Monoolein Cubic Phase Gels and Cubosomes Doped with Magnetic

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Monoolein cubic phase gels and cubosomes doped with magnetic nanoparticles - hybrid materials for controlled drug release Monika Szl#zak, Dorota Nieciecka, Aleksandra Joniec, Marek P#ka#a, Ewa Gorecka, Mélanie Emo, Marie Jose Stebe, Pawel G. Krysinski, and Renata Bilewicz ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12889 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on January 9, 2017

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ACS Applied Materials & Interfaces

Monoolein cubic phase gels and cubosomes doped with magnetic nanoparticles - hybrid materials for controlled drug release Monika Szlezak†, Dorota Nieciecka†, Aleksandra Joniec†, Marek Pękała†, Ewa Gorecka†, Mélanie Emo‡, Marie J. Stébé‡, Paweł Krysiński†, Renata Bilewicz†* †Faculty of Chemistry, University of Warsaw, Pasteura 1, PL 02-093 Warsaw, Poland ‡Université de Lorraine/CNRS, SRSMC, UMR7565, F 54506 Vandoeuvre-lès-Nancy cedex, France

Keywords: lipidic cubic phase, magnetocubosome, magnetic nanoparticles, liquid crystalline phase, doxorubicin ABSTRACT: Hybrid materials consisting of a monoolein lipidic cubic phase (LCP) incorporating two types of magnetic nanoparticles 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 magnetocubosomes. These two LCPs were characterized by SmallAngle X-ray Scattering (SAXS), cross-polarized microscopy, magnetic measurements and phase diagrams. The magnetic dopants were hydrophobic NPoleic and hydrophilic NPcitric nanoparticles, which were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and their influence 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 ACS Paragon Plus Environment


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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.

INTRODUCTION Recently, there has been increased interest in novel drug delivery systems (DDS) based on lipidic mesophases because of their large drug loading capacity, stability, biocompatibility, and sustained release of drugs.1,2,3 In the case of anticancer drugs, encapsulation of such drugs into the DDS would reduce the side effects caused by the drug, especially reducing toxic effects on healthy cells. Lipidic cubic and hexagonal phases are advantageous materials for drug delivery since they are stable in excess water and can accommodate relatively large loads (drug) due to a high surface area of ca. 400 m2/g.2–6 The highly structured reverse bicontinuous lipidic cubic phase (LCP), composed of highly curved lipid bilayers that are surrounded by two identical, non-intersecting aqueous channels, exhibits interesting properties for applications as a drug carrier.1,3,5,6 Because of their internal structure, LCPs can incorporate hydrophilic, amphiphilic and hydrophobic drugs. Hydrophobic drugs tend to partition into the lipid bilayer, while hydrophilic drugs reside preferentially in the aqueous channels.7–9 LCPs have therefore been extensively investigated as vehicles for the sustained release of bioactive molecules of various sizes and molecular weights. The location of the drug is an important parameter affecting the diffusion and release rate.10–16 Partitioning between the lipidic and aqueous compartments, which is related to the size and polarity of the drug, largely determines the release kinetics. 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 cell replication.17,18 The drug has low selectivity; it affects both healthy and diseased cells, causing a number of unwanted side effects. One of the approaches to reduce these effects relies on the lower pH of the tumor cell environment compared with that of healthy cells. Release profiles of Dox from LCP were monitored 2

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electrochemically by measuring the voltammetric peak current of doxorubicin reduction. It was found that drug release from the phase is pH-dependent. The pKa of doxorubicin is 8.2.19 Protonated doxorubicin, which carries a positive charge, is soluble in water, whereas the unprotonated drug prefers a hydrophobic lipidic environment, where its diffusion is much slower.15,16 Recently, we have voltammetrically evaluated the release kinetics of Dox by analyzing a decrease in its peak current over time. The results suggest that the release mechanism is in agreement with the Higuchi model at low pH, while at higher pH values, non-Fickian transport dominates.16 In the present study, we designed the following hybrid materials based on monoolein cubic mesophases containing two types of magnetic nanoparticles (NPs) differing in their stabilizing coatings around the magnetic core: hydrophilic - nickel-zinc ferrite nanoparticles stabilized with a citric acid adlayer (NPcitric) and lipophilic - iron oxide core nanoparticles coated with an oleic acid adlayer (NP oleic). The addition of magnetic nanoparticles to the mesophase would open new possibilities for directing a liquid crystal drug carrier to the desired target using a magnetic field,20–21 as well as for magnetic hyperthermia.22 The mesophase can be dispersed into nanoparticles - cubosomes,23 which have the same properties as the bulk phase but have lower density. Cubosomes doped with nano-objects are of interest not only as the drug delivery systems 3,24-29 but also as contrast agents for magnetic resonance imaging (MRI).30-31 We studied the physical and chemical properties of the hybrid materials as a thin film on the electrode surface and as cubic phase nanoparticles – cubosomes, both modified with the magnetic nanoparticles, NPcitric and NPoleic. The two types of NPs used, due to their different hydrophilic-hydrophobic properties, are located in different phase domains of the LCPs. All samples exhibited a double-diamond cubicPn3̅m phase at room and human body temperature, with some differences in the diameters of the water channels. For the mesophase doped with 2% w/w of hydrophilic NPcitric above 40 degrees, a switch from

