Synthesis and characterization of liposomes nano-composite-particles with hydrophobic magnetite as a MRI probe

Applied Surface Science 376 (2016) 252–260

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Synthesis and characterization of liposomes nano-composite-particles with hydrophobic magnetite as a MRI probe Limin Han, Xingping Zhou ∗ College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China

a r t i c l e

i n f o

Article history: Received 25 November 2015 Received in revised form 15 March 2016 Accepted 21 March 2016 Keywords: Nano-magnetic liposomes Hydrophobic Fe3 O4 nanoparticles Thin film dispersing method Oil-water interface method MRI

a b s t r a c t Nano-magnetic liposomes (MLs) consist of liposomes and magnetic nanoparticles (MNPs). Due to the active surfaces of liposomes, various functional groups can be attached for ligand-specific targeting. Here, we describe synthesis of magnetic nano-composite liposomes (HMLs) by a thin film dispersing method, based on hydrophobic magnetite (Fe3 O4 ) nanoparticles. The results showed that the particle diameter of the HMLs containing Fe3 O4 OA NPs at a final Fe loading of 11.02 g/mol phosphatidylcholine (POPC) mainly in a sandwich-structure was 125.3 ± 12.9 nm determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). While the initial Fe concentration in the solution varied from 0.25 to 3.0 mg/mL, an effective Fe3 O4 NPs loading was achieved, with encapsulation efficiency (EE%) from 91.0% to 71.0%. Subsequently, the HMLs were confirmed to be quite cytocompatible and hemocompatible in the applied concentration range by MTT and hemolysis assays. We also found that HMLs had more advantages than those liposomes with hydrophilic Fe3 O4 NPs by comparing their EE% and r2 relaxivity. Finally, it was concluded that the analyzed Fe concentration in HMLs was sufficient to produce a pronouncedly weak signal for MRI in vitro to enhance the contrast between tumors and normal tissues. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Tumor is a kind of commonly multiple diseases, and threatens human beings seriously as it is hardly cured in the later stage. It becomes quite important for controlling and diagnosing the disease earlier [1]. Magnetic resonance (MR) technique is a kind of clinically important noninvasive test methods to discover tumors earlier due to its outstanding advantages such as high sensitivity, spatial resolution, and strong specificity [2–4]. For MR imaging (MRI) of tumors, the use of contrast agents is necessary to increase the contrast between the lesion sites and normal tissues in order to promote the sensitivity and definition of diagnosis in the process of MRI. Recent advances in nanoscience and nanotechnology have developed various contrast agents for MRI applications, such as Gd(III)- or Mn(II)-based T1 MR and magnetic nanoparticlesbased T2 MR contrast agents [5–7]. Among the used contrast agents, super-paramagnetic magnetite (Fe3 O4 ) nanoparticles (NPs) have been widely developed as negative contrast agents that can reduce the T2 relaxation time of water protons and avoid some disadvantages of Gd-DTPA-based T1 MR contrast agents [2–4,8], such as a

∗ Corresponding author. E-mail address: [email protected] (X. Zhou). http://dx.doi.org/10.1016/j.apsusc.2016.03.164 0169-4332/© 2016 Elsevier B.V. All rights reserved.

large amount of clinical injection, great toxicity and side effects due to its high contents of the heavy metal elements [9]. The most important features for nano-materials to be used as contrast agents of MRI are biocompatibility, a long blood circulation time, and a good colloidal stability especially for Fe3 O4 NPs. The modification of hydrophilic and non-cytotoxic polymers on the surface of Fe3 O4 NPs has been proven to be an effective strategy to make the particles meet the above requirements [8,10,11]. At present, various polymers such as chitosan, dextran, and polyethylene glycol have been successfully coated onto the surfaces of Fe3 O4 NPs for biomedical applications [4,6,8]. However, there are still some disadvantages and challenges. For example, nanoparticles can trigger allergic reactions occasionally [12] and contrast agents can be eliminated by endocytosis of the organism in vivo [13–15]. It was reported that the above disadvantages could be avoided by embedding magnetic particles in liposomes [16]. As liposomes are a kind of artificial membranes similar to cell membrane, their biocompatibility enables them to be utilized as carriers, either for therapeutics or for diagnostics in vivo [17–19]. Also, liposomes as a kind of vesicular systems are formed when phospholipids are dispersed in aqueous solution, through self-assembling into one or more concentric bilayers surrounding an aqueous core [20]. In addition, liposomes are firmly bonded to the surfaces of magnetic nanoparticles previously modified by various materials (eg, dex-

