Letter www.acsami.org
Magnetically Actuated Wormlike Nanomotors for Controlled Cargo Release Min Liu,*,† Liqing Pan,† Hongguang Piao,† Hongyu Sun,‡ Xiufeng Huang,† Changde Peng,§ and Yiman Liu*,† †
College of Science, China Three Gorges University, Yichang 443002, Hubei, China National Center for Electron Microscopy, Tsinghua University, 100084 Beijing, China § School of Physics and Electronic Engineering, Jiangsu Normal University, Xuzhou 221116, Jiangsu China ‡
S Supporting Information *
ABSTRACT: Magnetically actuated nanomotor, which swims under externally applied magnetic fields, shows great promise for controlled cargo delivery and release in biological fluids. Here, we report an on-demand release of 6carboxyfluoresceins (FAM), a green fluorescein, from G-quadruplex DNA functionalized magnetically actuated wormlike nanomotors by applying an alternating magnetic field. This field-triggered FAM releasing process can be easily controlled by multiple parameters such as magnetic field, frequency, and exposure time. In addition, the experimental results and the theoretical simulation demonstrate that both a thermal and a nonthermal mechanism are involved in the cargo releasing process.
KEYWORDS: magnetic actuation, wormlike nanomotor, cargo delivery, CoFe2O4 nanoparticles, HeLa cells
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activator (t-PA)-mediated thrombolysis by elevating drug transport through a hydrodynamic convection. Such nanomotors display an active motion in a rotational magnetic field, thus, they can serve as an independent input to power the drug’s efficacy and develop a safer and more effective medical treatment. To date, the magnetically actuated nanomotors are mainly fabricated up with solid noble metal structures, like Au/ Ag/Ni nanowires or Ni/Ti or Ni/Au helixes. The rigid properties of such motors, however, limit their delivery capability, as cargo can only be linked or loaded on nanomotors’ outer surface. In addition, most of the previous studies were focused on how to bestow a magnetic propulsion force upon the passively moving nanomotors. The use of nanomotors for magnetic actuation, together with controlled cargo delivery and release, is rarely explored. In this letter, we describe a new nanomotor strategy for controlled cargo delivery and release under an externally applied magnetic field. The concept behind this strategy is illustrated in Scheme 1. The wormlike mesoporous silica nanotube (MSN) as a host platform offers an exceptionally high surface area,18,19 which enables the loading of diverse cargoes. The individual CoFe2O4 nanoparticles20,21 deposited onto the surface of MSNs can act as power sources by harvesting energy from the external magnetic field, allowing the nanomotors to
ynthetic nanomotors have received increasing attention over the past decade because of their considerable promise for diverse potential applications.1−5 In particular, the remarkable performance of such artificial machines makes them the active workhorses in biological environments and enables numerous functions,6 ranging from biopsy7 to precision microsurgery8 and from intracellular transport of vesicles9 to directed drug delivery.10 For instance, tremendous progress2,5−7 has been recently made on drug delivery based on chemically powered, self-propelled catalytic nanomotors. However, many catalytic nanomotors rely on the use of external peroxide fuel and often lead to incomplete pollutant degradation, hindering their applications in biologically relevant media. To address these limitations, researchers have been exploring fuel-free nanomotors propulsion mechanisms, such as the utilization of electrical,11 optical,12 ultrasound,4,13 and magnetic field.14,15 Magnetically actuated nanomotors, which swim under externally applied magnetic fields, are particularly promising for use in variety of in vitro, and more importantly, in vivo biomedical applications. For example, J. Wang et al. reported recently a magneto-acoustic hybrid nanomotor, which displays efficient propulsion in the presence of either magnetic or acoustic field16 without adding any chemical fuel. Such adaptive hybrid operation holds considerable promise for diverse practical biomedical application of fuel-free nanomachines. A recent work from Cheng et al. illustrated a magnetically powder nanomotor17 that can accelerate the tissue plasminogen © XXXX American Chemical Society
Received: September 22, 2015 Accepted: November 17, 2015
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DOI: 10.1021/acsami.5b08946 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Scheme 1. Schematic Illustration of Magnetically Actuated Nanomotor for Controlled Fam Release in HeLa Cells by Applying an Alternating Magnetic Field
Figure 1. SEM and TEM images of various nanostructures. (a−c) SEM of typical wormlike MSNs, and (d) its corresponding 3D reconstruction. (e− h) Representative TEM of wormlike MSNs. (i) 3D reconstruction of nanomotor and (j−l) representative TEM images.