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the cubic to the hexagonal phase takes place, which results in slower drug release from the mesophase. Drug release from the bicontinuous cubic phase is significantly faster than from the hexagonal phases.5 The release profiles of Dox were evaluated using electrochemical methods, which monitor the changes in drug concentration directly in the cubic phase layer covering the electrode. A comparison of structures and drug elution profiles reveals that the hybrid material with NPcitric changes the properties of the cubic phase. In the case of hydrophobic nanoparticles, the structure remains unaffected. All results show that the hybrid material with hydrophobic magnetic NPs is a promising matrix for drug delivery. Hybrid cubosome dispersions were also prepared. The magnetocubosomes containing both types of magnetic nanoparticles have magnetic properties similar to those of magnetic nanoparticles alone and can be considered for drug delivery directed by means of a magnetic field. EXPERIMENTAL SECTION Materials All chemicals were of the highest quality available commercially. Monoolein (1-oleoyl-rac-glycerol) (MO), MES sodium salt (2-(N-morpholino)-ethanesulfonic acid sodium salt), Doxorubicin (Dox) and Pluronic F-127 were purchased from Sigma-Aldrich and were used as received. A 0.2 M buffer was prepared by titrating MES with 0.2 M NaOH to pH 5.8. All solutions were prepared using Milli Q water (18.2 M cm-1), Millipore, Bedford, MA, USA. Hydrophilic nanoparticles were synthesized by using Fe(NO3)3·9H2O and TMAH (tetramethylammonium hydroxide) from Sigma and Ni(NO3)2·6H2O, Zn(NO3)2·6H2O, NaOH, HNO3, and KCl from POCH. Hydrophobic nanoparticles were synthesized by using FeCl3·6H2O, FeCl2·4H2O (Sigma-Aldrich), NH4OH (25% aqueous solution in H2O, Chempur, Poland) and oleic acid (>99% pure, Sigma Aldrich). Preparation of Cubic Phases and Cubosomes The non-doped LCP was prepared by adding an appropriate amount of distilled water to a glass vial with molten MO. The ratio of components was chosen on the basis of Qiu and Caffrey’s phase diagram 4


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for the MO/water mixture.32 The cubic phase doped with Dox was prepared similarly by first dissolving Dox in buffer and then adding to molten MO to obtain 59.5/0.5/40 (% w/w) for the MO/Dox/H2O phase. To prepare the LCPs loaded with hydrophilic nanoparticles (NPcitric), the NPcitric suspension or NPcitric/Dox solution was added to molten MO. In the case of hydrophobic nanoparticles, the appropriate amount of NP dispersion in hexane (NPoleic) was added to molten MO and sonicated. The mixture was left in a desiccator to 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 59.3/0.2/0.5/40 or 57.5/2/0.5/40 (% w/w) for MO/NP/H2O and MO/NP/Dox/H2O, respectively. To obtain homogeneous, transparent and viscous LCPs, all the samples were left for at least 24 h in tightly closed vials at room temperature in the dark. Phase diagrams of the obtained hybrid phases were evaluated. The obtained phases were used to prepare cubosomes23. First, 0.1 g of LCP phase with NPs (34.5 mg of NPs per gram of monoolein) was placed in a glass vial, and 1.85 ml of Pluronic F-127 solution (1%) in deionized water was added. The cubic phases were sonicated for 20 minutes in a bath-type sonicator. Synthesis of hydrophilic mixed ferrite nanoparticles (Ni0.5Zn0.5Fe2O4) Hydrophilic nanoparticles (NPcitric) were synthesized according to 33 with minor modifications. Briefly, the nanoferrites were prepared using the “bottom-up” technique by co-precipitation of nanoferrites from the solution of their precursors with a strong base according to the following equation: Ni2+ + Zn2+ + 4Fe3+ 16OH- → 2Ni0.5 Zn0.5 Fe2O4↓ + 8H2O First, the precursor solution was prepared by mixing the heated aqueous nitrate salts of 0.6667 M Fe 3+, 0.1667 M Ni2+ and 0.1667 M Zn2+ with a hot aqueous solution of sodium hydroxide. Then, in a reaction vessel, 40 ml of this solution was mixed with 8 ml 2 M HNO3 (to avoid hydrolysis), and 152 ml H2O was added; the resulting solution was heated with continuous stirring to 95°C under reflux. An aqueous solution of NaOH (400 ml, 0.75 M) was preheated to 95°C in a separate container and then transferred to the reaction vessel. A dark-brownish precipitate appeared, and the reaction was left heated for the 5