L. Han, X. Zhou / Applied Surface Science 376 (2016) 252–260

tran, folic acid, or oleic acid) [20] to avoid endocytosis of cells. Therefore, these former mentioned problems can be promisingly solved by using the liposomes entrapped with magnetic NPs. Magnetic liposomes were firstly proposed and obtained by Margolis et al. [21–23], and used to illustrate a kind of nanoscale magnetic phospholipids complex [24]. Recently, people tend to research the liposomes containing hydrophilic nano-magnetic particles mainly due to the easy operation for forming nanocomposite materials in the aqueous system [25–27], while studies on liposomes containing hydrophobic nano-magnetic particles in membranes are rarely reported. Then, for liposomes with hydrophilic nano-magnetic particles, there are still some deficiencies. For instance, the magnetic NPs can easily be leaked, leading to injurious effects on biological systems due to directly contacting human tissues. Bare magnetic NPs may gather to large secondary particles in most of human tissues, causing the cytotoxic activity of the body’s oxygen metabolism [28]. However, for liposomes entrapped with hydrophobic nano-magnetic particles, most of the side-effects from the magnetic cores can be eliminated, as the magnetic cores are completely separated from the aqueous environments, to access higher stability, better biocompatibility, and a strong magnetic response. Thus, to achieve this aim, in our approach, hydrophobic Fe3 O4 NPs were firstly synthesized at room temperature by a novel oil-water interface method, with a size comparable to the thickness of a lipid bilayer [29]. Also, liposomes embedded with the hydrophobic magnetic NPs (HMLs) were formed (Scheme 1). Their physicochemical properties were characterized, including Fe loading capacity, encapsulation efficiency, particle size, morphology, and zeta potential. Moreover, the biocompatibility of the HMLs was tested by MTT and hemolysis assays, and their magnetic properties were also investigated by MRI of Hela cells in vitro. 2. Methods and materials 2.1. Materials The following raw chemicals were used for preparing magnetic nanoparticles (Fe3 O4 NPs) and magnetoliposomes (HMLs). Ferric chloride hexahydrate (FeCl3 ·6H2 O, » 99.0%, AR), ferrous sulfate heptahydrate (FeSO4 ·7H2 O, > 99.0%, AR), oleic acid (OA, C18 H34 O2 , > 97.0%, AR), chloroform (CHCl3 , AR), ammonia solution (25–28%, AR), cyclohexane (C6 H12 , > 99.5%, AR), cholesterol (C26 H46 O, AR), methanol (CH4 O, AR), dimethyl sulfoxide (DMSO), 1,6-Diphenyl-1,3,5-hexatriene (DPH), and potassium thiocyanate (KSCN, > 99.0%), were purchased from Sinopharm Chemical Reagent Co., Ltd., China. 1, 2-diacyl-sn-glycero-3-phosphocholine (lecithin, phosphatidylcholine (POPC), > 90%) was acquired from Aladdin Industrial Corporation (Shanghai, China). MTT was gained from Shanghai Sango Biological Engineering Technology & Services Co., Ltd., China. Hela cells were obtained from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). Ultrapure water and nitrogen were used in the whole experiment process.

