Article pubs.acs.org/Langmuir
Stabilized Magnetic Cerasomes for Drug Delivery Zhong Cao,‡,§ Xiuli Yue,§ Xiaoda Li,§ and Zhifei Dai*,† †
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, People’s Republic of China Department of Biomedical Engineering, College of Engineering, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China § State Key Laboratory of Urban Water Resources and Environment, School of Sciences, Harbin Institute of Technology, Harbin 150080, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: Doxorubicin hydrochloride (DOX)-loaded magnetic cerasomes (DLMCs) were successfully constructed by loading both hydrophobic Fe3O4 nanoparticles (NPs) and antitumor drug DOX into the aqueous interior of cerasomes via facile one-step construction. A possible explanation is that the hydrophobic Fe3O4 NPs can be trapped inside the aqueous core of cerasomes through the formation of an intermediate Fe3O4/micelle complex. It was found that the loading content of Fe3O4 in DLMCs could reach the maximum at a Fe3O4/lipid molar ratio of 4:1. Moreover, DLMCs demonstrated high superparamagnetism and responded strongly to magnetic fields. In addition, DLMCs had a high encapsulation efficiency of 43.4 ± 4.7% and a high drug loading content of 3.2 ± 1.3%. In comparison to drug-loaded liposomes, DLMCs exhibited higher storage stability and better sustained release behavior. A cellular uptake study showed that the use of an external magnetic field enables a rapid and efficient uptake of DLMCs by cancer cells, resulting in higher capability to kill tumor cells than non-magnetic drug-loaded cerasomes. This study suggests that magnetic cerasome offers a potential and effective drug carrier for anticancer applications.
1. INTRODUCTION Because of the low selectivity of the anticancer drugs toward cancer cells, they were generally administered at a very high dose to achieve clinical effectiveness, which, however, may cause severe damage to healthy cells as well. Therefore, many studies have been conducted thus far to develop effective drug delivery systems that decrease the cytotoxicity while increase the cell internalization of free drugs.1,2 Especially, the targeted drug delivery systems are preferred because of their increased efficiency of drug delivery and decreased side effects. Because of their ultrafine size, biocompatibility, and superparamagnetic properties, magnetic nanoparticles (MNPs) have attracted extensive attention in the field of targeted drug delivery and other biomedical applications. Up to now, liposomal formulations have been found to be one of the most effective delivery systems for cytotoxic drugs. Recently, it has been reported that MNPs can be encapsulated into liposomes to obtain the so-called magnetic liposomes (MLs).3,4 Generally, MNPs with a hydrophobic nature were integrated directly into the phospholipid bilayer,5−7 while hydrophilic MNPs were encapsulated into the lumen of liposomes.8−10 As a versatile delivery system, magnetic liposomes possess excellent potential for drug delivery, magnetic resonance imaging (MRI), and hyperthermia treatment of cancer, in addition to good biocompatibility and easy chemical functionalization.11−13 Guided by an external magnetic field (MF), the drug-loaded magnetic liposomes can accumulate in the target tissue area, and the drug is then © 2013 American Chemical Society
released from the magnetic liposomes in a controllable way, avoiding too much impact on normal tissue. Despite the tremendous results obtained with magnetic liposomes, a few challenges remain when designing magnetic liposomes that are stable and long-circulating in vivo yet can be destabilized in a controlled fashion to facilitate drug release at the target site.7 For example, magnetic liposomes suffer from the classical issue of instability associated with lipid bilayers. The morphology of magnetic liposomes varies strongly with MNPs and lipid concentrations. They are prone to aggregate to form large vesicles in suspension, resulting in a short circulation time and inclusion leakage after intravenous administration.14,15 Therefore, we have a pressing need for the development of more stable magnetic liposomes. During the past decade, a hybrid liposomal cerasome (partially ceramic- or silica-coated liposome) has drawn much attention. In our previous work, we reported paclitaxel (PTX)and doxorubicin hydrochloride (DOX)-loaded cerasomes, which exhibited advanced controlled release behavior and much higher stability than the conventional liposomes.16−18 Because of the liposomal character, cerasomes can accommodate both hydrophobic and hydrophilic agents, enabling the design of multifunctional drug delivery systems. Moreover, because cerasomes have a less leaky membrane than convenReceived: May 23, 2013 Revised: October 23, 2013 Published: November 3, 2013 14976
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
Langmuir
Article
Scheme 1. Schematic Illustration of DLMCs
