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Monoolein Cubic Phase Gels and Cubosomes Doped with Magnetic


NanoparticlesHybrid Materials for Controlled Drug Release
Monika Szlezak, Dorota Nieciecka, Aleksandra Joniec, Marek Pkaa, Ewa Gorecka, Melanie Emo,
Marie J. Stebe, Pawe Krysinski, and Renata Bilewicz*,

Faculty of Chemistry, University of Warsaw, Pasteura 1, PL 02-093 Warsaw, Poland

Universite de Lorraine/CNRS, SRSMC, UMR7565, Vandoeuvre-les-Nancy CEDEX F54506, France


*
S Supporting Information

ABSTRACT: Hybrid materials consisting of a monoolein


lipidic cubic phase (LCP) incorporating two types of magnetic
nanoparticles (NP) were designed as addressable drug delivery
systems. The materials, prepared in the form of a gel, were
subsequently used as a macroscopic layer modifying an
electrode and, after dispersion to nanoscale, as magneto-
cubosomes. These two LCPs were characterized by small-angle
X-ray scattering (SAXS), cross-polarized microscopy, magnetic
measurements, and phase diagrams. The magnetic dopants were hydrophobic NPoleic and hydrophilic NPcitric, characterized by
dynamic light scattering (DLS) and transmission electron microscopy (TEM), and their inuence on the properties of the cubic
phases was investigated. The removal of the anticancer drug, Doxorubicin (Dox) from the hybrid cubic phase gels was studied by
electrochemical methods. The advantages of incorporating magnetic nanoparticles into the self-assembled lipid liquid crystalline
phases include the ability to address the cubic phase nanoparticle containing large amounts of drug and to control the kinetics of
the drug release.
KEYWORDS: lipidic cubic phase, magnetocubosome, magnetic nanoparticles, liquid crystalline phase, doxorubicin

INTRODUCTION
Recently, there has been increased interest in novel drug
Doxorubicin (Dox) is an antineoplastic agent widely used in
the treatment of several types of cancer. The drug binds to
double-stranded DNA, which results in the inhibition of tumor
delivery systems (DDS) based on lipidic mesophases because of
cell replication.17,18 The drug has low selectivity; it aects both
their large drug loading capacity, stability, biocompatibility, and
healthy and diseased cells, causing a number of unwanted side
sustained release of drugs.13 In the case of anticancer drugs,
eects. One of the approaches to reduce these eects relies on
encapsulation of such drugs into the DDS would reduce the
the lower pH of the tumor cell environment compared with
side eects caused by the drug, especially reducing toxic eects that of healthy cells. Release proles of Dox from LCP were
on healthy cells. Lipidic cubic and hexagonal phases are monitored electrochemically by measuring the voltammetric
advantageous materials for drug delivery since they are stable in peak current of doxorubicin reduction. It was found that drug
excess water and can accommodate relatively large loads (drug) release from the phase is pH-dependent. The pKa of
due to a high surface area of ca. 400 m2/g.26 The highly doxorubicin is 8.2.19 Protonated doxorubicin, which carries a
structured reverse bicontinuous lipidic cubic phase (LCP), positive charge, is soluble in water, whereas the unprotonated
composed of highly curved lipid bilayers surrounded by two drug prefers a hydrophobic lipidic environment, where its
identical, nonintersecting aqueous channels, exhibits interesting diusion is much slower.15,16 Recently, we have voltammetri-
properties for applications as a drug carrier.1,3,5,6 Because of cally evaluated the release kinetics of Dox by analyzing a
their internal structure, LCPs can incorporate hydrophilic, decrease in its peak current over time. The results suggest that
amphiphilic, and hydrophobic drugs. Hydrophobic drugs tend the release mechanism is in agreement with the Higuchi model
to partition into the lipid bilayer, while hydrophilic drugs reside at low pH, while at higher pH values, non-Fickian transport
preferentially in the aqueous channels.79 LCPs have therefore dominates.16
been extensively investigated as vehicles for the sustained In the present study, we designed the following hybrid
release of bioactive molecules of various sizes and molecular materials based on monoolein cubic mesophases containing
weights. The location of the drug is an important parameter two types of magnetic nanoparticles (NPs) diering in their
aecting the diusion and release rate.1016 Partitioning
between the lipidic and aqueous compartments, which is Received: October 11, 2016
related to the size and polarity of the drug, largely determines Accepted: December 28, 2016
the release kinetics. Published: December 28, 2016

