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 oley-lamine (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 and
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
the 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.
176
177
178
179
![Page 3: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/3.jpg)
ARTICLE IN PRESSG Model
IJP 13259 1–9
L. Philosof-Mazor et al. / International Journal of Pharmaceutics xxx (2013) xxx– xxx 3
F reparew n pard ht: GL
VN
2
ouIuesrmto
2
tcuviFad
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
ig. 1. IONPs-containing bolavesicles characterization. (A) Cryo-TEM image of the pithout IONPs; right: vesicles with encapsulated IONPs. Scale bar 50 nm; (C) Electrootted lines), and IONPs associated with bolavesicles (solid lines). Left: GLH-19, rig
esicle size and zeta potential were determined using a Zetasizerano ZS (Malvern Instruments, UK).
.5. Electron paramagnetic resonance (EPR)
EPR spectra of IONPs re-suspended in chloroform (in presencef oleic acid and oleylamine; i.e., the same form of IONPs that wassed for generation of the IONPs-containing formulations) or of the
ONPs-embedded bolavesicles resuspended in PBS were obtainedsing a Bruker EMX-220 X-band (� ∼ 9.4 GHz) EPR spectrometerquipped with an Oxford Instruments ESR 900 temperature acces-ories and an Agilent 53150A frequency counter. Spectra wereecorded at room temperature with the non-saturating incidenticrowave power 20 mW and the 100 KHz magnetic field modula-
ion of 0.2 mT amplitude. Processing of EPR spectra, determinationf spectral parameters were done using Bruker WIN-EPR software.
.6. Cryogenic transmission electron microscopy (cryo-TEM)
Specimens studied by cryo-TEM were prepared. Sample solu-ions (4 �L) were deposited on a glow discharged, 300 mesh, laceyarbon copper grids (Ted Pella, Redding, CA, USA). The excess liq-id was blotted and the specimen was vitrified in a Leica EM GP
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
itrification system in which the temperature and relative humid-ty are controlled. The samples were examined at −180 ◦C using aEI Tecnai 12 G2 TWIN TEM equipped with a Gatan 626 cold stage,nd the images were recorded (Gatan model 794 charge-coupledevice camera) at 120 kV in low-dose mode.
d IONPs. Scale bar 20 nm; (B) Cryo-TEM images of bolavesicles. Left: empty vesiclesamagnetic resonance (EPR) spectra of free IONPs (not associated with bolavesicles;H-20. The insets show the magnified peak areas.
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):
FI − F0
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
%FCR =F100
× 100 (1)
where FI is the fluorescence emission of the lipid/PDA vesiclesafter addition of the tested membrane-active compounds, F0 is the
225
226
227
![Page 4: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/4.jpg)
ING Model
I
4 ourna
flptfl
2
oDsscwwpAwDdt3abamoac
2
wRImpB3
2
(mccm3wfLChMpet
2
mCo
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
ARTICLEJP 13259 1–9
L. Philosof-Mazor et al. / International J
uorescence of the control sample (without addition of the com-ounds), and F100 is the fluorescence of a sample heated to producehe highest fluorescence emission of the red PDA phase minus theuorescence of the control sample.
.8. Fluorescence anisotropy
Giant vesicles (GUV’s) were prepared through the rapid evap-ration method (Moscho et al., 1996). Briefly, GUVs comprisingMPE and DMPG (1:1 mole ratio) were prepared through dis-
olving the lipid constituents in chloroform/ethanol (1:1, v/v),ubsequently adding to round-bottom flask (250 mL) containinghloroform (1 mL). The aqueous phase (5 mL of PBS buffer, pH 7.4)as then carefully added along the flask walls. The organic solventas removed in a rotary evaporator under reduced pressure (finalressure 40 mbar) at room temperature and 40 rpm rotation speed.fter evaporation for 4–5 min, an opalescent fluid was obtainedith a volume of approximately 5 mL. The fluorescence probe TMA-PH was incorporated into the DMPE/DMPG vesicles by adding theye dissolved in tetrahydrofuran (1 mM) to the vesicle solution tohe final concentration of 0.22% (molar ratio) and incubating for0 min at room temperature. 30 �L of bolavesicles (10 mg/mL) weredded to 30 �L of the TMA-DPH/DMPE/DMPG vesicles followedy addition of low ionic strength PBS buffer (NaCl/10, pH = 7.4) to
total volume of 1.0 mL. TMA-DPH fluorescence anisotropy waseasured at 428 nm (excitation 360 nm) on an FL920 spectroflu-
rimeter (Edinburgh Instruments, UK). Anisotropy values wereutomatically calculated by the spectrofluorimeter software usingonventional methodology.