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elevated to 40 ◦ C with vigorous agitation. Subsequently, 10 mL of concentrated ammonia was added dropwise and slowly. After that, the solution color was found to alter from orange to black, leading to a black precipitate. Ultrapure water was then added to wash the precipitates to remove un-reacted chemicals and this procedure was repeated for 5 times. Finally, the precipitate was re-dispersed in chloroform by ultrasonic treatment for 30 min and stored in refrigerator at 4 ◦ C. 2.3. Synthesis of liposomes containing hydrophobic magnetic nanoparticles (HMLs) HMLs were obtained by the film dispersing method [30]. In brief, 40 mg lecithin and 20 mg cholesterol were dissolved in 8.0 mL chloroform–methanol (2:1, V/V) solvent. Subsequently, Fe3 O4 OA NPs (0.5 mg/mL) were dispersed in chloroform and then transferred into the lecithin chloroform-methanol solution with ultrasonic treatment. Then, this suspension was evaporated at 45 ◦ C in a N2 stream. Lasting to the dryness point, the resulted thin film in the bottom of the flask was left in a vacuum chamber overnight to obtain a drier thin film. After that, the thin lipid film was hydrated with 20 mL ultrapure water and the liposomal suspension was stirred for 6 h in N2 atmosphere. Finally, the liposomal suspension was shocked for 15 min by ultrasonic treatment, followed by agitation of 30 min, and this operation cycle was repeated for 3 times to get the initial HMLs. Extrusion method was applied to get the HMLs with a high uniformity. Primarily, the liposomal suspension was filtered through a polycarbonate membrane of 220 nm and this operation was repeated for 5 times. Subsequently, the liposomal suspension after being filtered was centrifuged at 4 ◦ C for 10 min at 10,000 rpm for 3 times to remove the unsettled free small magnetic particles. Finally, the HMLs were re-suspended in 20 mL ultrapure water and stored at 4 ◦ C. 2.4. Chemical characterization methods The Fe3 O4 OA NPs and HMLs were characterized by using Fourier transform infrared spectroscopy (FT-IR, Avatar380, Thermoelectric group Co., Ltd., USA) and X-ray diffraction (XRD, D/max-2550/PC, Rigaku Corporation, Japan) with Cu k␣ radiation (␭ = 0.154 nm) at 40 kV and 200 mA to identify the dominant phase of the samples. The thermal behavior of the Fe3 O4 OA NPs was evaluated through a thermo gravimetric analyzer (TGA, F1 209, NETZSCH Instruments Co., Ltd., Germany). 2.5. Particle size and zeta potential measurements The particle size and size-distribution of the HMLs were valued by dynamic light scattering (DLS) with a standard 633 nm laser using the particle size analyzer (Zestasizer Nano ZS ZEN3600, Malvern Instruments, U. K.). The samples were filtered through a polycarbonate membrane of 220 nm before measurement. Zeta potential of the HMLs was analyzed in a PBS (pH 7.2) solution by the same instrument, and the samples prepared two weeks ago were also measured to investigate their stability.

2.2. Preparation of magnetic nanoparticles (Fe3 O4 NPs)

2.6. Microscopy observation and steady state fluoresence measurements

Fe3 O4 NPs were synthesized by the novel oil-water interface method. Briefly, FeCl3 ·6H2 O (1.38 g) and FeSO4 ·7H2 O (0.95 g) were fully dissolved in 8.5 mL ultrapure water. The resulting Fe salt solution was added into a three-necked flask containing 30 mL cyclohexane and then stirred for 30 min, followed by the dropwise addition of 1.0 mL oleic acid. Hereafter, the temperature was

Transmission electron microscopy (TEM) was applied with a JEOL 2010F transmission electron microscope (JEOL, Japan) at 200 kV to visualize the microscopy of the Fe3 O4 OA NPs and HMLs. For TEM observation of the HMLs, we took appropriate HMLs suspension and 1.0% phosphotungstic acid solution with equal volumes to mix well through ultrasonic treatment of 1 h.

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Scheme 1. Formation Process of HMLs for MR imaging.

1,6-Diphenyl-1,3,5-hexatriene (DPH) can assess specific alterations in the fluidity of lipid assemblies by being inserted into the liposomal membrance [20]. Based on that, the current HMLs and the liposomes containing hydrophilic nano-magnetic particles were compared with each other. In brief, DPH tertrahydrofuran solution was added into the chloroform-methanol solution when the phospholipid film was formed. The responsive fluorescence anisotropy was measured using a fluorescence spectrometer with polarization accessory unit. Steady-state fluorescence anisotropy (r) was calculated as: r = (I| − g I⊥ )/(I| + 2 g I⊥ ), in which g = I⊥ /I| .I| and I⊥ are the parallel and perpendicular fluorescence intensity of the components observed corresponding to the plane of polarization of the excitation beam, respectively [29]. 2.7. Determination of HMLs encapsulation efficiency (EE%) EE% is expressed as g Fe/mol POPC × 100 and this value is given by hydrophobic or hydrophilic Fe3 O4 NPs embedded in liposomes. Fe content of the liposomes was determined photometrically using KSCN [20]. In details, 50 ␮L of liposomal solution was mixed to 12.5 ␮L of Triton X-100 to break the liposomes and the Fe3 O4 OA NPs were released to give ferric ions with the aid of 0.563 mL concentrated HCl (37.0%). Hereafter, the samples were incubated for 3–5 min with 0.625 mL of 40 mM KSCN aqueous solution. The product of the reaction between the anion (SCN− ) and the Fe3+ was a red-colored complex-pentaaqua (thiocyanate-N) whose absorbance (A) at 480 nm was obtained by UV spectrophotometer. HMLs and Blank liposomes (BLs) were separately dealt with by using hydration fluid with quinine sulfate (0.1 mg/mL) to compare their drug loading capacity. A certain amount of Triton-X 100 was added to destroy the liposomes, and the absorbance (A) was mea-

sured by UV spectrophotometer at 388 nm. Finally, we calculated free quinine sulfate liposomes content according to the standard curve equation: EE% = (1-wfree /wtotal ) × 100%.

