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Fe3O4–silica core–shell nanoporous particles for high-capacity pH-triggered drug delivery
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X. F. Zhang,ab S. Mansouri,a L. Clime,a H. Q. Ly,d L. ’H. Yahiac and T. Veres*ab Received 21st March 2012, Accepted 6th May 2012 DOI: 10.1039/c2jm31749d We demonstrate a one-step procedure for the synthesis of Fe3O4–silica core–shell nanoparticles with hierarchically ultra-large pores independent of any post-treatment such as annealing and templatemolecule removal. The nanoporous silica shells with available amine groups were functionalized by clickable linkers to produce pH-sensitive amides for regulating the release of an anti-cancer drug, doxorubicin (DOX). The loading amount of DOX reached up to 13.2 mg per 100 mg nanoparticles, 74.2% of which can be effectively released after 63 h at body temperature and pH 5 with decreased side effects. Such excellent features of these nanoparticles appear to arise from the integrated hierarchically ultra-large open-porosities and a homogeneous dispersibility in aqueous solution that has a great potential for their use as drug delivery systems.
1 Introduction In recent years, magnetic nanoparticles have been mainly studied for their potential applications in a wide range of biomedical fields, such as magnetic resonance imaging (MRI), targeted drug delivery and magnetic separation. Currently, critical issues to be resolved are their stability and biocompatibility in the circulatory system, and surface functionalizations that conjugate the targeting spacers or therapeutic agents.1,2 Core–shell structures have been proposed in an effort to address the biochemical stability and biocompatibility issues, as well as to provide a template surface for the assembly of heterogeneous functions.3–5 Among all the potential candidates, nanoporous shells provide the distinct advantage of intrinsically higher surface areas, which are especially important when employed as drug carriers.616 In the present work, we report on a one-pot procedure for the synthesis of superparamagnetic core–shell Fe3O4–silica(porous) nanoparticles containing both amine groups in the hierarchically ultra-large porous silica shell. Compared with other synthesis procedures for porous silica structure,616 the porous silica shell in our case, with the pore size up to 10 nm, is in situ formed without any post-treatment. The hierarchically ultra-large pores ensure opened channels allowing a large-loading storage of guest molecules, while also acting as interceptors for slow diffusion of a Biomedical Engineering Department, McGill University, Quebec, Canada H3A 2B4 b Industrial Materials Institute, National Research Council of Canada, 75 Boul. de Mortagne, Boucherville, Qu ebec, Canada J4B 6Y4. E-mail:
[email protected]; Fax: +1 450 641-5105 c Ecole Polytechnique de Montr eal, Case Postale 6079, succursale CentreVille, Montr eal, Qu ebec, Canada H3C 3A7 d Department of Cardiovascular Medicine, Montreal Heart Institute, University of Montreal School of Medicine, Montreal, Quebec, Canada
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these molecules due to the disordered topology of pore channels. These advantages make these nanoparticles promising candidates for biomolecule delivery. By functionalizing the nanoparticles with 1,2-cyclohexanedicarboxylic anhydride as a click linker, we herein present the feasibility of coupling doxorubicin (DOX) via amides to the porous silica shells with a superior loading capacity up to 11.7 wt%. The coupled DOX molecules are relatively stable at neutral pH 7.4, but can be rapidly released in the range of pH 5.0–6.0 due to the hydrolysis of amide bonds.
2
Experimental and theoretical sections
2.1 Materials All reagents used in this study are commercially available. Oleic acid (OA, 90%), 1-hexanol anhydrous (99%), octyl ether (98%), ammonia solution (NH4OH, 28–30 wt% in water), Triton X100, hexane (95%), cyclohexane (99.5%), dimethyl sulfoxide (DMSO, 99%), 1,2-cis-cyclohexanedicarboxylic anhydride (98%), triethylamine (98%), tetraethoxysilane (TEOS, 99.999%), sodium hydroxide (99%), tetrachloroaurate(III) hydrate (99.99%), and DOX hydrochloride (98%) were purchased from Sigma-Aldrich Inc. Iron pentacarbonyl (99.5%) was purchased from Strem Chemicals, Inc. (Newburyport, MA) and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3), 3mercaptopropyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, N-ethylaminoisobutyltrimethoxysilane, Nphenylaminopropyltrimethoxysilane, n-butylaminopropyltrimethoxysilane, tetraethoxysilane, and N-[(trimethoxysilyl) propyl]poly(ethylenimine) were purchased from Gelest (Tullytown, PA). Fluorescamine was purchased from MP Biomedicals, LLC. J. Mater. Chem.
