Alternating Magnetic Field Controlled, Multifunctional Nano-Reservoirs: Intracellular Uptake and Improved Biocompatibility
© to the authors 2009
Received: 7 September 2009
Accepted: 5 October 2009
Published: 25 October 2009
Biocompatible magnetic nanoparticles hold great therapeutic potential, but conventional particles can be toxic. Here, we report the synthesis and alternating magnetic field dependent actuation of a remotely controllable, multifunctional nano-scale system and its marked biocompatibility with mammalian cells. Monodisperse, magnetic nanospheres based on thermo-sensitive polymer network poly(ethylene glycol) ethyl ether methacrylate-co-poly(ethylene glycol) methyl ether methacrylate were synthesized using free radical polymerization. Synthesized nanospheres have oscillating magnetic field induced thermo-reversible behavior; exhibiting desirable characteristics comparable to the widely used poly-N-isopropylacrylamide-based systems in shrinkage plus a broader volumetric transition range. Remote heating and model drug release were characterized for different field strengths. Nanospheres containing nanoparticles up to an iron concentration of 6 mM were readily taken up by neuron-like PC12 pheochromocytoma cells and had reduced toxicity compared to other surface modified magnetic nanocarriers. Furthermore, nanosphere exposure did not inhibit the extension of cellular processes (neurite outgrowth) even at high iron concentrations (6 mM), indicating minimal negative effects in cellular systems. Excellent intracellular uptake and enhanced biocompatibility coupled with the lack of deleterious effects on neurite outgrowth and prior Food and Drug Administration (FDA) approval of PEG-based carriers suggest increased therapeutic potential of this system for manipulating axon regeneration following nervous system injury.
Treatment potential for many biomedical conditions is limited by the lack of therapeutics that can be efficiently targeted and precisely controlled. For instance, functional recovery following neurotraumatic injury could be facilitated by therapeutics for guided axon regeneration . Axon growth can be directed by magnetic or electrical fields alone [2–4], but a more manipulable system that provides precisely tunable therapeutic delivery may offer enhanced potential to direct axon regeneration and guidance to targets. Nano-structured materials and smart surfaces provide great potential for developing such novel biomedical therapeutics [5–8]. However, several issues limit the use of conventional magnetic nanoparticles for biomedical applications. Magnetic nanoparticles alone are highly toxic, but excipients can facilitate their delivery to cells . Several groups have synthesized magnetic nanoparticle systems using smart polymers like poly (N-isopropylacrylamide) (PNIPAM) [10–12]. However, PNIPAM based systems have low biocompatibility because the NIPAM monomer is carcinogenic and teratogenic . Dimercaptosuccinic acid (DMSA) coated particles enhances intracellular uptake, but the surface functionalization does not improve the biocompatibility of the nanomagnets . Traditional polyethylene glycol (PEG) coating reduces toxicity, but it does not retain thermo-sensitive behavior or external tunability. Thus, development of a highly biocompatible, non-toxic and highly tunable nanoparticle system is necessary. Ideally, the constructed materials should: (1) minimize toxicity, (2) control intracellular functions like temperature or pH, and (3) elicit a fast response to external stimuli (e.g., ac magnetic field). A system that meets these goals possesses enhanced potential for combinatorial therapeutics. For example, simultaneous use of hyperthermia and low doses of chemotherapeutic agents decreases tumor growth by targeted cytotoxicity with reduced systemic effects .
For axon regeneration, a system that allows manipulation of cellular function or delivery of drugs through regulation of temperature and/or magnetic fields may be ideal. PNIPAM coated nanomagnets are attractive because of their perceived intelligence to external stimuli. For instance, they have a lower critical solution temperature (LCST) close to the normal physiological temperature (~33 °C), allowing size tunability and therefore controlled release of small molecules around 33–35 °C . However, it is necessary to tune the LCST in the range of 33–42 °C for certain biomedical applications requiring volumetric transition at higher temperatures. Recently, LCSTs of polyethylene glycol (PEG) based systems have been tailored between 24 and 42 °C by changing the molar ratio and molecular weight of the copolymers [17, 18]. It is also possible to regulate pressure/flow/temperature in micrometer or sub-micrometer range by using tunable magnetic nanocrystals embedded in thermo-sensitive polymer networks . Since PEG is non-toxic, anti-immunogenic and approved by the FDA [9, 17], the potential of magnetic modulation makes it an attractive alternative for overcoming traditional difficulties in actuating conventional micro- or nano-structures using chemical, mechanical or thermal excitation [20–22]. Although optical actuation may be used, its application for use in vivo is limited. Moreover, magnetic actuation could use remotely applied ac and dc fields to regulate intracellular temperature , control the release of drug from the tunable excipient by regulating swelling/shrinkage behavior  and manipulate cellular functions (e.g., axon growth) within tissues . Thus, using tunable PEG derivatives to encapsulate nanomagnets may facilitate the simultaneous regulation of localized temperature and sustained release of pharmaceuticals to the targeted cells in clinical practice.
