Cost-Effective Design of High-Magnetic Moment ... - ACS Publications

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Article

Cost-effective design of high-magnetic moment nanostructures for biotechnological applications Beatriz Mora, Arantza Perez-Valle, Carolina Redondo, Maria Dolores Boyano, and Rafael Morales ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16779 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Cost-effective design of high-magnetic moment nanostructures for biotechnological applications Beatriz Mora,† Arantza Perez-Valle,§ Carolina Redondo,† Maria Dolores Boyano,‡ and Rafael Morales*,∥,⊥ †

Department of Chemical-Physics, University of the Basque Country UPV/EHU, 48940 Leioa, Spain.

§

Department of Cell Biology and Histology, University of the Basque Country UPV/EHU, 48940 Leioa, Spain ‡

Department of Cell Biology and Histology, University of the Basque Country UPV/EHU, and Biocruces Health Research Institute, 48903 Barakaldo, Spain.

∥Department

of Chemical-Physics & BCMaterials, University of the Basque Country UPV/EHU, 48940 Leioa, Spain. ⊥IKERBASQUE,

Basque Foundation for Science, 48011 Bilbao, Spain.

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Disk-shaped magnetic nanostructures present distinctive features for novel biomedical applications. Fine tuning of geometry and dimensions is demanded to evaluate efficiency and capability of such applications. This work addresses cost-effective, versatile and maskless design of biocompatible high-magnetic moment elements at the sub-micrometer scale. Advantages and disadvantages of two high throughput fabrication routes using interference lithography were evaluated. Detrimental steps as the release process of nanodisks into aqueous solution were optimized to fully preserve the magnetic properties of the material. Then, cell viability of the nanostructures was assessed in primary melanoma cultures. No toxicity effects were observed, validating the potential of these nanostructures in biotechnological applications. The present methodology will allow the fabrication of magnetic nanoelements at the sub-micrometer scale with unique spin configurations, such as vortex state, synthetic antiferromagnets, or exchangecoupled heterostructures, and their use in biomedical techniques that require a remote actuation or a magneto-electric response.

KEYWORDS: magnetic nanostructures, interference lithography, magnetic vortex, cell viability, nanomedicine, biomedical applications.

1. INTRODUCTION Due to their unique properties, magnetic nanoparticles have yielded highly promising results in several areas of biomedical research such as drug delivery,1–3 magnetic hyperthermia,4–6 and contrast agents in magnetic resonance imaging, MRI.7–10


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Superparamagnetic nanoparticles synthesized by chemical procedures have been widely used for this purpose. These nanoparticles exhibit zero remanence, which is crucial for biomedical applications as it prevents particles from agglomeration when dispersed in solution.11 However, one of the biggest drawbacks for such nanoparticles, which are typically iron oxides, is their low magnetic moments. This represents a serious limitation of its efficiency.12 Higher magnetic moments could be achieved by increasing the size of the nanoparticles but superparamagnetic nanoparticles of higher dimensions would overcome the superparamagnetic limit giving rise to non-zero remanence, which can be translated into non-desirable particle aggregation. By contrast, top-down fabrication routes combining lithography and physical vapor deposition techniques allow the production of nanostructures of higher magnetization -magnetic moments per unit volume- since pure elements and alloys can be deposited.13 Moreover, magnetic nanostructures with unique spin configurations -like vortex state- and large dimensions can be obtained while preserving zero remanence. Hence, these routes provide high versatility for the design of magnetic configurations, playing with different sizes and geometries, and the significant advantage to grow multilayers of dissimilar materials with designed magnetic properties.14–16 Top-down fabrication routes yield flat magnetic nanostructures (hereinafter referred to as MNS) with strong shape anisotropy, in contrast to synthesized spherical magnetic nanoparticles (hereinafter referred to as MNP). When an alternating field is applied, MNS rotate to align the magnetic moment along the field direction. Consequently, the torque or the mechanic force they exert can be exploited to manipulate soft matter17 and are highly promising for a wide range of applications including biomedicine, rheology and optics.18–20 Specifically, there is increasing interest in the mechanical stimulation of biological cells. It is well known that cells convert



mechanical stimulus into biochemical signals through a mechanism called mechanotransduction. The ability of manipulating cell signal in a remote manner by means of external magnetic fields opens up a wide variety of possibilities for the development of new clinical treatments. By now, only MNP have been used for this purpose in areas including regenerative medicine21 and neurostimulation.22–24 The use of MNS might provide a significant advance due to their distinctive feature of non-isotropic elements. Bottom-up approaches have also been developed to synthesize non-isotropic particles, such as nanorods, nanowires, and cubic or hexagonal particles.18,25–27 However, top-down lithography techniques present a controlled way to produce magnetically anisotropic elements such as disks, ellipses, stripes, cups with any magnetic element or multiple materials.16,28 Therefore, MNS can be designed with specific magnetic properties and magnetic configurations in a wide range of sizes. On the contrary, in comparison with bottom-up techniques, top-down approaches offer lower production yields. An innovative application of MNS based on this mechanical stimulus was proposed as a promising cancer therapy. Kim et al. investigated the use of vortex state disks with 1 µm of diameter fabricated by photolithography for cancer cell destruction in vitro assays.29 The photolithographic technique has a limitation in the element size in the micrometer range. However, it is crucial for biomedical applications to scale down into the submicrometer scale. Submicrometric MNS can be achieved by means of alternative lithographic techniques.30–33 Among them, interference lithography (IL) is an excellent candidate. This technique enables to pattern large areas without the use of a mask or costly solid stamps allowing nanofabrication at significant lower-cost. It is worth noting the short patterning fabrication time and the reduced cost of a IL system compared to other maskless equipments.34 Moreover, IL is a versatile technique. Patterns of different sizes –few hundreds of nm in diameter- and geometries –disks,