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2.2 Synthesis of core–shell Fe3O4–silica(nanoporous) nanoparticles The oleic acid-coated Fe3O4 (Fe3O4–OA) nanoparticles were synthesized based on a well-known process.17 The core–shell Fe3O4–silica nanoparticles were fabricated by hydrolyzing TEOS in a water-in-oil microemulsion that contains the Fe3O4–OA nanoparticles as seeds. Briefly, Fe3O4–OA nanoparticles were first dispersed in cyclohexane, at a concentration of 1 mg ml1, and then 0.5 ml of the Fe3O4-containing cyclohexane dispersion were rapidly injected into a mixture of 1.77 g of Triton X-100, 1.6 ml of anhydrous 1-hexanol and 7 ml of cyclohexane under a strong vortex for about 1 h. Subsequently, 0.5 ml of ammonia solution (28–30% ammonia solution : water ¼ 1 : 4) were added in the above solution and shaken for another 1 h. Finally, 25 ml of TEOS were added, and the mixture was allowed to react for 24 h. The as-fabricated product was separated by centrifugation at 9000 rpm, washed with ethanol, and the silica shells are nonporous state (Fe3O4–silica nanoparticles). To synthesize the Fe3O4– silica(porous) nanoparticles, 25 ml of AEAP3 were injected into the above reaction mixture for another 24 h, and then the product was separated by the same procedures. To uncover the mechanism for the formation of a nanoporous structure we studied seven types of different silanes with various molecular structures, which were used in the second hydrolysis step of the process.
cells were placed in a medium without nanoparticles at the same cell density. The image-capture system consisted of a Nikon Eclipse microscope equipped with a filter set that has a scan range from 380–750 nm. Using a 20 objective, bright-field and fluorescence images were captured with a computer-controlled charged-coupled device (CCD) camera using the Simple PCI software imaging. 2.5 In vitro cell viability The cell viability test was carried out via the reduction of the MTT reagent (Invitrogen). After 48 h of culture with the Fe3O4– silica(porous) core–shell nanoparticles (1, 5 10, and 50 mg ml1), Fe3O4–silica(porous) core–shell nanoparticles functionalized with DOX (1, 5, 10, and 50 mg ml1) and free DOX (0.1, 0.5, 1, and 5 mg ml1), 100 ml of MTT dye solution (5 mg ml1 in phosphate buffer pH-7.4) was added to each well and incubated for 4 h at 37 C and 5% CO2. The medium was removed and formazan crystals were solubilized with 150 ml of dimethylsulphoxide (DMSO). Absorbance of each well was read using a spectrophotometer (Biotek, USA) at 540 nm and the relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated by [A]test/[A]control 100. Three replicates were measured, and the results presented as mean standard deviation. 2.6 Characterization methods
2.3 DOX loading and pH-regulated release 2 mg Fe3O4–silica(porous) nanoparticles were dissolved in 20 ml DMSO, followed by sonicating for 30 min. Triethylamine (100 ml) was subsequently added and magnetically stirred for 2 h. The grafted nanoparticles were separated by centrifugation at 9000 rpm, and mildly washed by DMSO three times. The grafted nanoparticles and DOX hydrochloride salt (1 mg) were dissolved in 20 ml DMSO solution, and magnetically stirred for 2 h. In order to remove the free DOX molecules, the DOX-coupled nanoparticles were separated by centrifugation and mildly washed by pH 7.4 phosphoric acidic buffer solutions three times. The release of DOX from coupled Fe3O4–silica(porous) nanoparticles was carried out at 37 C and at pH 7.4, 6.0 and 5.0 phosphoric acidic buffer solutions, respectively. The separated supernatant solution was monitored by UV-Vis spectra.