2016 American Chemical Society 2796 DOI: 10.1021/acsami.6b12889


ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

stabilizing coatings around the magnetic core: hydrophilic to molten MO. In the case of hydrophobic nanoparticles, the
nickelzinc ferrite nanoparticles stabilized with a citric acid appropriate amount of NP dispersion in hexane (NPoleic) was added to
adlayer (NPcitric) and lipophiliciron oxide core nanoparticles molten MO and sonicated. The mixture was left in a desiccator to
coated with an oleic acid adlayer (NPoleic). The addition of evaporate the solvent. Then, water or Dox solution was added. The
ratios of the hybrid systems were 59.8/0.2/40 or 58/2/40 and 59.3/
magnetic nanoparticles to the mesophase would open new 0.2/0.5/40 or 57.5/2/0.5/40 (% w/w) for MO/NP/H2O and MO/
possibilities for directing a liquid crystal drug carrier to the NP/Dox/H2O, respectively. To obtain homogeneous, transparent, and
desired target using a magnetic eld,20,21 as well as for magnetic viscous LCPs, all the samples were left for at least 24 h in tightly closed
hyperthermia.22 The mesophase can be dispersed into nano- vials at room temperature in the dark. Phase diagrams of the obtained
particles: cubosomes,23 which have the same properties as the hybrid phases were evaluated.
bulk phase but have lower density. Cubosomes doped with The obtained phases were used to prepare cubosomes.23 First, 0.1 g
nano-objects are of interest not only as the drug delivery of LCP phase with NPs (34.5 mg of NPs per gram of monoolein) was
systems3,2429 but also as contrast agents for magnetic placed in a glass vial, and 1.85 mL of Pluronic F-127 solution (1%) in
resonance imaging (MRI).30,31 deionized water was added. The cubic phases were sonicated for 20
min in a bath-type sonicator.
We studied the physical and chemical properties of the Synthesis of Hydrophilic Mixed Ferrite Nanoparticles
hybrid materials as a thin lm on the electrode surface and as (Ni0.5Zn0.5Fe2O4). Hydrophilic nanoparticles (NPcitric) were synthe-
cubic phase nanoparticles, i.e., cubosomes, both modied with sized according to ref 33 with minor modications. Briey, the
the magnetic nanoparticles, NPcitric and NPoleic. Due to their nanoferrites were prepared using the bottom-up technique by
dierent hydrophilic/hydrophobic properties, the two types of coprecipitation of nanoferrites from the solution of their precursors
NPs used are located in dierent phase domains of the LCPs. with a strong base according to the following equation:
All samples exhibited a double-diamond cubic Pn3m phase at Ni 2 + + Zn 2 + + 4Fe3 +16OH 2Ni 0.5Zn 0.5Fe2O4
+ 8H 2O
room and human body temperature, with some dierences in
the diameters of the water channels. For the mesophase doped First, the precursor solution was prepared by mixing the heated
with 2% w/w of hydrophilic NPcitric above 40 C, a switch from aqueous nitrate salts of 0.6667 M Fe3+, 0.1667 M Ni2+, and 0.1667 M
the cubic to the hexagonal phase takes place, which results in Zn2+ with a hot aqueous solution of sodium hydroxide. Then, in a
reaction vessel, 40 mL of this solution was mixed with 8 mL of 2 M
slower drug release from the mesophase. Drug release from the
HNO3 (to avoid hydrolysis), and 152 mL of H2O was added. The
bicontinuous cubic phase is signicantly faster than from the resulting solution was heated with continuous stirring to 95 C under
hexagonal phases.5 reux. An aqueous solution of NaOH (400 mL, 0.75 M) was
The release proles of Dox were evaluated using electro- preheated to 95 C in a separate container and then transferred to the
chemical methods, which monitor the changes in drug reaction vessel. A dark-brownish precipitate appeared, and the reaction
concentration directly in the cubic phase layer covering the was left heated for the next 12 h with stirring. Afterward, the reaction
electrode. A comparison of structures and drug elution proles was cooled to room temperature. Nanoparticles were precipitated with
reveals that the hybrid material with NPcitric changes the a magnet and washed three times with deionized water using
properties of the cubic phase. In the case of hydrophobic sedimentation and decantation in the eld of a permanent magnet.
The NP surfaces were treated with citrate ions by incubating for 1 h in
nanoparticles, the structure remains unaected. All results show
a 0.5 mg/mL citric acid solution with continuous stirring at 90 C.
that the hybrid material with hydrophobic magnetic NPs is a Then, the obtained suspension was cooled to room temperature and
promising matrix for drug delivery. Hybrid cubosome washed several times with an excess of deionized water under magnet-
dispersions were also prepared. The magnetocubosomes assisted sedimentation to remove the unbound citric acid. The
containing both types of magnetic nanoparticles have magnetic hydrophilic nanoparticles grafted with citric acid were located
properties similar to those of magnetic nanoparticles alone and primarily in the LCP aqueous domain.
can be considered for drug delivery directed by means of a The choice of these particular magnetic cores was intentional since
magnetic eld. it allowed control of the crystallite size and magnetic properties of the

mixed zincnickel ferrite (vide inf ra). This particular ferrite is


ferromagnetic, and the resultant magnetic moment comes solely
EXPERIMENTAL SECTION from nickel cations because the magnetic moments of Fe3+ ions are
Materials. All chemicals were of the highest quality available antiparallel, partially compensating for the overall magnetic moment of
commercially. Monoolein (1-oleoyl-rac-glycerol) (MO), MES sodium the NPs. Introduction of zinc further alters the structure of the
salt (2-(N-morpholino)-ethanesulfonic acid sodium salt), Doxorubicin nanocrystallites, allowing control of the magnetic properties of the
(Dox), and Pluronic F-127 were purchased from Sigma-Aldrich and nanoparticles.
were used as received. A 0.2 M buer was prepared by titrating MES Additionally, an introduction of metal ions of dierent ionic radii
with 0.2 M NaOH to pH 5.8. All solutions were prepared using Milli than Fe3+ (78.5 pm) alters the crystallographic structure due to the
Q water (18.2 M cm1, Millipore, Bedford, MA, USA). Hydrophilic ionic radii mismatch in the spinel structure of magnetite (Ni2+ 70 pm,
nanoparticles were synthesized by using Fe(NO3)39H2O and TMAH Zn2+ 74 pm), thereby aecting the size of the resulting nanoparticles.34
(tetramethylammonium hydroxide) from Sigma and Ni(NO3)26H2O, Synthesis of Hydrophobic Magnetite Nanoparticles (Fe3O4).
Zn(NO3)26H2O, NaOH, HNO3, and KCl from POCH. Hydrophobic Hydrophobic nanoparticles (NPoleic) were prepared based on the
nanoparticles were synthesized by using FeCl36H2O, FeCl24H2O description of the procedure from Mahdavi et al.35 The whole
(Sigma-Aldrich), NH4OH (25% aqueous solution in H2O, Chempur, synthesis was carried out in a three-necked ask under vigorous
Poland), and oleic acid (>99% pure, Sigma-Aldrich). stirring and an argon atmosphere. First, 0.023 mol of FeCl24H2O and
Preparation of Cubic Phases and Cubosomes. The nondoped 0.046 mol of FeCl36H2O were dissolved in 150 mL of deionized
LCP was prepared by adding an appropriate amount of distilled water water. The mixture was heated to 45 C, then 11 mL of 25% ammonia
to a glass vial with molten MO. The ratio of components was chosen added. Instantaneously, a black precipitate of nanoparticles appeared.
on the basis of Qiu and Careys phase diagram for the MO/water After 30 min, 3 mL of oleic acid was added, and the suspension was
mixture.32 The cubic phase doped with Dox was prepared similarly by heated to 80 C. After 1 h, the Fe3O4 nanoparticles coated with oleic
rst dissolving Dox in buer and then adding it to molten MO to acid, NPoleic, were decanted using magnetic separation and washed
obtain 59.5/0.5/40 (% w/w) for the MO/Dox/H2O phase. several times with deionized water and ethanol. After removal of the
To prepare the LCPs loaded with hydrophilic nanoparticles excess of oleic acid, the nanoparticles were suspended in ethanol and
(NPcitric), the NPcitric suspension or NPcitric/Dox solution was added centrifuged for 1 h at 13.4 krpm. Next, the ethanol was removed using