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ellipses, and lines- can be simply obtained by changing the incident angle of the laser beam and the exposure dose. For this reason, this technique embraces many uses in a variety of subjects, as energy conversion,35 biotemplating,36 electronics,37,38 photonics,39,40 and nanomedicine.41,42 Specifically, in biotecnological applications, both magnetic and non-magnetic nanostructures fabricated by IL could be used as carrier vehicle for drug delivery,43,44 nanorobots in biohybrid devices,45 cell labeling,46 and bioseparation.47 Moreover, this technique can be very practical for surface patterning in research topics like bacteria-driven propulsion,48 nanoparticle transport,49 antimicrobial materials,50,51 surface texturing for neurobiology applications and cell-substrate interactions.24,52

In this work, we report on this alternative to fabricate high-magnetic moment nanostructures suitable for biomedical applications, i.e. non-toxic particles with non-magnetic moment at remanence. A cost effective IL technique was used to pattern large areas (cm2), leading to high mass production of MNS for biomedical assays. The set up of the equipment makes easy to tailor the shape and the size of the nanoelements, from about 50 nm to a few microns, which enables to engineer MNS with specific magnetic properties. Namely, MNS with vortex spin configuration, which exhibit zero magnetization in absence of external magnetic field and rotate with alternating magnetic fields, were fabricated using positive and negative tone resists. Advantages and disadvantages of each route are discussed. A crucial step is the subsequent release of the MNS into aqueous solution. This chemical etching can modify, and even spoil, the magnetic properties of the nanoelements. This procedure was optimized for two distinctive sacrificial layers, Ge and Al. Finally, the cell viability of these MNS was assessed in vitro skin cancer cultures.



2. EXPERIMENTAL SECTION 2.1. Fabrication of magnetic nanostructures (MNS). Disk-shaped MNS were fabricated by interference lithography using positive and negative resists. Resist templates confer the shape and dimensions to the magnetic material deposited afterwards. Our IL system uses a Lloyd’s mirror interferometer with a He-Cd laser (λ = 325nm) as light source. A double exposure by the 90o rotation of the sample after the first exposure was needed to yield square arrays of dots and antidots for positive and negative resist, respectively. The set up of the equipment enables to easily adjust the periodicity of the array, and consequently the lateral size of the nanoelements, by simply rotating the sample holder and changing the angle of the incident light and controlling the exposure dose. Two fabrication routes were considered using either positive or negative resists. Positive resist route. Positive photoresist AZ MIR 701 (diluted 1:2 with the resist solvent AZ EBR) was spin coated at 6000 rpm for 1 minute onto a Si/Ti (15 nm) substrate and baked on a hotplate at 90 ºC for 60 seconds. The Ti (15 nm) layer was deposited onto the Si wafer to promote the positive resist adhesion. After that, a top antireflective layer AZ Aquatar was spin coated on top of the resist layer at 3000 rpm for 1 minute. A double exposure was performed with the sample rotated 90º respect to the first exposure (8 min each dose). Then, the resist was post-baked at 180 ºC for 1 minute and developed in AZ 726 MIF yielding a resist pillar array, Fig. 1(a). The periodicity of the resist template and the diameter of the resist pillars were characterized by scanning electron microscopy (SEM). A Permalloy (Ni80Fe20) thin film was deposited on the resist pattern by thermal evaporation with base pressure of over 10-7mbar, Fig.


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Nanodisks fabrication steps. Fabrication route using a positive resist-tone and antireflective coating (ARC) spin-coated onto a wafer: (a) exposure by IL and development of the positive resist, (b) thermal evaporation of magnetic materials, (c) lift-off process using NMP and detachment of the disks. Fabrication route using ARC and negative resist-tone coated onto a sacrificial layer of Ge deposited on a Si wafer: (d) exposure and development of IL negative-tone resist, (e) thermal evaporation of Ti/Permalloy/Ti, (f) lift-off process of the resist, (g) chemical etching with H2O2 and release of the disks. 1(b). Then, the Permalloy disks deposited on the top of the resist pillars were released into solution by removing the resist in a lift off process using 1-methy-2-pyrrolidonine (NMP), Fig. 1(c). Negative resist route. For this fabrication route, a sacrificial layer was placed between the Si wafer and the resist coating. For that purpose, a Ge layer was deposited onto the Si wafer by



thermal evaporation. The antireflective coating WIDE-8B (from Brewer Science) was spin coated onto the Si/Ge wafer at 5000 rpm for 60 seconds and baked on a hotplate in two steps: 40 seconds at 100 ºC and 60 seconds at 180 ºC. Later, the negative resist tone TSMR-IN027 (from Ohka) was spin coated on the ARC coating at 4000 rpm for 60 seconds followed by a baking step at 90 ºC for 90 seconds leading to a 280 nm resist stack. A double exposure by the 90º rotation of the sample after the first exposure was carried out using the IL system described above. The exposed resist was post- backed at 110 ºC for 1 minute and developed in AZ 736 MIF leading to a resist antidot array, Fig. 1(d). A Permalloy layer was deposited on the antidot pattern by thermal evaporation, Fig. 1(e). Then, the resist was removed in a lift of process with NMP yielding an array of Permalloy disks on the Ge buffer layer, Fig. 1(f). Finally, the Permalloy disks were released into solution by etching the Ge layer with hydrogen peroxide, Fig. 1(g). Since the fabrication strategy includes the use of a wet metal etchant (hydrogen peroxide), the magnetic material must be protected. For that purpose a Ti layer was deposited prior and after the Permalloy layer. Consequently, both sides of the disks were covered by Ti aiming to protect Permalloy disks from oxidation. Thickness and diameter of nanodisks were chosen to achieve a vortex state configuration according to the Permalloy phase diagram.53 2.2. Cytotoxicity assays. L-Glutamine, Phosphate Buffered Saline (PBS) and the XTT Cell Proliferation Kit II were all obtained from MERCK Chemicals and Life Science S.A. Dulbecco´s Modified Eagle´s Medium (DMEM) and the Penicillin/Streptomycin antibiotic solutions were purchased from Thermo Fisher Scientific Inc. Fetal Bovine Serum (FBS) was acquired from GE Healthcare.