The size and morphology of nanoparticles were analyzed using a Hitachi S-4700 transmission electron microscope (TEM) operated at a voltage of 30 kV. The microstructure and composition of the samples were characterized by high resolution TEM (HRTEM), selected area electron diffraction (SAED), and energy dispersive X-ray spectroscopy (EDS) on a JEOL 2010F (200 kV) transmission electron microscope. TEM samples were prepared by dropping 25 ml of particle dispersion in hexane on amorphous carbon coated copper grids, and drying under vacuum overnight. FTIR spectra were collected with a Nicolet Fourier spectrophotometer at wavenumbers between 600 and 4000 cm1. UV-Vis spectra were collected on a Perkin Elmer Lambda 950 spectrometer. Magnetic measurements of major hysteresis loops (MHLs) at different temperatures as well as zero-field cooled (ZFC) magnetization processes were performed with a Quantum Design PPMS model 6000 magnetometer.
2.4 Cell culture DOX release and the cytotoxicity of the Fe3O4–silica(porous) core–shell nanoparticles functionalised with DOX were evaluated using adenocarcinomic human alveolar basal epithelial cells (A549, American Type Culture Collection (ATCC), USA). The medium used was Ham’s F-12 (ATCC, USA) supplemented with penicillin (100 IU ml1), streptomycin (100 mg ml1) and 10% fetal bovine serum (FBS). The cells were cultured at a density of 1 105 cells per 1 ml of medium in 24-well culture plates at 37 C in a 5% CO2 atmosphere. After 20 h of culture, the medium in the wells was replaced with a fresh medium containing Fe3O4– silica(porous) core–shell nanoparticles (1, 5, 10 and 50 mg ml1), Fe3O4–silica(porous) core–shell nanoparticles functionalised with DOX (1, 5, 10 and 50 mg ml1) and DOX (0.1, 0.5 1, and 5 mg ml1), and was further cultured for 48 h. In control cultures, the J. Mater. Chem.
3
Results and discussion
3.1 Morphology and structures Fig. 1(a) and (b) show typical transmission electron microscopy (TEM) images of as-synthesized Fe3O4–OA (OA ¼ oleic acid) nanoparticles. The particles are seen to have a narrow size distribution and form a self-assembled super-lattice. The measurement of about 200 particles has shown that the particles are essentially spherical in shape, with a mean diameter of 15.1 nm. The corresponding fast Fourier transform (FFT) pattern [inset of Fig. 1(b)] in the region marked with a red square has a symmetrical lattice, indicating the single crystalline nature of the nanoparticles. Fig. 1(c) and (d) show the Fe3O4–silica nanoparticles with dense shells and the statistic particle This journal is ª The Royal Society of Chemistry 2012
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Fig. 1 (a) A self-assembly TEM image and (inset) the size distribution from more than 200 particles of Fe3O4–OA nanoparticles; (b) high-resolution TEM image and (inset) the fast Fourier transform pattern corresponding to the red square; (c) and (d) TEM images of core–shell Fe3O4–silica nanoparticles; and (e) and (f) TEM images of the Fe3O4–silica(porous) nanoparticles.
diameters of 56.2 0.09 nm. Fig. 1(e) and (f) show TEM images of Fe3O4–silica(porous) nanoparticles. The as-synthesized nanoparticles are all spherical in shape with an average total diameter of 65.5 0.06 nm. Compared with the structural features of Fe3O4–silica nanoparticles, it is worth noting that all the particles present nanoporous structures.
Fig. 2 (a) EDS element mapping of Fe3O4–silica(porous) nanoparticles; (b) line analysis along the axis of a single Fe3O4–silica(porous) nanoparticle, and (c) high angle annular dark field (HAADF) image of a Fe3O4–silica(porous) nanoparticle, indicating the hierarchically ultralarge pores with bicontinuous channels.