2.8. Biocompatibility test 2.8.1. Cytotoxicity assay To check the biocompatibility of HMLs in vitro, we selected Hela cells to investigate the cytotoxicity of the HMLs using MTT assay. MTT method is usually used to detect cell vitality and growth, mainly depending on the cell’s reduction capacity to the yellow tetrazolium salt into a colored formazan product [31]. Briefly, Hela cells were seeded in the 96-well plates at a density of 10,000 cells per well and cultured for 24 h in 200 ␮L of RPMI1640 medium with 10% FBS in a humidified atmosphere containing 5.0% CO2 . After overnight incubating to bring the cell to confluence, the cells were then incubated for 24 h in the fixed volume of fresh medium containing pure PBS buffer (control), and in the same volume of the HMLs with various concentrations (62.5, 125, 250, 375, and 500 ␮g/mL), respectively. Subsequently, this medium was replaced with 200 ␮L of fresh medium containing 20 ␮L of MTT solution (0.5 mg/mL in PBS buffer) to detect the metabolically active cells and the cells were continuously incubated for 4 h at 37 ◦ C. Afterwards, 150 ␮L of dimethyl sulphoxide (DMSO) was added to each well in order to dissolve the insoluble formazan crystals. Finally, the absorbance of the HMLs with some concentration in each well was detected by using an Enzyme-linked immune detector (Thermo scientific, USA) at 490 nm. Each sample was measured for 5 times to calculate the standard deviation.

Table 1 Zeta potentials and hydrodynamic sizes of HMLs with various Fe concentrations. Sample

(mg/ml)

Zeta potential value (mv)

1–0 1–1 1–2 1–3 1–4

blank 0.25 0.5 1.0 3.0

−3.970 ± 0.80 −26.21 ± 0.03 −26.38 ± 0.01 −26.40 ± 0.01 −29.37 ± 0.05

Hydrodynamic size (nm) 345.1 ± 10.5 132.1 ± 9.9 127.1 ± 2.2 125.2 ± 2.0 148.8 ± 3.0

Polydispersity index (PDI) 0.533 ± 0.043 0.190 ± 0.022 0.210 ± 0.005 0.241 ± 0.027 0.249 ± 0.003

L. Han, X. Zhou / Applied Surface Science 376 (2016) 252–260 Table 2 The DPH anisotropy loaded in liposomes in different temperatures. T(K)

Liposome type

Anisotropy of DPH (r)

218

BLs HMLs MLs-W

0.0859 ± 0.00133 0.0475 ± 0.00215 0.0846 ± 0.00128

318

BLs HMLs MLs-W

0.0422 ± 0.00063 0.0400 ± 0.00550 0.0428 ± 0.00168

Notes: BLs is blank liposomes; MLs-W is liposomes with hydrophilic Fe3 O4 NPs in the inner aqeous core; HMLs means loading with Hydrophobic Magnetic Fe3 O4 NPsLiposomes.

2.8.2. Hemolysis test Blood was collected from rats in a centrifuge tube (2.0 mL) with 0.05 mL of heparin solution (20 mg/mL) to prepare a fresh anticoagulant. Then, the fresh anticoagulant was centrifuged at 1000 rpm for 10 min and purified by successive rinsing with PBS buffer for 5 times. Then, 50 mL of PBS buffer solution was added to prepare the red blood cell suspension. We designed 8 parallel experiments. No. 1 to No. 6 tubes were respectively filled with 0.2 mL of blood diluent and 0.8 mL of PBS containing HMLs suspension with various concentrations (37.5–500 ␮g/mL). Then, 0.2 mL of blood diluent was added to No.7 tube with 0.8 mL of normal saline as a negative control and 0.2 mL of blood diluent was added to No.8 tube with 0.8 mL of ultrapure water as a positive control. After a gentle shaking of 30 min, the mixtures were incubated for 2 h in a 37 ◦ C water bath. Then, the samples were centrifuged at 10,000 rpm for 1 min and the hemolysis percentages of the HMLs were calculated with dividing the difference in the absorbance at 541 nm between the samples and the negative control by the difference in the absorbance at 541 nm between the positive and negative controls.