2797 DOI: 10.1021/acsami.6b12889


ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

a rotary evaporator and replaced with 100 mL of hexane. The lattice parameter of corresponding phase, and l is the lipid chain
hydrophobic nanoparticles stabilized with oleic acid were mainly length/monolayer thickness.
embedded in the lipidic part of the cubic phase; however, since they Finally, the water channel radius rw was obtained using equation:38
are also larger than the bilayer width, parts of the nanoparticle are
protruding into the aqueous channels (Scheme 1).36 rw = ( /2 )1/2 a l (4)
Polarized Microscopy. Polarized light microscopy (Olympus BX
Scheme 1. Characteristics over Length Scales of the 50) conducted with a heating/cooling stage was used for visual
Magnetic Nanoparticles, Cubic Mesophase, and inspection of the samples.
Magnetocubosomes Dynamic Light Scattering (DLS) and Zeta Potential. The
hydrodynamic diameter of the cubic phase particle dispersions was
determined using a Malvern Zetasizer instrument (Nano ZS, UK)
tted with a 4 mW HeNe laser ( = 632.8 nm) as the light source at
a scattering angle of 173. The solutions were equilibrated for 2 min
before measurement. The zeta potential was measured using the same
instrument. The values were reported as averages from 5 measure-
ments of each sample.
Thermogravimetric Analysis (TGA). Thermogravimetric analysis
of coated nanoparticles was performed at a temperature range of 25
600 C with a heating rate of 10 Cmin1 under oxygen using the
TGA Q-50 thermal analyzer (TA Instrument). From these measure-
ments, we evaluated that the amount of citric acid on NPcitric and oleic
acid on NPoleic was 25.0 and 24.6% w/w, respectively (Figure S1A).
Transmission Electron Microscopy (TEM). TEM measurements
(Libra 120 microscope, Zeiss) were used to assess the size of the
magnetic nanoparticles (Figure 1A,B). Before the measurement, a

METHODS
Small-Angle X-ray Scattering. The phase identity and structural
parameters of the lipidic samples were determined by small-angle X-
ray scattering (SAXS). The experiments were carried out with a
GADDS system (Bruker) using a 3-pinhole collimation system
working with Cu K radiation (CuK = 0.1542 nm), a Nanostar
system (Bruker) working with CuK radiation equipped with a Vantec
2000 area detector, a SAXSess mc2 instrument (Anton Paar) using a Figure 1. TEM images of the citrate-coated NPs (A) and oleic-
line collimation system and equipped with a sealed X-ray tube stabilized NPs (B); scale bars = 50 nm.
(PANalytical, CuK = 0.1542 nm), and a CCD detector (Princeton
Instruments). Samples were introduced into special thin glass
capillaries, which were immediately ame-sealed. SAXS spectra were drop of a sample suspension was placed on a Formvar-coated copper
recorded in the range of 1060 C. The scattering intensities I(q) grid and allowed to dry in the air. Hydrophilic nanoparticles NPcitric
were represented as a function of the magnitude of the scattering revealed an average diameter of 13 3 nm, while the average diameter
vector q = (4/) sin(), where 2 is the total scattering angle. The of hydrophobic NPoleic was ca. 7 2 nm (Figure S1B,C).
SAXS patterns obtained from the Bruker apparatus were analyzed with Cryo Scanning Electron Microscopy (Cryo-SEM). A cryo
the Bruker Topas 3 software. For the SAXS mc2 apparatus, all data scanning electron microscopy (cryo-SEM) image was taken using a
were corrected for background scattering from the empty capillary and Carl Zeiss Microscopy at a voltage 2 kV and a working distance of 2.6
for slit-smearing eects by a desmearing procedure from the suppliers mm. Sample was loaded into a holder and frozen in the liquid
software using the Lake method. nitrogen. The sample was transferred into the cryochamber, which was
The size of water channels was calculated based on the lattice held at 140 C. Then the sample was sublimed at 85 C for 23
parameter and the composition of cubic phases.37 Initially, the water min.
volume fraction was calculated by the equation: Magnetization Measurements. The magnetic properties of both
types of nanoparticles as a function of the magnetic eld at
w = c w /(c w + (1 c w)(w /l )) (1) temperatures ranging from 2 to 300 K were reported by us earlier.39,40
Those results were comparable, regardless of the surface modication
where w is the water volume fraction, cw is the water weight fraction,
(citric or oleic shell), and in general revealed typical ferromagnetic-like
w is the density of water =0.997 g/cm3, and l is the density of lipid =
behavior, with no hysteresis loop at temperatures close to room
0.942 g/cm3.
temperature. Here, we complemented our previous results with
The lipid volume fraction was determined from the equation:
magnetization measurements of NPs and hybrid materials performed
l = 1 w (2) with an automatic Faraday balance at a constant magnetic eld of 1 T.
The results are shown in Figure S2. Temperature was controlled with
The lipid chain length (l) was determined by solving the following an accuracy better than 0.5 K. The magnetization values for
equation:37 nanoparticles and hybrid materials were calculated using the weight
l = 2(l/a) + 4/3 (l/a)3 of nickelzinc ferrite and magnetite measured by TGA (Figure S1A).
(3)
The temperature variation in the mass magnetization of the
where is the ratio of the minimal surface in a unit cell to the quantity nanocrystalline NPcitric sample, containing nickelzinc ferrite, is
(unit cell volume)2/3, is the EulerPoincare characteristic, a is the plotted in Figure S2A and shows ferromagnetic ordering up to the

