Naturally Derived Iron Oxide Nanowires from Bacteria for Magnetically

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Naturally derived Iron oxide nanowires from bacteria for magnetically triggered drug release and cancer hyperthermia in 2D and 3D culture environments: Bacteria biofilm to potent cancer therapeutic Tushar Kumeria, Shaheer Maher, Ye Wang, Gagandeep Kaur, Luoshan Wang, Mason Erkelens, Peter Forward, Martin Francis Lambert, Andreas Evdokiou, and Dusan Losic Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00786 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

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Nature is capable of building highly unique and intricate nanostructures. This study demonstrates the ability of iron oxide nanowires obtained from bacteria biofilm as a triggered drug delivery and hyperthermia agent under 2D and 3D cell culture setups. 153x138mm (96 x 96 DPI)

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Naturally derived Iron oxide nanowires from bacteria for magnetically triggered drug release and cancer hyperthermia in 2D and 3D culture environments: Bacteria biofilm to potent cancer therapeutic Tushar Kumeria#1, Shaheer Maher#1,3, Ye Wang1,4, Gagandeep Kaur1,4, Luoshan Wang1, Mason Erkelens2, Peter Forward5, Martin F. Lambert2,* Andreas Evdokiou4* and Dusan Losic1*

# These authors contributed equally to this work.

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School of Chemical Engineering The University of Adelaide, Adelaide, S.A. 5005, Australia School of Civil, Environmental and Mining Engineering, The University of Adelaide, Adelaide SA 5005, Australia 3 Faculty of Pharmacy, Assiut University, Assiut, 71526, Egypt 4 Discipline of Surgery, Basil Hetzel Institute, The University of Adelaide, Adelaide, SA 5005, Australia 5 South Australian (SA) Water, Adelaide, SA-5005, Australia 2

*Corresponding Authors Prof. D Losic E-mail [email protected] Prof. M. F. Lambert E-mail: [email protected] Prof. A. Evdokiou E-mail: [email protected]

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Abstract Iron oxide nanowires produced by bacteria (Mariprofundus ferrooxydans) are demonstrated as new multifunctional drug carriers for triggered therapeutics release and cancer hyperthmia applications. Iron oxide nanowires are obtained from biofilm waste in the bore system used to pump saline groundwater into the River Murray, South Australia (Australia) and processed into individual nanowires with extensive magnetic properties. The drug carrier capabilities of these iron oxide nanowires (Bac-FeOxNWs) are assessed by loading anticancer drug (doxorubicin, Dox) followed by measuring its elution under sustained and triggered release conditions using alternating magnetic field (AMF). The cytotoxicity of Bac-FeOxNWs assessed in 2D (96 well plate) and 3D (Matrigel®) cell cultures using MDA-MB231-TXSA human breast cancer cells and mouse RAW 264.7 macrophage cells shows that Bac-FeOxNWs are biocompatible even at concentrations as high as 250 µg/mL after 24 h of incubation. Finally, we demonstrate the capabilities of Bac-FeOxNWs as potential hyperthermia agent in 3D culture setup. Application of AMF increased the local temperature by 14 °C resulting in approximately 34% decrease in cell viability. Our results demonstrate that these naturally produced nanowires in the form of biofilm can efficiently act as drug carriers with triggered payload release and magnetothermal heating features for potential anti-cancer therapeutics applications.

Keywords: Hyperthermia, iron oxide nanowires, 3D cell culture, AMF triggered release, Doxorubicin


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1. Introduction In the last two decades a plethora of new synthetic functional nanomaterials have been introduced and applied for biomedical applications, in particular cancer treatment. Nanomaterials of a wide range of shapes (i.e. spheres, tubes, cubes, rods, stars and so on)1-4 and material (i.e. silica, gold, silver, iron oxide and many others)5-7 have been proven for a variety of functions including payload delivery (i.e. drugs, proteins, antibodies, enzymes),8 diagnosis, hyperthermia, and others.6 Iron oxide based nanomaterials, without any reservations, have become very popular for biomedical applications recently.9-23 This is possible due to their pronounced magnetic properties that impart distinguished features as magnetic field guided tumour targeting, hyperthermia (i.e. heating due to applied alternating magnetic field), and MRI contrast enhancement.13,

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Concrete evidence of the popularity of iron oxide nanoparticles is the safe

approval by food and drug administration (FDA) of USA. In addition, commercial circulating tumour cells sorting kit (i.e. CellSearch®)24 and MRI contrast enhancement agent (i.e. Feridex®) based on superparamagnetic iron oxide nanoparticles are already in the market.25 Typically, iron oxide nanoparticles are prepared by co-precipitation of Fe2+ and Fe3+ salts protected by an organic coating to prevent aggregation but it leads to poor aqueous suspendability. This organic coating not only limits the biomedical applications of the iron oxide nanoparticles by low aqueous suspendability but also results in significant cytotoxicity even after phase transfer (i.e. replacing the organic coating with aqueous soluble coating, which always leaves organic residues in the formulation).26, 27 Some studies have reported on aqueous routes of iron oxide nanoparticles synthesis. However, these methods require harsh chemical treatments, high temperatures, and provide poor control over the size and morphology of the particles.


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Nature, on the other hand, has been known to prepare fascinating nanostructures with unique 2D and 3D complexity, different shapes, orders, patterns, chemical compositions and properties by simple and genetically controlled self-assembly process.28 The focus of this study is the iron oxide nanowires prepared by zetaproteo bacteria, Mariprofundus ferrooxydans on an iron (II) substrate, found in pipe-lines of bore systems used to pump saline ground water entering into the River Murray, South Australia and fixes the iron present in the environment into iron oxide as dark brown colored biofilm causing enormous problem by blocking of pipes, which require regular cleaning. A microscopy analysis of this biofilm revealed that this biofilm by Mariprofundus ferrooxydans species consists of twisted bundles of several helical shape iron oxide nanowires. The unique shape and morphology, and magnetic properties of these nanowires produced by bacteria (referred as Bac-FeOxNWs) offer great potential for many applications including drug delivery, especially for triggered drug delivery and cancer hyperthermia. Therefore, systematic studies to explore drug loading, drug release, cytotoxicity, and hyperthermia performance are required to assess this material for proposed applications. It is also worth mentioning that most studies have focused on obtaining the efficacy of these nanomaterials in a 2D culture setup (i.e. a well plate). However, all the tumors are confined in a 3D microenvironment and thus, making it necessary to truly understand the behavior of these nanoparticles in 3D environments. Although some studies have previously pointed this out

29,30

using nanoparticles like but no study has ever reported on behavior and triggered release and hyperthermia capabilities of iron oxide nano-structures, especially nanowires in a 3D culture setup. The aim of this pioneering study is to demonstrate drug loading, in-vitro release (i.e. sustained and triggered), cytotoxicity, and cancer hyperthermia properties of Bac-FeOxNWs in


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2D and 3D cell culture systems (Scheme 1). Doxorubicin was used as model cytotoxic drug to asses loading and in-vitro drug release characteristics of Bac-FeOxNWs under sustained and triggered release modes. Cytotoxicity, cell uptake, and drug delivery capabilities of BacFeOxNWs were assessed using breast cancer cell line MDA-MB231-TXSA and RAW 264.7 macrophage cells under 2D (i.e. 96 well plate) and 3D (i.e. Matrigel® gelatinous protein mixture) cell culture systems. To mimic the tissue microenvironment and understand the impact of the cell culture type on interaction of cells with nanomaterials in far greater details, we chose 3D type cell culture system (i.e. Matrigel®).30 Lastly, to demonstrate hyperthermia properties of Bac-FeOxNWs using MDA-MB231-TXSA cell lines under an alternating magnetic field (AMF) with at 230 KHz for 3D cell culture systems (Scheme 1).