obtained thin film was dispersed in 4 mL of pure water or DOX aqueous solution [10:1 (mol/mol) Si−lipid/DOX], followed by sonication for 5 min with a probe-type ultrasonic disintegrator to prepare MCs or DLMCs. Fluorescently labeled magnetic cerasomes (FMCs) were prepared by incorporation of 3% 1,2-dioleoyl-sn-glycero3-phosphoethanolamine-N-(7-nitro-2−1,3-benzoxadiazol-4-yl) (NBDDOPE) into cerasomes. The non-encapsulated Fe3O4 NPs were removed by centrifugation at 800 rpm. Then, the solution of DLMCs was separated from non-encapsulated DOX using strongly acidic cation-exchange resin. The encapsulated iron content was measured after digestion in concentrated HCl based on the determination of ferrous ions using a 1,10-phenanthroline colorimetric method.22 The iron(II) concentration was determined by an ultraviolet−visible (UV−vis) spectrophotometer at 510 nm (Cary 4000, Varian, Palo Alto, CA) based on a pre-established calibration curve at a concentration of 0.5−8.0 μg/ mL in 0.01 mol/L hydrochloric acid. From the iron concentration, the superparamagnetic iron oxide nanoparticle (SPION) concentration was calculated using the formula: [Fe] = 0.43[Fe3O4 NPs], obtained by control experiments. 2.3. Characterization of DLMCs. The hydrodynamic diameter (Dhy) and the ζ potential of DLMCs were analyzed using a 90Plus/BIMAS dynamic light scattering analyzer (Brookhaven Instruments, Holtsville, NY) at room temperature. The morphology of DLMCs was analyzed by transmission electron microscopy (TEM) using a Hitachi H-7650 transmission electron microscope. To prepare the TEM specimens, a drop of sample solution was placed on a copper grid and then negatively stained with aqueous solution of uranyl acetate (2 wt %). The magnetization of Fe3O4 NPs and DLMCs were performed in their solid state at room temperature using a vibrating sample magnetometer (VSM, Lake Shore 7307, Westerville, OH). Phase purity and identification of the samples were performed by X-ray diffraction (XRD) (Philips powder diffractometer PW1710). The infrared spectra of the lyophilized materials were recorded on a Varian spectrophotometer (Excalibur 3100, Varian, Palo Alto, CA). The KBr compressed pellet method was adopted for solid samples. Matrixassisted laser desorption/ionization time-of-flight (MALDI−TOF) mass spectrometry was performed on a Bruker ultraflextreme with dithranol (Aldrich, 97%) as the matrix. 2.4. Determination of Drug-Loading Content and Encapsulation Efficiency. To determine the DOX-loading content, preweighed freeze-dried DLMCs were redissolved in HCl solution (pH 3). Acid treatment also converted the iron oxide (i.e., magnetic
tional liposomes, it is likely to reduce passive release of encapsulated drugs. This provided us an opportunity to further develop a nanosystem combining the advantages of both cerasome carriers and MNPs in the present study.19 In this paper, we describe a convenient procedure to develop magnetic cerasomes (MCs) combining the advantages of both cerasome carriers and MNPs to overcome common problems related to current magnetic liposome technology. DOX-loaded magnetic cerasomes (DLMCs) were constructed by loading both Fe3O4 NPs and antitumor drug DOX into cerasomes in combination with the sol−gel process and self-assembly technique via one-step construction (Scheme 1). The obtained DLMCs were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Their encapsulation efficiency (EE), drug-loading content (DLC), and in vitro release were also analyzed. The cytotoxicity of DLMCs was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa cells.
2. EXPERIMENTAL SECTION 2.1. Preparation of Fe3O4 Nanoparticles (NPs). Oleic-acidmodified magnetic Fe3O4 NPs were prepared according to the published literature.7 Briefly, FeCl3·6H2O (0.08 mol) and FeSO4· 7H2O (0.04 mol) were dissolved in deionized water (50 mL) under nitrogen gas with mechanical stirring. Then, ammonium hydroxide (5 mL) was added to this solution with vigorous agitation. After 30 min, 3 mL of oleic acid was added to the mixture to modify the Fe3O4 NPs. The mixture was heated to 75 °C, refluxed under N2 protection for 1 h, and then cooled to room temperature. The Fe3O4 NPs were magnetically separated and washed with deionized water and ethanol 3 times and then dried at vacuum conditions.20 Finally, the obtained magnetite nanocrystals were dispersed in hexane and have a diameter of 9−11 nm. 2.2. Preparation of DLMCs. The Si−lipid N-[N-(3-triethoxysilyl)propylsuccinamoyl]dihexadecylamine was synthesized according to the reported method.21 Co-encapsulation of Fe3O4 NPs and DOX within cerasomes was performed using the conventional Bangham method. Briefly, Si−lipid and Fe3O4 at various Fe/C molar ratios were codissolved in 5 mL of chloroform. After removal of chloroform, the 14977
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
Langmuir
Article
Figure 1. TEM images of the (a and b) Fe3O4−DOX−micelle complex formed at a Fe/C molar ratio of 10:1 and DLMCs formed at Fe/C molar ratios of (c and d) 4:1 and (e and f) 1:1. particles) into iron chloride. The concentration of DOX was determined on fluorescence spectrophotometry using the corresponding standard calibration curve. The encapsulation efficiency (EE) and drug-loading content (DLC) of the DLMCs were evaluated by the following formulas:
EE (%) =
amount of DOX in vesicles × 100% initial amount of DOX
DLC (%) =
amount of DOX in vesicles × 100% total amount of vesicles
supernatants were discarded and cells were washed twice with PBS and incubated for another 20 h in freshly RPMI-1640 culture medium. Then, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 h of incubation. The precipitate was dissolved in dimethyl sulfoxide (DMSO) and analyzed on a microplate reader (BioRad, BIO-680) at 490 nm. Viability of the cells was measured by the MTT method as described previously.