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Fig. 2(a) and (b) show the energy dispersive X-ray spectroscopy (EDS) elemental mapping and the line-scan analysis of the elemental distribution of iron, oxygen, silicon and nitrogen along the axis of one nanoparticle. A core–shell feature can be clearly observed, indicating that the core is rich in iron and oxygen, while the shell is mainly made of silicon, oxygen and nitrogen. Fig. 2(c) shows the high angle annular dark field (HAADF) image of Fe3O4–silica(porous) nanoparticles, providing compelling evidence for the nanoporous structures, revealing the hierarchically ultra-large porosities with bicontinuous channels extended to the surface. Such excellent features can significantly improve the storage space of the guest molecules, as well as the slow diffusion ascribed to the internal steric hindrance of the hierarchical channels, offering a distinct advantage for their use as drug delivery vehicles. To reveal the formation mechanism of nanoporous silica shells, we carried out a comprehensive study by using another seven types of silanes with different molecular structures, as shown in Fig. 3. Although the mechanism of particle structures (dense, porous or hollow) affected by the various silanes is still not quite clear, we herein would like to make a speculation, according to the current experimental observations. In our two-step hydrolysis process of silica shell formation, the hydrophobic Fe3O4–OA nanoparticles were firstly activated by Triton X-100 molecules via polyethylene oxide groups to disperse in aqueous reaction cells, which provided the condition for the condensation of TEOS molecules on the surface of Fe3O4 nanoparticles. Under the effect of adsorbed Triton X-100 molecules, the hydrolyzed TEOS silanes could form a ‘soft’ shell consisting of incompletely condensed silica fragments and Triton X-100 molecules. These fragments would further react with subsequently added silane molecules (such as AEAP3). Depending on the steric hindrance of functional groups with various charges and backbones (such as NH2(CH2)2NH(CH2)3– for AEAP3) of silane molecules, the silica shells could be finally transformed into dense, porous, or even hollow structures when removing the Triton X-100 molecules by ethanol washing. J. Mater. Chem.
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Fig. 3 TEM images of Fe3O4–silica core–shell nanoparticles with different microstructures depending on the usage of various silanes as indicated: (a) 3-mercaptopropyltrimethoxysilane; (b) 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane; (c) N-ethylaminoisobutyltrimethoxysilane; (d) N-phenylaminopropyltrimethoxysilane; (e) n-butylaminopropyltrimethoxysilane; (f) tetraethoxysilane; (g) N-[(trimethoxysilyl)propyl]poly(ethylenimine); and (h) AEAP3. The scales in all images are 200 nm.
3.2 Magnetic properties Fig. 4(a)–(c) show the major hysteresis loops (MHLs) and corresponding enlargements of Fe3O4–OA, Fe3O4–silica and Fe3O4– silica(porous) nanoparticles at 10 and 300 K. The as-synthesized Fe3O4–OA nanoparticles, in Fig. 4(a), exhibit typical superparamagnetic behavior, consisting of Langevin-type curves of nearly zero coercive fields at room temperature. As expected, in the low temperature regime, blocked (ferromagnetic) particles become preponderant and the MHLs become slightly hysteretic, with an increased saturation magnetization (Ms) of 73 emu g1 and a coercive field (Hc) of 53 Oe. The chemical analysis results from Guelph Chemical Laboratories Ltd. show that the assynthesized Fe3O4–OA nanoparticles contain about 62.5 wt% iron, corresponding to 86.3 wt% Fe3O4 and 13.7 wt% OA regardless of other impurities. This corresponds to a net value of 84.5 emu g1 for normalized Fe3O4 nanoparticles, as previously reported by Sun et al.18 However, the organic species can be removed completely on silica coating, as seen from the fast Fourier transmission infrared (FTIR) spectra. In Fig. 4(b) and (c), the Ms values of Fe3O4–silica and Fe3O4–silica(porous) nanoparticles are 3.1 and 1.1 emu g1, corresponding to the nonmagnetic silica compositions of 96.3 wt% and 98.7 wt%, respectively. J. Mater. Chem.