2. Experimental Section 2.1 Materials and Chemicals: Raw bacteria biofilm waste was obtained from bore pipe number 3 of the bore system at River Murray and was provided by SA Water Pty. Ltd. (South Australia, Australia). Doxorubicin hydrochloride (DOX) solution (2 mg.mL-1) was purchased from Hospira Pty Ltd. (Australia). Dulbecco's modified Eagle's medium, fetal calf serum (FCS), penicillin/streptomycin, sodium pyruvate, trypsin and phosphate buffer solution (PBS) were purchased from Life Technologies Pty Ltd. (Australia). AlamarBlue® (Life Technologies Corporation) and D-Luciferin Firefly, potassium salt were purchased from Caliper Life Sciences, PerkinElmer (Australia). All chemicals and reagents were used as-received without further purification steps. Matrigel® was obtained from BD Biosciences (Australia) and used with any further dilutions. High purity mili-Q water Option Q–Purelabs (Australia) (18.2 MΩ) was used to prepare all solutions used throughout this study.


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2.2 Separation and characterization of Bacteria Nanowires: Bacteria biofilm samples were purified by suspend-centrifuge and washing cycles to remove organic and inorganic impurities. The purified iron oxide nanowire material was heated at high temperature (600 °C) to change the crystal phase and improve their magnetic properties. Finally, Bac-FeOxNWs were resuspended in ethanol followed by ultra-sonication to separate them into individual nanowires of required dimensions. Bac-FeOxNWs were characterized using a series of analytical techniques including scanning electron microscopy (SEM, FEI Quanta 450 FEG-SEM, USA), energy-dispersive Xray spectroscopy (EDX), X-ray diffraction (XRD, Rigaku MiniFlex 600, Japan), Fourier transform IR (FTIR) spectroscopy (NicoLET 6700 Thermo Fisher, Australia). The particle size and zeta potential were measured at room temperature with dynamic light scattering (ZetaSizer Nano, Malvern Instruments Ltd., Worcestershire, UK). 2.3 Doxorubicin Loading on Bac-FeOxNWs: Dox loading onto Bac-FeOxNWs was carried out at three different pH conditions (i.e. acidic: pH 3, Neutral: pH 7, and Basic: pH 9.5) to evaluate the effect of pH on loading conditions and obtain maximum loading. About 12 mg of BacFeOxNWs were weighed out in a plastic eppendorf tube and suspended in 300 µL of buffer solution of required pH by sonication. Next, 200 µL of Dox solution (i.e. 2 mg.mL-1) was added to these Bac-FeOxNWs and vortexed for 4 h followed by overnight incubation at room temperature. Next day, Dox loaded Bac-FeOxNWs were collected and washed three times with mili-Q water to remove any loosely bound surface drug. The supernatant and the wash solutions were collected to measure the amount of drug loaded per milligram of Bac-FeOxNWs according to the following equation. = ( − )/


(1)

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Where, DL is the amount of drug loaded (i.e. loading capacity in µg/mg of BacFeOxNWs), Mfeed is the amount of Dox in feed solution (i.e. 400 µg in this case), Mwash is the amount of Dox recovered during washing after overnight incubation, and Mparticles is the mass of the particle (i.e. 12 mg in this case). The loading efficiency was calculated using the formula: (%) = ( / ) ×

(2)

Where, Lefficiency is the Loading efficiency (%) and ML is the amount of drug loaded in 12 mg of Bac-FeOxNWs. After washing the Dox loaded Bac-FeOxNWs (i.e. Dox- Bac-FeOxNWs) were dried under vacuum and stored at 4 °C till further use. Same procedure was repeated for all the proposed pH conditions for Dox loading onto Bac-FeOxNWs. 2.4 In-vitro Dox Release from Bac-FeOxNWs: The release of Dox from Bac-FeOxNWs was carried out under passive triggered and active externally triggered environments. Passive triggered conditions include the release at two different pH values to mimic physiological milieu (i.e. PBS at pH 7.4) and local acidic environment around tumour (i.e. PBS at pH 5.5).20 The purpose of this part of the study is to assess the effect of pH on Dox release with desire to achieve a faster release under acidic pH conditions (i.e. tumour surrounding). For this, 2 mg of Dox- Bac-FeOxNWs were suspended in 1.5 mL of the release buffer (i.e. PBS at pH 5.5. and 7.4). On the first day to measure the burst release of Dox from Bac-FeOxNWs, 1 mL of release buffer was collected from the sample in progressively increasing time intervals for 6 h (i.e. every 15 min for fist 2 h, 30 min for next 2 h, and 60 min for next 2 h). Sustained release was measured by collecting the same volume of release buffer from the sample every 24 h till the entire loaded


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drug is released. Note that the release was carried out at 37 °C for all the samples unless otherwise mentioned. Active externally triggered release of Dox from Bac-FeOxNWs was assessed by exposing the Dox- Bac-FeOxNWs to alternating magnetic field (AMF). In detail, 2 mg of Dox- Bac-FeOxNWs were suspended in PBS (@ pH 7.4) and allowed to release the drug for 30 min initially under normal conditions (i.e. off condition) and release buffer was collected every 15 min. After this, the Dox- Bac-FeOxNWs were exposed to AMF for 30 min (i.e. on condition) during which an aliquot was collected every 15 min. After first on and off cycle the release under normal was carried out for 45 min and exposure to AMF was 45 min. We also measured the release under elevated temperature (i.e. 50 °C) in order to assess the effect of temperature on release under sustained release conditions, which could serve as a control for AMF based heating triggered release. Since Dox is inherently fluorescent, fluorescence spectroscopy (FLS, Fluoromax-4 Horiba Jobin Yvon, Japan) was used to measure the amount of drug loaded or released from Bac-FeOxNWs. For this, the fluorescence signal from Dox in the collected release time points was measured at 590 nm after excitation at 490 nm wavelength of light. The final concentration of Dox was calculated using a standard calibration curve prepared by measuring the corrected fluorescence intensity of known concentrations of Dox using the above described method. 2.5 Cell Lines and Cultures: Raw 264.7 (Mentioned as RAW cells) and MD-MBA231 TXSA (Mentioned as TXSA cells) cell lines were used for studying cytotoxicity of Bac-FeOxNWs and cultured as previously reported.31 Note that, the MD-MBA231 TXSA cell line used for this study is genetically modified to express green fluorescent protein (GFP) and produce luciferase


