1. Introduction
Curcumin is a polyphenolic compound that can be extracted from Turmeric (
Curcuma longa). This phytochemical is a liposoluble compound that has been showing a wide range of promising pharmacological properties, which include anti-inflammatory, antioxidant, antidiabetic, antimicrobial, and anticancer actions [
1,
2,
3,
4]. The consumption of this compound at levels up to 20 mg as a food additive is recognized as safe by the US Food and Drug Administration (FDA) [
5]. However, curcumin is chemically unstable, which is mainly related to solvolysis and oxidative degradation resulting in the production of some degradation products (e.g., vanillin, ferulic acid, and feruloylmethane). It is also sensitive to type I and type II reactions with molecular oxygen resulting in photochemical degradation [
6]. In addition, it presents a low water solubility (456 μg/L), poor absorption, and rapid metabolism and elimination, which limit its biomedical use and therapeutic effect, justifying the lack of success in clinical trials [
7,
8]. In the last years, the scientific community has been focused on the development of strategies that aim to improve curcumin’s ADMET properties (absorption, distribution, metabolism, excretion, and toxicology) and its specificity. Some of these drawbacks can be minimized by curcumin encapsulation in suitable nanosystems.
The use of drug delivery systems is one of the most employed and effective strategies to address drug bioavailability issues by protecting and carrying payloads to the target site. The nanocarriers include, for instance, micelles, microemulsions, nanogels, polymeric nanoparticles, carbon nanotubes, magnetic nanoparticles, and liposomes. Liposomes are lipid layered vesicles which can encapsulate a wide range of compounds and improve their solubility. They show a high biocompatibility, low toxicity, and low immunogenicity. The liposomes preparation method and variations in composition allow to modify their inherent features, such as size, circulation time in biological conditions, responsiveness to specific external and internal stimuli, and to control payload release. All the modifications must be accurately developed in order to take advantage of the uniqueness of cancer physiological features, since they include chemical (such as enzyme concentration and pH) and structural (capillary structure) differences compared to normal cells/tissues. Considering the leaky nature of tumor blood vessels, as a result of the presence of gaps within the range of 100 to 780 nm between the endothelial cells of tumor capillaries and the lack of lymphatic drainage, liposomes are an effective strategy for passive targeting. This phenomenon, known as the enhanced permeability and retention (EPR) effect, allows the extravasation of liposomes smaller than 400 nm, but it is more effective at sizes below 200 nm [
9,
10]. However, it is necessary to ensure that the liposomes are not rapidly captured by the liver and spleen phagocytic cells and pass as many times as possible through the target site. Polyethylene glycol (PEG) is a polymer that forms a hydrophilic shield on the liposome surface, which prevents liposome uptake by the reticuloendothelial system (RES), contributing for a prolonged systemic circulation. As a result, nonmodified liposomes and PEGylated liposomes have gained attention as an improved therapeutic drug delivery system for cancer therapy, resulting in some FDA-approved liposomal formulations. However, the associated defective blood perfusion in tumors leads to a low pH and oxygen (hypoxia) inside the solid tumors and surrounding tissues. These conditions difficult the therapeutic effect, either of radiotherapy (based on the creation of reactive oxygen species) or chemotherapy (since the drugs are not able to concentrate in the tumor at the aimed therapeutic dosage). Therefore, many efforts are being made to further improve liposomes’ therapeutic effectiveness.
Superparamagnetic nanoparticles are single domain magnetic materials with heating ability under an alternating magnetic field (AMF), and can be entrapped in liposomes (forming magnetoliposomes) and used as mediators for magnetic hyperthermia. This approach brings complementary advantages to lipid vesicles, since it allows one to selectively direct the nanosystems to the tumor by a magnetic gradient, a further increase of chemotherapeutic drugs concentration on tumor, and to directly overheat cancer cells under an AMF.