.9. Cell culture
b.End3 immortalized mouse brain capillary endothelium cellsere kindly provided by Prof. Philip Lazarovici (Institute for Drugesearch, School of Pharmacy, The Hebrew University of Jerusalem,
srael).The b.End3 cells were cultured in DMEM medium supple-ented with 10% fetal bovine serum, 2 mM l-glutamine, 100 IU/mL
enicillin and 100 �g/mL streptomycin (Biological Industries Ltd.,eit Haemek, Israel). The cells were maintained in an incubator at7 ◦C in a humidified atmosphere with 5% CO2.
.10. Internalization of CF by the cells in vitro
b.End3 cells were grown on 24-well plates or on coverslipsfor fluorescence-activated cell sorting (FACS) and fluorescence
icroscopy analysis, respectively). The medium was replaced withulture medium without serum and CF solution, or tested bolavesi-les (equivalent to 0.5 �g/mL CF), or equivalent volume of theedium were added to the cells and incubated for 5 h at 4 ◦C or at
7 ◦C. At the end of the incubation, cells were extensively washedith complete medium and with PBS, and were either detached
rom the plates using trypsin-EDTA solution (Biological Industriestd., BeitHaemek, Israel) and analyzed by FACS (FACSCalibur Flowytometer, BD Biosciences, USA), or fixed with 2.5% formalde-yde in PBS, washed twice with PBS, mounted on slides usingowiol-based mounting solution and analyzed using an Olym-
us FV1000-IX81 confocal microscope (Olympus, Tokyo, Japan)quipped with 60x oil objective. All the images were acquired usinghe same imaging settings and were not corrected or modified.
.11. Live confocal imaging
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
b.End3 cells were grown on 24-well plates, after 24 h, theedium was replaced with culture medium without serum and
F solution, or studied bolavesicles (equivalent to 0.5 �g/mL CF),r equivalent volume of the medium were added to the cells and
PRESSl of Pharmaceutics xxx (2013) xxx– xxx
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-
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
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
342
343
344
345
![Page 5: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/5.jpg)
ARTICLE IN PRESSG Model
IJP 13259 1–9
L. Philosof-Mazor et al. / International Journal of Pharmaceutics xxx (2013) xxx– xxx 5
GLH-19
GLH-20
OO
O(CH2)10 O
O
HO
NO
O O
O
OH
NO
OOCl Cl
O(CH )
OO
ONO
O
ClO
O
ON O
O
Cl
iphile
bbcicfnIicopbllrrcim
FetnssAnSakblmrsIn
3
bs(I
TI
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
HO
Scheme 1. Bolaamph
olaamphiphile composition. Specifically, in case of GLH-19olavesicles, the IONPs appear to localize in vicinity to the vesi-le membrane in a dispersed form, with part of the IONPs presentnside the bolavesicles, while the other part is outside the bolavesi-les (Fig. 1B). In contrast, when GLH-20 was used for vesicleormation, the IONPs appear as clusters inside the bolavesicles,ot in close vicinity to the membrane. These distinct forms of
ONP/bolavesicle association most likely reflect the different chem-cal structures of the bolaamphiphiles. Specifically, the positivelyharged choline moiety head group in GLH-19 is located at the endf the alkyl side-chain (see Scheme 1). The repulsion between theositive groups at the vesicle interface might allow the hydropho-ic IONPs to penetrate and reside in vicinity to the bolaamphiphile