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cell lines and culture conditions. Prior to the incubation with cells, MNS were sonicated for one minute and cleaned with sterile PBS. This process was repeated three times, and afterwards the MNS were autoclaved (Matachana Serie 500) at 120 ºC for 1 h 30 min. The A375 (CRL-1619) primary melanoma cell line was obtained from ATCC (American Type Culture Collection). They were routinely cultured as monolayers in 25 cm2 flasks with DMEM medium, supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-Glutamine and antibiotics (100 µg/ml Streptomycin and 100 IU/ml Penicillin). All the experiments were carried out at ≥90% viability and the cells were cultured in an 80-90% of confluence. The proliferation rate of these melanoma cells is between 6 and 12 hours.54 Cell viability. A375 cells were seeded in culture medium in flat bottom 96 well plates at the density of 10,000 cells per well. After allowing the cells to attach to the wells overnight, they were incubated with different concentrations of MNS (0-140 MNS per cell) for 24 h. XTT colorimetric assay was used to determine cell viability according to the manufacturer´s instructions. After 4 h of incubation, the absorbance was measured at 490 nm in a microplate reader (SynergyTM HT, BioTek). Untreated controls were also included and five replicate wells were analyzed for each condition. The cell viability percentage was calculated relative to controls: experimental absorbance / untreated control absorbance x100. Time-lapse. For a time-lapse experiment 200,000 A375 cells were seeded in culture medium in a 35 mm glass bottom dish and allowed to attach overnight. Cells were incubated with 140 MNS per cell. This culture was recorded by taking photos at a 20X magnification every 10 minutes during 24 h with the BioStation IMQ live cell time-lapse microscopy (Nikon Instruments B.V.).



3. RESULTS AND DISCUSSION 3.1. Morphological characterization. Scanning electron microscopy was used to verify the homogeneity of the arrays, the geometry and dimensions of the nanoelements in each step of the fabrication process. Fig. 2 shows the SEM images of the most representative steps of the two routes. Fig. 2(a) and (d) correspond to arrays of resist pillars and resist antidots obtained by IL using positive and negative resist tones, respectively. Fig. 2(b) and (e) show the samples after depositing the Permalloy layer and performing the lift-off process of the resist. In order to illustrate the process for the positive resist route, a SEM image was taken on the substrate when the lift-off was not fully completed Fig. 2(b) –for a SEM image of a complete lift-off see Fig. S1 in Electronic Supporting Information. The white dots correspond to the disks that were still on top of the resist pillars and the dark dots correspond to the holes in the Permalloy film that the resist pillars left after being removed by the solvent. For the negative resist route, after a successfully performed lift-off process, an array of Permalloy disks remained on the substrate, Fig. 2(e). Fig. 2(c) and (f) show the released disks obtained from the positive and the negative routes that were subsequently dried on a Si wafer. The surface topography of the disks was tested during fabrication by atomic force microscopy (AFM) and SEM. Fig. 3 shows microscopy images at different stages of the two fabrication routes. In case of positive resist, 25 nm of the magnetic material were grown on resist pillars, Fig. 3(a). The AFM scan reveals a curved profile of the top side of the pillars, Fig. 3(b). This profile yields concave nanodisks when released, as observed by SEM on dried MNS, Fig. 3(c).


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. SEM images of samples at the most representative steps of the two fabrication routes. Positive resist route: (a) array of resist pillars (dark dots), (b) substrate after a lift-off process not fully completed, (c) released disks dried on a Si substrate. Negative resist route: (d) array of resist antidots, (e) array of Permalloy disks after lift-off, (f) released disks dried on a Si substrate. The scale bar is 1µm. Concave nanostructures are the result of a Permalloy deposit on a top-curved positive resist pillars. However, the MNS thickness seems quite uniform over the lateral dimensions of the nanostructure.

By contrast, no curvature was observed on the disks fabricated by the negative resist route. Fig. 3(d) shows the AFM image of Permalloy disks attached to the substrate. Fig. 3(e) displays the AFM profile of a single Permalloy disk with 50 nm of height coated by around 10 nm of Ti on both sides. Released MNS from the negative resist route present a much flatter surface than those



Figure 3. Morphological characterization of Permalloy disks obtained from the positive and negative resist route. Disks from the positive resist route: (a) AFM image after

thermal evaporation of Permalloy on top of resist pillars, (b) AFM profile along the blue line in (a), (c) SEM image of nanodisks after lift-off of the resist. Disks from negative resist route: (d) AFM image after Permalloy deposition and lift-off of the resist, (e) AFM profile along the blue line in (d), (f) SEM image of nanodisks after etching of the sacrificial layer. from the positive resist, Fig. 3(f). The lack of curvature following this route is foreseeable since in this case the Permalloy layer was grown onto a flat Si/Ge wafer. Flat disks are desirable for having a better control of the magnetic properties, which strongly depend on the nanostructure geometry. Consequently, the negative resist route is more suitable for the fabrication of nanodisks with more complex magnetic configurations such as synthetic antiferromagnetic nanostructures (SAF) in which the role of the interfaces is crucial. However,


Page 12 of 29

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for the case of the vortex state Permalloy disks, the curvature of disks fabricated from the positive resist route does not appear to introduce a major problem, as discussed below.