Fig. 4(d)–(f) show the temperature-dependent zero-field-cooling (ZFC) and field-cooling (FC) magnetization curves of Fe3O4–OA, Fe3O4–silica and Fe3O4–silica(porous) nanoparticles, respectively, measured at an applied magnetic field of 50 Oe. The ZFC curve of Fe3O4–OA nanoparticles exhibits a broader maximum of 200 K (Tmax). The Tmax values of Fe3O4–silica and Fe3O4– silica(porous) nanoparticles become more obvious and shift to lower temperatures at 109 and 101 K, respectively, as the thicknesses of shells increase, although the Fe3O4 cores were not changed. For isolated, non-interacting nanoparticles, Tmax is normally related to the blocking temperature (TB) at which the particles undergo a phase transition from ferromagnetic to superparamagnetic. As for the ZFC analysis, the experimental curves were compared to a theoretical model based on the blocking behavior of assemblies of superparamagnetic nanoparticles.19
3.3 Surface chemistry In order to confirm the functional groups on the surface of nanoparticles, the FTIR spectra were collected on (a): Fe3O4– OA, (b): Fe3O4–silica and (c): Fe3O4–silica(porous) nanoparticles, as shown in Fig. 5. The absence of –OH vibrations This journal is ª The Royal Society of Chemistry 2012
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Fig. 4 (a–c) Hysteresis loops at 10 and 300 K of (a) Fe3O4–OA, (b) Fe3O4–silica and Fe3O4–silica(porous) nanoparticles after zero field cooling. (d–f) ZFC-FC magnetization curves of Fe3O4–OA, Fe3O4–silica and Fe3O4–silica(porous) nanoparticles under an applied magnetic field of 50 Oe. Insets in (a) and (b) are the enlargements of the graph near the origin. The inset in (c) is the photograph of Fe3O4–silica(porous) nanoparticles dispersed in DI water, with and without magnetic separation.
Fig. 5 FTIR spectra of (a) Fe3O4–OA, (b) Fe3O4–silica and (c) Fe3O4–silica(porous) nanoparticles.
at 3300 cm1 from acid groups indicates that all the OA molecules have reacted with the Fe3O4 surface and no physisorbed oleic acid molecules remained. This results in a selfassembled oleate monolayer around the surface of Fe3O4 nanoparticles. Thus, the Fe3O4–OA nanoparticles bear –CH3 groups at the free termini of OA chains, leading to a hydrophobic behavior. As shown in Fig. 5(b), asymmetric and symmetric stretching vibrations of ^Si–O–Si^ were observed at about 1080 and 798 cm1, respectively, corresponding to characteristic peaks of silica.20,21 The peaks at 3400, 1637 and 960 cm1 are assigned, respectively, to the stretching and deformation vibrations of adsorbed water molecules and the stretching mode Si–OH (hydroxyl groups).22–24 The FTIR spectrum of Fe3O4– silica(porous) nanoparticles in Fig. 5(c) shows that the characteristic peaks of silica at 1050 (asymmetric stretching ^Si– O–Si^) and 920 (symmetric stretching ^Si–O–Si^) shift to lower wavenumbers compared to that of Fe3O4–silica nanoparticles, which is ascribed to the effect of aminoethylaminopropyl groups. The broad peak at 3350 cm1 is due to an overlap of hydrogen-bonded O–H and N–H stretching. The peaks at 2900 cm1 are due to stretching vibrations This journal is ª The Royal Society of Chemistry 2012
of –CH2– bonds. The multiple peaks between 1300 and 1600 cm1 are consequences of the vibrations of amine groups.25 The FTIR analyses confirmed that the silica shells of Fe3O4–silica(porous) nanoparticles are in situ functionalized by hydroxyls, primary and secondary amine groups. 3.4 pH-triggered DOX release DOX is one of the most widely used chemotherapeutic drugs. However, it is limited by dose-dependent toxic side effects.26 Thus, targeted drug delivery, providing therapeutically effective drug release at the tumor site, exhibits immense potential to resolve this issue and improve the treatment of cancers.27,28 The coupling and pH-sensitive hydrolysis properties of DOX molecules with primary and secondary amine groups, via 1,2-cyclohexanedicarboxylic anhydride as a linker, have been reported previously.29,30 The amides with neighboring carboxylic acid groups are stable at neutral pH, while at a low pH they are negatively charged to regenerate the amine groups and release the free DOX molecules. In line with this concept, we developed a magnetically guided pH-triggered drug delivery carrier based J. Mater. Chem.