Even though magnetite and maghemite are the most studied nanoparticles for magnetic hyperthermia, they have been associated with a tendency to react with oxygen which results in the production of free radicals as reactive oxygen species (ROS), leading to oxidative damage in the human body [
11]. In order to be suitable for entrapping in liposomes and use in magnetic hyperthermia, magnetic nanoparticles must be biocompatible. The synthesis of particles without transition metals has been exploited. Spinel ferrites, MFe
2O
4 (where M = Fe, Mn, Zn, Co, Mg, Cu, Ni) present unique electric, optical, and magnetic properties. As a result, they have been applied, for instance, for contrast enhancement of magnetic resonance imaging and as magnetic carriers for drug delivery [
12]. Magnesium ferrite (MgFe
2O
4) is a class of compounds that crystalize into an inverse spinel structure with tetrahedral (A) and octahedral (B) sites, where cations distribution occurs. The magnitude of magnetization is highly sensitive to the synthesis methodology, to the distribution of magnetic ions in tetrahedral and octahedral sites, and to grain size and shape. The substitution of transition and diamagnetic metal ions is a strategy that can be followed to improve the magnetization of spinel ferrites [
13]. Calcium ferrite (CaFe
2O
4) nanoparticles are biocompatible, eco-friendly, and present a high thermal stability [
14]. Calcium-substituted magnesium ferrite nanoparticles (Ca
xMg
1−xFe
2O
4) not only can promote an increase in ferrite biocompatibility, but also improve the magnetic properties [
15,
16]. It is reported that upon doping with nonmagnetic Zn
2+ ions, magnesium ferrite nanoparticles showed an enhanced magnetization as a result of changes in ion site distribution [
17,
18].
In this work, three different proportions of calcium and magnesium were used to synthesize CaxMg1−xFe2O4 nanoparticles (x = 0.75, 0.25, 0.50), and their optical, structural, morphological, and magnetic properties were characterized. The mixed ferrites were included in magnetoliposomes, forming the magnetic core, either within an aqueous phase (aqueous magnetoliposomes, AMLs) or surrounded by a lipid bilayer without water (solid magnetoliposomes, SMLs). Curcumin was encapsulated in these novel nanosystems and its release rate was evaluated under an alternating magnetic field. The influence of magnetoliposomes PEGylation in curcumin location and release was also investigated. The developed nanosystems are promising for applications as curcumin nanocarriers for synergistic cancer therapy (using a combined approach of magnetic hyperthermia and chemotherapy), considering the already reported antitumor properties of curcumin.
3. Materials and Methods
Ultrapure water Milli-Q grade (MilliporeSigma, St. Louis, MO, USA) and spectroscopic grade solvents were used in all preparation steps.
3.1. Preparation of CaxMg1−xFe2O4 Ferrite Nanoparticles
Calcium-substituted magnesium ferrite nanoparticles (CaxMg1−xFe2O4) were prepared by coprecipitation method. First, an aqueous solution containing magnesium sulfate, hydrated calcium acetate at the corresponding molarity (for x = 0.25, 0.50 or 0.75), and iron (III) chloride hexahydrate was prepared, in a 1:2 molar ratio. The resulting mixture was added, drop by drop, to a 90 °C heated solution of sodium hydroxide at 5 M, under constant and vigorous magnetic stirring. The precipitated nanoparticles were washed by several cycles of centrifugation (5000 rpm for 5 min) and redispersed in water. Finally, the nanoparticles were calcined at 600 °C for 30 min.
3.2. Preparation of Magnetoliposomes and GUVs
For aqueous magnetoliposome (AML) preparation, where magnetic nanoparticles are encapsulated in liposomes, an ethanolic solution of egg yolk–phosphatidylcholine (Egg–PC, from Sigma-Aldrich, St. Louis, MO, USA), was injected, under vortex, onto an aqueous solution containing 0.1 mM of nanoparticles, to a final lipid concentration of 1 mM (ethanolic injection method [
58,
59]). Then, the ferrofluid was washed with water by magnetic decantation to eliminate the nonencapsulated nanoparticles. Curcumin was incorporated into AMLs by the coinjection method, as previously described [
22].
The preparation method of solid magnetoliposomes (SMLs), consisting in a lipid bilayer surrounding a cluster of magnetic nanoparticles, was previously developed by us and the formation of the liposomal structure has been confirmed in previous works [
22,
24], using the lipid DPPC (1,2-dipalmitoyl-
sn-glycero-3-phosphocholine, from Sigma-Aldrich, St. Louis, MO, USA). First, 10 μL of a solution of magnetic nanoparticles (0.02 mg/mL) were ultrasonicated for one minute at 189 W, and 3 mL of chloroform was added to the solution, resulting in the formation of nanoparticle clusters. To form the first lipid layer, the solution containing the clusters was heated up to 55 °C (above the melting temperature of DPPC, 41 °C [
46]) and 150 µL of a lipid methanolic solution (20 mM) was injected under vortexing. Magnetic decantation and several washing steps with ultrapure water were performed for purification, removing lipid aggregates and liposomes without a magnetic core. For the second lipid layer formation, an aqueous solution of the purified systems (3 mL) was heated up to 55 °C and 150 µL of a lipid methanolic solution (20 mM) was injected under vortexing. The resulting solid magnetoliposomes were then washed and purified with ultrapure water by centrifugation. Curcumin was incorporated by injection of an ethanolic solution, under vortexing, right before the formation of the second lipid layer.