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next 12 hours with stirring. Afterward, the reaction was cooled to room temperature. Nanoparticles were precipitated with a magnet and washed three times with deionized water using sedimentation and decantation in the field of a permanent magnet. The NP surfaces were treated with citrate ions by incubating for 1 hour in a 0.5 mg/ml citric acid solution with continuous stirring at 90˚C. Then, the obtained suspension was cooled to room temperature and washed several times with an excess of deionized water under magnet-assisted sedimentation to remove the unbound citric acid. The hydrophilic nanoparticles grafted with citric acid were located primarily in the LCP aqueous domain. The choice of these particular magnetic cores was intentional since it allowed control of the crystallite size and magnetic properties of the mixed, zinc-nickel ferrite (vide infra). This particular ferrite is ferromagnetic, and the resultant magnetic moment comes solely from nickel cations because the magnetic moments of Fe3+ ions are antiparallel, partially compensating for the overall magnetic moment of the NPs. Introduction of zinc further alters the structure of the nanocrystallites, allowing control of the magnetic properties of the nanoparticles. Additionally, an introduction of metal ions of larger ionic radii than Fe3+ (78.5 pm) alters the crystallographic structure due to the ionic radii mismatch in the spinel structure of magnetite (Ni 2+ 70 pm, Zn2+ 74 pm), thereby affecting the size of the resulting nanoparticles.34 Synthesis of Hydrophobic Magnetite Nanoparticles (Fe3O4) Hydrophobic nanoparticles (NPoleic) were prepared based on the description of the procedure from Mahdavi et al.35 The whole synthesis was carried out in a three-necked flask under vigorous stirring and an argon atmosphere. Firstly, 0.023 mol of FeCl2·4H2O and 0.046 mol of FeCl3·6H2O were dissolved in 150 ml of deionized water. The mixture was heated to 45°C and then 11 ml of 25% ammonia was added. Instantaneously, a black precipitate of nanoparticles appeared. After 30 minutes, 3 ml of oleic acid was added, and the suspension was heated to 80°C. After one hour, the Fe3O4 nanoparticles coated with oleic acid - NPoleic - were decanted using magnetic separation and washed several times with deionized water 6


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and ethanol. After removal of the excess of oleic acid, the nanoparticles were suspended in ethanol and centrifuged for one hour at 13.4 krpm. Next, the ethanol was removed using a rotary evaporator and replaced with 100 ml of hexane. The hydrophobic nanoparticles stabilized with oleic acid were mainly embedded in the lipidic part of the cubic phase, however since they are also larger than the bilayer width parts of the nanoparticle is protruding into the aqueous channels (Scheme 1). 36

Scheme 1: Characteristics over length scales of the magnetic nanoparticles, cubic mesophase and magnetocubosomes. METHODS Small-angle X-ray Scattering (SAXS)

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The phase identity and structural parameters of the lipidic samples were determined by SAXS. The experiments were carried out with a GADDS system (Bruker) using a 3-pinhole collimation system working with CuKα radiation (λ

Cu, Kα

= 0.1542 nm), a Nanostar system (Bruker) working with CuKα

radiation equipped with a Vantec 2000 area detector and a SAXSess mc² instrument (Anton Paar), using a line collimation system and equipped with a sealed X-Ray tube (PANalytical, λ

Cu, Kα

= 0.1542 nm)

and a CCD detector (Princeton Instruments). Samples were introduced into special thin glass capillaries, which were immediately flame-sealed. SAXS spectra were recorded in the range of 10 to 60°C. The scattering intensities I(q) were represented as a function of the magnitude of the scattering vector q = (4π/λ) sin(θ), where 2θ is the total scattering angle. The SAXS patterns obtained from the Bruker apparatus were analyzed with the Bruker Topas 3 software. For the SAXS mc² apparatus, all data were corrected for background scattering from the empty capillary and for slit-smearing effects by a desmearing procedure from the supplier’s software using the Lake method. The size of water channels was calculated based on the lattice parameter and the composition of cubic phases.37 Initially, the water volume fraction was calculated by the equation: φw = cw/(cw + (1-cw)(ρw/ρl))

(1)

where φw - water volume fraction, Cw - water weight fraction, ρw - density of water = 0.997 g/cm3, ρl - density of lipid = 0.942 g/cm3. The lipid volume fraction was determined from the equation: φl = 1- φw

(2)

The lipid chain length (l) was determined by solving the following equation37: φl = 2δ(l/a) + ⁴ /₃ πχ(l/a)3

(3)

where δ - ratio of the minimal surface in a unit cell to the quantity (unit cell volume)2/3, χ - Euler– Poincare´ characteristic, a - lattice parameter of corresponding phase, and l - lipid chain length/monolayer thickness. 8


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Finally, the water channel radius - rw was obtained using equation38: rw = (-δ/2π χ)1/2 a - l

(4)

Polarized Microscopy Polarized light microscopy (Olympus BX 50) conducted with a heating/cooling stage was used for visual inspection of the samples. 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) fitted with a 4 mW He–Ne laser (λ = 632.8 nm) as the light source at a scattering angle of 173o. The solutions were equilibrated for 2 minutes before measurement. The Zeta potential was measured using the same instrument. The values were reported as averages from 5 measurements 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°C·min−1 under oxygen using the TGA Q-50 thermal analyzer (TA Instrument). From these measurements, 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 S1 A). Transmission Electron Microscopy (TEM) TEM measurements (Libra 120 microscope, Zeiss) were used to assess the size of the magnetic nanoparticles (Figure 1 A, B). Before the measurement, a drop of a sample suspension was placed on a formvar®-coated copper grid and allowed to dry in the air. Hydrophilic nanoparticles NPcitric revealed an average diameter of 13±3 nm, while the average diameter of hydrophobic NPoleic was ca. 7±2 nm (Figure S1 B, C).