2798 DOI: 10.1021/acsami.6b12889


ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

Curie temperature of TC 267 C. This Curie temperature is These results prompted us to use both types of magnetic nanoparticles
comparable with the TC = 264 C of the polycrystalline Ni0.5Zn0.5 as prospective vectors incorporated inside the cubosomes loaded
Fe2O4.41 At room temperature, the magnetic moment per nickelzinc with doxorubicin, forming a nanoparticulate hybrid drug delivery
ferrite equals 1.5 B (Table 1, vide inf ra) and is smaller compared to system as described further.
Electrochemical Measurements. Electrochemical measurements
Table 1. Magnetic Moment per Molecule of Citric Acid- were performed using the CH Instruments bipotentiostat model
Coated NP, Oleic-Coated NP, Hybrid Cubic Phases, and CHI750B. The three-electrode system, consisting of a glassy carbon
electrode (GCE, A = 0.07 cm2), an Ag/AgCl reference electrode, and a
Hybrid Cubosomes at Room Temperature platinum foil as a counter electrode, was employed. Prior to the
magnetic moment (B) measurements, the GCE electrodes were polished with an alumina
slurry (1.0, 0.3, and 0.05 m) on a polishing cloth. The electrodes
NPcitric 1.5
were then rinsed with a direct stream of ultrapure water, sonicated in
phase with NPcitric 1.4 an ultrasonic bath, and left to air-dry. After cleaning, the working
cubosomes with NPcitric 1.5 electrodes were modied with a thin lm of the cubic phase.
NPoleic 2.3 Measurements at elevated temperatures were performed using a BVT
phase with NPoleic 2.7 MT-1 minithermostat. Argon-saturated solutions were obtained by
cubosomes with NPoleic 1.9 bubbling high-purity argon gas for 15 min into the solution and
continuing with a ow of pure gas (Ar) over the solution during
experiments. Release of Dox from LCPs deposited on a GCE was
the 3.17 B of the polycrystalline Ni0.5Zn0.5Fe2O4. The reduced monitored using dierential pulse voltammetry (DPV). For each type
magnetic moment of nanocrystalline ferrite is typically attributed to of hybrid cubic phase, triplicate experiments were performed.


the structural and magnetic disorder within the crystallite surface layer.
The temperature variation in the mass magnetization of the
nanocrystalline NPoleic sample, containing magnetite Fe3O4, is plotted
RESULTS AND DISCUSSION
in Figure S2B and shows the ferromagnetic ordering up to the Curie Structural Characterization of the Cubic Phases. To
temperature of TC 490 C, which is lower than the 585 C reported evaluate the eect of the addition of two types of magnetic
for the polycrystalline magnetite42 and the 580 C reported for the nanoparticles on the cubic phase properties, SAXS and cross-
magnetite Fe3O4 coated with citric acid.43 polarized microscopy experiments were carried out.
The magnetic moment per Fe3O4 equals 2.3 B at room Polarized light microscopy and SAXS are the most widely
temperature (Table 1, vide inf ra) and is reduced compared to the used techniques for characterization of liquid crystalline phases.
3.77 B of the polycrystalline.36 The minute magnetization hump
observed between 220 and 320 C may reveal some change in the Cubic phases are isotropic; therefore, a dark image appears
interparticle interaction caused by removal of the surface-coating oleic when viewed under a microscope equipped with cross-
layer, which is also observed in the same temperature range for the polarizers. Other structures found in the MO/H2O system,
TGA spectra (Figure S1A). An analogous eect has been reported for lamellar and hexagonal phases, are anisotropic and have a
citrate-stabilized Fe3O4 aqueous colloidal magnetic nanoparticles.43 characteristic textures when observed by polarized light

Figure 2. Left panel: Monooleinwater partial phase diagrams with 2% w/w of hydrophilic nanoparticles (A) and 2% w/w of hydrophobic
nanoparticles (C). Right panel: Comparison of SAXS spectra at human body temperature obtained from the bulk cubic phases with 40% w/w of
water: MO/NPcitric/H2O (B) and MO/NPoleic/H2O (D) with MO/H2O system.

2799 DOI: 10.1021/acsami.6b12889


ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

Figure 3. SAXS spectra of the various monoolein-based systems with 40 wt % of water at dierent temperatures: pure MO (A); with 2% w/w of
hydrophobic NPs (B); with 2% w/w of hydrophilic NPs (the intensities are represented on a logarithmic scale) (C). Polarized-light microscopy
images for phase with NPcitric at 25 C (Pn3m) (D), 40 C (Pn3m and H2 phases) (E), and 50 C (H2 phase) (F).