ayer, as depicted in Fig. 1B. In the case of GLH-20, the choline isocated further down in the bolaamphiphile alkyl chain (Scheme 1),esulting in a more condensed bolaaphiphile layer (or a strongerepulsion between the headgroups and the IONPs within the vesi-le membrane). Consequently, the IONPs appear to be localizednside the bolavesicle core rather than close to the bolaamphiphile
onolayer membrane.The electron paramagnetic resonance (EPR) data shown in
ig. 1C confirm that the IONPs are exposed to different molecularnvironments in the GLH-19 and GLH-20 bolavesicle formula-ions. EPR spectra of aqueous solutions containing control IONPsot associated with bolavesicles (Fig. 1C, dotted-line traces) con-ist of an intense, slightly asymmetric signal characteristic ofuper-paramagnetic single-domain NPs (Köseoglu et al., 2004).ssociation of the IONPs with the bolavesicles resulted in sig-ificant modulation of the EPR spectra (Fig. 1C, solid traces).pecifically, the EPR spectra of the IONPs/bolavesicles are notice-bly broadened, ascribed to inter-particle distance which is notinetically averaged, due to interaction of the IONPs with theolavesicles. Importantly, the spectral changes were clearly corre-
ated to the type of bolaamphiphile; the broad EPR component wasuch more dominant in GLH-20 vs. GLH-19 bolavesicles (Fig. 1C,
ight vs. left panels). This result corroborates the cryo-TEM datahown in Fig. 1B, pointing to more condensed association of theONPs inside the GLH-20 bolavesicles, resulting, most likely, in lessanoparticle mobility (and hence broadened EPR signal).
.2. Membrane interactions of IONPs-containing bolavesicles
To investigate the interactions of the new IONPs-containing
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
olavesicles with biological membranes, we applied fluorescencepectroscopy in conjunction with lipid bilayer model systemsFig. 2). Fig. 2A depicts a kinetic experiment in which theONPs-containing bolavesicles were incubated with biomimetic
able 1ONP/bolavesicle sizes and surface charges.
Bolavesicle composition Hydrodynamic diam(nm) (mean ± SEM)
GLH-19/cholesterol/CHEMS 127 ± 33
GLH-19/cholesterol/CHEMS + 0.5 mg/mL IONPs 114 ± 46
GLH-20/cholesterol/CHEMS 115 ± 46
GLH-20/cholesterol/CHEMS + 0.5 mg/mL IONPs 110 ± 60
2 10 O OH
s used in this study.
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).
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
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).
eter Poly dispersityindex (PDI)
Zeta potential, mV(mean ± SD)
0.054 41.4 ± 4.40.109 38.6 ± 1.10.109 32.4 ± 1.00.169 27.0 ± 2.9
434
435
436
437
![Page 6: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/6.jpg)
ARTICLE IN PRESSG Model
IJP 13259 1–9
6 L. Philosof-Mazor et al. / International Journal of Pharmaceutics xxx (2013) xxx– xxx
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.
(dwfaGltsa
tpGltbzai
3
catebuu2at(ufla
nCrdF
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
In similar fashion to the biomimetic lipid/PDA assay resultsFig. 2A), the fluorescence anisotropy data in Fig. 2B underscoreifferences both between GLH-19 and GLH-20 bolavesicles, asell as between the IONPs-containing bolavesicles and IONPs-
ree bolavesicles. Specifically, Fig. 2B shows a marked decrease innisotropy when the DPH-containing GUVs were incubated withLH-19 bolavesicles as compared to the GLH-20 bolavesicles. The
ower fluorescence anisotropy is indicative of higher mobility ofhe DPH dye, which echoes the PDA assay data (Fig. 2A) pointing toignificantly greater bilayer disruption by the GLH-19 bolavesicless compared to the GLH-20 bolavesicles.