(1)
3. RESULTS AND DISCUSSION 3.1. Fabrication and Characterization of MCs. DLMCs were fabricated by incorporating oleic-acid-modified Fe3O4 NPs and DOX into cerasomes at various Fe3O4/CFL (Fe/C) molar ratios according to the reported method.16−18 Upon ultrasonication, the liposomal membrane was formed by the selfassembly process and self-rigidified via the in situ sol−gel reaction (Si−OCH2CH3 + H2O → Si−OH + CH3CH2OH, followed by 2Si−OH → Si−O−Si + H2O) on the surface.16 The hydrolysis of the lipid head moiety formed the amphiphilic structure. The amphiphilic nature of the lipid provided the opportunity to form bilayer vesicles called “cerasome”. In addition, siloxane networks would be formed by intravesicular condensation among the silanol groups on the relatively hydrophobic membrane surface. Table S1 of the Supporting Information shows the diameters, ζ potential, DOX encapsulation efficiency, and Fe3O4-loading content of Fe3O4−DOX− micelles or DLMCs at different Fe/C ratios. As the Fe/C molar ratios decreased, the diameter of DLMC increased, demonstrating that the sizes could be finely adjusted by varying the Fe/C ratios. The ζ potentials ranged from −13.21 ± 5.6 to −19.23 ± 2.85 mV, indicating that cerasomes possessed comparable surface properties with silica NPs (see Table S1 of the Supporting Information). The negative charges on the surface of cerasomes ensured their good dispersibility and high colloidal stability in water. It was reported that the encapsulation of hydrophobic Fe3O4 NPs with phospholipids in aqueous environments formed the Fe3O4−micelle complex9,23 or the Fe3O4−liposome complex.24,25 In the Fe3O4− micelle complex, Fe3O4 is covered by a lipid monolayer, and in the Fe3O4−liposome state, Fe3O4 is incorporated into the lipid
(2)
2.5. In Vitro Drug Release. The in vitro drug release test was performed by suspending the DLMC solution in a dialysis bag, immersing in 20 mL of phosphate-buffered saline (PBS) buffer solution (pH 7.4), and shaking at 37 °C. At fixed time intervals, 2.0 mL of solution outside the dialysis bag was replaced with the same volume of fresh buffer solution for analysis.18 The release experiment lasted for 120 h. The DOX concentration was determined by a fluorescence spectrophotometer (Cary Eclipse, Varian, Palo Alto, CA) using the corresponding standard calibration curve. 2.6. In Vitro Evaluation of Cellular Uptake of FMCs by Confocal Laser Scanning Microscopy (CLSM) and Fluorescence-Activated Cell Sorter (FACS). The assay was carried out using the NBD-PE-labeled MCs, which were called FMCs. HeLa cells were incubated with various concentrations of FMCs with or without a strong Nd−Fe−B magnet (Ningbo Permanent Magnetics Co., Ltd., China; field strength, ∼0.42 T; positioned beneath the cell culture dishes). The cells were stained with 2-(4-amidinophenyl)-6indolecarbamidine dihydrochloride (DAPI) for 1 min, and the glass slides were rinsed with PBS 3 times. Imaging was performed with a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany), using the 64× oil immersion lens with argon laser at 488 nm to detect the nuclei and the He Ne laser at 543 nm to detect the FMCs. The cell pellet was analyzed for NBD-PE fluorescence using the FACS instrument (BD FACS Calibur, BD Biosciences) with the 488 nm line of an air-cooled argon laser as the excitation source. 2.7. Cytotoxicity Study. After HeLa cells were incubated with solutions of DOX, DOX-loaded cerasomes, and DLMCs, the cytotoxicity was evaluated by the tetrazolium-based colorimetric (MTT) assay. The cell culture plate was positioned under a permanent MF to impose magnetic influence for 2 h, followed by another 2 h of incubation without the MF. In control experiments, the cells were incubated for 4 h without the presence of a MF. Afterward, the 14978
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
Langmuir
Article
Figure 2. DLMCs: (a) optical photograph and (b) hysteresis loops.