propel efficiently in biological fluids. Subsequently, the surface of nanomotors is covered with a G-quadruplexes layer. Gquadruplexes22,23 are highly ordered DNA structures derived from G-rich sequences formed by tetrads of hydrogen-bonded guanine bases, which prevents leaking of the cargoes. Because of the negative charges on the sugar phosphate backbones, the G-quadruplexes can also provide additional stability to the nanomotors. As previously reported,23−25 external stimuli (i.e.,
temperature, ion concentration, pH value) can substantially affect the conformation and stability of theses G-quadruplex DNA. Thus, the payload molecules can be released to target locations when these DNA underwent a stimuli-responsive conformational change between a G-quadruplex structure and a random single-stranded structure. The payload releasing process can be easily controlled by tuning the magnetic field because of its thermal effect and the mechanical effect on B
DOI: 10.1021/acsami.5b08946 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 2. (a) Real-time fluorescence spectrum collected in the FAM releasing process. (b) Plot of fluorescence at 518 nm against the exposure time in different frequency magnetic fields. Data were obtained from four measurements and the error bars represent standard deviation.
nanomotors. Such magnetically actuated nanomotors possess several distinct advantages for biomedical use, including excellent biocompatibility, high loading capacity, and controllable releasing behavior. Because of this extremely attractive performance, the nanomotors are expected to offer considerable promise for directed drug delivery, biopsy, local diagnosis, or precision nanosurgery in biological systems. The superparamagnetic CoFe2O4 NPs were first synthesized via a classic coprecipitation process as we previously reported.21 The size of the CoFe2O4 NPs was controlled by tuning the concentration of NaBH4. The nanoparticles with an approximate diameter of 5−8 nm were chosen, since the size of nanoparticles should be small enough to minimize the potential blocking effects on the mesopore channels. TEM images confirmed the expected size and indicated a relatively good monodispersity (the inset of Figure S1). An energy-dispersive X-ray spectrum (EDX) was also collected to confirm the chemical composition of the particles (Figure S1a). The ferrum and cobalt signals can be unambiguously observed in the figure, illustrating the presence of both Co and Fe in the prepared nanoparticles. The magnetic hysteresis loop of CoFe2O4 NPs has been measured at room temperature (Figure S1b). The magnetic property analyzed from minor hysteresis loop physically indicates that the synthesized CoFe2O4 NPs in this study are nearly in the superparamagnetic state. Importantly, the saturation magnetization of value 104 emu/g is observed, which is significantly larger than its Fe3O4 counterpart. Such a high saturation magnetization makes the CoFe2O4 NPs a promising alternative to propel nanostructures with high efficiency. An optimized silica/Pluronic 123/ethanol system was used to fabricate wormlike MSNs. Here, we use wormlike MSNs as host platforms for three purposes: (i) Silica can be easily functionalized with biocompatible materials (G-quadruplex DNA in this case), allowing us to simultaneously improve the colloidal stability and biocompatibility of nanomotors; (ii) the mesopores of the wormlike MSNs can ensure much higher cargo loading capacity compared to solid nanoparticles because of their extremely high specific surface area and pore volume; (iii) the smooth wormlike morphology and the asymmetric structures of MSNs provide the flexibility to control their movement. The size and morphology of the MSNs was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As shown in Figure 1, MSNs are wormlike nanorods of 1−1.5 μm length, exhibiting uniform pore sizes of 8 nm and an aspect ratio AR = 5−6. The wormlike nanoparticles showed periodical “fringes” along their