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on the Fe3O4–silica(porous) nanoparticles. The Fe3O4–silica(porous) nanoparticles with primary and secondary amine groups were preliminarily functionalized by grafting 1,2-cyclohexanedicarboxylic anhydride molecules. The other side of 1,2cyclohexanedicarboxylic anhydride molecules was subsequently coupled to the amine groups of DOX molecules, forming the same amide bonds with neighboring carboxylic acid groups. Under normally physiological conditions (pH z 7), the coupled DOX molecules are quite stable, whereas mild acid hydrolysis can release them when they are transported in the vicinity of cancerous tissues (pH 5–6).30–32 The characteristic peaks of DOX molecules at 450–550 nm in UV-vis spectra are used to confirm the coupling and release processes. Concentration of the released DOX was examined by comparing the normalized absorbance intensity of separated supernatant solutions. Based on the intensity at 504 nm, the amount of coupled DOX was estimated at 13.2 mg per 100 mg nanoparticles, while the released amount of DOX molecules, at pH 5.0 for 63 h, was estimated to be about 9.8 mg per 100 mg nanoparticles. As a comparison, we also carried out the measurement of the loading capacity of DOX molecules in the Fe3O4–silica nanoparticles with dense silica shells, confirming that the maximum loading capacity is only 1.2 mg per 100 mg nanoparticles. It is clearly indicated that, in the case of Fe3O4– silica(porous) nanoparticles, DOX molecules are loaded not only on the surface of silica shells, but also on the interior pore/ channel surfaces. Fig. 6 shows the release kinetic of DOX molecules as a function of the pH and release time. It indicates that at a pH of 7.4 only 3.8% of the initial DOX molecules’ payload was released after 10 h. This low loss of the initial charge replies that the amide bonds were stable enough in order to retain the DOX molecules. As the pH decreased, a sharp increase was measured in the concentration of the DOX molecules released in the supernatant solution. The concentrations released after 10 h at pH 6 and 5 were estimated to be 46% and 76% respectively. These data revealed that the release process of DOX from the Fe3O4–silica(porous) nanoparticles is pH-triggered and can be well controlled. Conversely, in Fig. 6, we see that the DOX release process exhibits a bimodal release pattern. It seems that the early-time window, from 0 to 3.25 h, has different dynamics than the latetime interval, from 3.25 to 10 h, for both pH 6.0 and 5.0. Obviously, the origin of this bimodal behavior is related to two
Fig. 6 Release profiles of DOX molecules from Fe3O4–silica(porous) nanoparticles in buffer solutions of pH 5.0, 6.0 and 7.4 at 37 C.
J. Mater. Chem.
different release kinetics, that have different diffusion coefficients. A similar bimodal release behavior has been observed in other pH-regulated nanocarriers such as porous silicon nanoparticles with a nanovalve system with a cyclodextrin cap33 and mesoporous silica nanoparticles with ZnO nanolids.34 In these systems, the release is mainly controlled by both the pH-dependent hydrolysis kinetics and the diffusion (or dissolution) of cyclodextrin nanovalves (or ZnO nanolids). In comparison, the bimodal release behavior in the Fe3O4–silica(porous) nanoparticles indicates that the diffusion of released DOX molecules could also be affected by the hierarchical architecture of nanochannels. The hierarchical nanochannels act as interceptors to slow the diffusion which is similar to the role of nanovalves. Such a feature is important as the hierarchical porous carriers proposed herein can release their cargo within a specific timescale of interest. 3.5 Delivery of DOX-nanoparticles into cells The therapeutic efficacies of Fe3O4–silica(porous) nanoparticles functionalized with DOX were tested using A549 cells, adenocarcinomic human alveolar basal epithelial cells. After incubation for 48 h, an MTT assay was performed to evaluate the viability of A549 cells, as shown in Fig. 7. These results indicate that the Fe3O4–silica(porous) nanoparticles are relatively nontoxic at lower concentrations (1, 5 and 10 mg ml1) with around 70% cell viability, whereas both free DOX and Fe3O4–silica(porous)–DOX nanoparticles exhibited a significant loss of cell viability, most notably at 50 mg ml1. However, at the same DOX concentration of 0.5, 1 and 5 mg ml1, Fe3O4–silica(porous)– DOX nanoparticles show higher viability compared with free DOX. The cytotoxicity effect was further confirmed by microscopic visualization of the cell morphology change after treatment with the nanoparticles and/or DOX. Fig. 8 shows the morphologies of A549 cells treated with Fe3O4–silica(porous) nanoparticles, Fe3O4–silica(porous)–DOX nanoparticles and free DOX, respectively. Cells incubated with DOX became rounded and non-adherent, indicative of the fact that they have undergone apoptosis. In contrast, no rounded and detached cells can be visualized in both control cells and cells treated with Fe3O4–silica(porous) and Fe3O4–silica(porous)–DOX nanoparticles. The loaded DOX molecules are visualized hidden in the
Fig. 7 Cell viability percentage after 48 h of incubation with Fe3O4– silica(porous), Fe3O4–silica(porous)–DOX nanoparticles, and free DOX with various concentrations.