In this study, we report synthesis and actuation of low toxicity magnetic nanospheres and assessed their effect on PC12 cell viability and morphology. Copolymers of 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate (PEGETH2MA-co-PEGMA) based hydrogels were synthesized to encapsulate ferromagnetic nano-particles. The polymeric shell acts as the reservoir of the drug molecules, while the magnetic core acts as nano-source of heat when exposed to the ac magnetic field. To our knowledge, this is the first time that ac field modulated remote actuation, model drug release and cytotoxicity is assessed for monodisperse, novel nanospheres that are made of all biocompatible and tunable polymers.
Scanning and Transmission Electron Microscopy
Sphere morphology was assessed using an FEI QUANTA 200 scanning electron microscope (SEM). A Philips EM 420 transmission electron microscope (TEM) was used to simultaneously observe polymer encapsulation and the embedded magnetic nanoparticles (core–shell structure). Cryo-immobilization was performed to prevent the nanospheres from collapsing during electron microscopy. Accelerating voltage during SEM imaging was kept between 5 and 20 kV; TEM analysis was performed with 120 kV electron beam.
Light Scattering and Magnetic Measurements
For the dynamic light scattering (DLS) experiment, a laser light scattering spectrometer (ALV, Germany) equipped with an ALV-5000 digital time correlator was used with a helium–neon laser (Uniphase 1145P, wavelength of 632.8 nm) as the light source. The hydrodynamic radius distribution of the nanospheres in water was measured at a scattering angle of 60°. A homemade magnetic field generator was designed to extract the AC field induced heating response. This device consists of an AC signal generator, a power amplifier, and a copper coil (diameter 25 mm, coil quality factor ~70). The coil was an element of a resonant RLC circuit with a self-inductance of 48 μH. The field strength was calculated using a high-frequency current probe (Tektronix) and an oscilloscope (Agilent). Custom software (written in LabView) controls the signal. Magnetic property [M(H)] of the nanospheres was measured using a Lakeshore model 7300 vibrating sample magnetometer (VSM) at ambient temperature.
Cell Culture and Treatment
PC12 rat pheochromocytoma cells (ATCC, Manassas, VA) were used to assess nanosphere biocompatibility. Cells were routinely cultured at 37 °C in 5% CO2 in F-12 nutrient mixture with Kaighn’s modification (F12 K) containing 2.5% fetal bovine serum and 15% horse serum (both from Invitrogen, Carlsbad, CA). For experiments, cells were plated at 10,000 cells/cm2 on glass coverslips in 24-well tissue culture plates and allowed to grow for 48 h. Cultures were washed twice with phosphate buffered saline (PBS) and placed into serum- and phosphate-free HEPES-buffered Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) to prevent particle aggregation. Nanospheres were added at final iron concentrations (assessed by TGA analysis) of 0, 0.34, 3.4, 6.0 or 16 mM for 48 h.
Assessment of Particle Internalization
PC12 cells were exposed for 48 h at 37 °C to fluorescent nanospheres containing magnetic nanoparticles at an iron concentration of 3.4 mM. Cultures were washed with PBS to remove extracellular nanoparticles, and cells were incubated for 24 more hours at 37 °C. Cultures were fixed for 20 min in 4.0% paraformaldehyde and 2.0% glutaraldehyde in PBS at room temperature. Images were captured through a 64× objective in differential interference contrast (DIC) and tetramethylrhodamine isothiocyanate (TRITC, Ex = 568 nm, Em = 585/40 nm) channels using a Zeiss Axiovert 200 M microscope (Zeiss, Thornwood, NJ) fitted with a Yokogawa CSU-10 confocal scanner and a Hamamatsu camera (McBain Instruments, Simi Valley, CA).
Assessment of Cell Viability
After exposure to magnetic nanospheres, cells were washed twice in PBS and viability was assessed using the Live/Dead viability/cytotoxicity assay (Invitrogen, Carlsbad, CA), according to the manufacturer’s directions. In brief, cultures were double-labeled with calcein AM, which permeates cell membranes and becomes fluorescent when exposed to esterase activity in living cells, and ethidium homodimer-1 (EthD1), which is excluded by the plasma membrane from living cells but crosses the compromised membrane of damaged cells to bind nucleic acids and fluoresce. Digital images were captured through a 10× objective on a Zeiss Aviovert 200 M microscopy (Zeiss, Thornwood, NJ) in fluorescein isothiocyanate (FITC, Ex = 480/30 nm, Em = 535/40 nm) and TRITC (Ex = 540/25 nm, Em = 605/55 nm) channels for calcein AM and EthD1, respectively. Only non-aggregated single cells (at least 300 cells/condition) were quantified, and the percent of viable cells was calculated. Significant differences between magnetic nanospheres containing different concentrations of iron were determined by ANOVA with subsequent Tukey post-hoc tests. Nuclear morphology was assessed using confocal images captured through a 64× objective from cells labeled with 4′,6-diamidino-2-phenylindole (DAPI, Ex = 405 nm, Em = 450/35 nm), following exposure to nanospheres containing magnetic nanoparticles at different concentrations of iron.