2.9. T2 relaxivity measurement and In vitro MR imaging experiments 2.9.1. T2 relaxivity measurement T2 relaxation time was measured by a 0.5T NMI20-Analyst NMR Analyzing and Imaging system (Shanghai Niumag Corporation, China). The samples were diluted in water with Fe concentration in the range of 0.0025–0.05 mM. The instrumental parameters were set at a point resolution of 156 mm × 156 mm, a section thickness of 0.6 mm, TR of 4000 ms, and TE of 60 ms. The T2 relaxivity was calculated by a linear fit of the inverse T2 (1/T2 ) relaxation time as a function of Fe concentration.

2.9.2. In vitro MR imaging experiments Hela cells were seeded in 6-well plates at a density of 3.0 × 106 cells per well in 2.0 mL of RPMI 1640 medium and incubated at 37 ◦ C in atmosphere of 5.0% CO2 . After overnight culturing to bring the cells to confluence, the medium was replaced with 2.0 mL fresh medium containing PBS buffer (control), or HMLs at different Fe concentrations (0.15, 0.3, 0.6 and 0.75 mM), and then the cells were incubated at 37 ◦ C in the atmosphere of 5.0% CO2 for 6 h. After the cells were washed with PBS buffer solution for 5 times, they were then trypsinized, centrifuged, and re-suspended in 1.0 mL PBS (containing 0.5% agarose) in a 2.0 mL Eppendorf tube before MR imaging. T2 MR imaging of the cell suspension in each sample tube was performed by a 0.5 T NMI20-Analyst NMR Analyzing and Imaging system (Shanghai Niumag Corporation) using a wrist receiver coil with a CPMG sequence and the instrumental parameters were set according to those for T2 relaxivity measurement.

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3. Results and discussion 3.1. Characterization of Fe3 O4 OA NPs by XRD and TGA XRD (Fig. A.1) of OA-coated Fe3 O4 NPs showed that the lattice spacing at 2-theta angle range of 21.3, 33.2, 36.7, 41.2, 53.2 and 61.5◦ matched well to those from the JCDS card (220, 311, 400, 422, 511, and 440 planes of magnetite Fe3 O4 crystals), respectively [11,32,33]. To identify OA on the surfaces of Fe3 O4 NPs, chemical structure of the synthesized-COO- conjugate was characterized by TGA and FT-IR (Fig. A.2). The FT-IR data of OA-coated Fe3 O4 NPs revealed the OA adsorption on magnetic particles. The absorption bands of OA were evident (Fig. A.2a). After OA coating on surfaces of magnetic particles, the spectra displayed the characteristic bands of OA at 2921.92, 2853.17, 1525.43, and 1416.25 cm−1 [11]. In addition, the characteristic bands at 1525.43 and 1416.25 cm−1 were attributed to the asymmetric and symmetric COO stretches, indicating the attachment of oleic acid chain to the surfaces of Fe3 O4 NPs [34]. Fig. A.2b illustrates TGA curves of the OA Fe3 O4 NPs and blank Fe3 O4 NPs. It can be seen that the thermogram of OA Fe3 O4 NPs exhibits three different stages in comparison to the blank Fe3 O4 NPs. Water, ethanol, and cyclohexane are evaporated in the range of 30–240 ◦ C which is considered as the first stage. The decomposition stage around 300–400 ◦ C in the TGA curve is attributed to the interaction between the OA and the Fe3 O4 NPs which postpones the decomposition of OA. Actually, the Fe3 O4 OA NPs exhibit a weight loss of 11% due to OA coating. The residue at 700–800 ◦ C is ascribed to the combination of OA and Fe3 O4 NPs. Thus, the weight percentage of the Fe3 O4 OA NPs is calculated from the weight residue of the NPs at 700–800 ◦ C. The formed Fe3 O4 OA NPs are stably dispersed in common organic solvents. 3.2. Characterization of Fe3 O4 OA NPs and HMLs by TEM and DLS TEM has been used to observe the morphology and size distribution of the formed Fe3 O4 OA NPs and HMLs (Fig. 1). It is clear that the size of the spherical NPs is 4.4 ± 1.3 nm for Fe3 O4 OA NPs (Fig. 1(a)) and 125.3 ± 12.9 nm for HMLs obtained with 0.5 mg/mL Fe concentration (Fig. 1(b)), respectively. It is also found that the Fe3 O4 OA grains (the dark dots) are tightly packed in the lipid bilayer. As shown in Table 1, for hydrodynamic size of the samples, it is revealed that the average sizes of BLs and HMLs containing different Fe concentrations (0.25, 0.50, 1.0 and 3.0 mg/mL) are 345.1, 132.1, 127.1, 125.2, and 148.8 nm, respectively. Also, from the TEM images shown in Fig. 2, the HMLs with an average size of 123.3 nm are almost in a spherical shape when the initial Fe concentration embedded in liposomes was 0.5–2.0 mg/mL. By comparing size and morphology of the HMLs with the composite liposomes embedded with hydrophilic Fe3 O4 NPs, the hydrophobic properties of the magnetic particles affected markedly the particle size and polydispersity of liposomes, caused by the rendering of the inner magnetic nanoparticles to avoid the fusion among the molecules of liposomes. In addition, the size of the HMLs decreased with the increase in hydrophobic magnetic particles (Fig. A.3). Also, zeta potential measurement was employed to investigate potential changes in particle surfaces for BLs and HMLs. As shown in Table 1, the BLs and HMLs are negatively charged with surface potential of −3.97 and −27.09 mv, respectively. The negative charge of the HMLs may be due to OA coating on Fe3 O4 , further demonstrating that Fe3 O4 OA NPs are successfully embedded into liposomes [20]. Additionally, the location of Fe3 O4 OA NPs in the lipid bilayer was also observed from the TEM results (Fig. A.4). As shown in Fig.