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Fig. 8 Bright-field microscopic images of the cells incubated after 48 h with Fe3O4–silica(porous), Fe3O4–silica(porous)–DOX nanoparticles, and free DOX with various concentrations.
hierarchically ultra-large channels of the Fe3O4–silica(porous) nanoparticles, which reduces their effective exposure to cells, and therefore results in a lower toxic effect. Fluorescence images were used to determine intracellular localization and accumulation of DOX in A549 cells. DOX is localized in their cytoplasm and subcellular compartment as indicated by the clearly visible red fluorescence of DOX in Fig. 9. However, rounded and non-adherent cells could be found in free
DOX treated cells. This implies that the Fe3O4–silica(porous)– DOX nanoparticles could be directly uptaken by cells with reduced toxicity. Moreover, the uniform fluorescence intensity and distribution in each cell indicates that the Fe3O4–silica(porous)–DOX nanoparticles have a good dispersibility in the medium and no aggregation occurs when interacted with cells, which is attributed to a combined consequence of the nearly zero magnetic interaction and the surface charged hydroxyls.
4
Fig. 9 (a and c) Bright-field and (b and d) fluorescence microscopic images of DOX release detection of (a and b) 10 mg ml1 Fe3O4–silica(porous)–DOX nanoparticles and (c and d) 1 mg mg1 free DOX in cells with incubation of 48 h.
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Conclusions
We report a one-pot synthesis for producing water soluble, amino-functionalized Fe3O4–silica(porous) core–shell nanoparticles with a 15.1 nm Fe3O4 core and a 20 nm nanoporous silica shell. The porous silica shell was characterized by high angle annular dark field imaging technology, revealing the hierarchically ultra-large pores and their channels extended to the surface. We further developed and demonstrated a chemical protocol to covalently couple and release DOX via dual regulations of the pH-dependent hydrolysis kinetics of the amides and the hierarchical nanochannels that act as interceptors for slow diffusion. The largest amount of released DOX at pH 5 after 63 h was about 9.8 mg for 100 mg Fe3O4–silica(porous) nanoparticles, and 76% of them can be effectively released after 10 h. At pH 7.4, only 3.8% and 9% were released after 10 and 63 h, respectively. The nanoparticles synthesized herein show a saturated magnetization of 1.1 emu g1. Based on a theoretical model, the temperature-dependent magnetization processes point toward a nearly zero magnetic interaction between superparamagnetic Fe3O4 cores due to the steric hindrance of the shells, contributing to good dispersibility. Such significant features meet the desirable requirements for a drug delivery system while providing the possibility of effectively tracking the drug carriers using MRI technology. J. Mater. Chem.
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Acknowledgements The work was jointly supported by the NSERC-CRD grant, the Canadian Institutes of Health Research and the National Research Council of Canada, Industrial Materials Institute (IMI-NRC). The authors would like to thank Franc¸ois Normandin from IMI-NRC for the help with magnetic measurements and Dr Gianluigi Botton from McMaster University Center for Electron Microscopy for his help with the TEM characterization. We are grateful to Nitric Medical Devices Inc. for the financial support and to Dr Blaise Gilbert and Dr Omar Quraishi for the insightful advice for the use of the magnetic carriers for biomedical applications. We would like to thank Dr Edward Sacher for technical revision of the manuscript.
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