length (indicated by arrows in Figure 1f), which represent the ordered hexagonal pore arrangements. The representative magnified TEM images of MSNs are presented in Figure 1g, h, confirming the mesopore alignment and the mesochannel structure. To drive the MSNs by an external magnetic field, masses of CoFe2O4 NPs were successfully deposited on the MSNs, as shown in Figure 1j−l. The density of attachment can be easily controlled by tuning the dose of CoFe2O4 NPs. In this case, the mole ratio of CoFe2O4 to MSNs is estimated to be around 1−1.5 × 104:1 by statistical analysis of TEM data. The resulting nanomotors (MSNs@ CoFe2O4 NPs) are macroscopically magnetic (Figure S2), which should facilitate the controlled cargo delivery and release. To investigate the cargo releasing process, we chose 6carboxyfluoresceins (FAM),26 a green fluorescein, as a model payload because of its well-characterized spectral characteristics. The FAM molecules were initially loaded into the nanochannels via free diffusion. The optimal mole ratio of FAM to MSNs in the sample was estimated to be 1 × 106:1. The maximum amount of FAM that a MSN can load was determined to be roughly 1 × 10−17 to 1 × 10−15 g by comparing the fluorescence with a standard sample, demonstrating the remarkably high loading capacity of such MSNs. After loading the FAM molecules, the nanosystems were functionalized with G-quadruplex DNA (G-quadruplexes), as depicted in Scheme 1. Generally, G-quadruplexes could fold into successive G-quartets structures, which prevents leaking of the FAM molecules. This functionalization was confirmed by UV−vis spectrum and zeta potential results (Figure S3). After G-quadruplexes functionalization, the nanosystems were exposed to an alternating magnetic field (0.5 mT) with a frequency of 100 Hz. The fluorescence spectra were collected in the releasing process, and the results are presented in Figure 2a. Initially, the fluorescence of FAM was quenched completely by the high concentration of CoFe2O4 nanoparticles in the mesopores. Thus, in the figure, no discernible spectrum is observed within the first 16 min, suggesting an energy accumulation stage during which period no FAM molecules have been distinctly released. From 16 to 60 min, the fluorescence intensity is increased steeply, confirming the effective field-triggered release of FAM molecules. After 60 min, the fluorescence intensity reaches a maximum because the releasing process was saturated. The fluorescence microscopy images presented in Figure S4b, c further confirmed the release results. The releasing process is strongly dependent on the frequency of the applied magnetic field. Figure 2b shows a quantitative C
DOI: 10.1021/acsami.5b08946 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces plot of averaged fluorescence intensity at 518 nm as a function of exposure time under different magnetic fields. It is clear from these data that, under a 100 Hz magnetic field, the fluorescence intensity begins to increase after 16 min of continuous radiation and approaches saturation after ∼60 min, as we discussed above. While in the lower frequency fields, it would take longer to reach fluorescence saturation. As an example, under a 5 Hz magnetic field, the fluorescence increase was sustained for at least 330 min, indicating a much slower release process. As a control experiment, a sample that was not treated with an external magnetic field was used. As expected, no discernible spectrum was produced during the first several hours, verifying that no FAM molecules were released (Table S1). But 5 days later, an intense fluorescence can also be detected in the control sample, likely due to the slow-leaking of FAM molecules in PBS buffer. Additionally, the influence of the field strength to the releasing process is also investigated. As is summarized in Table S1, a fast release process was detected under a strong magnetic field (0.5 mT), whereas application of weaker fields resulted in a rapid fall in the FAM release. To use these nanomotors for molecular release in live cells, we incubated the nanomotors with FAM molecules with HeLa cell for 2 h. To facilitate cell uptake, we placed a magnet under the culture dish. After incubation, one culture dish was exposed to an alternating magnetic field (0.5 mT, 100 Hz) for 1 h. The cells were imaged using both fluorescence and bright-filed microscopy. As shown in Figure 3a, an intense green