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enzyme. The MDA-MB-231 derivative cell line, MDA-MB-231-TXSA was kindly provided by Dr Toshiyuki Yoneda (formerly at University of Texas Health Sciences Centre, San Antonio, Texas). Both the cell lines were cultured in 75 cm2 culture flasks (Corning Inc. Life Sciences) using Dulbecco’s modified Eagle’s culture medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamine (2 mM), 1% penicillin (Pen: 100 IU.mL-1), 1% streptomycin (Strep: 100 µg.mL-1) and 1% sodium pyruvate. The culture was maintained at 37 °C in a 5% CO2 and 95% relative humidity. The cells were passaged every 2-3 days to make sure all the cell studies are carried out during their growth phase of cell life-cycle. RAW cells 264.7 from passages 25-35 and TXSA cells from passage numbers from 18-27 where used throughout the whole study. Note that, all the reported experiments were repeated minimum of three times to carry out statistical analysis. 2.6 Cytotoxicity of Bac-FeOxNWs in 2D Culture Setup: The RAW and TXSA cells cultured in 75 cm2 culture flask were each harvested using trypsin-EDTA-PBS. These harvested cells were seeded in a 96-well culture plate (Greiner, Bio-One) at 10000 cells/well in 200 µL growth medium in each well and incubated overnight prior to addition of Bac-FeOxNWs to promote surface attachment. Dose-dependent cytotoxicity of Bac-FeOxNWs was investigated with five different concentrations of Bac-FeOxNWs (i.e. 10, 25, 50, 100 and 250 µg.mL-1) over a course of 7 days. Bac-FeOxNWs were sterilized by suspending in 70% ethanol followed by washing with sterile PBS and final suspension was prepared in sterile PBS for all the cytotoxicity studies. Untreated cells (i.e. simply grown in culture media without any particles or drug) and Dox treated cells (i.e. cells cultured in media containing 50 µg.mL-1 Dox) were marked as positive and negative controls, respectively.


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Alamar blue assay was used to quantify the viability of RAW cells while the viable TXSA cells were quantified using luciferase assay. Alamar blue assay relies on reduction of non-fluorescent reagent into a fluorescent species by live cells. Therefore, the fluorescence signal is proportional to the number of the viable cells. For Alamar blue assay, the growth media from the cells cultured for specified time point was carefully removed and washed with PBS (100 µL). Next, 200 µL of Alamar blue reagent (diluted 1:10 v/v in PBS) was added to each well and incubated at 37 °C around 15 min. The fluorescence reduced Alamar reagent was measured at 590 nm using a 530 nm excitation wavelength in a plate reader (Fluostar OPTIMA, BMG Labtech). The viable TXSA cells were quantified using bioluminescence originating from metabolisation of firefly dluciferin through luciferase assay. As described before the studied TXSA cells are genetically modified to produce luciferase, which metabolically catalyse d-lucifern firefly potassium salt in presence of ATP to give out bluish-green bioluminescence. For luciferase assay after the specific time point (i.e. 1 to 7 days of Bac-FeOxNWs incubation), the culture media was carefully removed. Next, 100 µL of d-luciferin solution (@150 µg.mL-1) was added to each well and incubated for approximately 15 min at 37 °C followed by the bioluminescence measurement using plate reader. The drug delivery capability of Bac-FeOxNWs was also assessed in-vitro using the same cell lines (i.e. Raw and TXSA) and quantification assays (i.e. Alamar blue for Raw and Luciferase for TXSA). For this, Bac-FeOxNWs loaded with Dox at basic pH via the described method (vide supra) under sterile conditions were suspended in PBS. Both the cell lines were cultured in the same manner and seeded in the 96-well plate with same concentration as mentions above (i.e. 10000 cells/well). Dox-Bac-FeOxNWs were


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suspended in PBS and introduced into the well plate at three different concentrations (i.e. 50, 100, and 250 µg.mL-1). Pure Dox solution at equivalent concentrations (to Dox-BacFeOxNWs) was used as control. Note that, similar protocol was used to investigate the biocompatibility of Bac-FeOxNWs against Mammary fibroblast cells in 2D culture setup. 2.7 Cytotoxicity of Bac-FeOxNWs in 3D Culture Setup: 3D cell culture was carried out using Matrigel® matrix. Note that, Matrige® is a gelatinous protein mixture secreted by EngelbrethHolm-Swarm (EHS) mouse sarcoma cells, which is artificially loaded with proteins and growth factors for use as matrix for 3D cell culture. Matrigel® exists in liquid form at 4 °C whereas it forms a gel-like matrix when heated to 37 °C. The gel-like Matrigel® can sustain a 3D geometry inside a 96-well plate and is used as 3D cell culture system. For cell culture in Matrigel®, both Raw and TXSA cells were harvested and 10000 cells were mixed with Matrigel®. Next, 100 µL of this cell-Matrigel® mixture was seeded in a 96-well plate and incubated at 37 °C for the gel to solidify and cell to proliferate. After one day of incubation under the aforementioned conditions three different concentrations of Bac-FeOxNWs (i.e. 50, 100, and 250 µg.mL-1) were carefully injected into the gel. The cell viability was measured again using the same viability assays after one and seven days of incubation, respectively. Similarly, the Dox release capabilities of BacFeOxNWs in the gel were evaluated by injecting same concentrations of Bac-FeOxNWs loaded with Dox. 2.8 Hyperthermia in Cancer Cells using Bac-FeOxNWs: Magnetothermal heating (i.e. hyperthermia) capabilities of the Bac-FeOxNWs were investigated using a custom built AMF generation setup operating at 230 kHz, operated at 2A and 15 V to provide a power output of 30 W. Schematic illustration of the AMF setup is provided in Scheme 1 and Figure S1 (Supporting Information). Briefly, 300 µL of Bac-FeOxNWs suspension in PBS (@ 30 mg


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mL-1, pH 7.4) was added to polycarbonate container and exposed to AMF by placing in the water cooled copper coil (Figure S5, Supporting Information). The local change in temperature was measured by a fluoroptic thermometer. The specific absorption rate (SAR), a measure of the amount of radio frequency energy absorbed by the material when exposed to AMF, is a typical method for characterization of heating capabilities of magnetic nanomaterials. SAR of BacFeOxNWs was calculated according to equation (Eq. 1) defined in Supporting Information.19, 3235