3.2. Magnetic characterization The negative resist route makes possible to magnetically characterize the array of Permalloy disks before being released into solution. This advantage is due to the fact that the array of Permalloy disks is placed on a non-magnetic layer (Ge) and represents the only magnetic contribution to the total magnetic signal -the Permalloy layer deposited on the resist was removed in the lift off process. The possibility to magnetically characterize the array of Permalloy disks is important for the fabrication process since it enables to determine the geometrical conditions that lead to magnetic vortex-state configuration. Permalloy disk arrays obtained from the negative resist route with diameter of 650 nm and thickness of 50 nm were magnetically characterized by magneto-optical Kerr effect (MOKE) at room temperature. The obtained hysteresis loops from MNS attached to the Si/Ge substrate, Fig. 1(f), revealed the existence of spin-vortex configuration, Fig. 4(a). The abrupt reduction of the magnetization decreasing the magnetic field, around H=225 Oe, indicates the creation of a vortex state close to the edge of the nanodisk.55 The vortex core is placed at the center of the nanostructure at zero field, as indicated by the null remanence of the hysteresis loop. Larger magnetic fields towards negative saturation induce the vortex state annihilation at H=-700 Oe.



Figure 4. Magnetic characterization of the Permalloy MNS. Negative route: hysteresis loops of (a) array of Py disks attached to the substrate, (b) released disks dried on a Si substrate. (c) Positive route: hysteresis loop of released disks dried on a Si substrate.

Released magnetic disks fabricated by either positive or negative resist route were magnetically characterized by superconducting quantum interference device (SQUID) at room temperature. After the release process, the disks were washed and dispersed in deionized water. The preparation of the sample consisted on drying a droplet of the disks water suspension on a Si wafer. The magnetic response of dried disks fabricated by the negative resist route, Fig. 1(g), is shown in Fig. 4(b). The disks do not exhibit the same hysteresis loops when attached to a substrate as when released and dried. These differences in magnetic behavior might be due to the little aggregation originated during the drying process.56 While Fig. 4(a) can be assumed as the magnetic response of individual disks in a vortex state (neighboring interactions are negligible), Fig. 4(b) accounts for nanodisks in a vortex state with strong neighboring interactions due to the stack formation in the drying process (see Fig. 2). The disks obtained from the positive resist route can be only characterized once they are released into solution. Before detachment, Permalloy disks deposited on top of the resist pillars coexist with the Permalloy layer deposited onto the Si/Ti wafer giving rise to two magnetic


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


contributions to the total signal. The SQUID magnetization curve of dried disks with 450 nm of diameter and 25 nm of thickness is shown in Fig. 4(c). Hysteresis loops of the released disks obtained from both negative and positive resist routes appeared to be equivalent. Therefore, we conclude that the Permalloy disks fabricated by the positive resist route also presented spinvortex configuration. This spin configuration was expected for the MNS diameter and thickness according to the phase diagram of Permalloy nanostructures.53 A magnetic response of MNS in water solution to alternating magnetic fields can be observed in video01 -Electronic Supplementary Information.

3.3. Optimization of the MNS release process Although at this point the negative resist route seems to be the most preferable strategy among the routes we propose for the fabrication of MNS, we should consider the fact that the negative route requires wet etching of the sacrificial layer by H2O2 that could also oxidize the magnetic material and deteriorate the magnetic properties of the MNS. As described below, a Ti layer was deposited prior and after the Permalloy layer to protect the sides of the disk from oxidation but with this approach the sidewalls of the disk remained unprotected. Considering that there might


(a) H2O2(30%)

2.5h

H2O2(30%)

H2O2(15%)

0

30

(b) 1.0

1h

1h 10

20

30

Magnetization

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 29

20

KOH (30%)

0.5

10

0.0 0

% of Magnetization reduction

1

2

3

0 4

% Magnetization reduction


Time (h)

Figure 5. Magnetization reduction test of: (a) Ti/PermalloyTi disks in an aqueous solution of H2O2 at concentrations of 15% and 30% H2O2, and time periods of 2.5 and 1 hour. (b) Unprotected Permalloy layer in an aqueous solution of 30% KOH.

Normalized magnetization (solid squares), and percentage of magnetization reduction (solid circles).

be certain oxidation in the wet etching step of the negative resist route, the effects of the H2O2 on our MNS was investigated by immersing arrays of Ti/Pemalloy/Ti disks deposited on Si wafers in aqueous solutions of H2O2 (no sacrificial layer was deposited for this study, therefore the MNS remain always attached to the Si substrate). Fig. 5(a) shows the magnetization reduction that the arrays of Ti/Permalloy/Ti disks experiment after their immersion in aqueous solutions at concentrations 15% and 30% H2O2. The percentage of magnetization reduction was calculated from the saturation magnetization of SQUID hysteresis loops before and after the immersion in the chemical solution. This reduction in the saturation magnetization corresponds to the proportion of Permalloy that was laterally oxidized in the nanodisks. Decreasing the duration of the experiment from 2.5 hours to 1 hour the oxidation reduces from 27% to 16%. The dilution of peroxide has also an important effect. For the same period of time (1 hour) the magnetization reduction can be decreased down to 8% for concentration of 15% H2O2. It is worth


Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


noting that despite the oxidation of the lateral sides of the disks, the stability of the vortex is not altered. The SQUID magnetization curve after immersion resembles the same shape than the hysteresis loop before immersion, Fig. 4(a), but with a reduced value of the saturation magnetization (see original hysteresis loops in Fig. ES2 -Electronic Supplementary Information). In order to further improve MNS protection against oxidation and preserve their magnetic performance we considered other materials as sacrificial layers. Aluminum was a good candidate, thermally evaporated Al layers can be easily removed in an aqueous solution of KOH (potassium hydroxide) and provide excellent results for the fabrication of Permalloy nanostructures in solution. Similarly to the oxidation study with H2O2, the effect of KOH on Permalloy layers was tested in the wet etching step. In this case we did not use Ti/Permalloy/Ti disks grown onto Si wafers because KOH also etched Si and detached part of the disks from the substrate. Thus, the oxidation test of the magnetic material in 30% KOH solutions was performed with unprotected Permalloy thin films. As seeing in Fig. 5(b), despite the Permalloy thin film being completely unprotected, little reduction of the magnetization was observed at the first hours of the experiment. The saturation magnetization decreases only (6.7%) for immersions longer than 3 h and less than 2% for almost 1 h. Consequently, the etching process of the sacrificial layer in the negative resist route can be significantly optimized using an Al buffer layer and a KOH solution as etchant. This recipe provides an excellent approach to fabricate and preserve high-moment MNS suitable for biotechnical applications, with unique magnetic properties and submicrometric dimensions.

3.4. Cell viability study



Figure 6. Melanoma cells viability: (a)-(b) Evolution of the cell culture of A375 cells and MNS (140 MNS/Cell) over time (0, 24 h). Images were taken every 10 minutes for up to 24 hours. The scale bar is 30 µm. (c) Viable cells percentage after 24 hours of incubation of different concentrations of MNS (0-140 MNS/Cell) with A375 cells. Data are present as Mean ± SD (standard deviation).

The toxicity of Permalloy nanodisks protected on both sides by a thin Ti layer was assessed in primary melanoma cultures. A375 skin cancer cells were incubated for 24 hours with nanodisks of 650 nm in diameter and 50 nm in thickness. XTT assays were evaluated for concentrations of 0 (control), 40, 60, and 140 MNS per cell. Optical microscope images taken every 10 min for 24 hours provide evidence that cells introduce the nanostructures present in the culture medium without suffering any negative effects; the cancer cells continue to replicate and move normally (see recorded video in Electronic Supplementary Information). Fig. 6(a) displays two images at 0 h and 24 h for an initial concentration of 140 MNS/cell. These nanodisks are innocuous when no magnetic film is applied, which turns them into excellent candidates for studying new therapeutic options for cancer treatment.


Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. CONCLUSIONS In summary, we present two different routes to fabricate biocompatible magnetic nanostructures in solution preserving their high-magnetic moment. Both routes provide a costeffective, versatile and high throughput method to design functional magnetic elements at the sub-micrometer scale with unique spin configurations. Interference lithography with positive resist requires fewer steps but has the disadvantages of poor adhesion to Si wafers and production of curved nanostructures. On the contrary, a negative resist route allows a complete magnetic characterization and flat nanostructures. The side effects of the chemical etching of the sacrificial layers were mitigated to preserve the full performance of the MNS. In vitro cell viability assays demonstrate that these MNS are not toxic for skin cancer cells, which opens new possibilities to explore magnetic field-driven effects in living cells and to improve detection limits in nanomagnetism-based sensor devices. Besides the vortex state of the present MNS, more complex magnetic configurations can be achieved with this technique. Taking advantage of deposition methods of thin film magnetic heterostructures, new kind of magnetic particles can be fabricated and exploited in medical applications in a cost-effective way.

ASSOCIATED CONTENT Additional movies and figures in Electronic Supporting Information: Video S1: A movie of MNS in water rotating by an alternating magnetic field. The video captures the light scattering of a laser beam crossing an eppendorf with released nanodisks in water. An alternating magnetic field is applied parallel to the laser beam. With no magnetic field, nanodisks are randomly oriented and the light scattering is high (bright). When magnetic field is



on, MNS align parallel to the field reducing the reflective light (dim). Low frequency is applied at the beginning. Then a higher frequency is set after few seconds. •

Video-ES1-water-released-MNS-driven-by-alternating-magnetic-fields (AVI)

Video ES2: A movie recording cellular uptake of magnetic nanostructures by melanona cancer cells compresses an in vitro culture for 24 h. Frames were taken every 10 min. •

Video-ES2-in-vitro-cancer-cells-with-MNS-24h-every-10min-A375 (AVI)

Figure S1: SEM image of a complete lift-off with positive resist Figure S2: SQUID hysteresis loops before and after H2O2 oxidation

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENT This project has received funding from Spanish grants AEI-MINECO FIS2016-76058, UE FEDER "Una manera de hacer Europa" and AEI-MINECO FIS2013-45469, the European Union´s Horizon 2020 research and innovation programme under the Marie Sklodowsks-Curie grant agreement No 734801, and the Basque government grant IT970-16. The authors thank for technical and human support provided by the Laser Facility and the Magnetic Measurements unit of SGIker UPV/EHU and European funding (ERDF and ESF). R.M. is also grateful to Prof. Antonio Gómez (UPV/EHU) for his initial tests with tumoral cells and Dr. Andrey Svalov (UPV/EHU) for providing Si/Ti substrates. REFERENCES (1)

Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991.