Figure 3. DLMCs: (a) XRD patterns and (b) FTIR spectra.
interior, while the others are empty (Figure 1e). The picture taken at higher magnification (Figure 1f) clearly shows the mixture of Fe3O4-loaded vesicles and Fe3O4 NP-free vesicles, which formed because of the fact that the amount of Fe3O4 NPs was not enough to fully fill in cerasomes. The excess of lipid likely caused the formation of blank vesicles. The mean diameter of blank cerasome was 178.6 ± 12.6 nm, suggesting that the addition of Fe3O4 NPs to cerasome had no apparent effect on the DLMC size. The EE of DOX increased with a decreasing ratio of Fe/C, which was evaluated to be 21.7 ± 5.4% at the Fe/C ratio of 10:1 and 51.6 ± 5.9% at the Fe/C ratio of 0:10, respectively. DOX is water-soluble; therefore, it is believed that most DOX molecules are present in the core domain of cerasomes. Actually, there was a very small proportion of DOX molecules that could exist in the cerasomal bilayer membrane because of their amphiphilic nature or attachment on the cerasome surface via electrostatic interaction. The iron concentration was routinely determined using the 1,10-phenanthroline method.22 DLMCs prepared at different Fe/C ratios led to different Fe3O4-loading contents. Most importantly, the Fe3O4-loading content reached a maximum (approximately 0.69 mg/mL) at a Fe/C molar ratio of 4:1. In addition, the DLMCs obtained at a Fe/C molar ratio of 4:1 have a good drug loading content of 3.2 ± 1.3% and a high EE of 43.4 ± 4.7%, which is close to the highest value of 50.6 ± 5.9% at the Fe/C ratio of 0:10. Therefore, the Fe/C molar ratio of 4:1 was chosen for the fabrication of DLMCs for further studies. Superparamagnetism is important in drug delivery. As seen in Figure 2a, DLMCs in water showed fast response to the external MF and could be redispersed quickly with a slight
bilayer of liposomes. The cerasome bilayer thickness is about 4 nm.26 The hydrophobic Fe3O4 surface should be fully covered by the lipid monolayer because hydrophobic Fe3O4 NPs larger than 5 nm could not stay in the lipid bilayer as a result of a high energy penalty caused by water exposure of the hydrophobic part. This could result in the formation of the Fe3O4−micelle complex composed of a Fe3O4 core and a hybrid bilayer shell.27 As shown in Table S1 of the Supporting Information, the diameter of the obtained particles decreased as the Fe/C molar ratios increased. DLS measurements showed that the particles prepared at the Fe/C molar ratio of 10:1 had a diameter of 28.5 ± 9.6 nm. As the molar ratio of Fe/C increases, the Fe3O4loading content increases. Therefore, it is reasonable that Fe3O4 NPs are prone to form the Fe3O4−micelle complex, instead of being incorporated into the bilayer membrane of cerasomes at the Fe/C ratio of 10:1. The Si-lipid-stabilized Fe3O4−micelle complex is monodispersed in water. TEM images show that such a Fe3O4−micelle complex had a diameter ranging from 9 to 26.5 nm (panels a and b of Figure 1). Interestingly, after the Fe/C mole ratio was reduced to 4:1, the diameter of DLMCs increased to 169.8 ± 7.6 nm and Fe3O4 NPs were distributed in the aqueous compartment of vesicles according to TEM images (panels c and d of Figure 1). The entrapped Fe3O4 NPs existed as dark dots in the vesicles, maintaining the morphological properties of Fe3O4 NPs with a diameter close to 9 nm (Figure 1d). A possible explanation is that, at a lower molar ratio of Fe/ C, the hydrophobic Fe3O4 NPs can be entrapped into the aqueous interior of cerasomes through the formation of aqueous dispersible Fe3O4−Si−lipid complexes. When the Fe/C mole ratio decreases to 1:1, the TEM image shows that about half of the vesicles contain Fe3O4 NPs in their aqueous 14979
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
Langmuir
Article
Figure 4. (a) Hydrodynamic diameter (Dhy) and (b) DOX retention of DLMCs and DLMLs after storage for 1 month at 4 °C and (c) in vitro release of DOX from four formulations at 37 °C.