Hyperthermia in cells was executed by exposing a 3D TXSA cell culture block containing the

abovementioned concentrations of Bac-FeOxNWs (cylinder of dimensions, 5 mm d and 2 mm h) to AMF for 10 min. After the exposure, this culture block was subjected to luciferase assay to measure the viability. A 3D TXSA cell culture block was used as a positive control in this case. Note that, hyperthermia was carried out only for 3D type cell culture system as it mimics the tumour morphology. 2.9 Transmission Electron Microscopy of Cells Incubated with Bac-FeOxNWs: Transmission electron microscopy (TEM) was used to develop a deeper understanding about the uptake of Bac-FeOxNWs by both Raw and TXSA cells. TEM cross-section samples were prepared using the typical cell fixing procedure. Briefly, 1 x 106 cells were seeded and allowed to attach in each well of a 6-well plate (Greiner Bio-One). Next, 50 µg.mL-1 of Bac-FeOxNWs were added and incubated for 24 h, followed by removal of the culture media and washing with sterile PBS. Cells were then trypsinized and collected as a pallet by centrifugation at 1300 rpm for 5 mins. The collected cell pallets were fixed by treating with a solution of 4 vol % paraformaldehyde and 1.25 vol % glutaraldehyde overnight. The fixed cells were treated in a 2 vol % osmium tetroxide solution for 30 min followed by dehydration and embedding in epoxy resin. Microtome was used to cut Ultrathin cell section of 70 nm that were stained with uranyl


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acetate and lead citrate. These ultrathin slices were carefully picked up on TEM sample grid and imaged using TEM (FEI Tecnai G2 Spirit TEM, USA) at 100 kV. 2.10 Optical Microscopy Images of Bac-FeOxNWs Incubated with Cells: Optical and fluorescence microscopy was utilized to get general information about the interaction of cells with Bac-FeOxNWs. For this 20000 cells were seeded in 8-well chamber slide (Thermo Fisher) and allowed to attach overnight. The following day, Bac-FeOxNWs (50 µg.mL-1) were added and incubated for 24 h. 2.11 Statistical Analysis: All the results presented in this study are statistically treated and expressed as mean ± standard deviation (SD) of at least three independent experiments. Two tailed student T-test was also used to analyse the data, wherever required. The analysis was carried out using OriginPro 2015 (OriginLabs Corp. USA). The level for significance was set to p < 0.05 for all the comparisons.

3. Results and Discussions 3.1. Structural, chemical, magnetic properties of Bac-FeOxNWs Scanning Electron Microscopy (SEM) images of the Bac-FeOxNWs obtained by washing of bacteria biofilm and subsequent annealing are presented in Figure 1a-b. Iron oxide nanowire with length of 550-850 nm and aspect ratio of 36.29 ± 7.23 are clearly visible in the SEM image (Figure 1a-b). Notice that the Bac-FeOxNWs before annealing displayed an entangled helical like structure comprising of several nanowires (Figure S2a, Supporting Information). The average size of Bac-FeOxNWs (Pristine-Bac-FeOxNWs: P-Bac-FeOxNWs) before annealing was up to 10 µm, whereas it reduced to around 674 ± 92 nm after annealing, as measured by the SEM image analysis. Transmission electron microscopy (TEM) image of Bac-FeOxNWs after 13 ACS Paragon Plus Environment

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annealing is provided in Figure 1c. TEM images clearly show wire like structure of BacFeOxNWs. Currently, the potential application of these nanowires could be in localized tumor therapy as the size represents a constraint on their systemic delivery. However, it is worth mentioning that the size of these nanowires can be further reduced by tuning the annealing temperature and sonication time or power to obtain nanowires of desired dimensions for systemic delivery. A TEM image of pristine Bac-FeOxNWs is provided in Figure S2b (Supporting Information). The size of Bac-FeOxNWs measured from SEM and TEM images are in good agreement with size obtained from DLS (Figure 1d). The zeta-potential of BacFeOxNWs at pH 7.4 was measured to be -24 ± 3.7 mV, which is expected. It should be noted that negative value of zeta-potential.is due to the presence of hydroxyl groups on the surface and pays role in formation of stable suspension in aqueous media for administration and postprocessing (i.e. surface functionalization with positively charged reagents), drug loading, and others.20 Bac-FeOxNWs display crystallization, whereas P-Bac-FeOxNWs do not show any as confirmed using XRD analysis presented in Figure 2a and Figure S2c (Supporting Information) for Bac-FeOxNWs and P-Bac-FeOxNWs, respectively. Bac-FeOxNWs showed clear crystallization with peaks at 24.2, 33.3, 35.7, 40.96, 49.5, 54.14, 57.6, 62.5, and 64.2. The 2-theta values of peaks, Miller indices and d-spacing values are marked in Figure 2a while the later displays only amorphous structure. The XRD spectrum suggests that the Bac-FeOxNWs were primarily composed of Fe2O3 (Hematite phase).36 The specific surface area of the BacFeOxNWs was determined using nitrogen adsorption-desorption isotherm (Figure 2b). The nitrogen adsorption-desorption plot reveal a type II isotherms with a distinctive hysteresis loops of type H3. This type of isotherm is characteristic of disordered and interconnected structures.37,

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The specific surface area of 9.97 m2.g-1 was measured by BET data analysis.


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Chemical nature of Bac-FeOxNWs was evaluated using FTIR (Figure S2d, Supporting Information). FTIR shows a broad band around 3400 and 1630 cm-1 corresponding to stretching of the surface hydroxyl groups and a small peak around 535 cm-1 is generally related to alphaFe2O3.39 Magnetic properties of Bac-FeOxNWs were investigated by simple visual assessment. Figure 2b shows Bac-FeOxNWs suspension at time 0 and 2 sec on exposure to a permanent magnet. It can be seen that Bac-FeOxNWs get collected at the magnet and suspension becomes clear in a few sec. A video is provided as evidence in the supporting information (Video V1) to prove that the Bac-FeOxNWs are highly magnetic. Furthermore, superconducting quantum interference device (SQUID) magnetometry measurements (Figure S3a, Supporting Information) were performed to determine the magnetic saturation of Bac-FeOxNWs. The BacFeOxNWs shows nearly single phase magnetic behavior with an extraordinarily large coercivity (almost 20 kOe) and the magnetic saturation around 8 emu.g−1 of Fe. The Néelian and Brownian relaxation have been reported as the primary mechanism of heat generation rather than hysteretic losses in alpha iron oxide nanomaterials. Specific absorption rate (SAR) was calculated to be around 0.115 W g-1 of Fe (at 230 kHz and 30 W power output) from the temperature-time curve and the equations presented in Figure S3b (Supporting Information).32 3.2. Dox loading and release from Bac-FeOxNWs The drug loading and in-vitro release properties of Bac-FeOxNWs were investigated using a model anti-cancer chemotherapeutic agent, doxorubicin hydrochloride (Dox). Dox is an anthracycline antitumor antibiotic, used for treatment of a variety of tumors (e.g. breast, gastric, lung, thyroid, ovarian, multiple myeloma, sarcoma, and pediatric cancers). Dox loading capacity and loading efficiency under different pH conditions (i.e. acidic, neutral, and basic) are summarized in Table 1. Notice that, the loading capacity under different pH conditions shows