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Cryo Scanning Electron Microscopy (Cryo-SEM) Cryo-SEM image was taken using a Carl Zeiss Microscopy at a voltage 2 kV and a working distance of 2.6 mm. Sample was loaded into a holder and frozen in the liquid nitrogen. The sample was transferred into the cryochamber, which was held at −140 °C. Then the sample was sublimed at −85 °C for 2– 3 min.

Figure 1. TEM images of the citrate-coated NPs (A) and oleic-stabilized NPs (B), scale bars – 50 nm. Magnetization Measurements The magnetic properties of both types of nanoparticles as a function of the magnetic field at temperatures ranging from 2 to 300 K were reported by us earlier.39,40 Those results were comparable, regardless of the surface modification (citric or oleic shell), and in general, revealed typical ferromagnetic-like behavior, with no hysteresis loop at temperatures close to room temperature. Here, we complemented our previous results with magnetization measurements of NPs and hybrid materials performed with an automatic Faraday balance at a constant magnetic field of 1 Tesla. The results are shown in Figure S2. Temperature was controlled with an accuracy better than 0.5 K. The magnetization values for nanoparticles and hybrid materials were calculated using the weight of nickel-zinc ferrite and magnetite measured by TGA (Figure S1 A).

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The temperature variation in the mass magnetization of the nanocrystalline NPcitric sample, containing nickel-zinc ferrite, is plotted in Figure S2 A and shows ferromagnetic ordering up to the Curie temperature of Tc267°C. This Curie temperature is comparable with the Tc=264°C of the polycrystalline Ni0.5Zn0.5 Fe2O4.41 At room temperature, the magnetic moment per nickel-zinc ferrite equals 1.5B (Table 1 , vide infra) and is smaller compared to the 3.17 B of the polycrystalline Ni0.5Zn0.5Fe2O4. The reduced magnetic moment of nanocrystalline ferrite is typically attributed to the structural and magnetic disorder within the crystallite surface layer. The temperature variation in the mass magnetization of the nanocrystalline NP oleic sample, containing magnetite Fe3O4, is plotted in Figure S2 B and shows the ferromagnetic ordering up to the Curie temperature of Tc490°C, which is lower than the 585°C reported for the polycrystalline magnetite42 and the 580°C reported for the magnetite Fe3O4 coated with citric acid.43 The magnetic moment per Fe3O4 equals 2.3 B at room temperature (Table 1, vide infra) and is reduced compared to the 3.77 B of the polycrystalline.36 The minute magnetization hump observed between 220 and 320°C may reveal some change in the interparticle interaction caused by removal of the surface-coating oleic layer, which is also observed in the same temperature range for the TGA spectra (Figure S1 A). An analogous effect has been reported for citrate-stabilized Fe3O4 aqueous colloidal magnetic nanoparticles.43 These results prompted us to use both types of magnetic nanoparticles as prospective “vectors” incorporated inside the cubosomes loaded with doxorubicin, forming a nanoparticulate hybrid drug delivery system as described further.

Table 1. Magnetic Moment per Molecule of Citric Acid-coated NP, Oleic-coated NP, Hybrid Cubic Phases and Hybrid Cubosomes at Room Temperature. Magnetic moment [B] 11



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NPcitric

1.5

Phase with NPcitric

1.4

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Cubosomes with NPcitric 1.5 NPoleic

2.3

Phase with NPoleic

2.7

Cubosomes with NPoleic 1.9 Electrochemical Measurements Electrochemical measurements were performed using the CH Instruments bipotentiostat model CHI750B. The three-electrode system, consisting of a glassy carbon electrode (GCE, A = 0.07 cm2), an Ag/AgCl reference electrode and a platinum foil as a counter electrode, was employed. Prior to the measurements, the GCE electrodes were polished with an alumina slurry (1.0, 0.3 and 0.05 μm) on a polishing cloth. The electrodes were then rinsed with a direct stream of ultrapure water, sonicated in an ultrasonic bath and then left to air dry. After cleaning, the working electrodes were modified with a thin film of the cubic phase. Measurements at elevated temperatures were performed using a BVT MT-1 minithermostat. Argon-saturated solutions were obtained by bubbling high-purity argon gas for 15 min into the solution and continuing with a flow of pure gas (Ar) over the solution during experiments. Release of Dox from LCPs deposited on a GCE was monitored using differential pulse voltammetry (DPV). For each type of hybrid cubic phase, triplicate experiments were performed. RESULTS AND DISCUSSION Structural Characterization of the Cubic Phases To evaluate the effect of the addition of two types of magnetic nanoparticles on the cubic phase properties, Small-Angle X-ray Scattering (SAXS) and cross-polarized microscopy experiments were carried out.