The fluorescence anisotropy data in Fig. 2B also highlighthe significant impact on membrane interactions of IONPs incor-oration within the bolavesicles. Indeed, for both GLH-19 andLH-20, the IONPs-containing bolavesicles gave rise to markedly
ower fluorescence anisotropy of DPH following incubation withhe DPH-TMA/lipid GUVs, compared to the respective IONPs-freeolavesicles. This result reflects more pronounced lipid reorgani-ation induced by binding of the IONPs-containing bolavesiclesnd again corroborates the interpretation of the PDA assay datan Fig. 2A.
.3. Cell uptake of IONPs-containing bolavesicles
The biophysical experiments in Fig. 2 demonstrate more effi-ient membrane interactions of the IONPs-containing bolavesicless compared to their IONPs-free counterparts. We further inves-igated whether this difference is still apparent in experimentsxamining the interaction of IONPs-containing and IONPs-freeolavesicles with brain capillary endothelial cells. To this end, wesed murine b.End3 cells, which are among the most extensivelysed cell lines for brain uptake and permeability studies (Li et al.,010). These cultured cells possess many features that are char-cteristic to the BBB (e.g., monolayer formation that expresseshe tight junction proteins ZO-1, ZO-2, occludin and claudin-5)Brown et al., 2007). Previously, we used b.End3 cells to analyzeptake and intracellular fate of bolavesicles encapsulating a modeluorescently-labeled protein (BSA-FITC) (Dakwar et al., 2012) and
fluorescent marker (carboxyfluorescein, CF) (Popov et al., 2012).b.End3 cells were used here to determine the extent of inter-
alization of the bolavesicles encapsulating CF as compared to free
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
F by fluorescence activated cell sorting (FACS) at 4 ◦C and 37 ◦C,espectively (Fig. 3). The FACS data clearly show that the cellsid not internalize free CF at both temperatures (blue curves inig. 3). This outcome is expected since CF is negatively charged at
physiological pH and does not interact with the negatively chargedplasma membrane of the cells. Incubation of the CF-loaded bolavesi-cles with the cells at 4 ◦C resulted in little internalization of thedye, as can be seen from the shift of the FACS curves to the right(Fig. 3A,C). This shift was substantially higher at 37 ◦C indicatingthat the uptake of the bolavesicles by the cells can be energy-dependent. The FACS data also show that the uptake of GLH-19bolavesicles appears to be more efficient at 37 ◦C than that of GLH-20 bolavesicles (Fig. 3B and D), which is consistent with the morepronounced interactions of GLH-19 bolavesicles with membranes,as discussed above (Fig. 2). It also should be noted that association ofIONPs with the bolavesicles appeared to enhance the uptake of thebolavesicles by the cells, particularly in case of the GLH-20 bolavesi-cles (Fig. 3C and D), although to a small extent (see the slight shift ofthe green curves to the right, as compared to the orange curves, anda small population of highly-fluorescent cells in the green curve onpanel C of Fig. 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 within thecells. Importantly, free IONPs [not encapsulated within bolavesicles]rapidly aggregate in solution and do not permeate into cells.
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 signifi-cant amount of CF fluorescence was seen in the cytoplasm of thecells. By comparison, in the case of GLH-20 bolavesicles after 5 h,smaller amount of the fluorescent dye accumulated inside the cellsand a substantial number of (IONPs-containing and IONPs-free)bolavesicles were associated with the cell membranes (appear-ing as punctuated green fluorescence). This result is indicative ofgreater membrane permeation by GLH-19 bolavesicles, and consis-tent with the biophysical experiments discussed above (Fig. 2).