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negligible variation with values 33.19 ± 0.05 µg.mg-1 for pH-3.5, 32.97 ± 0.06 µg.mg-1 for pH7.4, and 31.73 ± 0.01 µg.mg-1 for pH 8.5. This high loading capacity is due to the synergistic of effect of high surface that leads to enhanced physical adsorption and electrostatic interaction between Dox and Bac-FeOxNWs. The Dox loading capacity and efficiency of Bac-FeOxNWs was found to be either comparable or better than other iron oxide nanomaterials as summarized in Table S1, (Supporting Information). Significantly high loading efficiencies (i.e. > 95%) can be attributed to high surface area and surface roughness of the Bac-FeOxNWs, reported by BET analysis. Drug loading was confirmed by zeta potential measurement, which increased to -12.4 ± 1.12 from -24.7 ± 0.2, indicating successful loading of Dox. An ideal anti-tumour therapeutic should preferably have maximum drug release either in the vicinity or inside the tumour cells to avoid damage to healthy tissue. Therefore, nano-carriers that display accelerated release at the target site and minimum indiscriminate release over healthy tissues are highly desirable. In-vitro drug release from Bac-FeOxNWs was carried out at two different pH conditions including neutral (i.e. pH 7.4) and slightly acidic (pH 5.5) to simulate healthy tissue and local tumour environment, respectively (Figure 3). The results indicate that drug release rate of Dox was faster under acidic pH conditions in comparison to the neutral pH environment. Figure 3b presents the burst release plot of Dox from Bac-FeOxNWs (i.e. yellow highlighted area in Figure 3a), which shows a similar trend with around 30% and 50% of loaded drug released in first 6 h under neutral and acidic pH conditions, respectively. Under acidic pH conditions almost all (100%) the loaded drug is released from the sample in approximately 30 days whereas only around 80% of the loaded drug was released under neutral pH conditions. This pH dependent in-vitro release of Dox payload is an important result in achieving selective release of drug at target tumour site because it can minimize the amount of


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premature drug release during circulation (pH 7.4), yet provide a sufficient amount of drug to effectively destroy the cancer cells once the nanocarriers are in local tumour environment or internalized and enter the endocytic pathway (pH ∼4.5 to 6.5). Our explanation is that faster Dox release in mild acidic (i.e. similar to tumour environment) is likely due to weaker interaction between Dox and the hydroxyl groups on Bac-FeOxNWs surface, and the re-protonation of the amino group of Dox.20 Notice that the drug release displays a bi-phasic release profile showing an initial burst phase during the first 6 h followed by a sustained release stage. This type of release profile is beneficial for cancer therapy since burst release phase would eliminate majority of the cancer cells at the first stage of action followed by a sustained dose of the anti-cancer therapeutic over a prolonged period to maintain the drug release with in the effective therapeutic window, prevent dose related side-effects, and recurrence of the tumour. The release of Dox from Bac-FeOxNWs could due to be diffusion of the drug in the eluting medium because of the present concentration gradient. Externally triggered release of Dox was carried out by periodically exposing the Dox-loaded Bac-FeOxNWs to AMF (230 kHz) and subsequent spectroscopic measurements during the time course. AMF triggered release of Dox in the form of rate of release of Dox is presented in Figure 3c. The AMF on or off periods are clearly labeled. The increase in rate of release of Dox during the on period of AMF confirms that the release of Dox from Bac-FeOxNWs can be triggered by the magnetic field. AMF trigger was supplied at variable periods of time (i.e. either 30 min or 45 min) resulting in a burst Dox release each time from Bac-FeOxNWs. The average rate of Dox release during AMF off period was 0.012 ± 0.0059 µg/min. This rate during the AMF on period was 0.025 ± 0.0031 µg/min, which is twice as much as during the AMF period. This triggered release is believed to be caused by localized heating. To prove this hypothesis, we


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performed drug release experiment at elevated temperature (i.e. 50 °C) (Figure S4, Supporting Information). The Dox release was carried out for two weeks and it resulted in a significantly higher amount of drug elution. Approximately 65% of the total loaded Dox was eluted from BacFeOxNWs at 37 °C in 14 days, whereas this release amount was approximately 87% for BacFeOxNWs incubated at 50 °C. These results show that Bac-FeOxNWs not only are capable of holding drug in their surface and release it over an extended period of time but can be triggered to release their payload at will as per the requirements of the therapy. 3.3. Biocompatibility and Cytotoxicity of Bac-FeOxNWs using 2d and 3D cell cultures We explored the biocompatibility and cytotoxicity of the Bac-FeOxNWs (i.e. unloaded and Dox loaded Bac-FeOxNWs) in two different types of cultures; 2D and 3D culture. Note that, 3D culture setup was chosen as a proof-of-concept to demonstrate the ability and activity of BacFeOxNWs in environments similar to actual tumour tissues. A large number of studies have evaluated iron oxide nanoparticles for their cytotoxicity in 2D cultures. However, 2D culture setup does not provide comprehensive information about the way cells interact with nanomaterials under real tissue milieu, which is a 3D structure.29, 30 naturally produced 1D iron oxide materials (i.e. nanowires). Biocompatibility and cytotoxicity of Bac-FeOxNWs was evaluated with two different cell lines including RAW 264.7 macrophages (i.e. a type of immune cells) and MDA-MB-231-TXSA cells (i.e. a type of breast cancer cells) (Figure 4 a-b). Cell toxicity of Bac-FeOxNWs was measured for day 1 and day 7 after incubation of cells with BacFeOxNWs at different concentrations (i.e. from 10, 25, 50, 100 and 250 µg.mL-1). As noted above, cell viability was determined by Alamar blue and luciferase activity assay shows no apparent toxicity of Bac-FeOxNWs after one day of incubation is observed with cells at all the doses. However, the Bac-FeOxNWs displayed noticeable toxicity towards both the cell lines


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after seven days of incubation at higher dosages (Figure 4c and d). In this regard, the viability of RAW cells at the highest concentration (i.e. 250 µg mL-1) of Bac-FeOxNWs decreased to around 68%, whereas TXSA cells reported significant cell toxicity at two higher dosages (i.e. 100, and 250 µg.mL-1) with viability of approximately 75% and 58%, respectively. This toxicity of the Bac-FeOxNWs at higher dosages could be attributed to high cell membrane coverage (i.e. could result in asphyxiation and low nutrient transport to cells), generation of reactive oxygen species (ROS), and the interaction of Bac-FeOxNWs with cell membrane. Optical microscopy images of the Bac-FeOxNWs incubated with cells are provided in Figure 5, which show no structural or morphological changes in cells after incubation. Interestingly, we observed that large quantities of Bac-FeOxNWs localized around the cells due to surface charge of the Bac-FeOxNWs. This feature of Bac-FeOxNWs could be beneficial as the drug loaded Bac-FeOxNWs can locally unload their payload on or around cell surface. This localization of Bac-FeOxNWs in/around the cells was more pronounced for RAW cells, which is believed to be due to their phagocytic nature (i.e. engulfment of foreign object and removal). Short term biocompatibility and cytotoxicity (i.e. for 24 h) of Bac-FeOxNWs was also evaluated with another fibroblast cell line (i.e. mammary fibroblast cells). The measurements were made using Alamar blue assay and the results are presented in Figure S5a (Supporting Information). Similar to the other two cell lines, mammary fibroblasts did not show any toxicity on incubation with Bac-FeOxNWs. The 3D cell culture was carried out using Matrigel® as the matrix to explore the cytotoxicity, drug carrier efficacy, and the magnetic field assisted hyperthermia of Bac-FeOxNWs. First, the cytotoxicity of Bac-FeOxNWs was assessed at two different concentrations (i.e. 50 and 100 µg.mL-1) that showed almost 100% cell survivability (Figure 6).