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Polarized light microscopy and SAXS are the most widely used techniques for characterization of liquid crystalline phases. Cubic phases are isotropic, and therefore, a dark image appears when viewed under a microscope equipped with cross-polarizers. Other structures found in the MO/H2O system lamellar and hexagonal phases - are anisotropic and have a characteristic textures when observed by polarized light microscopy. The space group of the phases and the structural parameters can then be determined by SAXS. A monoolein-water phase diagram has been described in the literature,32,45,46 and therefore, the influence of the incorporation of 2% w/w of NPcitric and NPoleic was investigated. The phase diagrams were determined in the range of 10 to 60°C for a water content varying between 20 and 40% w/w. By adding hydrophilic nanoparticles that are located mainly in the water channels of the cubic phases, substantial changes in the phase diagram in the studied region can be observed (Figure 2 A). Below 40°C, the sequence of liquid crystals remains unchanged, except for the lamellar phase. Both the Ia3d and Pn3̅m cubic phases are less temperature-stable. Indeed, the Ia3d cubic phase melts at 45°C, and the Pn3̅m phase disappears at approximately 60°C, while for MO with hydrophobic NPs, these two phases are stable over 60°C. Interestingly, a phase transition from Pn3̅m to H2 occurs at 40°C for a water content of 40% w/w, as shown on the SAXS spectra and confirmed by the texture of the samples observed with an optical microscope equipped with cross polarizers (Figure 3), whereas the transition appears at 90-100°C for pure MO in a limited range of water content (15 to 25% w/w of water). The decrease in the phase transition temperature (Pn3̅m cubic phase - H2) may be induced by the presence of the hydrophilic NPs that swell the water channels. Therefore, the lattice axes are increased, and this results in a reduced curvature of the bilayer and a less pronounced wedge shape for the molecules. This behavior favors the formation of a reverse hexagonal phase.45 By adding hydrophobic nanoparticles that are solubilized in the hydrophobic chains, no significant modification of the phase behavior was observed (Figure 2 C). Indeed, in comparison with pure MO, the same sequence of liquid crystals 13



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(lamellar phase – Ia3d cubic phase – Pn3̅m cubic phase) was obtained as a function of the water composition, and the stable areas associated with the temperature and concentration are similar. This is an important observation for future applications of various forms of mesophases as drug delivery platforms with NPs acting as delivery “vectors”.

Figure 2. Left panel: Monoolein-water 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.

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Figure 3. SAXS spectra of the various monoolein-based systems with 40 wt.% of water at different temperatures: pure MO (A); with 2% w/w of hydrophobic NPs (B); with 2% w/w of hydrophilic NPs (C) (the intensities are represented on a logarithmic scale); and pictures of polarized-light microscopy for phase with NPcitric at 25°C (Pn3̅m) (D), 40°C (Pn3̅m and H2 phases) (E) and 50°C (H2 phase) (F). Comparing the SAXS experiments of samples with 40% w/w of water, the spectra of 2% w/w and 0.2% w/w NPs (Figures 2B, D) at human body temperature are similar to that of pure MO and present a cubic structure that corresponds to the Pn3̅m space group, as confirmed by the relative positions of the reflection lines 1:√³/₂ :√2:√3:2:√⁹ /₂ . For the hybrid phase with 0.2% w/w of citric NPs, the peaks are shifted to lower q values (Figure 2B), while for the phase with 2% w/w NPcitric, the situation is opposite. For NPoleic, no shift of the peaks is observed (Figure 2D), which indicates that the incorporation of 15



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hydrophobic NPs does not affect the organization of lipidic mesophases. Moreover, for the SAXS spectra (Figure 2, 3), it should be noted that for samples with NPs, diffusion at small q values is observed, due to the presence of NPs in the system. Increasing the temperature induces a shift of the peaks towards higher q values for hydrophobic NPs, and the Pn3̅m cubic phase is maintained (Figure 3 B), as observed for pure MO (Figure 3 A). For the samples with 2% w/w of hydrophilic NPs, the reverse hexagonal phase H2 appears at 40°C, as confirmed by the relative positions of the reflection lines 1:√3:2 on the SAXS spectra (Figure 3 C), which is characteristic of the hexagonal symmetry. For the samples with 0.2% w/w of hydrophilic NPs, the peaks are shifted to higher q values, and no phase transition is observed in the temperature range from 25°C to 60°C (data not shown). The calculated values of the lattice parameter, lipid length and diameter of water channels are reported in Table 2. The values confirm the observations of the SAXS spectra. At room temperature, the addition of 0.2% w/w of hydrophilic NPs leads to an increase in the water channel width by 0.3 nm. The addition of 2% w/w of NPcitric leads to a decrease in both the lattice parameter and channel width by 0.6 and 0.3 nm, respectively. The addition of hydrophobic NPs has no influence on the lattice parameter compared to pure MO. As expected, after increasing the temperature, the lattice parameter and the diameter of water channels decrease for both NPs similar to the pure MO phase.32 The incorporation of doxorubicin into MO does not significantly affect the lattice parameter and size of water channels since the drug molecule is small (ca. 1-5 nm) and is easily accommodated in the structure (Table S3).15 However, the incorporation of Dox into hybrid materials leads to an increase in the lattice parameter and the width of the water channels, which is more pronounced in the case of 2% w/w of hydrophilic nanoparticles.