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
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 the
522
523
524
525
![Page 7: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/7.jpg)
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
ARTICLE IN PRESSG Model
IJP 13259 1–9
L. Philosof-Mazor et al. / International Journal of Pharmaceutics xxx (2013) xxx– xxx 7
Fig. 3. b.End3 cell uptake of IONP loaded bolavesicles analyzed by FACS. The cells were i(right). At the end of the incubation the cells were extensively washed and analyzed by F
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.
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
ncubated with the vesicles or with control solutions for 5 h at 4 ◦C (left) or at 37 ◦CACS.
bolavesicles following uptake by the cells. In a dramatic contrast,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 assessed inthis study following 5 h in vitro incubation. For the purpose of tar-geted delivery, much shorter time periods would likely be sufficientand therefore the intracellular fate of the vesicles in vivo may becompletely different. Indeed, we previously observed substantialbrain accumulation of a fluorescent dye when delivered encap-sulated within GLH-20 bolavesicles at 30 min after intravenousadministration (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 free
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
IONPs (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
558
559
560
561
![Page 8: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/8.jpg)
ARTICLE ING Model
IJP 13259 1–9
8 L. Philosof-Mazor et al. / International Journa
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 incubatedwtv
as
4
nCawelp
tettptftpa
eaoscliatfi
amip
Q5
Li, G., Simon, M.J., Cancel, L.M., Shi, Z.D., Ji, X., Tarbell, J.M., Morrison 3rd., B., Fu, B.M.,
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
ith IONPs-containing bolavesicles (GLH-20). Rapid migration of the cells towardhe externally placed magnet was recorded; Bottom row: Cells incubated with con-entional (IONPs-free) bolavesicles (GLH-20). No cell movement was observed.
nd propensity of oleic-acid-coated IONPs to aggregate in aqueousolutions, that may limit their accessibility to the cells.
. Conclusions
We describe a novel formulation comprising iron oxideanoparticles (IONPs) associated with bolaamphiphile vesicles.haracterization of the IONPs-containing bolavesicles using EPRnd cryo-TEM (Fig. 1) confirmed that the IONPs were associatedith the bolavesicles. Interestingly, the IONPs interacted differ-
ntly with GLH-19 and GLH-20 in the vesicular environments, mostikely reflecting the distinct chemical structures of the two bolaam-hiphiles.
The incorporation of IONPs within the bolavesicles was showno significantly enhance their interactions with membrane bilay-rs in model systems. Specifically, more pronounced binding tohe bilayer interface and higher bilayer fluidity were induced byhe membrane-interacting IONPs-containing bolavesicles as com-ared to the IONPs-free bolavesicles. This outcome possibly relateso bolaamphiphile reorganization within the vesicular membraneollowing embedding of the IONPs, leading to higher exposure ofhe bolaamphiphiles’ positively charged moieties and consequentronounced interactions with the cell plasma membranes (whichre usually negatively charged).
The studied bolavesicle-based formulations were efficientlyndocytosed by the b.End3 brain endothelial cells, even in thebsence of the magnetic field, leading to efficient accumulationf the encapsulated materials in these cells. These observationsuggest that IONPs-containing bolavesicles might be excellentandidates for transport of different molecular cargoes through bio-ogical barriers. Specifically, the outcomes of this study indicate thatnteraction with IONPs-containing bolavesicles leads to significantssociation/accumulation of the IONPs with the cells. As a result,hese cells can be spatially manipulated using an external magneticeld.
Thus, the new IONPs/bolavesicle assembly might be used as
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
drug delivery and targeting vehicle. The encapsulated IONPsay help to target drug-loaded bolavesicles to specific region
n vivo by an external magnetic field. Subsequently, it could beossible to attain triggered bolavesicle decapsulation and drug
PRESSl of Pharmaceutics xxx (2013) xxx– xxx
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.
Uncited references
Lesieur et al. (2011) and Soenen et al. (2009).
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.
References
Arruebo, M., Fernández-Pacheco, R., Ibarra, M.R., Santamaría, J., 2007. Magneticnanoparticles for drug delivery. Nano Today 2, 22–32.
