Elsevier

Biomaterials

Volume 31, Issue 6, February 2010, Pages 1307-1315
Biomaterials

Nanoparticles functionalised with recombinant single chain Fv antibody fragments (scFv) for the magnetic resonance imaging of cancer cells

https://doi.org/10.1016/j.biomaterials.2009.10.036Get rights and content

Abstract

Superparamagnetic iron oxide nanoparticles (SPIONs) can substantially improve the sensitivity of magnetic resonance imaging (MRI). We propose that SPIONs could be used to target and image cancer cells if functionalised with recombinant single chain Fv antibody fragments (scFv). We tested our hypothesis by generating antibody-functionalised (abf) SPIONs using a scFv specific for carcinoembryonic antigen (CEA), an oncofoetal cell surface protein. SPIONs of different hydrodynamic diameter and surface chemistry were investigated and targeting was confirmed by ELISA, cellular iron uptake, confocal laser scanning microscopy (CLSM) and MRI. Results demonstrated that abf-SPIONs bound specifically to CEA-expressing human tumour cells, generating selective image contrast on MRI. In addition, we observed that the cellular interaction of the abf-SPIONs was influenced by hydrodynamic size and surface coating. The results indicate that abf-SPIONs have potential for cancer-specific MRI.

Introduction

The high spatial resolution of magnetic resonance imaging (MRI) is ideally suited for detection of cancer [1] and assessment of response to therapy [2]. However, MRI has found limited application in tumour imaging due to a lack of sensitivity [3]. Advances in nanotechnology and in particular the use of superparamagnetic iron oxide nanoparticles (SPIONs) have the potential to address this limitation. SPIONs, due to their large magnetic moments, significantly increase MRI R1 and R2 relaxivities, leading to a marked reduction in T1 and T2 times [4], thus enabling sensitive visualisation in vivo. Clinical application of SPIONs as contrast agents has been demonstrated with two products: EndoremĀ® and ResovistĀ® [5]. These SPIONs are not tumour specific per se but instead provide positive contrast in tumours based on their uptake by healthy phagocytic cells in preference to cancerous cells.

We have recently used the agent EndoremĀ® for cell labelling [6] and magnetic targeting to specific sites [7]. In this study, we aimed to achieve specificity by functionalising SPIONs with recombinant single chain Fv (scFv) antibodies. ScFvs have potential advantages over whole antibodies. First, with a molecular weight of ca. 30Ā kDa, scFvs are one-fifth the size of whole IgG antibodies [8] and yet they retain full antigen binding capacity. Thus, even when functionalised with scFvs, the relatively small diameter of the SPION is maintained. Second, unlike whole antibodies, scFvs do not contain the Fc constant domain and therefore are not able to trigger potentially harmful immune responses [8]. Third, scFvs are readily available inĀ recombinant form and can be generated for clinical use in non-mammalian systems [9], [10] with site-specific tags for purification and attachment.

The targeting potential of the scFv antibody functionalised SPIONs (abf-SPIONs) to tumour cells was assessed using sm3E, a high affinity scFv reactive to carcinoembryonic antigen (CEA) [11], [12]. Three different SPIONs were used to investigate the effect of surface coating and size on the abf-SPIONs targeting potential (Table 1). To test the influence of surface coating, we compared 50Ā nm abf-SPIONs coated with either dextran alone or with dextran plus polyethylene glycol (PEG). To test the influence of hydrodynamic particle size, we compared 20Ā nm and 50Ā nm abf-SPIONs, both coated with dextran plus PEG. The uptake of the abf-SPIONs was studied using a CEA+ve colorectal cancer cell line and a CEAāˆ’ve melanoma cell line, unmodified SPIONs were used as negative controls for specific uptake.

Section snippets

Superparamagnetic iron oxide nanoparticles

Details of the three SPIONs investigated are shown in Table 1, which gives information on hydrodynamic particle size, composition and coating as provided by the manufacturer. All three SPIONs were formulated as multi-domain iron crystal structures comprising of 35% (w/w) magnetite embedded in a matrix of dextran (40Ā kDa). According to the manufacturer description, for PEGylated SPIONs, 300Ā Da PEG chains were absorbed onto the dextran matrix. For simplicity, we named the SPIONs as follows: d50

Functionalisation of SPIONs

A total of 150Ā mg of monomeric sm3E was generated from a 10Ā l fermentation after purification by EBA-IMAC followed by SEC (Fig.Ā 2A). The material obtained gave a single band of ca. 27Ā kDa in SDS-PAGE that is in agreement with the molecular mass as deduced from the sm3E-His amino acid sequence (Fig.Ā 2B). Immunoreactivity with CEA was confirmed by ELISA (Fig.Ā 2C).

The purified sm3E was covalently conjugated to d50 SPIONs via dextran and to the PEGylated SPIONs via carboxylated PEG. Attachment and

Discussion

We have investigated the potential of scFvs to target SPIONs to cancer cells. We propose that these SPIONs could play a key role in tumour delivery of therapeutics and non-invasive monitoring of therapeutic response. In this study, we have tested the targeting potential of scFv-functionalised SPIONs by measuring their specificity and pattern of uptake in cancer cells and their MRI conspicuity in vitro following targeting to cancer cells. To achieve this targeting we conjugated the SPIONs to

Conclusions

We have demonstrated the functionality of three different SPIONs with the anti-CEA sm3E scFv and shown their potential as selective imaging contrast agents. Size and charge of the SPIONs were found to be important factors in the cellular behaviour of the SPIONs. The abf-d50 was shown to bind selectively to the cell membrane compared to the abf-PEGd50, which showed intracellular uptake and some non-specificity in particular with the CEAāˆ’ve A375M cell line. Both of these effects are believed to

Acknowledgements

Kim Vigor was supported by RAFT (Restoration of Appearance and Function Trust). The Authors would also like to thank Cancer Research UK, the UCL Cancer Institute Research Trust, the Rosetrees Trust, the UCL Experimental Cancer Medicine Centre, the UCL/UCLH Comprehensive Biomedical Research Centre of the NIHR, the UCL Institute of Child Health Research Appeal Trust, EPSRC (Engineering and Physical Sciences Research Council) Nanotechnology Grand Challenge Grant, Kings College London and UCL

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