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Table 2. Results of SAXS Measurements for Monoolein and Hybrid LCPs Depending on the Phase Composition and Temperature: Phase Identity, Lattice Parameter a, Lipid Length l, and Water Channel Diameters dw. Phase

Phase

composition

T

Symmetry

a (nm)

l (nm)

dw (nm)

(% w/w)

(°C)

MO/H2O

25

Pn3̅m

9.5

1.6

4.2

60/40

35

Pn3̅m

9.3

1.6

4.1

40

Pn3̅m+Water

9.0

1.5

4.0

45

Pn3̅m+Water

8.7

1.5

3.8

MO/NPcitric/H2O

25

Pn3̅m

10.1

1.7

4.5

59.8/0.2/40

36

Pn3̅m

9.7

1.7

4.3

40

Pn3̅m+Water

9.3

1.6

4.1

44

Pn3̅m+Water

8.9

1.5

4.0

MO/NPcitric/H2O

25

Pn3̅m

8.9

1.5

3.9

58/2/40

36

Pn3̅m

8.5

1.5

3.8

40

Pn3̅m and

8.3

1.4

3.7

trace H2

nd

nd

nd

Pn3̅m

8.1

nd

nd

and H2

6.0

nd

nd

44

MO/NPoleic/H2O

25

Pn3̅m

9.4

1.6

4.2

59.8/0.2/40

35

Pn3̅m

9.3

1.6

4.1

40

Pn3̅m+Water

9.2

1.6

4.1

45

Pn3̅m+Water

8.8

1.5

3.9

17



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MO/NPoleic/H2O

25

Pn3̅m

9.6

1.6

4.2

58/2/40

36

Pn3̅m

9.3

1.6

4.1

40

Pn3̅m+Water

9.0

1.5

4.0

44

Pn3̅m+Water

8.7

1.5

3.8

Properties of Cubosomes Nanoparticles of the cubic phase – cubosomes – were prepared, and their characteristics were evaluated (Figure S4, S5). The diameter of cubosome dispersions, in the absence/presence of NPs, was obtained from the DLS measurements. The cubic particle sizes are similar, between 110 and 150 nm, which is suitable for a drug delivery system (Table 3, Figure S4 A). With the DLS technique, we can perform measurements in the natural environment of the samples – water solutions. Lipidic particles are rather fragile, and preparation of samples for TEM measurements can change/destroy the structure. Nevertheless, we used TEM imaging to confirm the presence of NPs inside the cubosomes. Figure S4 presents TEM images of cubosomes with hydrophilic NPcitric [C] and hydrophobic NPoleic [D] nanoparticles (please note the different scale bars on these figures). The cubosomes are visible as gray discs with much darker dots of iron oxide nanoparticles because NPs have a much higher electron density than lipids. Table 3. Characteristic Values of Cubosomes: Zeta potential, Size and Polydispersity Index (PDI). Cubosomes Cubosomes with NPcitric Cubosomes with NPoleic Zeta potential [mV]

-18.9±4.4

-17.3±1.3

-19.6±2.3

Size [nm]

111±10

137±8

150±13

PDI

0.35

0.26

0.33

18


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Cryo-SEM image shows the spherical morphology of the cubosomes (Figure S4 B). The cubic nature of the LCP dispersion was characterized by SAXS. The SAXS pattern for MO cubosomes shows the local cubic structure with Im3̅m space group (the relative positions of the diffraction signals are: √2:√4:√6) (Figure S5). The crystallographic lattice parameter is 14.6 nm. It was reported earlier that sometimes during ultrasonic dispersion of material the Pn3̅m cubic phase underwent transition to the cubic structure with Im3̅m symmetry.47-50 In the case of cubosomes with NPoleic, the scattering from nanoparticles masks completely the first Bragg reflection and also partly the second Bragg peak. However, the position of the third peak is clearly visible and at the same q value as the corresponding peak for MO cubosomes, which confirms that incorporation of NPs does not affect the structure of cubosomes (Figure S5 - inset), similarly to the case of bulk cubic phases (Figures 2 and 3). Since the zeta potential for cubosomes with or without NPs is quite similar (see Table 3), and the zeta potential for NPs alone is much higher (-39 mV for NPcitric), we can conclude that the nanoparticles remain inside the lipidic particle and not on the surface. The cubosomes in the TEM images were larger in size than those observed with DLS, which was likely caused by flattening during the drying process. Doxorubicin Release Profile from Cubic Phases with Nanoparticles The drug release profiles of the pure monoolein and hybrid phases were investigated based on the changes in the DPV voltammograms with time (Figure 4, S6). The voltammetric experiments were conducted at room temperature and at 37°C and 44°C under an argon atmosphere. The electrodes were covered with a thin film (approximately 1 mm) of cubic phase and immersed in a 0.2 M buffer solution with pH 5.8 because the cancer cell environment is shown to have a lower pH than that of normal cells, which is ca. 7.4.51 Doxorubicin is electroactive because its molecules have quinone- and hydroquinone-type redox centers, which undergo reduction and oxidation in a 2e-/2H+ process (Schemes S7 and Figure S8).52 The magnetic nanoparticles, because of their size, should not block the access of Dox to the electrode, 19