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Fig. 1. TEM micrographs and size distribution histograms of (a) Fe3 O4 OA NPs without liposomes (50 ␮g/mL); (b) HMLs with loading of Fe3 O4 concentration. The mass ratio of Fe3 O4 OA NPs to lecithin in HMLs preparation was 0.04:1.

OA NPs at 0.5 mg/mL in Fe

Fig. 2. TEM micrographs of HMLs with various Fe concentrations (a: 0.5 mg/mL, b: 1.0 mg/mL, c: 2.0 mg/mL, d: 3.0 mg/mL).

A.4, the HMLs are formed in single chamber (white arrows) and sandwich-structured (black arrows) liposomes. The results are similar to those shown as type and in Scheme 1 [20,29,33,35,36]. As shown in Fig. A.4a,b,c (black arrows) and 4d, for most of the HMLs, fusion phenomenon occurs for forming sandwich-structured magnetoliposomes and Fe3 O4 OA NPs were embedded into the

lipid bilayers of multicellular liposomes to produce the structure displayed as Type in Scheme 1. In addition to the morphology characterization above, the fluorescence anisotropy is applied to further verify the location of Fe3 O4 OA NPs in the liposomes. Generally, the relatively more flexible structure with better membrane fluidity gives the higher rate

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Table 3 Fe quantification of HMLs with different initial Fe concentrations via colorimetric assay using KSCN and encapsulation efficiency estimation of purified HMLs. Sample (mg/mL) 2–1 2–2 2–3 2–4

Initial Fe loading of HMLs (g Fe/mol POPC) 0.25 0.5 1.0 3.0

6.4 ± 0.06 13.0 ± 0.27 25.0 ± 0.86 77.0 ± 5.60

Final Fe loading of HMLs (g Fe/mol POPC) 5.83 ± 0.09 11.02 ± 0.12 19.5.0 ± 0.93 55.2 ± 4.30

EE (%) 91 ± 1.0 84 ± 4.3 78 ± 3.8 71 ± 6.9

Notes: Percentage of encapsulation efficiency was calculated from the ratio of the Fe concentration in HMLs before and after purification (EE% expressed as g Fe/mol POPC × 100).

Fig. 3. (a) Hela cells viability from MTT assay after incubation with HMLs and Fe3 O4 OA NPs containing different Fe concentration (0–500 ␮g/mL) for 24 h. Fe3 O4 OA NPs were transferred to the aqueous solution and hela cells treated with PBS were used as control. (b) Hemolysis results of the HMLs at different Fe concentrations (37.5, 75, 150, 300, and 500 ␮g/mL). Water and PBS were used as a positive and negative control, respectively.