Table 2. Results of SAXS Measurements for Monoolein and Hybrid LCPs Depending on the Phase Composition and
Temperaturea
phase composition (% w/w) T (C) phase symmetry a (nm) l (nm) dw (nm)
25 Pn3m 9.5 1.6 4.2
35 Pn3m 9.3 1.6 4.1
MO/H2O 60/40
40 Pn3m+water 9.0 1.5 4.0
45 Pn3m+water 8.7 1.5 3.8
25 Pn3m 10.1 1.7 4.5
36 Pn3m 9.7 1.7 4.3
MO/NPcitric/H2O 59.8/0.2/40
40 Pn3m+water 9.3 1.6 4.1
44 Pn3m+water 8.9 1.5 4.0
25 Pn3m 8.9 1.5 3.9
36 Pn3m 8.5 1.5 3.8
8.3 1.4 3.7
MO/NPcitric/H2O 58/2/40 40 Pn3m and trace H2
nd nd nd
8.1 nd nd
44 Pn3m and H2
6.0 nd nd
25 Pn3m 9.4 1.6 4.2
35 Pn3m 9.3 1.6 4.1
MO/NPoleic/H2O 59.8/0.2/40
40 Pn3m+water 9.2 1.6 4.1
45 Pn3m+water 8.8 1.5 3.9
25 Pn3m 9.6 1.6 4.2
36 Pn3m 9.3 1.6 4.1
MO/NPoleic/H2O 58/2/40
40 Pn3m+water 9.0 1.5 4.0
44 Pn3m+water 8.7 1.5 3.8
a
Phase identity, lattice parameter a, lipid length l, and water channel diameters dw.

microscopy. The space group of the phases and the structural content varying between 20 and 40% w/w. By adding
parameters can then be determined by SAXS. hydrophilic nanoparticles that are located mainly in the water
A monooleinwater phase diagram has been described in the channels of the cubic phases, substantial changes in the phase
literature;32,4446 therefore, the inuence of the incorporation diagram in the studied region can be observed (Figure 2A).
of 2% w/w of NPcitric and NPoleic was investigated. The phase Below 40 C, the sequence of liquid crystals remains
diagrams were determined in the range of 1060 C for a water unchanged, except for the lamellar phase. Both the Ia3d and
2800 DOI: 10.1021/acsami.6b12889
ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

Pn3m cubic phases are less temperature-stable. Indeed, Ia3d The incorporation of doxorubicin into MO does not
cubic phase melts at 45 C, and Pn3m phase disappears at signicantly aect the lattice parameter and size of water
approximately 60 C. However, for MO with hydrophobic NPs, channels since the drug molecule is small (ca. 15 nm) and is
these two phases are stable over 60 C. Interestingly, a phase easily accommodated in the structure (Table S3).15 However,
transition from Pn3m to H2 occurs at 40 C for a water content the incorporation of Dox into hybrid materials leads to an
of 40% w/w, as shown on the SAXS spectra and conrmed by increase in the lattice parameter and the width of the water
the texture of the samples observed with an optical microscope channels, which is more pronounced in the case of 2% w/w of
equipped with cross polarizers (Figure 3), whereas the hydrophilic nanoparticles.
transition appears at 90100 C for pure MO in a limited Properties of Cubosomes. Nanoparticles of the cubic
range of water content (1525% w/w of water). The decrease phasecubosomeswere prepared, and their characteristics
in the phase transition temperature (Pn3m cubic phaseH2) were evaluated (Figures S4 and S5). The diameter of cubosome
may be induced by the presence of the hydrophilic NPs that dispersions, in the absence/presence of NPs, was obtained from
swell the water channels. Therefore, the lattice axes are the DLS measurements. The cubic particle sizes are similar,
increased, and this results in a reduced curvature of the bilayer between 110 and 150 nm, which is suitable for a drug delivery
and a less pronounced wedge shape for the molecules. This system (Table 3 and Figure S4A). With the DLS technique, we
behavior favors the formation of a reverse hexagonal phase.45
By adding hydrophobic nanoparticles that are solubilized in the Table 3. Characteristic Values of Cubosomes
hydrophobic chains, no signicant modication of the phase
cubosomes with cubosomes with
behavior was observed (Figure 2C). Indeed, in comparison cubosomes NPcitric NPoleic
with pure MO, the same sequence of liquid crystals (lamellar zeta potential (mV) 18.9 4.4 17.3 1.3 19.6 2.3
phaseIa3d cubic phasePn3m cubic phase) was obtained as a size (nm) 111 10 137 8 150 13
function of the water composition, and the stable areas polydispersity index 0.35 0.26 0.33
associated with the temperature and concentration are similar. (PDI)
This is an important observation for future applications of
various forms of mesophases as drug delivery platforms with can perform measurements in the natural environment of the
NPs acting as delivery vectors. sampleswater solutions. Lipidic particles are rather fragile, and