increased during the formulation processing and storage, leading to rapid uptake by the reticuloendothelial system (RES) and, thus, short half-life. Thus, it is critical to control and maintain liposomes to be small and uniform in size while developing a viable pharmaceutical product. As shown in panels a and b of Figure 4, DLMCs showed almost no change in the particle size and retained about 68.9 ± 6.9% of the initially encapsulated DOX after being stored for 1 month at 4 °C, indicating no drug precipitation, no aggregation, nor fusion of the DLMCs after long-term storage. In contrast, the diameter of DLMLs increased from less than 200 nm to 2.2 μm. After 1 month of storage, DLMLs retained only 33.2 ± 4.8% of the initially encapsulated DOX because of the aggregation/fusion of unstable liposomes. The results indicate that the storage stability of DLMCs is much higher than that of DLMLs. The MALDI−TOF mass spectrum (see Figure S1 of the Supporting Information) suggests that the formation of the siloxane network on the vesicular surface of DLMCs contributes to its higher stability. A wide range of lipid oligomers from monomer to nonamer was detected in the DLMC solution after 10 h of incubation. All of the detectable lipid oligomer species of the DLMCs are listed in Table S2 of the Supporting Information. Their observed molecular weights are consistent with the calculated molecular weights. Subsequently, kinetics of the in vitro drug release from these prepared vesicular dispersions was studied by dialyzing in physiological solution and measuring the time required for DOX molecules to go through the dialysis membrane (Figure 4c). The free DOX without any nanocarriers completed the release from the dialysis bag within 10 h. Approximately 92.32% DOX was released from DLMLs within 22 h. On the contrary,
shake after removal of the MF. There was no hysteresis loop on the two saturation magnetization curves measured at 300 K (Figure 2b), which means that both Fe3O4 NPs and DLMCs possess strong superparamagnetism. The saturation magnetization value (Ms) of DLMCs was 25.95 emu g−1, lower than that of Fe3O4 NPs (69.65 emu g−1). The lower value is attributed to the overall reduction in the total ferrite content in DLMCs. It is worth noting that the DLMCs still exhibit high magnetization, indicating its suitability for magnetic drug delivery and targeting. Figure 3a shows the XRD patterns of Fe3O4 and DLMCs. The position of diffraction peaks and their relative intensity are in accordance with those of Fe3O4 [Joint Committee on Powder Diffraction Standards (JCPDS) number 19-0629]. The broad diffraction peak at 2θ = 20−26° can be assigned to the amorphous silica shell (JCPDS number 29-0085). The XRD patterns of DLMCs showed typical diffraction peaks of both Fe3O4 and silica, suggesting the successful encapsulation of Fe3O4 into cerasomes, despite the fact that it caused an obvious decrease in the intensities of the corresponding Fe 3O 4 diffraction peaks. In the FTIR spectrum of DLMCs, the peaks that appeared at 578 and 998 cm−1 were characteristic vibrations of Fe−O and C−O bonds, respectively, indicating the presence of Fe3O4 NPs and DOX (Figure 3b). It further confirmed the successful fabrication of DLMCs. Obviously, these results indicated that the intrinsic magnetic properties of the ferrofluid were well-maintained in cerasomes. 3.2. Storage Stability and Release Behavior of DLMCs. It is widely known that the size of vesicles plays a critical role in their tissue distribution and clearance rate from blood. Conventional liposomes become instable when their size 14980
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
Langmuir
Article
Figure 5. CLSM images of HeLa cells incubated with cerasomes for 4 h: (a−e) FCs, (f−j) FMCs in the absence of a permanent MF, and (k−o) FMCs in the presence of a permanent MF. Cell nuclei were stained with DAPI. Scale bar = 20 μm.
As seen in panels k−o of Figure 5, it is worth noting that FMCs showed remarkably brighter fluorescence when an external MF was applied. Staining of tumor cell nuclei with DAPI shows that FMCs inside cells were distributed throughout the entire cytoplasm mainly as green spots, indicating lysosomal localization according to our previous work.29 That work has clearly showed that similar cerasomes without drug/SPIO loading were entrapped inside lysosomes after endocytosis. Lyso-Tracker green (DND-26) were used as a lysosome tracker, and fluorescent cerasome inside the cells was distributed in the cytoplasm but mainly localized in the lysosomes as red spots. Encapsulation of drug/SPIO did not obviously change their morphology and size. Therefore, it should be reasonable to deduce that the drug-/SPIO-loaded cerasomes undertake the same endocytosis pathway, resulting in lysosomal entrapment. This implies that external magnetic stimulus greatly enhanced the cellular uptake of FMCs. Even though the cellular uptake processes are likely the same for magnetic and non-magnetic NPs, there is some evidence that the magnetic NPs are prone to attach on the surface of the cell, and then, an external MF can enhance their cell uptake.30 In vitro evaluation of the cellular uptake of cerasome formulations was also performed by FACS analysis (see Figure S2 of the Supporting Information). There was no significant difference in the uptake by HeLa cells between FCs and FMCs with an increasing concentration without applying an external MF. Nevertheless, significantly higher fluorescence intensity was observed when an external MF was applied during HeLa cell incubation with FMCs. HeLa cells incubated with FMCs under a MF showed a mean fluorescence intensity (MFI), which was approximately 8- and 3-fold higher than that of cells incubated with FCs and FMCs without applying a MF, respectively. The FACS analysis provided additional evidence that magnetic targeting effectively enhanced the cellular uptake of FMCs at various concentrations, well consistent with the