of DPH’s rotational motion with lower anisotropy [29]. As shown in Table 2, the DPH anisotropy for MLs-W is similar to that for BLs at 278 K, revealing that the immediate environment of DPH in MLs-W is not influenced by the hydrophilic Fe3 O4 NPs which are located in the inner aqueous core of liposomes. However, the anisotropy value of DPH in HMLs is lower than those of BLs and MLs-W at the same temperatures. Combined with the results above, the obvious influence from the hydrophobic Fe3 O4 NPs is probably due to that the NPs are encapsulated within the lipid bilayers so as to disrupt the original regular arrangement of lipid molecules. When the temperature rises to 318 K, there is no obvious difference among DPH anisotropy value for all samples, probably caused by the liquid crystal state of the lipid in the film.

cannot promote the absolute yield. Additionally, we have also found that EE% of quinine sulfate in BLs is 90.0%, while it is about 82.0% with the HMLs containing 0.5 mg/mL Fe, probably caused by the interaction of liposomes with the magnetic nanoparticles and quinine sulfate. Encapsulation efficiency of liposomes with hydrophilic Fe3 O4 NPs was also investigated as a control. Only low encapsulation efficiencies of 8.0% and 22.0% were obtained by using initial Fe concentrations of 3.0 and 0.25 mg/mL, respectively. Therefore, the magnetic liposomes with hydrophobic Fe3 O4 NPs possess larger EE% and Fe content than those with hydrophilic Fe3 O4 NPs, displaying the significance of the thin film dispersing method.

3.3. Magnetic liposomes encapsulation efficiency

3.4. Cytotoxicity of materials

Immediately after extrusion, un-encapsulated magnetic nanoparticles were removed by centrifugation. Then, Fe content of HMLs was determined by a colorimetric test using KSCN, and the encapsulation efficiency (EE%) was calculated. In addition, the quinine sulfate concentration in the liposomes was determined by the standard linear equation, and the EE% was calculated using the formula as described previously. The corresponding results are shown in Table 3. We can see that the EE% decreases with increasing concentration of Fe3 O4 NPs used for loading. Also, the highest encapsulation efficiency of 91.0% is found for the HMLs with 0.25 mg/mL of Fe concentration. This is in good agreement with the results obtained by Sabate et al. that the EE% is inversely proportional to the initially applied Fe content [37]. However, for the HMLs with 3.0 mg/mL of Fe concentration, the high EE% is still achieved up to 70.0%, much larger than that in the range of 20–60% reported by others [38]. Regardless of this fact, the absolute amount of Fe embedded into liposomes is also enough high. Further increase in Fe concentration (over 3.0 mg Fe/mL)

For biomedical materials, cytocompatibility is an important indicator for their application. Hence, it is essential to assess the cytotoxicity of the HMLs before applying them to MR imaging and that of Fe3 O4 OA NPs as well for comparison. After incubation of Hela cells with HMLs and Fe3 O4 OA NPs at Fe concentration of 62.5, 125, 250, 375, and 500 ␮g mL for 24 h, MTT assay is applied to evaluate the cell viability. For the HMLs, as shown in Fig. 3(a), the viability of the cells is close to 100% with no obvious change in comparison to the PBS control. However, the cell viability in the case of Fe3 O4 OA NPs is less than 80%, obviously lower than that for the PBS control, indicating their decisive cytotoxicity. Therefore, it can be concluded that the HMLs have no obvious cytotoxic effects to Hela cells at the proper concentration range. More importantly, it is quite significant that Fe3 O4 OA NPs embedded in liposomes can effectively reduce the toxicity from the magnetic particles, showing the availability to their in vivo MR imaging application. With no doubt, location of the magnetic particles in such a special structure with the aid of liposomes plays a key role.

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Fig. 4. (a) Linear fit of 1/T2 of the HMLs and liposomes with hydrophilic Fe3 O4 NPs with Fe concentrations (0.003, 0.006, 0.01, 0.02, 0.03, and 0.05 mM), TE=60 ms. (b) Demonstration of magnetic properties of HMLs.

Fig. 5. (a)T2 -weighted MR images and (b) signal intensity analysis of Hela cells after treated with PBS and samples with different Fe concentrations (0.15, 0.3, 0.6, and 0.75 mM).

3.5. Hemolysis assay Hemocompatibility is a key point for in vivo applications of nanoprobes. The vitro hemolysis test of the HMLs was carried out by a V–530 ultraviolet spectrophotometer. As shown in Fig. 3(b), the result is similar to the PBS (negative control). In contrast, the red cells exposed to water (positive control) display apparent hemolytic behaviors. Hemolysis rates with different Fe concentrations (500, 300, 150, 75, and 37.5 ␮g/mL) have been quantified based on the absorbance of the supernatant at 541 nm. The hemolysis percentages of the HMLs are 3.6%, 2.5%, 1.9%, 1.8%, and 1.7%, respectively. Since they are all less than 5.0% in the analyzed

concentration range (37.5–500 ␮g/mL), the outstanding hemocompatibility is achieved [8].