Giant Unilamellar Vesicles (GUVs) were used as models of cell membranes and were prepared using a procedure previously described [
44,
45], using a 1 mM solution of L-α-phosphatidylcholine (Soybean lecithin, from Sigma-Aldrich, St. Louis, MO, USA).
3.3. Characterization of Nanoparticles and Magnetoliposomes
Magnetization measurements were carried out in a MPMS3 SQUID magnetometer MPMS5XL (Quantum Design Inc., San Diego, CA, USA). The hysteresis cycles (magnetization versus magnetic field) of the samples were measured in the convenient field range for each sample. The measurement method was by DC extraction or VSM oscillation at a frequency of 14 Hz. A specific magnetic field correction for the trapped flux in the superconducting coil was made achieving an accuracy of residual less than 2 Oe.
X-Ray diffraction (XRD) analyses were performed in a conventional Philips PW 1710 (Royal Philips, Amsterdam, The Netherlands) diffractometer, operating with CuKα radiation, in a Bragg–Brentano configuration.
HR-TEM images were obtained at C.A.C.T.I (Centro de Apoio Científico e Tecnolóxico á Investigación), Vigo (Spain) using a Transmission Electron Microscope JEOL JEM 2010F (JEOL Ltd., Tokyo, Japan) operating at 200 kV. TEM images were processed using ImageJ software, version 1.52 p (National Institutes of Health (NIH), Bethesda, MD, USA).
Hydrodynamic diameters, polydispersity, and zeta potential values were determined in a Dynamic Light Scattering (DLS) equipment NANO ZS Malvern Zetasizer (Malvern Panalytical Ltd., Malvern, UK) at 25 °C, performing five independent measurements for each sample.
3.4. Spectroscopic Measurements
Absorption spectra were obtained in a Shimadzu UV-3101PC UV–Vis-NIR (Shimadzu Corporation, Kyoto, Japan) spectrophotometer. Fluorescence spectra were recorded in a Fluorolog 3 spectrofluorimeter (HORIBA Jobin Yvon IBH Ltd., Glasgow, UK), all spectra being corrected for the instrumental response. The steady-state fluorescence anisotropy, r, was measured in the latter equipment, using Glan–Thompson polarizers.
FRET assays were carried out as previously reported [
22,
24,
41], allowing confirming the formation of the double lipid layer in SMLs and the interactions with model membranes. The detailed procedure is described in the
Supplementary Material. The fluorescence quantum yield of the dye NBD (donor) in magnetoliposomes (containing the different magnetic nanoparticles) was determined by the standard method [
60,
61], using the labeled lipid NBD-C
12-HPC in lipid membranes as reference, with Φ
r = 0.32 at 25 °C [
62].
3.5. Drug Release Studies
In total, 0.5 mL of curcumin-loaded magnetoliposomes solution was placed in Amicon® Ultra-0.5 mL centrifugal filters with 0.1 µm pore size (under mild shaking conditions) containing a GUVs solution in the bottom container, with GUVs being used as acceptor membrane models for curcumin. At 30 min intervals (for 4.5 h), 200 µL were collected from the acceptor compartment for assessing curcumin concentration, and an equal volume of fresh GUVs were added. The fluorescence of the collected solution was measured and the cumulative release of curcumin was calculated using calibration curves previously obtained. Three independent release assays were performed.
Since magnetoliposomes were envisioned as magnetic responsive nanosystems, the release profile of curcumin was also assessed under the actuation of an AMF. The AMF was generated in a custom-designed solenoid device (800 turns per meter, length: 31 cm and internal diameter: 4.8 cm) by applying an alternating electric current. A magnetic field of 2.98 mT at 1000 kHz was used. Release profile assays were performed in triplicate.