(2)

Noh, S.; Moon, S. H.; Shin, T.-H.; Lim, Y.; Cheon, J. Recent Advances of MagnetoThermal Capabilities of Nanoparticles: From Design Principles to Biomedical Applications. Nano Today 2017, 13, 61.

(3)

Voronin, D. V.; Sindeeva, O. A.; Kurochkin, M. A.; Mayorova, O.; Fedosov, I. V.; Semyachkina-Glushkovskaya, O.; Gorin, D. A.; Tuchin, V. V.; Sukhorukov, G. B. In Vitro and in Vivo Visualization and Trapping of Fluorescent Magnetic Microcapsules in a Bloodstream. ACS Appl. Mater. Interfaces 2017, 9 (8), 6885.

(4)

Revia, R. A.; Zhang, M. Magnetite Nanoparticles for Cancer Diagnosis, Treatment, and Treatment Monitoring: Recent Advances. Mater.Today 2016, 19 (3), 157.



(5)

Silva, A. C.; Oliveira, T. R.; Mamani, J. B.; Malheiros, S. M. F.; Malavolta, L.; Pavon, L. F.; Sibov, T. T.; Amaro, E.; Tannús, A.; Vidoto, E. L. G.; Martins, M. J.; Santos, R. S.; Gamarra, L. F. Application of Hyperthermia Induced by Superparamagnetic Iron Oxide Nanoparticles in Glioma Treatment. Int. J. Nanomed. 2011, 6, 591.

(6)

Grillo, R.; Gallo, J.; Stroppa, D. G.; Carbó-Argibay, E.; Lima, R.; Fraceto, L. F.; BañobreLópez, M. Sub-Micrometer Magnetic Nanocomposites: Insights into the Effect of Magnetic Nanoparticles Interactions on the Optimization of SAR and MRI Performance. ACS Appl. Mater. Interfaces 2016, 8 (39), 25777.

(7)

Rosen, J. E.; Chan, L.; Shieh, D.-B.; Gu, F. X. Iron Oxide Nanoparticles for Targeted Cancer Imaging and Diagnostics. Nanomed.-Nanotechnol. Biol. Med. 2012, 8 (3), 275.

(8)

Zabow, G.; Dodd, S.; Moreland, J.; Koretsky, A. Micro-Engineered Local Field Control for High-Sensitivity Multispectral MRI. Nature 2008, 453 (7198), 1058.

(9)

Nandwana, V.; Ryoo, S. R.; Kanthala, S.; De, M.; Chou, S. S.; Prasad, P. V.; Dravid, V. P. Engineered Theranostic Magnetic Nanostructures: Role of Composition and Surface Coating on Magnetic Resonance Imaging Contrast and Thermal Activation. ACS Appl. Mater. Interfaces 2016, 8 (11), 6953.

(10)

Ortgies, D. H.; De La Cueva, L.; Del Rosal, B.; Sanz-Rodríguez, F.; Fernández, N.; Iglesias-De La Cruz, M. C.; Salas, G.; Cabrera, D.; Teran, F. J.; Jaque, D.; Martín Rodríguez, E. In Vivo Deep Tissue Fluorescence and Magnetic Imaging Employing Hybrid Nanostructures. ACS Appl. Mater. Interfaces 2016, 8 (2), 1406.

(11)

Note that zero remanence prevents agglomeration due to magnetic interactions. However,


Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


other non-magnetic interactions, like van der Waals forces, can lead to a cluster formation of nanoparticles. (12)

Dutz, S.; Hergt, R. Magnetic Particle Hyperthermia—a Promising Tumour Therapy? Nanotechnology 2014, 25 (45), 452001.

(13)

Hu, W.; Wilson, R. J.; Koh, A.; Fu, A.; Faranesh, A. Z.; Earhart, C. M.; Osterfeld, S. J.; Han, S.-J.; Xu, L.; Guccione, S.; Sinclair, R.; Wang, S. X. High-Moment Antiferromagnetic Nanoparticles with Tunable Magnetic Properties. Adv. Mater. 2008, 20 (8), 1479.

(14)

Van Roosbroeck, R.; Van Roy, W.; Stakenborg, T.; Trekker, J.; D’Hollander, A.; Dresselaers, T.; Himmelreich, U.; Lammertyn, J.; Lagae, L. Synthetic Antiferromagnetic Nanoparticles as Potential Contrast Agents in MRI. ACS Nano 2014, 8 (3), 2269.

(15)

Vemulkar, T.; Mansell, R.; Petit, D. C. M. C.; Cowburn, R. P.; Lesniak, M. S. Highly Tunable Perpendicularly Magnetized Synthetic Antiferromagnets for Biotechnology Applications. Appl. Phys. Lett. 2015, 107 (1), 12403.

(16)

Martín, J. I.; Nogués, J.; Liu, K.; Vicent, J. L.; Schuller, I. K. Ordered Magnetic Nanostructures: Fabrication and Properties. J. Magn. Magn. Mater. 2003, 256 (1–3), 449.

(17)

Erb, R. M.; Martin, J. J.; Soheilian, R.; Pan, C.; Barber, J. R. Actuating Soft Matter with Magnetic Torque. Adv. Funct. Mater. 2016, 26 (22), 3859.

(18)

Fratila, R. M.; Rivera-Fernández, S.; de la Fuente, J. M. Shape Matters: Synthesis and Biomedical Applications of High Aspect Ratio Magnetic Nanomaterials. Nanoscale 2015, 7 (18), 8233.