3.6. T2 enhancing capability evaluation Fe3 O4 NPs are known to be used as T2 negative contrast agents, which are able to decrease the MRI signal intensity by dephasing transverse magnetization and reducing the transverse time value of T2 -weighted imaging. To evaluate T2 enhancing capability of the HMLs and liposomes with hydrophilic Fe3 O4 NPs, we measured the transverse relaxivity (r2 , the transverse relaxation rate per mM of Fe) and the results are shown in Fig. 4(a). Obviously, the T2 relax-

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ation time of HMLs decreases as Fe concentration increases and the trend is well fit by a straight line within the analyzed range of Fe concentration, and the r2 of HMLs is calculated to be 98.5 mM−1 s−1 . However, for the liposomes with hydrophilic Fe3 O4 NPs, the T2 relaxation time is calculated to be 79.6 mM−1 s−1 and reported to be 45.7 mM−1 s−1 by G. Bealle et al. [39], obviously lower than for HMLs, further exhibiting the significance of hydrophobic Fe3 O4 NPs embedded in such a special structure. Fig. 4(b) demonstrates magnetic properties of the HMLs. The HMLs are strongly attracted by an external magnetic field due to their Fe3 O4 OA NPs, revealing that Fe3 O4 OA NPs are still magnetically sensitive while they are embedded in the liposomes. 3.7. MR imaging of tumor cells in vitro To further confirm the targeting ability to tumor cells and inspect the MR imaging performance of the HMLs, Hela cells were treated with the HML suspensions with various Fe concentrations (0.15, 0.3, 0.6, and 0.75 mM) at 37 ◦ C in atmosphere of 5.0% CO2 for 6 h. Liposomes with hydrophilic Fe3 O4 NPs were also tested for comparison. From the T2 -weighted MR scanning images shown in Fig. 5(a), the MR signal intensity decreases with the increase of Fe concentration. However, the decrease trend of MR signal in case of hydrophilic Fe3 O4 NPs is much weaker than that for the HMLs. Then, by drawing the T2 MR signal intensity of the Hela cells as a function of Fe concentration, as shown in Fig. 5(b), we can clearly find the varying trend. This trend is mainly due to the decrease in signal intensities of the MR scans while the actual Fe concentration increases, followed by the gradually enhanced contrast. It is also easy to understand that the weak contrast in the case of the liposomes containing hydrophilic Fe3 O4 NPs with inefficient encapsulation and low effective Fe concentration. In a word, these results further affirm the HMLs to be a good T2 negative contrast agent as they can markedly affect the MR signal in comparison to the liposomes with hydrophilic Fe3 O4 NPs. 4. Conclusions In summary, the purpose of this study is to synthesize and characterize HMLs with adequate properities for MRI. The HMLs embedded with hydrophobic magnetite NPs have been successfully synthesized by a film dispersing method. The obtained HMLs are mainly in a sandwich-structure and the magnetite content of HMLs can be adjusted by controlling the mass ratio of lecithin to Fe3 O4 OA NPs. As a kind of MRI agent, many advantages for the HMLs have been confirmed, such as good biocompatibility, high encapsulation efficiency, excellent magnetic properties, and prominent r2 relaxivity. Firstly, by comparing HMLs and liposomes with hydrophilic Fe3 O4 NPs, we have found EE% of liposomes with hydrophilic Fe3 O4 NPs is far lower than that of HMLs. Secondly, T2 relaxation time measurements have showed that the HMLs have a r2 relaxivity of 98.50 mM−1 s−1 , much higher than that of the liposmes with hydrophilic Fe3 O4 NPs. Thirdly, the Fe content in the HMLs is sufficient to produce a pronounced weaken signal in MRI in vitro. In a word, it is concluded that the HMLs can be used as an efficient nanoprobe for MR imaging of tumor in vitro and vivo. Potential applications for HMLs include tumor-targeting therapy and drug/gene delivery to tumors [10,11]. Acknowledgements The authors are thankful for support from hemolysis assay of Professor Hong research group and the technical supporting from Professor Shi research group in cell trials and targeted MR imaging of tumor cells in vitro.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsusc.2016.03. 164.

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