Preliminary Results of Betulinic Acid-Loaded Magnetoliposomes-a Potential Approach to Increase Therapeutic Efficacy in Melanoma

CLAUDIA GEANINA FARCAS1,2, ELENA ALINA MOACA1*, RAZVAN DRAGOI3*, DELIA BERCEANU VADUVA3*, IASMINA MARCOVICI1, CIPRIAN VALENTIN MIHALI4, FELICIA LOGHIN2 1Victor Babes University of Medicine and Pharmacy Timisoara, Faculty of Pharmacy, 2 Eftimie Murgu Sq., 300041, Romania 2Iuliu Hatieganu University of Medicine and Pharmacy Cluj-Napoca, Faculty of Pharmacy, Dept of Toxicology, 8 Victor Babes Str., 400012, Cluj-Napoca, Romania 3Victor Babes University of Medicine and Pharmacy Timisoara, Faculty of Medicine, 2 Eftimie Murgu Sq., 300041, Romania 4Vasile Goldis Western University of Arad, Institute of Life Sciences, George Emil Palade Electron Microscopy Center, 86 Liviu Rebreanu Str., 310414, Romania

Despite all these features, BA possesses a major inconvenience due to its low solubility in the biological hydrophilic medium, leading to poor bioavailability [11]. Derivatization of two key positions of the chemical structure of BA, marked red in figure 1 -C 3 hydroxyl, C 28 carboxyl, has been shown to potentiate the cytotoxic effect of BA and even increase its hydrophilicity, thus enhancing its bioavailability. However, the purification process of these derivatives is difficult to achieve [12].
incorporation of BA into liposomes [15], BA functionalized carbon nanotubes [16], thus this topic has become of great interest among researchers around the world.
Liposomes embedding nanoparticles with magnetic properties (MNPs) are generically entitled magnetoliposomes [17]. Due to superparamagnetic features, MNPs can be used for magnetically induced hyperthermia, as drug delivery tools, or as magnetic resonance imaging (MRI) agents [18], hence the magnetoliposome platform can also benefit from the aforementioned features [17]. Among all MNPs, the most commonly used structure is Fe 3 O 4 -magnetite (part of the iron oxide magnetic nanoparticles group -IOMNPs) due to its biocompatibility, high saturation magnetization and good colloidal stability if the surface is properly coated [19]. The use of a coating agent is necessary because due to their large surface area to volume ratio, van der Walls forces and/or by application of an external magnetic field, IOMNPs can agglomerate leading to large cluster formation [20].
However, to the best of our knowledge, no study has so far reported the incorporation of BA within magnetoliposome structure. This new formulation would not only improve BA's bioavailability but could offer new therapeutic approaches as an alternative to the classical chemotherapy, by merging BA therapeutic activity with the superparamagnetic behaviour of IOMNPs. In this study, Fe 3 O 4 nanoparticles coated with citric acid (Fe 3 O 4 @CA) were used for the synthesis of magnetoliposomes. Through the coating process, nanoparticles have acquired hydrophilicity and colloidal stability, possessing a hydrodynamic diameter of 101nm (data not shown), thus overcoming undesirable effects that are characteristic of uncoated nanoparticles: agglomeration and rapid elimination by macrophages.
Herein, we present the development of a magnetoliposome structure that incorporates BA into the phospholipid bilayer and Fe 3 O 4 @CA into the aqueous core. In the last years, several types of formulations have been developed to improve the bioavailability of BA, such as: BA nanoemulsion [9], incorporation of BA into cyclodextrins [13], cocrystals formation of BA and vitamin C [14], The preliminary results of the effects induced by the magnetoliposome platform, as well as the blank liposomal structures, on two melanoma cell lines (murine melanoma B164A5 cells and human melanoma A375 cells) and one healthy cell line (mice epidermis JB6 Cl 41-5a cells) are also discussed.
The aim of the study is to provide basis information regarding development of smart multifunctional systems, such as magnetically-guided magnetoliposomes which can be controlled to reach the tumour site by applying an alternating magnetic field (AMF) followed by magnetically induced hyperthermia -able to alter the structure of liposomes, emerging BA triggered release. The cell lines used in this study JB6 Cl 41-5a (ATCC ® CRL-2010 ) and A375 (ATCC ® CRL-1619) were acquired from American Type Culture Collection (ATCC) and B164A5 vial (code 94042254) was purchased from Sigma-Aldrich (Munich, Germany). All the cell lines were maintained in standard conditions (liquid nitrogen) until the experiments begun.

Experimental part
Preparation of magnetic iron oxide nanoparticles (IOMNPs) The IOMNPs were synthesized using the combustion method [21] using iron nitrate nonahydrate (Fe(NO 3 ) 3 ·9H 2 O -as the oxidizing agent and citric acid monohydrated C 6 H 8 O 7 ·H 2 O -as reducing agent. The obtained powder was hand grinded, washed with hot distilled water (60-70°C) and dried.The dried Fe 3 O 4 nanoparticles were coated with citric acid (CA) and then dispersed in water at basic pH thus obtained the biocompatible colloidal suspension.