Brown, R.C., Morris, A.P., O‘Neil, R.G., 2007. Tight junction protein expression andbarrier properties of immortalized mouse brain microvessel endothelial cells.Brain Res. 1130, 17–30.
Chorny, M., Fishbein, I., Forbes, S., Alferiev, I., 2011. Magnetic nanoparticles fortargeted vascular delivery. IUBMB Life 63, 613–620.
Colombo, M., Carregal-Romero, S., Casula, M.F., Gutierrez, L., Morales, M.P., Bohm,I.B., Heverhagen, J.T., Prosperi, D., Parak, W.J., 2012. Biological applications ofmagnetic nanoparticles. Chem. Soc. Rev. 41, 4306–4334.
Dakwar, G.R., Abu Hammad, I., Popov, M., Linder, C., Grinberg, S., Heldman, E., Stepen-sky, D., 2012. Delivery of proteins to the brain by bolaamphiphilic nano-sizedvesicles. J. Control. Release 160, 315–321.
Fuhrhop, J.H., Wang, T., 2004. Bolaamphiphiles. Chem. Rev. 104, 2901–2937.Gao, J., Gu, H., Xu, B., 2009. Multifunctional magnetic nanoparticles: design, synthe-
sis, and biomedical applications. Accounts Chem. Res. 42, 1097–1107.Grinberg, S., Kipnis, N., Linder, C., Kolot, V., Heldman, E., 2010. Asymmetric bolaam-
phiphiles from vernonia oil designed for drug delivery. Eur. J. Lipid. Sci. Technol.112, 137–151.
Grinberg, S., Kolot, V., Linder, C., Shaubi, E., Kas’yanov, V., Deckelbaum, R.J., Heldman,E., 2008. Synthesis of novel cationic bolaamphiphiles from vernonia oil and theiraggregated structures. Chem. Phys. Lipids 153, 85–97.
Huang, C., Soenen, S.J., Rejman, J., Trekker, J., Chengxun, L., Lagae, L., Ceelen, W.,Wilhelm, C., Demeester, J., De Smedt, S.C., 2012. Magnetic electrospun fibers forcancer therapy. Adv. Funct. Mater. 22, 2479–2486.
Jelinek, R., Kolusheva, S., 2001. Polymerized lipid vesicles as colorimetric biosensorsfor biotechnological applications. Biotechnol. Adv. 19, 109–118.
Kim, B.H., Lee, N., Kim, H., An, K., Park, Y.I., Choi, Y., Shin, K., Lee, Y., Kwon, S.G., Na,H.B., Park, J.G., Ahn, T.Y., Kim, Y.W., Moon, W.K., Choi, S.H., Hyeon, T., 2011. Large-scale synthesis of uniform and extremely small-sized iron oxide nanoparticlesfor high-resolution T1 magnetic resonance imaging contrast agents. J. Am. Chem.Soc. 133, 12624–12631.
Kolusheva, S., Boyer, L., Jelinek, R., 2000a. A colorimetric assay for rapid screeningof antimicrobial peptides. Nat. Biotechnol. 18, 225–227.
Kolusheva, S., Shahal, T., Jelinek, R., 2000b. Peptide-membrane interactions studiedby a new phospholipid/polydiacetylene colorimetric vesicle assay. Biochemistry39, 15851–15859.
Köseoglu, Y., Yıldız, F., Kim, D.K., Muhammed, M., Aktas , B., 2004. EPR studies onNa-oleate coated Fe3O4 nanoparticles. Phys. Stat. Sol. (c) 1, 3511–3515.
Lee, N., Hyeon, T., 2012. Designed synthesis of uniformly sized iron oxide nanopar-ticles for efficient magnetic resonance imaging contrast agents. Chem. Soc. Rev.41, 2575–2589.
Lentz, B.R., 1989. Membrane fluidity as detected by diphenylhexatriene probes.Chem. Phys. Lipids 50, 171–190.