Comparing the SAXS experiments of samples with 40% w/w preparation of samples for TEM measurements can change/
of water, the spectra of 2% w/w and 0.2% w/w NPs (Figures destroy the structure. Nevertheless, we used TEM imaging to
2B,D) at human body temperature are similar to that of pure conrm the presence of NPs inside the cubosomes. Figure S4
MO and present a cubic structure that corresponds to the presents TEM images of cubosomes with hydrophilic NPcitric
Pn3m space group, as conrmed by the relative positions of the (Figure S4C) and hydrophobic NPoleic (Figure S4D) nano-
reection lines 1:3/2:2:3:2:9/2. For the hybrid phase particles (please note the dierent scale bars on these gures).
with 0.2% w/w of citric NPs, the peaks are shifted to lower q The cubosomes are visible as gray discs with much darker dots
values (Figure 2B), while for the phase with 2% w/w NPcitric, of iron oxide nanoparticles because NPs have a much higher
the situation is opposite. For NPoleic, no shift of the peaks is electron density than lipids.
observed (Figure 2D), which indicates that the incorporation of Cryo-SEM image shows the spherical morphology of the
hydrophobic NPs does not aect the organization of lipidic cubosomes (Figure S4B). The cubic nature of the LCP
mesophases. Moreover, for the SAXS spectra (Figure 2, 3), it dispersion was characterized by SAXS. The SAXS pattern for
should be noted that for samples with NPs, diusion at small q MO cubosomes shows the local cubic structure with Im3m
values is observed, due to the presence of NPs in the system. space group (the relative positions of the diraction signals are
Increasing the temperature induces a shift of the peaks 2:4:6) (Figure S5). The crystallographic lattice param-
toward higher q values for hydrophobic NPs, and the Pn3m eter is 14.6 nm. It was reported earlier that sometimes during
cubic phase is maintained (Figure 3B), as observed for pure ultrasonic dispersion of material the Pn3m cubic phase
MO (Figure 3A). For the samples with 2% w/w of hydrophilic underwent transition to the cubic structure with Im3m
NPs, the reverse hexagonal phase H2 appears at 40 C, as symmetry.4750 In the case of cubosomes with NPoleic, the
conrmed by the relative positions of the reection lines scattering from nanoparticles masks completely the rst Bragg
1:3:2 on the SAXS spectra (Figure 3C), which is character- reection and also partly the second Bragg peak. However, the
istic of the hexagonal symmetry. For the samples with 0.2% w/ position of the third peak is clearly visible and at the same q
w of hydrophilic NPs, the peaks are shifted to higher q values, value as the corresponding peak for MO cubosomes, which
and no phase transition is observed in the temperature range conrms that incorporation of NPs does not aect the structure
from 25 to 60 C (data not shown). of cubosomes (Figure S5, inset), similar to the case of bulk
The calculated values of the lattice parameter, lipid length cubic phases (Figures 2 and 3).
and diameter of water channels are reported in Table 2. The Since the zeta potential for cubosomes with or without NPs
values conrm the observations of the SAXS spectra. At room is quite similar (see Table 3) and the zeta potential for NPs
temperature, the addition of 0.2% w/w of hydrophilic NPs alone is much higher (39 mV for NPcitric), we can conclude
leads to an increase in the water channel width by 0.3 nm. The that the nanoparticles remain inside the lipidic particle and not
addition of 2% w/w of NPcitric leads to a decrease in both the on the surface. The cubosomes in the TEM images were larger
lattice parameter and channel width by 0.6 and 0.3 nm, in size than those observed with DLS, which was likely caused
respectively. The addition of hydrophobic NPs has no inuence by attening during the drying process.
on the lattice parameter compared to pure MO. As expected, Doxorubicin Release Prole from Cubic Phases with
after increasing the temperature, the lattice parameter and the Nanoparticles. The drug release proles of the pure
diameter of water channels decrease for both NPs similar to the monoolein and hybrid phases were investigated based on the
pure MO phase.32 changes in the DPV voltammograms with time (Figures 4 and
2801 DOI: 10.1021/acsami.6b12889
ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