Preparation of betulinic acid-loaded magnetoliposomes
Betulinic acid-loaded magnetoliposomes (BA-Fe 3 O 4 @Lip) were obtained by preparing a blank liposomal solution (Lip) of 0.1 mM lipid mixture using the lipid film hydration method [22]. The lipid mixture was composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol and PEG-2000 in a molar ratio 90:10:0.4 and dissolved in chloroform by magnetic stirring at 40°C for 1 h, followed by drying at oven (40°C) overnight. Afterwards, the lipid film was hydrated with ultrapure water containing (NH 4 ) 2 SO 4 . For the synthesis of blank magnetoliposomes (Fe 3 O 4 @Lip) an amount of filtered liposomal suspension (Lip) was magnetic stirring at 45°C and the biocompatible colloidal suspension based on iron oxide magnetic nanoparticles (Fe 3 O 4 @CA) was dropwise added. Total lipid to magnetic colloidal suspension ratio was 4:1, according to the study of Kulshrestha and co-workers [23]. Finally, BA-Fe 3 O 4 @Lip sample was obtained by mixing an amount of liposomal suspension (Lip) with a chloroform solution based on betulinic acid (10:1 m/m). After that, the colloidal suspension of Fe 3 O 4 @CA was dropwise added (4:1 v/v) [23]. All liposomal suspensions were shocked for 1h using a Q Sonica ultrasound device, at 50% amplitude. After sonication, samples were centrifuged at 4°C for 10 min at 6000 rpm for 5 times, re-suspended in ultrapure water and shocked for 10 min at 50% amplitude. After that, the liposomal suspensions were filtered once through a hydrophilic membrane of 220 nm, and stored at 4°C until further use.
No instability signs (flocculation/agglomeration) were observed for almost one month if stored in appropriate conditions (4 o C).

Characterization of BA-Fe 3 O 4 @Lip Thermal analysis
To determine the phase transition temperature of BA-Fe 3 O 4 @Lip the sample was placed in aluminum crucibles under artificial air flow of 20 mL/min, and thermally scanned from 25 -1000 °C at a heating rate of 10 °C/min using a TG/DSC thermal analyzer (Netzsch STA 449 C instrument (Selb, Germany).

Structural characterization
Scanning electron microscopy (SEM) was employed to evaluate the morphology of BA-Fe 3 O 4 @Lip and the energy dispersive X-ray analysis (EDAX) was used in order to obtain the elemental composition of the sample. SEM-EDAX analysis was carried out with EDX detection on scanning electron microscope (SEM), using an EDAX detector (ZAF Quantification-Standardless, Element Normalized) with FEI Quanta 250 microscope (Eindhoven, Holland).

Cell viability evaluation by the means of AlamarBlue (AB) assay
Assessment of the cytotoxic potential of test samples was performed as previously described [24]. 1x106 cells/ well were seeded in 96-well plates and incubated overnight. The next day, the old medium was replaced with a fresh one containing the test compounds (Lip, Fe 3 O 4 @Lip, BA-Fe 3 O 4 @Lip and BA) to a final concentration equivalent to 5 and 25µM BA. BA dissolved in DMSO was also tested and was used as a positive control. After a period of 24/48 h of stimulation, 20µL AlamarBlue solution (AB -0.01%) was added in each well and incubated for 3h. In the final step, the absorbance of the wells was determined at 570 nm and 600 nm wavelengths, with a microplate reader (xMark TM Microplate, Biorad).

Cell morphology assessment
The morphological alterations induced by test samples on the three cell lines (JB6 Cl 41-5a, A375, B164A5) were assessed by taking pictures of the cells stimulated with the test compounds, at different intervals of time with an inverted Olympus IX73 microscope documented with an integrated DP74 camera (Olympus, Tokyo, Japan). The most severe morphological changes were observed 24h post-stimulation and are presented in fig. 5-7.

Statistical analysis
The statistical program used in this study was GraphPad Prism 5. Bar graphs are expressed as the mean values ± SD of three independent experiments. One-way ANOVA test followed by Tukey's post-test were employed to determine the statistical differences between experimental and control groups (* p <0.05; ** p<0.01; *** p<0.001)

Results and discussion
Physio-chemical characterization of BA-Fe 3 O 4 @Lip Thermogravimetric (TG) and differential scanning calorimetry (DSC) Thermal analysis and phase transition temperature of BA-Fe 3 O 4 @Lip are depicted in figure 2. From the graph, it can be observed that the phase transition of BA-Fe 3 O 4 @Lip from gel to liquid crystalline occurred at 39.437°C. This temperature could be achieved in vitro or in vivo by using Fe 3 O 4 nanoparticles as tools for inducing mild hyperthermia, following exposure to an external magnetic field due to magnetic hysteresis loss [18]. In the phase transition, the release of BA from the liposome nanosphere may emerge. Thus, it could be stated that the release of BA could be thermally controlled.