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regardless of their surface coatings. To prove this, we performed cyclic voltammetry experiments on GCE electrodes modified with a cubic phase alone or a hybrid system with hydrophilic NP citric or hydrophobic NPoleic magnetic nanoparticles. Since the peak current depends on the square root of the scan rate, the process remained diffusion controlled (Figure S8). The best method for checking the type of transport of Dox to the electrode surface is to use the Korsmeyer-Peppas model.53 This model is described by the equation: Mt/M∞ = ktn

(5)

where Mt/M∞ is a fraction of drug released at time t, k is the release rate constant and n is the release exponent. The n value gives information about the different release mechanisms of the drug (see Table 4). To determine the value of n, the logarithm of 0.89, to Super Case-II transport. For the simplest diffusion-controlled process (Fickian), where n≈0.5, the Korsmeyer-Peppas approach reduces to the so-called Higuchi model, which describes drug release from semi-solid matrices.53 The Higuchi model describes the diffusion process of drug release with the following expression: Mt/M∞ = kH√t

(6)

where kH is the Higuchi constant. Incorporation of 0.2% w/w NPcitric results in an increase in Dox removal without a change in peak potential, -0.550 V (Figure S6). Furthermore, 50% Dox (T50) is released after 50 min with 0.2% NPcitric, whereas without nanoparticles, it is 60 min. For the phase with 2% NPcitric, the DPV peak currents are five times smaller, and at -0.770 V, an additional signal appears, which might be related to the attraction 20


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of NPcitric and Dox in the aqueous channels (Figure S6). This result strongly suggests that positively charged Dox molecules interact electrostatically with the negatively charged NPcitric (the zeta potential of NPcitric is -39 mV due to the presence of negatively charged carboxyl groups on the surface of nanoparticles). After 350 min, as much as 75% of the drug remains in the cubic phase film (Figure 4A, S6). UV-Vis measurements at 480 nm performed for Dox and Dox with NPcitric in 0.02 M MES at pH 5.8 confirmed the influence of NPcitric [data not shown]. For NPoleic, no changes in the Dox voltammograms were observed, and the peak currents remain almost unaffected for the 0.2% and 2% NPoleic dopants (Figure S6). T50 occurs after 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 for the MO phase.

Figure 4. Release profile of Dox from LCPs (40% w/w of water) with and without nanoparticles at pH 5.8 and room temperature. A – hybrid system with hydrophilic nanoparticles, 1- MO phase, 2a - phase with 0.2% w/w NPcitric, 2b – phase with 2% w/w NPcitric; B – hybrid system with lipophilic nanoparticles, 1- MO phase, 2a – phase with 0.2% w/w NPoleic, 2b – phase with 2% w/w NPoleic. The proposed hybrid materials could be considered for combined therapies, for example for hyperthermia with chemotherapy. Considering that the temperature for hyperthermia is in the range of 21



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Page 22 of 33

41–46°C, we measured drug elution at 37°C and 44°C to verify the behavior of the hybrid phase with 2% w/w of NPs under these conditions (Figure 5, S9) (Table 4).

Figure 5. Comparison of Dox release profiles 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. At human body temperature (37°C), the drug is removed three times faster when 2% w/w NPcitric is added than at room temperature (Table 4), but at 44°C, the drug is released two times faster compared with room temperature. Release of molecules is faster at higher temperatures, which can be explained by accelerated diffusion of the molecules at elevated temperatures. The decrease in the release rate of Dox observed at 44°C compared to 37°C is ascribed to the phase transition to the reverse hexagonal phase (Figure 2B, 3, S9), for which transport is slower.3,5 In the presence of 2% w/w NPoleic, the release of drug at room temperature and higher temperatures is comparable (Table 4). In all cases, the value of T50 is 30 min. The NPs of both types are larger than the lipid bilayer width and are entrapped within the lipid matrix 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 22


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hydrophobic NPoleic interact mainly with the monoolein of the lipidic part of the cubic phase and not with the drug located in the aqueous channels. 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 model. The same situation is found for the phase with 2% w/w of NPoleic at room temperature, but when increasing the temperature, the influence of the matrix is observed (n deviates from 0.5). In the case of 2% w/w of NPcitric incorporated into the phase, clearly non-Fickian transport contributes to the transport, and the process cannot be described by the Higuchi model even at room temperature (Table 4). Table 4. Release Characteristics of Doxorubicin from Neat and Hybrid LCPs. LCP

Korsmeyer-Peppas

Higuchi

N

K[%/h-n]

R2

kH[%/h-1/2]

RH2

0.50

48.1

0.883

89.5

0.992

0.50

56.8

0.995

71.0

0.926

T=25°C

1.61

3.20

0.982

nd

nd

T=37°C

1.62

7.12

0.927

nd

nd

T=44°C

1.72

3.31

0.921

nd

nd

0.50

53.2

MO LCP T=25°C LCP + 0.2% NPcitric T=25°C LCP + 2% NPcitric

LCP + 0.2% NPoleic T=25°C

0.971

LCP + 2% NPoleic

23


81.1

0.933


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Page 24 of 33

T=25°C

0.48

63.6

0.911

82.1

0.931

T=37°C

0.59

70.6

0.925

93.0

0.992

T=44°C

0.75

77.4

0.950

nd

nd

Magnetic Properties of Hybrid Systems Magnetic characterization was conducted for nanoparticles covered with the citric/oleic layer, for the cubic phases containing 2% w/w of nanoparticles and for the cubosomes with nanoparticles. Initially, it was observed that both the cubic phase and the cubosome suspension reacted to the magnetic field of a neodymium magnet, involving a movement of particles towards the magnetic field.