only 61.26 and 46.57% of the loaded drug were released from DLMCs and DLCs over 120 h, respectively. DLMCs (pH 5 or 7.4) released slightly faster than DLCs because loading magnetic NPs into cerasomes could reduce the stability of cerasomes. However, DLMCs were more stable than DLMLs. The rate of DOX release was almost not affected by the pH values (pH 5 versus 7.4) of the release media, which indicates that the DOX release from the cerasomes compared to conventional liposomes slowed as well inside cells. The slower release rates of DLMCs and DLCs are attributed to the siloxane networks on the cerasome surface that block the drug-releasing channels (see Figure S1 of the Supporting Information). Sustained release is important for not only maintaining therapeotic efficacy but also preventing cancer from relapsing and developing drug resistance.28 The interesting drug-loading and -release properties of the magnetic DLMCs make them a promising candidate for external magnetic-field-guided cancer therapy in vivo, which is a well-known emerging field of biomedicine. 3.3. Cellular Uptake Study. CLSM was used to study the cellular uptake and localization of the fluorescently labeled particles in HeLa cell lines. The cells incubated with fluorescently labeled cerasomes (FCs) exhibited barely detectable green fluorescence, even when the concentration reached maximum (400 μM), indicating a lower cellular uptake of FCs (panels a−e of Figure 5). In contrast, the fluorescence of magnetic FMCs was stronger than non-magnetic FCs at the same concentration. Moreover, the uptake of FMCs by HeLa cells increased notably with increasing concentrations of FMCs after 4 h of incubation. Cells incubated with FMCs without a MF showed weak green fluorescence around the cell nuclei at low concentrations (panels f−h of Figure 5f∼h), but much more intense fluorescence was observed when treated with higher concentrations (250−400 μM) (panels i and j of Figure 5). 14981
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
Langmuir
Article
Figure 6. Antiproliferative effects of cerasomes and MCs: (a) non-magnetic field and (b) with a MF after 24 h of incubation of HeLa cells.
results obtained by CLSM images. Therefore, it can be concluded that a tight and sustained interaction between FMCs and the cell surface has been induced by the external MF. In addition to the magnetic targeting potential, the presence of magnetic NPs may also turn the cerasomes into a magnetic resonance imaging (MRI)-visible drug delivery system, i.e., the so-called theranostic nanomedicine for simultaneous drug therapy and monitoring of pathological progress and therapeutic efficacy with MRI. Moreover, incorporation of MNPs also brings about potential for magnetic hyperthermia in combination with anticaner drug therapy. We will conduct further studies to explore these potentials. 3.4. Inhibitory Effects of Released DOX on Cell Proliferation. Upon exposure of HeLa cells to free vesicles of cerasomes and MCs at various lipid concentrations ranging from 0 to 650 μM, no appreciable cell growth inhibition was observed, regardless of an external MF (panels a and b of Figure 6). The results suggest that all of these free vesicles are highly biocompatible, and they have no cytotoxicity to HeLa cells, which enabled us to better understand whether DOX release from cerasomes was the cause for the antiproliferative effects observed in the subsequent cell study. In cell viability studies, the cytotoxicities of free DOX, DLCs, DLMCs, and DLMCs + MF were compared to further verify the efficacy of magnetic targeting (Figure 7). All four DOX formulations exhibited clear cytotoxicity in a PTX concentration-dependent manner in HeLa cells. Cell viability
decreased with an increasing drug concentration. In comparison to non-magnetic DLCs, magnetic DLMCs showed slightly higher cytotoxicity in HeLa cells. At the highest concentration of 20 μM in the experiment, the cell viability was evaluated to be 63.89 ± 3.54 and 56.49 ± 3.41% for DLCs and DLMCs, respectively. Interestingly, a major increase in cytotoxicity was observed for DLMCs when an external MF was applied. It showed a cell viability of 27.28 ± 6.89% at 20 μM DOX concentration, much lower than that of DLMCs without an external MF (p < 0.05). It revealed the effective targeting of MCs to cancer cells when an external MF was applied. These data are consistent with the results obtained in the cell uptake studies. The magnetic targeting strategy is believed to provide an option to guide the accumulation of DLMCs in tumor tissues and facilitate their internalization into tumor cells. Eventually, DOX will be released from the DLMCs inside HeLa cells and further diffuse into the nuclei from cytosol, interrupting DNA replication and, thus, leading to cell death.31
4. CONCLUSION The drug-loaded MCs of DLMCs with high stability and sustained drug release properties have been successfully developed by loading both DOX and Fe3O4 NPs into cerasomes. The obtained magnetic DLMCs showed strong response to MF, which enabled the efficient uptake of cerasomes into tumor cells, resulting in a higher capability to kill tumor cells than non-magnetic DLCs. In addition, it is convenient to attach targeting ligands with silane-coupler chemistry, allowing for dual targeting of the drug-loaded cerasomes to desired sites to reduce unwanted side effects. It makes MC technology very attractive for controlled drug delivery and targeted cancer therapy applications. As far as we know, this is the first report regarding an improved cytotoxic effect of anticancer drugs using a cerasome-based drug delivery system upon exposure to an external MF.
