Transferrin-conjugated quasi-cubic SPIONs for cellular receptor profiling and detection of brain cancer

doi:10.1016/j.snb.2019.126737

Sensors and Actuators B: Chemical

Volume 297, 15 October 2019, 126737

https://doi.org/10.1016/j.snb.2019.126737 Get rights and content

Highlights

•
A solution-based precipitation method for large-scale synthesis of water-dispersible quasi-cubic γ-Fe₂O₃ SPIONs is shown.
•
In-situ production of sodium oleate through a saponification reaction allows morphological control of the SPIONs.
•
SPIONs act as enzyme-mimic catalysts with their activity dependent on their concentration, pH, temperature, and the concentration of substrates.
•
The conjugation of transferrin to NanoZyme catalysts allows profiling of cellular transferrin receptor and detection of U87MG glioblastoma cells.

Abstract

The high biocompatibility, unique magnetic properties and significant biomedical potential of superparamagnetic iron oxide nanoparticles (SPIONs) have spurred a major interest in developing new strategies to synthesise aqueous-phase dispersible SPIONs. In parallel, the rapidly growing field of enzyme-mimicking catalytic nanomaterials, referred to as NanoZymes, has seen immense interest in utilising the unique properties of NanoZymes for new applications. Here, we report a new strategy for the large-scale synthesis of water dispersible quasi-cubic γ-Fe₂O₃ SPIONs. This strategy provides a unique way to control the morphological characteristics of SPIONs through the in-situ formation of sodium oleate complex. Notably, our approach eliminates the need for high temperatures, complex cleaning protocols and post-synthesis phase-transfer strategies that are typically required during the well-established thermal decomposition synthesis approaches. We further establish the peroxidase-mimic NanoZyme activity of these quasi-cubic SPIONs and use them as an effective transducer platform to generate a unique colour through efficient oxidation of peroxidase substrates. By combining these inorganic enzymes with transferrin (Tf) as a brain cancer-specific targeting agent, we create a simple yet effective colorimetric biosensing platform that allows us to monitor the cellular expression profile of transferrin receptors (TfR). The difference in the expression profile of TfR in the U87MG glioblastoma cells and the fibroblast cells generates a distinct tonality of the colorimetric response. This not only allows cell surface receptor profiling but also allows robust identification of brain cancer cells. The high accuracy and precision of the proposed biosensor outlines the robustness of the NanoZyme-based catalysts in colorimetric detection of various targets.

Graphical abstract

Introduction

The widespread applicability of magnetic nanomaterials across biomedical imaging, hyperthermia, catalysis, sensing, magnetic separation and data storage has seen ongoing interest in developing new strategies to fabricate these materials [[1], [2], [3]]. In particular, superparamagnetic iron oxide nanoparticles (SPIONs) offer unique magnetic properties and high biocompatibility making them desirable candidates for biological applications [[1], [2], [3], [4], [5], [6], [7], [8], [9]]. For such applications, it is important to achieve good control over several nanoparticle characteristics, such as low polydispersity, uniform morphology, high magnetisation, good biocompatibility, and high aqueous stability. With an aim to achieve these properties, synthesis of SPIONs is regularly attempted across aqueous and organic solvents, with each solvent offering unique advantages and limitations [[1], [2], [3],[5], [6], [7],[9], [10], [11], [12], [13], [14]]. Aqueous routes although offer a simple method for the synthesis of SPIONs, their major drawback is poor control over nanoparticle size and morphology [[13], [14], [15]]. In contrast, thermal decomposition of iron alkoxides at elevated temperatures in the presence of high boiling point non-polar solvents produces high-quality nearly monodisperse SPIONs [2]. However, in this case, a rigid coating of long-chain fatty acids and their thermal by-products is formed on the surface of the SPIONs, making SPIONs hydrophobic. This limitation, particularly in the context of biological applications, demands for an additional surface modification strategy to transfer particles from the organic to the aqueous phase, which is not without its own challenges [15]. Thermal decomposition methods face another major challenge – to ensure that the SPIONs of characteristic sizes and morphologies can be reproducibly produced, the use of high-purity sodium oleate of consistent quality during synthesis is critical [16]. Sodium oleate tends to decompose during storage and therefore high-quality sodium oleate becomes impracticably expensive for iron oxide synthesis (sodium oleate of >99% purity is over 1000 times more expensive than of >82% purity). These on-going challenges with the synthesis of biologically-relevant SPIONs pointed us to an interesting thought – if sodium oleate could be produced in situ during aqueous precipitation synthesis, it may provide a reproducible morphological control over SPIONs directly in an aqueous environment. This is one of the aspects presented in this manuscript.

Further, while SPIONs have seen numerous applications across biomedical technologies, there has been a rapidly emerging interest to explore the enzyme-mimic catalytic ability of non-organic nanoparticles [10,[17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]]. Such catalysts are referred to as ‘NanoZymes’. In contrast to natural enzymes where a complex three-dimensional folding for the organic enzyme is critical for its catalytic activity, in case of inorganic NanoZymes, the nanoparticle surface atoms drive the reaction. This allows NanoZymes to overcome several disadvantages associated with natural enzymes, particularly those associated with the operational stability across wider pH and temperature ranges, lower production cost, design flexibility, and wider substrate scope [23]. Such interesting capabilities of NanoZymes have seen their use in applications ranging from environmental monitoring to biosensor development, control of microbial activity, and pro-drug activation [3,5,[9], [10], [11],[17], [18], [19], [20], [21], [22], [23], [24],[26], [27], [28], [29], [30], [31], [32], [33], [34], [35]]. Considering that the enzyme-mimic catalytic efficiency of NanoZymes is strongly governed by the nanoparticle composition, size, shape and surface characteristics [10,23], the second aspect of our work studies the enzyme-mimic activity of high-quality water-dispersible SPIONs of quasi-cubic morphology obtained from the aqueous precipitation route involving in situ synthesis of sodium oleate.

