Targeted magnetic nanoparticle hyperthermia for the treatment of oral cancer.

INTRODUCTION
Patients with oral squamous cell carcinoma currently experience a five year survival rate of approximately 60% with conventional surgical, chemotherapy and radiotherapy treatments. Magnetic hyperthermia offers an alternative treatment method by utilising the heating properties of magnetic nanoparticles to produce thermo-ablation of the tumour site when exposed to an alternating magnetic field. In this study we investigate in vitro if targeted magnetic hyperthermia offers a potential treatment for oral squamous cell carcinoma.


MATERIALS AND METHODS
Magnetic iron oxide nanoparticles, with a biocompatible silica coating, were produced and conjugated with antibodies to target integrin αvβ6, a well-characterised oral squamous cell carcinoma biomarker. Utilising the heating properties of the magnetic nanoparticles we exposed them to an alternating magnetic field to produce thermo-ablation of tumour cells either negative for or over-expressing integrin αvβ6.


RESULTS
The cell surface biomarker, αvβ6 integrin, was upregulated in tissue biopsies from oral squamous cell carcinoma patients compared to normal tissue. Functionalisation of the silica coating with anti-αvβ6 antibodies enabled direct targeting of the nanoparticles to αvβ6-overexpressing cells and applying thermal therapy significantly increased killing of the targeted tumour cells compared to control cells.


CONCLUSION
Combining antibody-targeting magnetic nanoparticles with thermal-ablation offers a promising therapy for the targeted treatment of oral squamous cell carcinoma. This article is protected by copyright. All rights reserved.


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Introduction:
Oral squamous cell carcinoma (OSCC) is the twelfth most common cancer worldwide, accounting for 300,000 new cases every year (1). Despite advances in conventional treatment, including surgery and radio-or chemo-therapy, the prognosis for OSCC remains poor. Survival rates have not improved over the past few decades with currently only a 60% 5 year survival rate (2). As with other cancers, there is a need to develop novel targeted methods, to not only improve efficiency but to reduce unwanted off-target side-effects that often cause considerable reduction in the quality of life for the patient.
An alternative potential cancer treatment, thermal ablation, involves applying heat (>50°C) to cause irreversible cell damage and necrosis of tumour cells (3,4). A current challenge is delivering this heat selectively to only the site of the tumour and minimising damage of healthy tissue elsewhere. One potential solution is through the use of magnetic nanoparticles (MNP). These particles emit thermal energy when exposed to a rapidly alternating magnetic field in a process termed magnetic hyperthermia (5). This process allows the heating of cells that are closely localised to the MNP (6). In addition, the high surface area of MNP means they can be readily functionalised to enhance their biocompatibility or to immobilise drugs, or targeting agents such as antibodies. Functionalisation of the surface towards cancer cells has the potential to generate a more specific cell killing whilst simultaneously reducing off target effects on healthy tissue (7).
Cancerous cells which display unique or highly upregulated biomarkers offer the opportunity to target treatments and diagnostics to the site they are needed, and bring about a reduction in offtarget side-effects (8). One such example is the heterodimeric transme integrin. This integrin is expressed on epithelial cells (9) and is highly upregulated during wound healing, as well as on the leading edge of OSCC (10,11) T integrin in OSCC is associated with a poor prognosis and increased disease progression (12,13). Cells

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This article is protected by copyright. All rights reserved. overbasement membrane and increased cell migration (14,15). This is a direct resul its ligand, causing the spread and development of cancer (9). The overtumours makes it an ideal molecule for the targeting of treatments in OSCC. By functionalising with MNP, and therefore the hyperthermia treatment, can be localised to areas of tumour growth, offering a directed therapy with the potential for significantly reduced side-effects and minimised damage to healthy tissue.
In this study we produced and characterised iron oxide MNP with a biocompatible silica shell, and conjugated them with an antibody against MNP were targeted specifically towards OSCC cells over-survival determined after inducing magnetic hyperthermia. We found that specific targeting of the particles increased the effectiveness of the hyperthermic treatment by increasing cell death compared to non-conjugated MNP and hyperthermic treatment alone.