Assessment of Cell Morphology, Cytoskeleton and Neurite Outgrowth
Following nanosphere exposure, cultures were washed twice with PBS and placed into serum-free F-12 K for 72 h with or without 100 ng/mL nerve growth factor β subunit (β-NGF, Sigma–Aldrich, St. Louis, MO) being added daily. Cultures were fixed in 4.0% paraformaldehyde and 2.0% glutaraldehyde in PBS, washed twice in PBS, and blocked for 30 min in PBS containing 1.5% normal donkey serum and 0.1% Triton X-100 at room temperature. Samples were double labeled for filamentous actin and tubulin by sequential exposure to 3.3 μM Texas Red-X phalloidin (30 minutes, Invitrogen, Carlsbad, CA) and rabbit anti-βIII tubulin (1:200, overnight, Abcam, Cambridge MA). Tubulin immunoreactivity was visualized by Alexafluor 488-conjugated donkey anti-rabbit secondary antibodies (1:200, 1 h, Invitrogen, Carlsbad, CA). Digital images were captured though a 40× objective using DIC optics, and through a 64× objective in TRITC (Ex = 568 nm, Em = 585/40 nm) and FITC (Ex = 488 nm, Em = 520/35 nm) channels on a Zeiss Axiovert 200 M microscope with confocal attachments (McBain Instruments, Simi Valley, CA).
Neurite outgrowth was assessed using the percent of neurite-bearing cells, determined from phase contrast images (five images/condition/experiment) captured through a 40× objective on a Zeiss Axiovert 200 M microscope. For this analysis, a neurite was defined as a cellular extension that exceeded 10 μm. Only non-aggregated cells where the cell and all its neurites were included in the image were quantified, and imaged fields were systematically selected from each quadrant and the center of each coverslip to ensure a representative sample. Differences between experimental conditions were determined by ANOVA with subsequent Tukey post-hoc test using a significance level of α = 0.05.
Results and Discussion
It was important to design a system where the polymer encapsulates the magnetic nanoparticles to facilitate solubility and bioavailability and also provides the potential to conjugate bioactive peptides, antibodies, oligonucleotides or drugs. Thus, a key feature was to design a PEG-based system with a tunable LCST close to physiological temperature. Based on previous reports [17, 18], we chose a molar ratio of PEGMA to PEGETH2MA of 20%. Unlike single domain crystals, multi-domain magnetic particles exhibit primarily hysteresis loss-induced heating inside the ac field exposure with low frequency. Hysteresis loss induced heating is preferred over relaxation loss because the former is easier to tune by the controlled modulation of the oscillating magnetic field, especially considering the sharp hydrodynamic radius change that may occur around the LCST and severely impact the Brownian relaxation loss. To maximize the energy absorption, nanomagnet size was chosen to be in the range of 25–30 nm, close to the ferromagnetic exchange length (27 nm) for magnetite .
Physiochemical Characterization of Magnetic Nanospheres
Response to Alternating Magnetic Field and Drug Release
Magnetic Nanosphere Internalization into Neuron-Like Cells
Magnetic Nanospheres are Minimally Toxic to Neuron-Like Cells
Magnetic Nanoparticles Minimally Affect Morphology and Neurite Outgrowth
In summary, we report the synthesis, actuation and dose-dependent modulation of a multifunctional nano-scale system consisting of all FDA-approved bio-polymers. Based on these results, we conclude that the designed nanomagnets possess dual capability of regulating temperature and tuning size, i.e., the mesh density by remote controlled actuation. These special features carry the potential for synergistic application of heat and sustained release of small molecules in therapeutics. Moreover, compared to currently available systems, cytotoxicity is remarkably reduced by the stealth polymer encapsulation around the magnetic core and allowing higher intracellular concentrations of magnetic nanoparticles. One exciting application for this system may be using either unaltered or derivatized nanospheres in conjunction with magnetic fields to manipulate axon growth. The neurite growth pattern seen at high nanosphere concentrations is especially promising for further studies with effect on primary neurons in terms of axon regeneration following nervous system injury.
Supported by the TWU Department of Biology, the SEMO Physics and Engineering Physics Department, and grants from the TWU Research Enhancement and Summer Stipend programs (to DLH), the Texas Higher Education Coordinating Board Closing the Gaps program (to DLH), and the SEMO Grants and Research Funding Committee (to SG). We sincerely appreciate Dr. Zhibing Hu’s generosity for laboratory access.
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