minimize the heat transfer between the sample and the surrounding environment, we loaded the measured samples in a homemade thermally isolated sample stage (Figure S4d). As is clearly demonstrated by the data in Figure S5, the nanomotors with CoFe2O4 nanoparticles were heated remotely via an alternating magnetic field (0.5 mT, 100 Hz), which reduced the stability of G-quadruplexes and changed their conformation partially, thus resulting in the cargo release. Nevertheless, in lower-frequency cases (5, 10, 20, 40 Hz), the very small increases in nanomotors surface temperature (Figure S5) are observed, suggesting that a nonthermal mechanism may be involved in the cargoes releasing process. We hypothesize that the nanometers could oscillate in an alternating magnetic field,26,29,30 and thus throw cargoes out of the nanochannels, which significantly affect the cargoes releasing process. On the basis of this hypothesis, we simulated the oscillation process of a nanomotor in a low frequency magnetic field using the finiteelement method (Videos S1 and S2). The dielectric function for Co determined by Johnson and Christy was used and the nanomotors were assumed to be immersed in H2O. In fact, the nanomotors subjected to a magnetic field H⃗ will therefore experience a driving torque τ = m⃗ × H⃗ , where m⃗ is the nanomotor magnetic moment, proportional to both the saturation magnetization Ms and the magnetic susceptibility.29,30 For simplicity, this scenario does not take into account the torque τ of the nanomotor. The simulated results demonstrated that applying an alternating magnetic field can create a strong oscillation, which probably enhance the cargo release efficacy. To further validate this nonthermal mechanism, we compared the present results with the releasing process in an open sample container (a cell culture dish in a 30 °C water bath), and the results are summarized in Table S1. Interestingly, for constant temperature, all of the releasing processes can still reach saturation although they need longer times than in the thermally isolated system. This means that the controlled FAM release can still be achieved even eliminating the thermal effect on nanomotors, which further confirms the nonthermal mechanism. In conclusion, we have demonstrated a magnetically actuated wormlike nanometer for intracellular cargoes delivery and release. FAM molecules as model payloads, initially loaded into mesoporous pore channel of nanomotors, were released into HeLa cells within 1 h by applying an alternating magnetic field (0.5 mT,100 Hz). The field-triggered FAM releasing process can be easily controlled by multiply parameters such as magnetic field, frequency, and exposure time. Besides, the experimental results and the theoretical simulation reveal that both a thermal and a nonthermal mechanism were responsible for the release. Such nanomotors can be remotely manipulated by an external magnetic field regardless of whether there are intervening structures, which is quite useful in drug delivery applications. In addition, the nanomotors exhibit several remarkable properties, such as excellent biocompatibility, extremely high specific surface area and pore volume, as well as smooth wormlike morphology. These attractive capabilities indicate that the nanomotor may find significant practical utilities in a variety of diagnostic, therapeutic, and biomedical applications.
Figure 3. Controlled FAM release in HeLa cells. (a) Fluorescence image, (b) bright-field image, and (c) merge image of HeLa cells under an alternating magnetic field (0.5 mT, 1 h). In contrast, no obvious fluorescence was observed in (d−f) the control cells, which were not treated with an alternating magnetic field.
fluorescence in HeLa Cells is observed after magnetic field exposure, demonstrating that the FAM molecules were released from the nanomotors successfully. In contrast, no discernible fluorescence is observed for the control experiment in which the cell dish was not treated with an external magnetic field exhibits, verifying that FAM release does not occur in this case (Figure 3d). During the experiment, no obvious cells death was found, indicating that the lower-frequency magnetic fields (