Table 4. Release Characteristics of Doxorubicin from Neat


and Hybrid LCPs
KorsmeyerPeppas Higuchi
LCP N K (%/hn) R2 kH (%/h1/2) RH2
MO LCP
T = 25 C 0.50 48.1 0.883 89.5 0.992
LCP + 0.2% NPcitric
T = 25 C 0.50 56.8 0.995 71.0 0.926
LCP + 2% NPcitric
T = 25 C 1.61 3.20 0.982 nd nd
T = 37 C 1.62 7.12 0.927 nd nd
Figure 4. Release proles of Dox from LCPs (40% w/w of water) with T = 44 C 1.72 3.31 0.921 nd nd
and without nanoparticles at pH 5.8 and room temperature. (A) LCP + 0.2% NPoleic
Hybrid system with hydrophilic nanoparticles: 1, MO phase, 2a, phase T = 25 C 0.50 53.2 0.971 81.1 0.933
with 0.2% w/w NPcitric, 2b, phase with 2% w/w NPcitric; (B) hybrid
LCP + 2% NPoleic
system with lipophilic nanoparticles: 1, MO phase, 2a, phase with 0.2%
T = 25 C 0.48 63.6 0.911 82.1 0.931
w/w NPoleic, 2b, phase with 2% w/w NPoleic.
T = 37 C 0.59 70.6 0.925 93.0 0.992
T = 44 C 0.75 77.4 0.950 nd nd
S6). The voltammetric experiments were conducted at room
temperature and at 37 and 44 C under an argon atmosphere. Incorporation of 0.2% w/w NPcitric results in an increase in
The electrodes were covered with a thin lm (approximately 1 Dox removal without a change in peak potential, 0.550 V
mm) of cubic phase and immersed in a 0.2 M buer solution of (Figure S6). Furthermore, 50% Dox (T50) is released after 50
pH 5.8 because the cancer cell environment is shown to have a min with 0.2% NPcitric, whereas without nanoparticles, it is 60
pH lower than that of normal cells, which is ca. 7.4.51 min. For the phase with 2% NPcitric, the DPV peak currents are
Doxorubicin is electroactive because its molecules have ve times smaller, and at 0.770 V, an additional signal
quinone- and hydroquinone-type redox centers, which undergo appears, which might be related to the attraction of NPcitric and
reduction and oxidation in a 2e/2H+ process (Scheme S7 and Dox in the aqueous channels (Figure S6). This result strongly
Figure S8).52 The magnetic nanoparticles, because of their size, suggests that positively charged Dox molecules interact
should not block the access of Dox to the electrode, regardless electrostatically with the negatively charged NPcitric (the zeta
of their surface coatings. To prove this, we performed cyclic potential of NPcitric is 39 mV due to the presence of negatively
voltammetry experiments on GCE electrodes modied with a charged carboxyl groups on the surface of nanoparticles). After
cubic phase alone or a hybrid system with hydrophilic NPcitric or 350 min, as much as 75% of the drug remains in the cubic
hydrophobic NPoleic magnetic nanoparticles. Since the peak phase lm (Figures 4A and S6). UVvis measurements at 480
current depends on the square root of the scan rate, the process nm performed for Dox and Dox with NPcitric in 0.02 M MES at
remained diusion controlled (Figure S8). The best method for pH 5.8 conrmed the inuence of NPcitric (data not shown).
checking the type of transport of Dox to the electrode surface is For NPoleic, no changes in the Dox voltammograms were
to use the KorsmeyerPeppas model.53 This model is observed, and the peak currents remain almost unaected for
described by the equation: the 0.2% and 2% NPoleic dopants (Figure S6). T50 occurs after
M t /M = kt n (5) 50 min for 0.2% NPoleic and 30 min for 2% NPoleic in the phase.
The total elution of Dox is maintained at the same level as that
where Mt/M is a fraction of drug released at time t, k is the for the MO phase.
release rate constant, and n is the release exponent. The n value The proposed hybrid materials could be considered for
gives information about the dierent release mechanisms of the combined therapies, for example, for hyperthermia with
drug (see Table 4). To determine the value of n, the logarithm chemotherapy. Considering that the temperature for hyper-
of <60% drug release data was tted versus the log of time. In thermia is in the range of 4146 C, we measured drug elution
the case of a cylindrical surface of a drug-containing carrier, the at 37 and 44 C to verify the behavior of the hybrid phase with
value of n = 0.45 means that the diusion mechanism 2% w/w of NPs under these conditions (Figures 5 and S9;
corresponds to Ficks laws. For n between 0.45 and 0.89, the Table 4).
transport is non-Fickian and can be regarded as anomalous At human body temperature (37 C), the drug is removed
transport, indicating diusion-controlled drug release and three times faster than at room temperature when 2% w/w
swelling-controlled drug release. When the exponent n = NPcitric is added (Table 4), but at 44 C, the drug is released
0.89, the drug release corresponds to Case-II transport, which two times faster compared with release at room temperature.
corresponds to zero-order release kinetics, and for n > 0.89, it Release of molecules is faster at higher temperatures, which can
corresponds to Super Case-II transport. be explained by accelerated diusion of the molecules at
For the simplest diusion-controlled process (Fickian), elevated temperatures. The decrease in the release rate of Dox
where n 0.5, the KorsmeyerPeppas approach reduces to observed at 44 C compared to that at 37 C is ascribed to the
the so-called Higuchi model, which describes drug release from phase transition to the reverse hexagonal phase (Figures 2B, 3,
semisolid matrices.53 The Higuchi model describes the and S9), for which transport is slower.3,5 In the presence of 2%
diusion process of drug release with the following expression: w/w NPoleic, the release of drug at room temperature and
M t /M = kH t higher temperatures is comparable (Table 4). In all cases, the
(6)
value of T50 is 30 min. The NPs of both types are larger than
where kH is the Higuchi constant. the lipid bilayer width and are entrapped within the lipid matrix
2802 DOI: 10.1021/acsami.6b12889
ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

Figure 5. Comparison of Dox release proles at room and human


body temperature and 44 C for LCPs (40% w/w of water) doped
with 2% w/w of nanoparticles at pH 5.8: (A) hybrid system with
hydrophilic nanoparticles NPcitric; (B) hybrid system with lipophilic
nanoparticles NPoleic. Figure 6. Temperature variation of magnetization for mesophases and
for cubosomes with NPs: (A) NPcitric; (B) NPoleic.
protruding into the aqueous phase of the channels. The results
shown above favor the material with hydrophobic magnetic
NPs as the drug delivery system. This is understood since nanoparticles and the bulk cubic phases. This conrms that the
hydrophobic NPoleic interact mainly with the monoolein of the magnetic properties of nanoparticles are conserved in the
lipidic part of the cubic phase and not with the drug located in dispersed systems studied, meaning that they can be exploited
the aqueous channels. for magnetic drug targeting, hyperthermia treatment, or
magnetic resonance imaging.