■
ASSOCIATED CONTENT
S Supporting Information *
Fe3O4−DOX−micelles or DLMCs fabricated at different Fe3O4/cerasome-forming lipid (Fe/C) molar ratios (Table S1), MALDI−TOF−MS spectrum of DLMCs (Figure S1), detectable species of lipid oligomers for DLMCs as evaluated by MALDI−TOF−MS spectrum (Table S2), and intracellular uptake of HeLa cells incubated with (a) control, (b) FCs, (c) FMCs, and (d) FMCs under permanent MF measured by FACS analysis (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 7. Antiproliferative effects of four DOX formulations of DOX, DLCs, DLMCs, and DLMCs + MF at various concentrations after 24 h of incubation of HeLa cells (mean ± SD; n = 3). 14982
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
Langmuir
■
Article
(14) Wang, B.; Zhang, L. F.; Bae, S. C.; Granick, S. Nanoparticleinduced surface reconstruction of phospholipid membranes. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18171−18175. (15) Plassat, V.; Martina, M. S.; Barratt, G.; Menager, C.; Lesieur, S. Sterically stabilized superparamagnetic liposomes for MR imaging and cancer therapy: Pharmacokinetics and biodistribution. Int. J. Pharm. 2007, 344, 118−127. (16) Cao, Z.; Ma, Y.; Yue, X. L.; Li, S. Z.; Dai, Z. F.; Kikuchi, J. Stabilized liposomal nanohybrid cerasomes for drug delivery applications. Chem. Commun. 2010, 46, 5265−5267. (17) Jin, Y. S.; Yue, X. L.; Zhang, Q. Y.; Wu, X. Y.; Cao, Z.; Dai, Z. F. Cerasomal doxorubicin with long-term storage stability and controllable sustained release. Acta Biomater. 2012, 8, 3372−3380. (18) Cao, Z.; Yue, X. L.; Jin, Y. S.; Wu, X. Y.; Dai, Z. F. Modulation of release of paclitaxel from composite cerasomes. Colloids Surf., B 2012, 98, 97−104. (19) Oliveira, H.; Encarnación, P.; Julie, T.; Olivier, S.; Edurne, B.; Sébastien, L. Magnetic field triggered drug release from polymersomes for cancer therapeutics. J. Controlled Release 2013, 169, 165−170. (20) Luo, Z.; Wang, Y. M.; Hong, R. Y.; Li, H. Z. Preparation and characterization of Fe3O4/ethiodized-oil magnetic fluids used in arterial embolization hyperthermia. Chin. J. Chem. 2012, 30, 854−860. (21) Katagiri, K.; Hashizume, M.; Ariga, K.; Terashima, T.; Kikuchi, J. Preparation and characterization of a novel organic-inorganic nanohybrid “cerasome” formed with a liposomal membrane and silicate surface. Chem.Eur. J. 2007, 13, 5272−5281. (22) IM, K.; DL, L.; TS, L. Reaction of ferrous and ferric iron with 1,10-phenanthroline III. The ferrous monophenanthroline comples and the colorimetric determination of phenanthroline. J. Am. Chem. Soc. 1950, 72, 2173−2177. (23) van Tilborg, G. A. F.; Mulder, W. J. M.; Deckers, N.; Storm, G.; Reutelingsperger, C. P. M.; Strijkers, G. J.; Nicolay, K. Annexin A5functionalized bimodal lipid-based contrast agents for the detection of apoptosis. Bioconjugate Chem. 2006, 17, 741−749. (24) Kuznetsov, A. A.; Filippov, V. I.; Alyautdin, R. N.; Torshina, N. L.; Kuznetsov, O. A. Application of magnetic liposomes for magnetically guided transport of muscle relaxants and anti-cancer photodynamic drugs. J. Magn. Magn. Mater. 2001, 225, 95−100. (25) Dwivedi, N.; Arunagirinathan, M. A.; Sharma, S.; Bellare, J. Nanoferrite embedded magnetocochleate microstructures to encapsulate insulin macromolecules. J. Phys. Chem. B 2009, 113, 13782− 13787. (26) Katagiri, K.; Ariga, K.; Kikuchi, J. Preparation of organic− inorganic hybrid vesicle “cerasome” derived from artificial lipid with alkoxysilyl head. Chem. Lett. 1999, 28, 661−662. (27) Giri, J.; Thakurta, S. G.; Bellare, J.; Nigam, A. K.; Bahadur, D. Preparation and characterization of phospholipid stabilized uniform sized magnetite nanoparticles. J. Magn. Magn. Mater. 2005, 293, 62− 68. (28) Nooter, K.; Stoter, G. Molecular mechanisms of multidrug resistance in cancer chemotherapy. Pathol. Res. Pract. 1996, 192, 768− 780. (29) Ma, Y.; Dai, Z. F.; Gao, Y. G.; Cao, Z.; Zha, Z. B.; Yue, X. L.; Kikuchi, J. I. Liposomal architecture boosts biocompatibility of nanohybrid cerasomes. Nanotoxicology 2011, 5, 622−635. (30) Plank, C.; Zelphati, O.; Mykhaylyk, O. Magnetically enhanced nucleic acid delivery. Ten years of magnetofectionProgress and prospects. Adv. Drug Delivery Rev. 2011, 63, 1300−1331. (31) Kim, J.; Lee, J. E.; Lee, S. H.; Yu, J. H.; Lee, J. H.; Park, T. G.; Hyeon, T. Designed fabrication of a multifunctional polymer nanomedical platform for simultaneous cancer-targeted imaging and magnetically guided drug delivery. Adv. Mater. 2008, 20, 478−483.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. URL: http://bme.pku.edu.cn/ ∼daizhifei. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
■
REFERENCES
This research was financially supported by the State Key Program of the National Natural Science of China (81230036), the National Natural Science Foundation of China (21273014 and 81101142), the National Natural Science Foundation for Distinguished Young Scholars (81225011), and the Natural Science Foundation of Guangdong Province (S2011040005922).