Figure 6. Temperature variation of magnetization for mesophases and for cubosomes with NPs [A NPcitric, B - NPoleic]. 24


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Both nanocrystalline nickel-zinc ferrite and magnetite covered with the citric/oleic layer were incorporated into the cubic phases. Figure 6 shows that mass magnetization decreases with temperature. At room temperature, the magnetic moment per nanocrystallite contained in the bulk cubic phase with NPcitric and NPoleic equals 1.4 and 2.7B (Table 1), respectively. As expected, these results confirm that the magnetic particles of the ferrite and magnetite still retain the magnetic moment in the cubic phases. The minute magnetization enhancement observed between 70°C and 110°C seems to be related to evaporation of water; however, the detailed mechanism is not understood at this time. The temperature variation of magnetization was also measured for the cubosomes (Figure 6). Qualitatively, the temperature variation is similar to that of the cubic phase. The absolute magnetization values are reduced approximately 3 and 12 times compared to the corresponding bulk cubic phases. When taking into account the content of magnetic nanoparticles in the cubosomes, the magnetic moments are found to be 1.5B and 1.9B for the ferrite (NPcitric) and magnetite (NPoleic) systems, respectively. These magnetic moment values are comparable to the values obtained for the nanoparticles and the bulk cubic phases. This confirms that the magnetic properties of nanoparticles are conserved in the dispersed systems studied, meaning that they can be exploited for magnetic drug targeting, hyperthermia treatment or magnetic resonance imaging. CONCLUSIONS The cubic phase is a promising material for the delivery of hydrophilic and hydrophobic drugs. Insertion of magnetic nanoparticles can extend its applications in medicine. We presented and characterized hybrid materials based on a monoolein cubic phase doped with two types of magnetic nanoparticles - hydrophilic and hydrophobic. The system was tested as a cancer drug delivery platform, and therefore, doxorubicin was chosen as an effective drug, which is, however, highly toxic to healthy cells. We showed that the Pn3̅m cubic phase structure is retained upon addition of 0.2% to 2% w/w magnetic nanoparticles and 0.5% w/w Dox. The lipidic hybrid material exhibited magnetic properties 25



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Page 26 of 33

and thus could be moved by the application of a magnetic field. The drug release profiles from the phases were established using differential pulse voltammetry (DPV). Introduction of 0.2% of nanoparticles results in removal of more drug by ca. 10% compared with the pure monoolein cubic phase. Adding 2% w/w of hydrophilic magnetic nanoparticles leads to a threefold slower release of the drug because the negative charge of these nanoparticles makes them reside mainly in the aqueous channels, and attractive interactions with the positively charged drug retain it in these channels. On the other hand, the presence of 2% w/w of hydrophobic NPs has almost no effect on the channel transport of the drug since the NPs are located mainly in the lipidic part of the cubic phase. Temperature increases the rate of drug release; however, in case of NPcitric, it is 53% at 37°C, whereas for NPoleic it is increased to 85%. This result is interesting from the view-point of magnetothermal applications of the system. Effective drug delivery systems should exhibit a large internal surface where the drug can be accumulated and should release the drug only in the cancer cell environment. Interactions with the drug are not favorable, and this is observed in the case of LCP with 2% w/w hydrophilic magnetic nanoparticles. In the case of hydrophobic nanoparticles, the T50 value is 50 minutes for the 0.2% NPoleic phase and 30 minutes for material with 2% NPoleic. At human body temperature, there was no change in the elution rate of drug for the phase with 2% w/w of NPoleic. All these results favor the material with hydrophobic magnetic NPs as a promising matrix for a drug delivery system. Hybrid cubosome dispersions were also prepared. The TEM images confirmed that the nanoparticles are inside the cubosomes. Hybrid materials containing both types of nanoparticles have similar magnetic properties and can be directed using a magnetic field. ASSOCIATED CONTENT Supporting Information

26


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Additional supporting information on the properties of magnetic nanoparticles (TGA, magnetization (S1 and S2) LCP properties (SAXS and DLS of cubosomes (S3 and S4), electrochemical properties of Doxorubicin and release profiles at different temperatures (S5-S8). AUTHOR INFORMATION Corresponding Author *Prof. Renata Bilewicz, Faculty of Chemistry, University of Warsaw, Pasteura 1, PL 02-093 Warsaw, Poland [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval for the final version of the manuscript. Notes The authors have no conflicts of interest to declare ACKNOWLEDGMENT This work was supported by Sinergia project no. CRSII2_154451 financed by the Swiss National Science Foundation. REFERENCES 1.

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Monoolein Cubic Phase Gels and Cubosomes Doped with Magnetic

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