Results obtained for Dox in systems containing 0.2% w/w of
both types of NPs embedded in the cubic mesophase suggest
that the release mechanism proceeds according to the Higuchi CONCLUSIONS
model. The same situation is found for the phase with 2% w/w The cubic phase is a promising material for the delivery of
of NPoleic at room temperature, but when increasing the hydrophilic and hydrophobic drugs. Insertion of magnetic
temperature, the inuence of the matrix is observed (n deviates nanoparticles can extend its applications in medicine. We
from 0.5). In the case of 2% w/w of NPcitric incorporated into presented and characterized hybrid materials based on a
the phase, clearly non-Fickian transport contributes to the monoolein cubic phase doped with two types of magnetic
transport, and the process cannot be described by the Higuchi nanoparticles: hydrophilic and hydrophobic. The system was
model even at room temperature (Table 4). tested as a cancer drug delivery platform; therefore, doxorubicin
Magnetic Properties of Hybrid Systems. Magnetic was chosen as an eective drug, which is, however, highly toxic
characterization was conducted for nanoparticles covered with to healthy cells. We showed that the Pn3m cubic phase
the citric/oleic layer, for the cubic phases containing 2% w/w of structure is retained upon addition of 0.22% w/w magnetic
nanoparticles, and for the cubosomes with nanoparticles. nanoparticles and 0.5% w/w Dox. The lipidic hybrid material
Initially, it was observed that both the cubic phase and the exhibited magnetic properties and thus could be moved by the
cubosome suspension reacted to the magnetic eld of a application of a magnetic eld. The drug release proles from
neodymium magnet, involving a movement of particles toward the phases were established using dierential pulse voltammetry
the magnetic eld. (DPV). Introduction of 0.2% w/w nanoparticles results in
Both nanocrystalline nickelzinc ferrite and magnetite removal of more drug by ca. 10% compared with the pure
covered with the citric/oleic layer were incorporated into the monoolein cubic phase. Adding 2% w/w hydrophilic magnetic
cubic phases. Figure 6 shows that mass magnetization decreases nanoparticles leads to a 3-fold slower release of the drug
with temperature. At room temperature, the magnetic moment because the negative charge of these nanoparticles makes them
per nanocrystallite contained in the bulk cubic phase with reside mainly in the aqueous channels, and attractive
NPcitric and NPoleic equals 1.4 B and 2.7 B (Table 1), interactions with the positively charged drug retain it in these
respectively. As expected, these results conrm that the channels. However, the presence of 2% w/w hydrophobic NPs
magnetic particles of the ferrite and magnetite still retain the has almost no eect on the channel transport of the drug since
magnetic moment in the cubic phases. The minute magnet- the NPs are located mainly in the lipidic part of the cubic phase.
ization enhancement observed between 70 and 110 C Temperature increases the rate of drug release: In case of
seems to be related to evaporation of water; however, the NPcitric, it is 53% at 37 C, whereas for NPoleic it is increased to
detailed mechanism is not understood at this time. 85%. This result is interesting from the viewpoint of
The temperature variation of magnetization was also magnetothermal applications of the system. Eective drug
measured for the cubosomes (Figure 6). Qualitatively, the delivery systems should exhibit a large internal surface where
temperature variation is similar to that of the cubic phase. The the drug can be accumulated and should release the drug only
absolute magnetization values are reduced approximately 3 and in the cancer cell environment. Interactions with the drug are
12 times compared to those of the corresponding bulk cubic not favorable, and this is observed in the case of LCP with 2%
phases. When taking into account the content of magnetic w/w hydrophilic magnetic nanoparticles. In the case of
nanoparticles in the cubosomes, the magnetic moments are hydrophobic nanoparticles, the T50 value is 50 min for the
found to be 1.5B and 1.9B for the ferrite (NPcitric) and 0.2% w/w NPoleic phase and 30 min for material with 2% w/w
magnetite (NPoleic) systems, respectively. These magnetic NPoleic. At human body temperature, there was no change in
moment values are comparable to the values obtained for the the elution rate of drug for the phase with 2% w/w NPoleic. All
2803 DOI: 10.1021/acsami.6b12889
ACS Appl. Mater. Interfaces 2017, 9, 27962805
ACS Applied Materials & Interfaces Research Article

these results favor the material with hydrophobic magnetic NPs (12) Clogston, J.; Craciun, G.; Hart, D. J.; Caffrey, M. Controlling
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similar magnetic properties and can be directed using a
(14) Fraser, S. J.; Mulet, X.; Hawley, A.; Separovic, F.; Polyzos, A.
magnetic eld.

Controlling Nanostructure and Lattice Parameter of the Inverse


Bicontinuous Cubic Phases in Functionalised Phytantriol Dispersions.
ASSOCIATED CONTENT J. Colloid Interface Sci. 2013, 408, 117124.
*
S Supporting Information (15) Nazaruk, E.; Szlezak, M.; Gorecka, E.; Bilewicz, R.; Osornio, Y.
The Supporting Information is available free of charge on the M.; Uebelhart, P.; Landau, E. M. Design and Assembly of pH-Sensitive
ACS Publications website at DOI: 10.1021/acsami.6b12889. Lipidic Cubic Phase Matrices for Drug Release. Langmuir 2014, 30,
13831390.
Properties of magnetic nanoparticles: TGA, magnet- (16) Nazaruk, E.; Miszta, P.; Filipek, S.; Gorecka, E.; Landau, E. M.;
ization (S1 and S2), LCP properties (SAXS and DLS of Bilewicz, R. Lyotropic Cubic Phases for Drug Delivery: Diffusion and
cubosomes (S3S5), electrochemical properties of Sustained Release from the Mesophase Evaluated by Electrochemical
Doxorubicin, and release proles at dierent temper- Methods. Langmuir 2015, 31, 1275312761.
atures (S5S9) (PDF) (17) Calendi, E.; Di Marco, A.; Reggiani, M.; Scarpinato, B.;

Valentini, L. On Physico-Chemical Interactions Between Daunomycin


AUTHOR INFORMATION and Nucleic Acids. Biochim. Biophys. Acta, Nucleic Acids Protein Synth.
1965, 103, 2549.
Corresponding Author (18) Quiles, J. L.; Huertas, J. R.; Battino, M.; Mataix, J.; Ramirez-
*E-mail: bilewicz@chem.uw.edu.pl. Tortosa, M. C. Antioxidant Nutrients and Adriamycin Toxicity.
ORCID Toxicology 2002, 180, 7995.
Renata Bilewicz: 0000-0003-0058-3691 (19) Burke, T. G.; Morin, M. J.; Sartorelli, A. C.; Lane, P. E.; Tritton,
T. R. Function of the Anthracycline Amino Group in Cellular
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The authors declare no competing nancial interest.

(20) Vallooran, J. J.; Handschin, S.; Bolisetty, S.; Mezzenga, R.


Twofold Light and Magnetic Responsive Behavior in Nanoparticle
ACKNOWLEDGMENTS Lyotropic Liquid Crystal Systems. Langmuir 2012, 28, 55895595.
This work was supported by Sinergia project no. (21) Vallooran, J. J.; Bolisetty, S.; Mezzenga, R. Macroscopic
CRSII2_154451 nanced by the Swiss National Science Alignment of Lyotropic Liquid Crystals Using Magnetic Nanoparticles.
Foundation. Adv. Mater. 2011, 23, 39323937.

(22) Jeyadevan, B. Present Status and Prospects of Magnetite


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2805 DOI: 10.1021/acsami.6b12889


ACS Appl. Mater. Interfaces 2017, 9, 27962805

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