(1) Pradhan, P.; Giri, J.; Rieken, F.; Koch, C.; Mykhaylyk, O.; Doblinger, M.; Banerjee, R.; Bahadur, D.; Plank, C. Targeted temperature sensitive magnetic liposomes for thermo-chemotherapy. J. Controlled Release 2010, 142, 108−121. (2) Ma, Y.; Liang, X. L.; Tong, S.; Bao, G.; Ren, Q. S.; Dai, Z. F. Gold nanoshell nanomicelles for potential magnetic resonance imaging, light-triggered drug release, and photothermal therapy. Adv. Funct. Mater. 2013, 23, 815−822. (3) Wijaya, A.; Hamad-Schifferli, K. High-density encapsulation of Fe3O4 nanoparticles in lipid vesicles. Langmuir 2007, 23, 9546−9550. (4) Hanuš, J.; Ullrich, M.; Dohnal, J.; Singh, M.; Štěpánek, F. Remotely controlled diffusion from magnetic liposome microgels. Langmuir 2013, 29, 4381−4387. (5) Katagiri, K.; Imai, Y.; Koumoto, K.; Kaiden, T.; Kono, K.; Aoshima, S. Magnetoresponsive on-demand release of hybrid liposomes formed from Fe3O4 nanoparticles and thermosensitive block copolymers. Small 2011, 7, 1683−1689. (6) Amstad, E.; Kohlbrecher, J.; Muller, E.; Schweizer, T.; Textor, M.; Reimhult, E. Triggered release from liposomes through magnetic actuation of iron oxide nanoparticle containing membranes. Nano Lett. 2011, 11, 1664−1670. (7) Chen, Y. J.; Bose, A.; Bothun, G. D. Controlled release from bilayer-decorated magnetoliposomes via electromagnetic heating. Acs Nano 2010, 4, 3215−3221. (8) Mikhaylov, G.; Mikac, U.; Magaeva, A. A.; Itin, V. I.; Naiden, E. P.; Psakhye, I.; Babes, L.; Reinheckel, T.; Peters, C.; Zeiser, R.; Bogyo, M.; Turk, V.; Psakhye, S. G.; Turk, B.; Vasiljeva, O. Ferri-liposomes as an MRI-visible drug-delivery system for targeting tumours and their microenvironment. Nat. Nanotechnol. 2011, 6, 594−602. (9) Namiki, Y.; Namiki, T.; Yoshida, H.; Ishii, Y.; Tsubota, A.; Koido, S.; Nariai, K.; Mitsunaga, M.; Yanagisawa, S.; Kashiwagi, H.; Mabashi, Y.; Yumoto, Y.; Hoshina, S.; Fujise, K.; Tada, N. A novel magnetic crystal−lipid nanostructure for magnetically guided in vivo gene delivery. Nat. Nanotechnol. 2009, 4, 598−606. (10) Lesieur, S.; Grabielle-Madelmont, C.; Menager, C.; Cabuil, V.; Dadhi, D.; Pierrot, P.; Edwards, K. Evidence of surfactant-induced formation of transient pores in lipid bilayers by using magnetic-fluidloaded liposomes. J. Am. Chem. Soc. 2003, 125, 5266−5267. (11) Chertok, B.; David, A. E.; Yang, V. C. Polyethyleneiminemodified iron oxide nanoparticles for brain tumor drug delivery using magnetic targeting and intra-carotid administration. Biomaterials 2010, 31, 6317−6324. (12) Veiseh, O.; Gunn, J. W.; Zhang, M. Q. Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging. Adv. Drug Delivery Rev. 2010, 62, 284−304. (13) Pallab, P.; Rinti, B.; Dhirendra, B.; Christian, K.; Olga, M.; Christian, P. Targeted magnetic liposomes loaded with doxorubicin liposomes. Methods Mol. Biol. 2010, 605, 279−293. 14983
dx.doi.org/10.1021/la401965a | Langmuir 2013, 29, 14976−14983