Lesieur, S., Gazeau, F., Luciani, N., Ménager, C., Wilhelm, C., 2011. Multifunctionalnanovectors based on magnetic nanoparticles coupled with biological vesiclesor synthetic liposomes. J. Mater. Chem. 21, 14387.
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
2010. Permeability of endothelial and astrocyte cocultures: in vitro blood–brainbarrier models for drug delivery studies. Ann. Biomed. Eng. 38, 2499–2511.
Moscho, A., Orwar, O., Chiu, D.T., Modi, B.P., Zare, R.N., 1996. Rapid preparation ofgiant unilamellar vesicles. P. Natl. Acad. Sci. USA 93, 11443–11447.
666
667
668
669
![Page 9: Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: New vehicles for magnetically targeted drug delivery](https://reader031.vdocuments.net/reader031/viewer/2022020614/575093521a28abbf6baf1cdc/html5/thumbnails/9.jpg)
ING Model
I
ourna
P
P
R
R
S
agents: physicochemical characteristics and applications in MR imaging. Eur.
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
ARTICLEJP 13259 1–9
L. Philosof-Mazor et al. / International J
opov, M., Grinberg, S., Linder, C., Waner, T., Levi-Hevroni, B., Deckelbaum, R.J.,Heldman, E., 2012. Site-directed decapsulation of bolaamphiphilic vesicles withenzymatic cleavable surface groups. J. Control. Release 160, 306–314.
opov, M., Linder, C., Deckelbaum, R.J., Grinberg, S., Hansen, I.H., Shaubi, E., Waner,T., Heldman, E., 2010. Cationic vesicles from novel bolaamphiphilic compounds.J. Liposomes. Res. 20, 147–159.
aifman, O., Kolusheva, S., Comin, M.J., Kedei, N., Lewin, N.E., Blumberg, P.M., Mar-quez, V.E., Jelinek, R., 2010. Membrane anchoring of diacylglycerol lactonessubstituted with rigid hydrophobic acyl domains correlates with biologicalactivities. FEBS J. 277, 233–243.
eddy, L.H., Arias, J.L., Nicolas, J., Couvreur, P., 2012. Magnetic nanoparticles: design
Please cite this article in press as: Philosof-Mazor, L., et al., Bolaamphiphimagnetically targeted drug delivery. Int J Pharmaceut (2013), http://dx.do
and characterization toxicity and biocompatibility, pharmaceutical and biomed-ical applications. Chem. Rev. 112, 5818–5878.
oenen, S.J., Hodenius, M., De Cuyper, M., 2009. Magnetoliposomes: versatile inno-vative nanocolloids for use in biotechnology and biomedicine. Nanomedicine –UK 4, 177–191.
PRESSl of Pharmaceutics xxx (2013) xxx– xxx 9
Stern, J., Freisleben, H.J., Janku, S., Ring, K., 1992. Black lipid membranes oftetraether lipids from Thermoplasma acidophilum. Biochim. Biophys. Acta 1128,227–236.
Veiseh, O., Gunn, J.W., Zhang, M., 2010. Design and fabrication of magnetic nanopar-ticles for targeted drug delivery and imaging. Adv. Drug. Deliver. Rev. 62,284–304.
Wang, Y.X., 2011. Superparamagnetic iron oxide based MRI contrast agents:current status of clinical application. Quant. Imaging Med. Surg. 1,35–40.
Wang, Y.X., Hussain, S.M., Krestin, G.P., 2001. Superparamagnetic iron oxide contrast
lic vesicles encapsulating iron oxide nanoparticles: New vehicles fori.org/10.1016/j.ijpharm.2013.04.017
Radiol. 11, 2319–2331.Yallapu, M.M., Othman, S.F., Curtis, E.T., Gupta, B.K., Jaggi, M., Chauhan, S.C., 2011.
Multi-functional magnetic nanoparticles for magnetic resonance imaging andcancer therapy. Biomaterials 32, 1890–1905.
696
697
698
699