(19)

Page 24 of 29

Liu, W.; Bao, Y.; Lam, M. L.; Xu, T.; Xie, K.; Man, H. S.; Chan, E. Y.; Zhu, N.; Lam, R. H. W.; Chen, T. H. Nanowire Magnetoscope Reveals a Cellular Torque with Left-Right Bias. ACS Nano 2016, 10 (8), 7409.

(20)

Li, F.; Lu, J.; Kong, X.; Hyeon, T.; Ling, D. Dynamic Nanoparticle Assemblies for Biomedical Applications. Adv. Mater. 2017, 29 (14), 1605897.

(21)

Santos, L. J.; Reis, R. L.; Gomes, M. E. Harnessing Magnetic-Mechano Actuation in Regenerative Medicine and Tissue Engineering. Trends Biotechnol. 2015, 33 (8), 471.

(22)

Colombo, E.; Feyen, P.; Antognazza, M. R.; Lanzani, G.; Benfenati, F. Nanoparticles: A Challenging Vehicle for Neural Stimulation. Front. Neurosci. 2016, 10 (MAR), 105.

(23)

Kunze, A.; Tseng, P.; Godzich, C.; Murray, C.; Caputo, A.; Schweizer, F. E.; Carlo, D. Di. Engineering Cortical Neuron Polarity with Nanomagnets on a Chip. ACS Nano 2015, 9 (4), 3664.

(24)

Singh, A. V.; Gharat, T.; Batuwangala, M.; Park, B.-W.; Endlein, T.; Sitti, M. ThreeDimensional

Patterning

in

Biomedicine:

Importance

and

Applications

in

Neuropharmacology. J. Biomed. Mater. Res. 2017, 1. (25)

Bauer, L. A.; Birenbaum, N. S.; Meyer, G. J. Biological Applications of High Aspect Ratio Nanoparticles. J. Mater. Chem. 2004, 14 (4), 517.

(26)

Brollo, M. E. F.; López-Ruiz, R.; Muraca, D.; Figueroa, S. J. A.; Pirota, K. R.; Knobel, M. Compact Ag@Fe3O4 Core-Shell Nanoparticles by Means of Single-Step Thermal Decomposition Reaction. Sci. Rep. 2014, 4, 1.

(27)

Silva, M. P.; Drummond, A. L.; Aquino, V. R. R.; Silva, L. P.; Azevedo, R. B.; Sales, M.


Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


J. A.; Morais, P. C.; Bakuzis, A. F.; Sousa, M. H. Facile Green Synthesis of Nanomagnets for Modulating Magnetohyperthermia: Tailoring Size, Shape and Phase. RSC Adv. 2017, 7 (75), 47669. (28)

Kovylina, M.; Alayo, N.; Conde-Rubio, A.; Borrisé, X.; Hibbard, G.; Labarta, A.; Batlle, X.; Pérez-Murano, F. Au Cylindrical Nanocup: A Geometrically, Tunable Optical Nanoresonator. Appl. Phys. Lett. 2015, 107 (3), 33102.

(29)

Kim, D.-H.; Rozhkova, E. A.; Ulasov, I. V.; Bader, S. D.; Rajh, T.; Lesniak, M. S.; Novosad, V. Biofunctionalized Magnetic-Vortex Microdiscs for Targeted Cancer-Cell Destruction. Nat. Mater. 2010, 9 (2), 165.

(30)

Luo, Y.; Misra, V. Large-Area Long-Range Ordered Anisotropic Magnetic Nanostructure Fabrication by Photolithography. Nanotechnology 2006, 17 (19), 4909.

(31)

Guo, L. J. Nanoimprint Lithography: Methods and Material Requirements. Adv. Mater. 2007, 19 (4), 495.

(32)

Cheung, C. L.; Nikolić, R. J.; Reinhardt, C. E.; Wang, T. F. Fabrication of Nanopillars by Nanosphere Lithography. Nanotechnology 2006, 17 (5), 1339.

(33)

Ross, C. A.; Hwang, M.; Shima, M.; Cheng, J. Y.; Farhoud, M.; Savas, T. A.; Smith, H. I.; Schwarzacher, W.; Ross, F. M.; Redjdal, M.; Humphrey, F. B. Micromagnetic Behavior of Electrodeposited Cylinder Arrays. Phys. Rev. B 2002, 65 (14), 144417.

(34)

Lu, C.; Lipson, R. H. Interference Lithography: A Powerful Tool for Fabricating Periodic Structures. Laser Photon. Rev. 2010, 4 (4), 568.

(35)

Chen, X.; Lin, P.; Yan, X.; Bai, Z.; Yuan, H.; Shen, Y.; Liu, Y.; Zhang, G.; Zhang, Z.;



Zhang, Y. Three-Dimensional Ordered ZnO/Cu2O Nanoheterojunctions for Efficient Metal-Oxide Solar Cells. ACS Appl. Mater. Interfaces 2015, 7 (5), 3216. (36)

Galloway, J. M.; Bird, S. M.; Talbot, J. E.; Shepley, P. M.; Bradley, R. C.; El-Zubir, O.; Allwood, D. A.; Leggett, G. J.; Miles, J. J.; Staniland, S. S.; Critchley, K. Nano- and Micro-Patterning Biotemplated Magnetic CoPt Arrays. Nanoscale 2016, 8 (22), 11738.

(37)

Liddle, J. A.; Gallatin, G. M. Lithography, Metrology and Nanomanufacturing. Nanoscale 2011, 3 (7), 2679.

(38)

Ma, Z.; Jiang, C.; Yuan, W.; He, Y. Large-Scale Patterning of Hydrophobic Silicon Nanostructure Arrays Fabricated by Dual Lithography and Deep Reactive Ion Etching. Nano-Micro Lett. 2013, 5 (1), 7.