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3.4. In-vitro drug carrier capabilities of Bac-FeOxNWs using 2d and 3d cell cultures. The in-vitro payload delivery and therapeutic efficacy of Dox loaded Bac-FeOxNWs was determined by incubating the formulation with breast cancer cells (TXSA) (Figure 7). Three different dosages of Dox loaded Bac-FeOxNWs were tested with TXSA including 50, 100, and 250 µg.mL-1 (with 33.2 µg.mg-1 loading capacity, which means total of 1.7, 3.3, and 8.3 µg of Dox). The therapeutic efficacy of Bac-FeOxNWs was compared with pure Dox at three equivalent dosages of 5, 10, and 20 µL (@ 500 µg.mL-1, which means 2.5, 5, and 10 µg of Dox). The cell viability for the control group (i.e. no therapeutic agent) was recoded at 100% whereas for pure Dox and Dox loaded Bac-FeOxNWs displayed significant cell death with cell viability 30% or less for all the investigated samples. An optical microscope image of TXSA cells incubated with Dox loaded Bac-FeOxNWs after 24 h of incubation is provided in the insert of Figure 7 showing almost complete destruction of all the present cells. Interestingly, but not surprisingly, Bac-FeOxNWs display a better drug efficacy than free drug even at lower total administered dosage. This could be attributed to localization of Bac-FeOxNWs in the vicinity of cells that leads to localized delivery of drug directly to the cells (Figure 5). Internalization of Bac-FeOxNWs by cells also plays a key role in higher efficacy of Dox loaded Bac-FeOxNWs in comparison to pure Dox. Drug carrier properties of Bac-FeOxNWs were also tested in-vitro with RAW cells with only a single dosage (i.e. total Dox amount of 2.5 and 3.3 µg for pure Dox and Dox-Bac-FeOxNWs, respectively). The cell viability results for RAW cells are presented in Figure S5b (Supporting Information), which show similar trend as shown for incubation of Dox-Bac-FeOxNWs with TXSA cells with 54% and 51% cell survivability for pure Dox and Dox-Bac-FeOxNWs, respectively. The interaction and internalization of Bac-FeOxNWs (@ 50 µg.mL-1) by macrophage cells (i.e RAW) and breast cancer cells (i.e. TXSA) was confirmed by


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transmission electron microscopy. Ultrathin section of RAW and TXSA cells imaged under TEM are presented in Figure 8. Figure 8a-b show TEM image of RAW and TXSA cells that were not incubated with Bac-FeOxNWs (i.e. controls). The cells show no sign of stress or change in morphology of major organelles (i.e. mitochondria, endoplasmic reticulum, etc.) appeared regular in their morphology. Figure 8c-d show the TEM images of RAW cells and TXSA (breast cancer) cells incubated with Bac-FeOxNWs for 24 h, respectively. In case of RAW cells (Figure 8c), it is clearly visible that all the internalized Bac-FeOxNWs were localized inside autophagic cellular vacuoles. Similarly, Bac-FeOxNWs internalized by TXSA cells were entrapped in the cellular autophagic vacuoles. Although Bac-FeOxNWs induced autophagy activation, the major cell organelles did not show any signs of stress. Previous studies have reported that high aspect ratio (HAR) materials tend to induce autophagy, mainly in RAW cells due to their phagocytic nature.31 Drug carrier capabilities and AMF induced heating capabilities of Bac-FeOxNWs under 3D culture settings are presented in Figure 9. The amount of Dox loaded Bac-FeOxNWs injected in the Matrigel® had a clear effect of cell viability. In this regard, three different amounts of Dox loaded Bac-FeOxNWs (50, 100, 250 µg.mL-1) were injected in the Matrigel® loaded with TXSA cells and their viability after 24 h was assessed using luciferase assay, as presented in Figure 9. The cell viability was recorded at around 88%, 77%, and 56% for 50, 100, and 250 µg.mL-1 of Dox loaded Bac-FeOxNWs. A positive control with 10 µL of pure Dox (@ 500 µg.mL-1) presented in Figure 9 resulted in significant cell death (around 80%). This is the first study to investigate cytotoxicity and drug carrier capabilities of iron oxide based nanomaterials in 3D culture setup. It is worth noticing that the drug carrier efficacy of BacFeOxNWs reduced significantly in the 3D type cell culture setup in comparison to 2D type cell


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culture setup. The cell viability values increased from 18% and 7% to 78% and 57% for 100 and 250 µg mL-1 concentrations of Dox loaded Bac-FeOxNWs, respectively. Similarly, the cytotoxicity for pure Dox was observed to be lower with viability going from around 13% to 20%. Note that, nearly all the cells are embedded in a 3D microenvironment in a tissue. However, most in-vitro cell cytotoxicity studies ignore this fact. This report shows that the cell microenvironment (i.e. culture setup) plays a key role in determining its response to a particular therapy. Based on these observations, we decided to assess the efficacy of AMF induced hyperthermia using Bac-FeOxNWs in a 3D type cell culture setup as it closely mimics the morphology of a tissue, where cells preside. The effect of AMF induced hyperthermia was carried out by culturing the cells in a Matrigel® matrix in a sterilized eppendorf tube and Bac-FeOxNWs (50 µg.mL-1) were injected into the 3D culture. After 10 min of exposure to AMF, the viability was measured by luciferase assay, which was measured to be around 66%. This is a significant decrease in cell viability in comparison to the control proving the potential applicability of Bac-FeOxNWs as hyperthermia agent in a 3D cell culture environment that exists in the physiological milieu. A recent study reported cell viability to be around 55% in case of HeLa cells incubated with PEGylated iron oxide nanoparticles after 1 h of AMF exposure.17 Another study reported a 20% decrease in tumour mass with gadolinium doped iron oxide nanoparticles when exposed to AMF for 30 min.32 The results suggest that BacFeOxNWs display better hyperthermia capabilities in comparison to the previous reports. We believe this could be due to the rod like shape that can damage the cell membrane during their migration in the Matrigel®. In addition, it is reported that wire or rod like nanostructures tends to concentrate the electromagnetic field at their tips, which results in field enhancement. This


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results in excessive heating at the tips of Bac-FeOxNWs resulting in greater hyperthermia efficiency.