Materials and Methods
All materials used were purchased from Sigma-Aldrich Company Ltd (Dorset, UK), unless otherwise stated.

Synthesis and coating of magnetic nanoparticles
Iron oxide nanoparticles were synthesised using room temperature co-precipitation with a ferric to

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The colour change of the supernatant at 585 nm was recorded. MNP without the conjugated anti-

Cell culture
Two OSCC cell lines with defined integrin expression were selected; VB6 (over-expressing high levels

D S W H
integrin subunits), (Health Protection Agency Culture Collections, Salisbury, UK)(16) and cultured as previously described (14). Briefly, cells were cultured in flavin and adenine-enriched medium

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view IIIu camera with associated Cell^D software (Olympus Soft Imaging Solutions, GmbH, Munster, Germany).

Flow cytometry
Cells were removed from tissue culture flasks non-enzymatically, pelleted, and re-suspended in cold PBS supplemented with 1% BSA and 0.1% sodium azide (FACS buffer). Cells (1 X 10 6 ) were incubated with 10 µg/ml anti-G I G

Treatment of OSCC cells with silica magnetic nanoparticles
VB6 cells were seeded onto a 35 mm cell culture plate at a density of 1.5 X 10 5 cells and incubated -MNP ( MNP) or vehicle control (uMNP). Cells were exposed to an alternating magnetic field (9.7 mT) for 10 minutes, along with appropriate controls. Cells were re-incubated for 24 and 48 hours after treatment before being analysed for cell viability using an alamarBlue® assay (Thermo Fisher, Waltham, Massachusetts, USA).

Analysis of cells after magnetic hyperthermia treatment
Cell metabolism was measured by adding alamarBlue® solution to cells G M C CO 2 before removing the reagent and subjecting it to centrifugation at 1800 x g for 5 minutes. The alamarBlue® solution was transferred into a 96 well plate and the fluorescence measured using a spectrophotometer at a wavelength of 585 nm. After

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removal of the alamarBlue® solution, the cell plates were washed with PBS and fresh medium added before being re-C CO 2 .

Statistics
Data are presented as mean values ± standard deviation (SD) of three independent experiments (n=3), unless otherwise stated. Statistical comparisons were performed using GraphPad Prism v7.00 (GraphPad Software, La Jolla, CA, USA). Group-wise comparisons were carried out using one-way independent analysis of variance (ANOVA) with Tuke and differences considered significant when p<0.05.

Production and characterisation of magnetic nanoparticles
MNP or iron oxide were synthesised using a simple room temperature coprecipitation of ferrous and ferric iron in sodium hydroxide. The solution of iron salts was added to a large excess of sodium hydroxide via a controlled dropwise addition. The resulting black precipitate was magnetically recovered, before coating with an amine terminated silica shell using conventional sol gel chemistry.
The presence of surface exposed amine groups allows for convenient functionalisation later. The size and morphology of the MNP were analysed using TEM both before and after the coating step, allowing measurement of both the size of the iron oxide core and the thickness of the silica shell.
The mean diameter of the uncoated MNP was found to be 8 + 6 nm (Figure 1, A), and after coating, a uniform 6 nm thick layer of lower electron density was visible surrounding the MNP (Figure 1, B, C).

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coated MNP revealed a coercivity of 13 Oe (Figure 1,E) and a saturation magnetisation of 56 emu/g.
Due to their size and distribution it is likely that the sample comprises a mixed population of superparamagnetic and single domain ferrimagnetic particles. We therefore exposed an 8 mg/ml aqueous suspension of the coated MNP to a rapidly alternating magnetic field (frequency of 174 kHz, magnetic field strength of 97 Oe) and recorded the heat rise of the solution using a fibre optic temperature probe. The calculated specific absorption rate (SAR) from the heating curve was 21.75 W/g with an intrinsic loss parameter (ILP) of 2.09 nHm 2 /k (Figure 1, F) (18). We wanted to exploit this hyperthermic potential for OSCC cell killing and to enhance any cell killing effects by specifically targeting the particles to OSCC cells.