Structural characterization
The EDAX analysis showed that only C, O, N, P, S and Fe were present in the sample (table 1). The resulting chemical species in EDAX analysis were expressed in weight percent (Wt %) or atomic percent (At %).
By scanning electron microscopy -SEM ( fig. 3) it has been established the details regarding the morphology and ultrastructure of the BA-Fe 3 O 4 @Lip.
From figure 3, it can be observed that the structure of BA-Fe 3 O 4 @Lip containing Fe 3 O 4 nanoparticles shows spherical structures without any inner water pool.
Cell viability assessment by the means of AlamarBlue assay As shown in figure 4, the viability of the healthy JB6 Cl 41-5a cells exposed to the lowest concentration of the liposomal structures (5 µM) was not significantly affected,  The blank liposomal structures (Lip, Fe 3 O 4 @Lip) manifested cell viability above 60% in the case of both cell lines, after treatment with the highest concentration (25µM). The effect induced by test compounds respected almost the same tendency of cell viability decrease on both melanoma cell lines (B164A5 and A375), yet A375 cells seemed to be more sensitive. Present data showed that the highest concentration (25µM) of BA-Fe 3 O 4 @Lip caused a significant inhibitory activity on both melanoma cell lines, displaying a viability rate of 24% on B164A5 cells versus 7.80% viability percentage in the case of A375 cells, after 24 h post-stimulation. However, both cell lines (A375, B164A5) manifested a slight cell viability growth after 48h, B164A5 cells expressing a viability percentage of 38%, compared to a viability rate of 31.5% expressed by A375 cells. The trend of cell viability growth after 48h of stimulation was observed by almost all test samples, except BA which exhibited a higher cytotoxic activity after 48h, compared with the one recorded after 24h posttreatment ( fig. 5).
Thus, we assume that the cytotoxic effect induced by BA-Fe 3 O 4 @Lip may be due to BA embedded in the magnetoliposome structure. Association of the BA wellknown cytotoxic potential with the applications IOMNPs in biotechnology (hyperthermia, drug controlled release) can lead to a synergistic anti-tumor activity.

Cell morphology after treatment with test samples
The most severe morphological alterations of the test cell lines (JB6 Cl 41-5a, B164A5, A375) were recorded 24h post-stimulation and are depicted in figures 6-8.
The healthy mice epidermis JB6 Cl 41-5a cells exposed to the lowest concentration of liposomal samples (5 mM) presented normal morphological aspects, epithelial-like with a confluence above 90%. However, the cells treated with BA at the same concentration of 5 M exhibited slight morphological alterations, indicated by the red arrows in figure 6.
The cells stimulated with test samples at the highest tested concentration (25 mM) induced some morphological changes, but still no more substantial than those induced by the same concentration of BA, used as positive control.
As shown in figure 7 and figure 8, both melanoma cell lines (B164A5 and A375 cells) manifested no altered morphological aspects after treatment with the lowest   . Clares and co-workers reported nanosized 5fluorouracil-loaded magnetoliposomes with good in vitro compatibility and features for creating hyperthermic conditions to release 5-fluorouracil from the thermosensitive magnetoliposome structure [25]. Beside hyperthermia and drug controlled release, recent studies reported the efficacy of magnetoliposomes as contrast agents: in a rat model of liver ischemia-reperfusion [26] or in a diabetic rat model for labeling pancreatic islets [27].
In a comprehensive literature review, Allen and Cullis explained the importance of including cholesterol in the liposome structure, considering that this compound improves the phospholipid bilayer's stiffness, thereby reducing drug leakage from the liposomes [28]. Moreover, the same study presented the advantages of using ammonium sulfate for obtaining remote loading of drugs within liposomes, by transforming the drugs in weak bases or weak base prodrugs. Other studies discussed the importance of using PEGylated liposomes to enhance drug encapsulation efficiency [29], along with a better stability [30]. The synthesis process employed in this study respected all the aforementioned details described in the literature to achieve an improved formula of magnetoliposomes.

Conclusions
The experiments conducted in this study provide basic evidence of the massive potential of magnetoliposomes as thermally controlled structures, displaying selective cytotoxicity on murine melanoma B164A5 cells and human melanoma A375 cells, showing slight inhibitory activity on mice epidermis JB6 Cl 41-5a cells viability. The magnetoliposome platform may offer a multitude of multifunctional biomedical applications, such as combining hyperthermia with drug controlled release. Nevertheless, in vitro and in vivo experiments should continue to be performed.