4. Conclusion This study demonstrated potential application of iron oxide nanowires from a biofilm waste produced by bacteria (Mariprofundus ferrooxydans) for cancer therapy. The structural and magnetic characterization revealed their nanoscale structure, aspect ratio and magnetic properties that are comparable with artificial NW, applicable for drug delivery and cell internationalization. The Bac-FeOxNWs demonstrated very high drug loading capacity (i.e. >33 µg.mg-1) and efficiency (i.e. > 99%) for a model anti-cancer drug Doxorubicn (Dox), and a sustained release for extended periods (i.e. over 30 days) with both passive (i.e. pH) and active (i.e. AMF) responsive feature to control the rate of release as required. The Bac-FeOxNWs proved to be biocompatible up to concentrations as high as 250 µg.mL-1 with two cell lines including macrophage cells and breast cancer cells under 2D (i.e. 96-well plate) and 3D (Matrigel®) type cell culture setup. Our results demonstrated that the in-vitro therapeutic efficacy therapeutic efficacy for Bac-FeOxNWs loaded with Dox was lower in case of 3D type cell culture setup in comparison to 2D type cell culture setup. Therefore, it is important to carefully optimize required dosage and culture setups to achieve targeted biocompatibility and therapeutic efficacy of nanomaterials, which is closest to the actual physiological settings. Lastly, the AMF induced hyperthermia application of Bac-FeOxNWs in a 3D type cell culture setup showed approximately 36% decrease in cell viability for breast cancer cells after 10 min of exposure to AMF at 230 kHz with 30 W power output. These findings demonstrate that Bac-FeOxNWs is a promising naturally produced magnetic nanomaterial with range unique features as passive and


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active trigger response and AMF induced hyperthermia for cancer therapeutic applications. We believe that with carefully selected targeting moiety (i.e. folic acid, antibodies etc.) that homes to the tumor site, this naturally derived nano-carrier could lead to more exciting applications such as targeted drug delivery and MRI contrast agents.

Supporting Information Schematic illustration of AMF setup, SEM, TEM, XRD, FTIR, Temperature versus time cure, Temperature dependent Dox release, and dose dependent cell viability for Mammary fibroblasts and RAW 264.7 macrophage cells is provided in the supporting information. Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This research was supported by funding support in part from SA Water (SAWA157252). Authors thank for the financial support provided by the Australian Research Council (ARC) through the grants DP120101680, FT110100711, DE140100549, and the School of Chemical Engineering, The University of Adelaide. Thanks to Dr Ali Karami and Prof Christophe from School of Electrical Engineering, The University of Adelaide for building the AMF set-up. Thanks to Dr Krishna Kant (The University of Adelaide) for and R. Woodward (University of Western Australia) for performing Squid measurements.

References [1] Chithrani B. D.; Ghazani, A. A.; Chan, W. C. W. Nano Lett. 2006, 6, 662-668. [2] Champion, J. A. and Mitragotri, S. Proc. Natl. Acad. Sci. 2006, 103, 4930-4934.


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[3] Gratton, S. E.; Ropp, P. A.; Pohlhaus, P. D.; Luft, J. C.; Madden, V. J.; Napier, M. E.; DeSimone, J. M. Proc. Natl. Acad. Sci. 2008, 105, 11613-11618. [4] Fadeel B. and Garcia-Bennett, A. E. Adv. Drug Deliv. Rev. 2010, 62, 362-374. [5] Chung, T. H.; Wu, S. H.; Yao, M.; C.; Lu, W.; Lin, Y. S.; Hung, Y.; Mou, C. Y.; Chen, Y. C.; Huang, D. M. Biomaterials 2008, 28, 2959-2966. [6] Liu, S.; Zhang, Z.; Wang, Y.; Wang, F.; Han, M. Y. Talanta 2005, 6,7 456-461. [7] Gu, L.; Fang, R. H.; Sailor, M. J.; Park, J. H. ACS Nano 2012, 6, 4947-4954. [8] Jiang, S.; Eltoukhy, A. A.; Love, K. T.; Langer, R.; Anderson, D. G. Nano Lett. 2013, 13, 1059-1064. [9] Park, S.; Son, Y. J.; Leong, K. W.; Yoo, H. S. Nano Today 2012, 7, 76-84. [10] Son, S. J.; Reichel, J.; He, B.; Schuchman, M.; Lee, S. B. J. Am. Chem. Soc. 2005, 127 7316-7317. [11] Abidian, M. R.; Kim, D.H.; Martin, D. C. Adv. Mater. 2006, 18, 405-409. [12] Park, J. H.; von Maltzahn, G.; Zhang, L.; Schwartz, M. P.; Ruoslahti, E., Bhatia, S. N.; Sailor, M. J. Adv. Mater. 2008, 20, 1630-1635. [13] Gupta A. K. and Gupta M. Biomaterials 2005, 26, 3995-34021. [14] Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N. Chem. Rev. 2008, 108, 2064-2110. [15] Nguyen, D. H.; Lee, J. S.; Choi, J. H.; Park, K. M.; Lee Y.; Park, K. D. Acta Biomater. 2016, 35, 109-117. [16] Kim, J.; Lee, J. E.; Lee, J.; Yu, J. H.; Kim, B. C.; An, K.; Hwang, Y.; Shin, C. H.; Park, J. G. J.; Kim, J. Am. Chem. Soc. 2006, 128, 688-689. [17] Quinto, C. A.; Mohindra, P.; Tong, S.; Bao, G. Nanoscale 2015, 7, 12728-12736.


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[18] Šmejkalová, D.; Nešporová, K.; Huerta-Angeles, G.; Syrovátka, J.; Jirák, D.; Gálisová, A.; Velebný, V. Biomacromolecules 2014, 15, 4012-4020. [19] Sonvico, F.; Mornet, S.; Vasseur, S.; Dubernet, C.; Jaillard, D.; Degrouard, J.; Hoebeke, J.; Duguet, E.; Colombo, P.; Couvreur, P. Bioconjugate Chemistry 2005, 16, 1181-1188. [20] Maeng, J. H.; Lee, D. H.; Jung, K. H.; Bae, Y. H.; Park, I. S.; Jeong, S.; Jeon, Y. S.; Shim, C. K.; Kim, W.; Kim, J. Biomaterials 2010, 31, 4995-5006. [21] Nowicka, A. M.; Kowalczyk, A.; Jarzebinska, A.; Donten, M.; Krysinski, P.; Stojek, Z.; Augustin, E.; Mazerska, Z., Biomacromolecules 2013, 14, 828-833. [22] Yu, M. K.; Jeong, Y. Y.; Park, J.; Park, S.; Kim, J. W.; Min, J. J.; Kim, K.; Jon, S. Angew. Chemie Int. Ed. 2008, 47, 5362-5365. [23] Carregal-Romero, S.; Guardia, P.; Yu, X.; Hartmann, R.; Pellegrino, T.; Parak, W. J. Nanoscale 2015, 7, 570-576. [24] Scher, H. I.; Morris, M. J.; Basch, E.; Heller, G. J. Clin. Oncol. 2011, 29, 3695-3704. [25] Kostura, L.; Kraitchman, D. L.; Mackay, A. M.; Pittenger, M. F.; Bulte, J. W.; NMR in Biomed. 2004, 17, 513-517. [26] Wang, Y.; Wong, J. F.; Teng, X.; Lin, X. Z.; Yang, H. Nano Lett. 2003, 3, 1555-1559. [27] Kim, J.; Piao, Y.; Hyeon, T. Chem. Soc. Rev. 2009, 38, 372-390. [28] Melton, E. D.; Swanner, E. D.; Behrens, S.; Schmidt, C.; Kappler, A. Nat. Rev. Microbiol. 2014, 12, 797-808. [29] Gelain, F.; Bottai, D.; Vescovi, A.; Zhang, S. PloS one, 2006, 1, e119. [30] Sodunke, T. R.; Turner, K. K.; Caldwell, S. A.; McBride, K. W.; Reginato, M. J.; Noh, H. M. Biomaterials 2007, 28, 4006-4016. [31] Wang, Y.; Santos, A.; Kaur, G.; Evdokiou, A.; Losic, D. Biomaterials 2014, 35, 5517-5526.