(39)

Jin, M.; van Wolferen, H.; Wormeester, H.; van den Berg, A.; Carlen, E. T. Large-Area Nanogap Plasmon Resonator Arrays for Plasmonics Applications. Nanoscale 2012, 4 (15), 4712.

(40)

Shukla, S.; Baev, A.; Jee, H.; Hu, R.; Burzynski, R.; Yoon, Y. K.; Prasad, P. N. LargeArea, near-Infrared (IR) Photonic Crystals with Colloidal Gold Nanoparticles Embedding. ACS Appl. Mater. Interfaces 2010, 2 (4), 1242.

(41)

Lamers, E.; Walboomers, X. F.; Domanski, M.; Prodanov, L.; Melis, J.; Luttge, R.; Winnubst, L.; Anderson, J. M.; Gardeniers, H. J. G. E.; Jansen, J. A. In Vitro and in Vivo Evaluation of the Inflammatory Response to Nanoscale Grooved Substrates. Nanomed.Nanotechnol. Biol. Med. 2012, 8 (3), 308.

(42)

Zhu, M.; Zhou, L.; Li, B.; Dawood, M. K.; Wan, G.; Lai, C. Q.; Cheng, H.; Leong, K. C.;


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Rajagopalan, R.; Too, H. P.; Choi, W. K. Creation of Nanostructures by Interference Lithography for Modulation of Cell Behavior. Nanoscale 2011, 3 (7), 2723. (43)

Timko, B. P.; Dvir, T.; Kohane, D. S. Remotely Triggerable Drug Delivery Systems. Adv. Mater. 2010, 22 (44), 4925.

(44)

Singh, A. V.; Hosseinidoust, Z.; Park, B.-W.; Yasa, O.; Sitti, M. Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active Cargo Delivery. ACS Nano 2017, 11 (10), 9759.

(45)

Singh, A. V.; Sitti, M. Targeted Drug Delivery and Imaging Using Mobile Milli/Microrobots: A Promising Future Towards Theranostic Pharmaceutical Design. Curr. Pharm. Des. 2016, 22 (11), 1418.

(46)

Chen, A.; Byvank, T.; Chang, W.-J.; Bharde, A.; Vieira, G.; Miller, B. L.; Chalmers, J. J.; Bashir, R.; Sooryakumar, R. On-Chip Magnetic Separation and Encapsulation of Cells in Droplets. Lab Chip 2013, 13 (6), 1172.

(47)

Kim, J.; Piao, Y.; Lee, N.; Park, Y. Il; Lee, I.-H.; Lee, J.-H.; Paik, S. R.; Hyeon, T. Magnetic Nanocomposite Spheres Decorated with NiO Nanoparticles for a Magnetically Recyclable Protein Separation System. Adv. Mater. 2010, 22 (1), 57.

(48)

Singh, A. V.; Sitti, M. Bacteria-Driven Particles: Patterned and Specific Attachment of Bacteria on Biohybrid Bacteria-Driven Microswimmers. Adv. Heal. Mater. 2016, 5 (18), 2325.

(49)

Holzinger, D.; Koch, I.; Burgard, S.; Ehresmann, A. Directed Magnetic Particle Transport above Artificial Magnetic Domains Due to Dynamic Magnetic Potential Energy



Landscape Transformation. ACS Nano 2015, 9 (7), 7323. (50)

Singh, A. V.; Vyas, V.; Patil, R.; Sharma, V.; Scopelliti, P. E.; Bongiorno, G.; Podestà, A.; Lenardi, C.; Gade, W. N.; Milani, P. Quantitative Characterization of the Influence of the Nanoscale Morphology of Nanostructured Surfaces on Bacterial Adhesion and Biofilm Formation. PLoS One 2011, 6 (9), e25029.

(51)

Rizzello, L.; Cingolani, R.; Pompa, P. P. Nanotechnology Tools for Antibacterial Materials. Nanomedicine 2013, 8 (5), 807.

(52)

Singh, A. V.; Patil, R.; Thombre, D. K.; Gade, W. N. Micro-Nanopatterning as Tool to Study the Role of Physicochemical Properties on Cell-Surface Interactions. J. Biomed. Mater. Res. 2013, 101A (10), 3019.

(53)

Cowburn, R. P.; Koltsov, D. K.; Adeyeye, A. O.; Welland, M. E.; Tricker, D. M. SingleDomain Circular Nanomagnets. Phys. Rev. Lett. 1999, 83 (5), 1042.

(54)

Benga, G. Basic Studies on Gene Therapy of Human Malignant Melanoma by Use of the Human Interferon β Gene Entrapped in Cationic Multilamellar Liposomes. J. Cell. Mol. Med. 2001, 5 (4), 402.

(55)

Guslienko, K. Y.; Novosad, V.; Otani, Y.; Shima, H.; Fukamichi, K. Magnetization Reversal due to Vortex Nucleation, Displacement, and Annihilation in Submicron Ferromagnetic Dot Arrays. Phys. Rev. B 2002, 65 (2), 24414.

(56)

Mahl, D.; Diendorf, J.; Meyer-Zaika, W.; Epple, M. Possibilities and Limitations of Different Analytical Methods for the Size Determination of a Bimodal Dispersion of Metallic Nanoparticles. Colloid Surf. A-Physicochem. Eng. Asp. 2011, 377 (1–3), 386.


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Table of Contents


Cost-Effective Design of High-Magnetic Moment ... - ACS Publications

Recommend Documents