Next, we explored the applicability of the NanoZyme activity of SPIONs for the profiling of receptors expressed on the surface of mammalian cells. As a proof-of-concept, we chose transferrin receptors (TfR) that are considered a reliable target for nanoparticle-mediated drug delivery for brain cancer [[36], [37], [38], [39], [40]] due to their overexpression in glioblastoma cells [37,38,[41], [42], [43]]. The overexpression of TfR in brain cancer cells is due to the involvement of TfR in iron uptake, as cancer cells require a considerably higher concentration of iron for the growth and proliferation of the tumour [44]. Considering the importance of brain cancer [45,46] and current reliance on time-intensive traditional technologies for glioblastoma detection (e.g., through observing the morphological changes in cells via cytology or histopathology or imaging techniques such as mammography, magnetic resonance imaging, endoscopy and computed tomography [31,[47], [48], [49], [50], [51], [52]]; a simple strategy for the profiling of TfR presented here may offer new opportunities in reliably detecting brain cancer cells.

Overall, in this work, we have developed a simple method for the large-scale synthesis of oleate-capped quasi-cubic SPIONs that remain colloidally stable at high concentrations in an aqueous environment. The ability to control the morphology of these particles during an aqueous precipitation strategy is credited to in-situ formation of sodium oleate through a saponification reaction during the synthesis. The peroxidase-mimic activity of these SPIONs was then combined with a Tf moiety as a recognition element to create a colorimetric biosensor that provides a simple way to obtain the cellular expression profile of TfR, thus aiding in the identification of U87MG brain cancer cells. The difference in the expression profile of TfR in U87MG in contrast to fibroblast cells allows a distinct tonality in the colorimetric response to accurately identify TfR overexpression in U87MG glioblastoma cells. The ability to use the development of colour to identify cancer cells outlines the robustness and effectiveness of the current NanoZyme-based colorimetric platform. The simple colorimetric NanoZyme assay to visually detect cancer cells may offer the potential to be translated into field-deployable devices. For instance, the ability to visually detect color change can aid in the development of a sensor similar to a pregnancy strip that can allow an easy detection tool for cancer. Moreover, an easy to use assay for cellular receptor profiling can also reduce assay time. Recently, the World Health Organisation identified a new criteria for the design of in vitro diagnostic tests – the ASSURED criteria specifically in the context of HIV (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users) [53]. These criteria are in fact applicable in a broader context and the current sensing platform fits well within this framework for detecting cancer.

Section snippets

Materials

Ferrous chloride tetrahydrate (FeCl₂.4H₂O), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), Dulbecco’s modified eagle medium (DMEM), 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), phosphate buffer saline (PBS) tablets, sodium hydroxide (NaOH), oleic acid (90%), dimethyl sulfoxide, ethanol and transferrin (Tf – human holotransferrin, product no. T0665) were purchased from Sigma Aldrich,

Results and discussion

Highly stable, water-dispersible quasi-cubic γ-Fe₂O₃ nanoparticles were synthesized using a one-pot precipitation method, in which the in-situ synthesis of sodium oleate during the reaction allows a facile control over the nanoparticle morphology (experimental details in supporting information). Briefly, the process (Fig. 1a) involves mixing the ethanolic solution of NaOH with an ethanolic solution of oleic acid to produce fresh sodium oleate via a saponification reaction. On addition of an

Conclusion

This work, for the first time demonstrates a new solution-based approach for the large-scale synthesis of oleate-capped quasi-cubic SPIONs that are dispersible in aqueous phase without any requirement of surface modification. The in-situ formation of sodium oleate through a saponification reaction during nanoparticle synthesis allows a facile control over particle morphology. The high biocompatibility of synthesised SPIONs allowed us to use them for the development of a simple colorimetric

Acknowledgments

P.W. and M.S. thank the Commonwealth of Australia for Australian Postgraduate Awards. V. B. thanks the Australian Research Council for a Future Fellowship (FT140101285). R. R. acknowledges RMIT University for the Vice Chancellor Fellowship. V.B. and R.R. acknowledge ARC for research support though an ARC Discovery (DP170103477) grant. The authors acknowledge the generous support of the Ian Potter Foundation for establishing Sir Ian Potter NanoBioSensing Facility at RMIT University. The authors

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Transferrin-conjugated quasi-cubic SPIONs for cellular receptor profiling and detection of brain cancer

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Results and discussion

Conclusion

Acknowledgments

J. Biosci. Bioeng.

Appl. Mater. Today

Adv. Drug Delivery Rev.

Biosens. Bioelectron.

Biomater.

Clinical Immunol.

Int. J. Biochem. Cell B.

J. Clin. Neurosci.

Cell

Trends Biotechnol.

Biomater.

Appl. Surf. Sci.

Int. J. Pharm.

Environ. Pollut.

Phys. Rev. Lett.

Nat. Mater.

J. Phys. D.

PLoS One

J. Phys. D.

J. Phys. D.

Theranostics

Dalton Tans.

RSC Adv.

Chem. Rev.

Chem. Rev.

Nanoscale Res. Lett.

Sci. Technol. Adv. Mater.

Nat. Nanotechnol.

ACS Appl. Nano Mater.

Chem. Commun. (Camb.)

Chem. Commun. (Camb.)

Langmuir