OSCC
Using immunohistochemistry, we confirmed as a suitable biomarker to differentiate between

MNP functionalisation with anti-
The MNP were functionalised with antiamine groups of the antibody with the amine terminated surface of the silica shell. This method provides a stable, covalent linkage between the targeting molecule and the MNP. Using a modified magnetic particle-based ELISA, the antibody functionalised particles provided a positive absorbance

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Discussion
More effective treatments against OSCC are urgently needed to improve outcomes and survival rates for patients. We have therefore, for the first time, brought together the emerging field of magnetic hyperthermia for cell heating and destruction, with an under exploited biomarker for OSCC, resulting in a promising new targeted treatment approach.
Iron oxide nanoparticles have been the subject of many studies into their use as magnetic hyperthermia agents, due to their intrinsic magnetic properties and biocompatibility (19). In particular, the iron oxide magnetite has favourable magnetic properties for hyperthermia treatment, but this is dependent on the size and shape of the particles used. We wanted to produce magnetite

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nanoparticles with suitable characteristics to allow them to be readily taken up into cells, and, crucially, still possess sufficient heating potential for cell killing. Previous studies which have investigated cellular uptake of iron oxide nanoparticles, suggest that the smaller the particles the higher the level of uptake (19) . This clearly needs to be balanced against the heating potential, whereby larger particles generally display greater levels of magnetically induced heating (20). To this end, we employed a synthesis of magnetite using a simple room temperature co-precipitation of Fe 2+ /Fe 3+ ions. The TEM particle sizing, powder XRD pattern, and coercivity are all consistent with small 8-12 nm magnetite nanoparticles. The measured SAR value matches that observed for similarly sized magnetite nanoparticles which achieve hyperthermia via Néel and Brownian modes (5).
We encapsulated the particles within an amine terminated silica shell in order to preserve the magnetite core from oxidation, minimise any unwanted cytotoxicity, and provide a functionalised surface for convenient conjugation to antibodies (21). In addition, silica coating has previously been shown to improve cellular uptake of iron oxide nanoparticles (22).
In agreement with others, t by OSCC tumour cells, making it a suitable target molecule for antibody-directed therapy (23). Being able to specifically target such MNP to cancer cells, allows for efficient heating and destruction of tumour cells whilst minimising effects on healthy tissue. We have demonstrated this in vitro, where MNP displaying an antiantibody resulted in the death of 85% of VB6 cells with only a single 10 minute exposure to an alternating magnetic field. This is in contrast to only a 20% reduction in cell viability negative (H357) cells exposed to the same regime. A further reduction in cell viability, for cultures exposed to both MNP and the alternating magnetic field, in both cell lines 48 hours after treatment compared to 24 hours was also observed. applied. This also confirms that the presence of both the antibody and over expressed receptor are required in order to access higher levels of cell killing.
In summary, we have synthesised and tested a MHT agent which is targeted towards VB6 cells associated with OSCC. Our results indicate efficient and significant cell killing when exposed to an alternating magnetic field, and only when the VB6 antibody is adequately paired with its cognate antigen. This promising in vitro study indicates that through careful pairing of antigen, antibody and nanoparticle, the killing potential of OSCC by magnetic hyperthemia can be significantly enhanced. increase of MNP when exposed to an alternating magnetic field. Scale bar = 40 nm.   H357 and VB6 cell monolayers were exposed to either anti-v 6 conjugated MNP, un-conjugated MNP or no particles for 1 hour before exposure to an alternating magnetic field for 10 minutes. Cells were cultured for a further 24