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Scheme 1.

Scheme 1. Schematic illustration of two methods for applying Bac-FeOxNWs as effective cancer therapy agent. (a) Pristine Bac-FeOxNWs as a multi strand helix obtained as a biofilm from the bore pipe from river Murray in South Australia. (b) Smaller sized Bac-FeOxNWs obtained by annealing at 600 °C followed by ultrasonication. (c) Bac-FeOxNWs incubated cells induced to AMF using the home made AMF setup that results in heating (i.e. hyperthermia based death of cells) of cells as represented by red aura. (d)Model drug (doxorubicin) loading onto Bac-FeOxNWs by overnight incubation. (e) Their In-vitro delivery and internalization into breast cancer cells (MDA-MB231-TXSA)


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Figure 1.

Figure 1. Scanning electron microscopy images of (a) Low magnification SEM image of BacFeOxNWs after annealing at 600 °C. (b) High magnification image of the same. (c) TEM image of Bac-FeOxNWs after annealing 600 °C. (d) DLS size measurement plot displaying Gaussian bell shaped curve with peak at around 750 nm and polydispersity index of 0.293.


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Figure 2.

Figure 2. (a) X-ray diffraction spectrum of Bac-FeOxNWs annealed at 600 °C showing the diffraction peaks with their corresponding Miller indices and d spacing. (b) Nitrogen adsorptiondesorption plot for determination of surface area of Bac-FeOxNWs. The inset shows digital photographs of Bac-FeOxNWs suspended in water before (t =0) and after (t=2) exposure to permanent magnet. Here, t is the time in seconds, when images were taken in respect to placement of the permanent magnet. 30 ACS Paragon Plus Environment

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Figure 3.

Figure 3. Dox release from Bac-FeOxNWs over the period of (a) 30 days showing a bi-phasic release profile with a burst release (yellow part) and a long-term sustained release. (b) Burst release part of the total release curve showing initial release for first 6 h. (c) AMF triggered release of Dox from BacNW operating at 230 kHz shown as Dox release rate. Light blue bars present the rate of release of Dox during AMF off and the red bars indicate the time period when AMF was switched on. Red shaded area indicates the average Dox release during AMF on, whereas blue shaded area is the rate of Dox release when AMF was off. 31 ACS Paragon Plus Environment

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Biomacromolecules

Figure 4.

Figure 4. The biocompatibility and cytotoxicity of Bac-FeOxNWs using 2 D culture is presented in terms of cell viability. The cell viability for two cell lines; RAW 264.7 (a and c) and MDAMB231-TXSA (b and d) is provided for incubation time of 1 and 7 days to a range of BacFeOxNWs concentration. The data was statistically analysed to obtain the standard deviation (SD) and level of significance for 2- tailed student T-Test was set at a p value < 0.05 for ** in comparison to control group (n = 4 = SD).


Biomacromolecules

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Figure 5.

Figure 5. Optical microscopy images of RAW 264.7 (left panels) and MDA-MB231-TXSA cells (right panels) at five different Bac-FeOxNWs concentrations ranging from 0 to 250 µg mL-1 (the direction of the black arrow on the left indicates increasing concentration).


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Biomacromolecules

Figure 6.

Figure 6. Cell cytotoxicity of Bac-FeOxNWs against TXSA cells in a 3D cell culture setup. Dose response of TXSA cells for three different concentrations of Bac-FeOxNWs injected into the 3D cell culture setup.


Biomacromolecules

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Figure 7.

Figure 7. The Drug carrying and delivery capabilities of Bac-FeOxNWs instigated under 2D culture setup with TXSA cells is presented for a number of different concentrations of pure Dox and Dox loaded Bac-FeOxNWs. The data was statistically analyzed to obtain the standard deviation (SD) and level of significance for 2-tailed student T-Test was set at a p value < 0.05 for ** in comparison to control group (n = 4). The insert provides an optical microscope image of TXSA cells incubated with 100 µg of Dox loaded Bac-FeOxNWs after 24 h. Note that the unit for concentration of Bac-FeOxNWs is µg mL-1, it is µL for pure Dox).


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Biomacromolecules

Figure 8.

Figure 8. Transmission electron microscopy images of macrophage cells and breast cancer cells. (a) RAW cells that were not treated with Bac-FeOxNWs. (b) TXSA cells that were not treated with Bac-FeOxNWs. (c) RAW cells incubated with 100 µg.mL-1 Bac-FeOxNWs for 24 h. and (d) TXSA cells incubated with 100 µg.mL-1 Bac-FeOxNWs for 24 h.


Biomacromolecules

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Figure 9.

Figure 9. Cell cytotoxicity of Bac-FeOxNWs against TXSA cells in a 3D cell culture setup. (a) Dose response of TXSA cells for three different concentrations of Bac-FeOxNWs injected into the 3D cell culture setup. and (b) In-vitro Dox delivery capabilities of Bac-FeOxNWs in 3D cell culture setup at four different dosages including a negative control as injection of 10 µL of pure Dox (at 500 µg mL-1). The last column in the plot is the viable cell count for the 3D cell culture setup containing Bac-FeOxNWs when exposed to AMF for 10 min. (Note that the unit for concentration of Bac-FeOxNWs is µg mL-1, whereas it is µL for pure Dox as marked in the figure). The data was statistically analysed to obtain the standard deviation (SD) and level of significance for 1-way student T-Test was set at a p value < 0.05 for ** in comparison to control group (n = 4 = SD).


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Biomacromolecules

Table 1. Dox loading capacity and loading efficiency of Bac-FeOxNWs at different pH values.

pH

Loading Capacity (µg mg-1)

Loading Efficiency (%)

3.5

33.19 ± 0.0313

99.5826258

7.4

32.97 ± 0.0381

98.9117774

8.5

31.73 ± 0.0077

95.1744584


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