Elsevier

Biomaterials

Volume 32, Issue 8, March 2011, Pages 2141-2148
Biomaterials

Affibody-based nanoprobes for HER2-expressing cell and tumor imaging

https://doi.org/10.1016/j.biomaterials.2010.11.053Get rights and content

Abstract

This article reports the affibody-based nanoprobes specifically target and image human epidermal growth factor receptor type 2 (HER2)-expressing cells and tumors. The affibody molecules are a promising class of targeting ligands with simple, robust, and precise structure and high affinity. Using near-infrared (NIR) quantum dots (QDs) and iron oxide (IO) nanoparticles as two representative nanomaterials, we designed anti–HER2 affibody molecules with a N-terminus cysteine residue (Cysteine-ZHER2:342) and precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates. The in vitro and in vivo study showed the conjugates are highly specific to target and image HER2-expressing cells and tumors. This work indicated the nanoparticle-affibody conjugates may be excellent candidates as targeting probes for molecular imaging and diagnosis.

Introduction

This article describes the use of affibody molecules as targeting proteins to conjugate with nanoparticles (quantum dots (QDs) or iron oxide (IO) nanoparticles) for imaging of human epidermal growth factor receptor type 2 (HER2) expressing cells and tumors. Small proteins as platforms for the development of molecular imaging probes have attracted significant interest because of their favorable properties, such as high affinity and specificity, small size, facile synthesis and preparation, and rapid blood clearance [1]. Affibody molecules, the engineered small protein scaffolds with 58-amino acid residues and a three-helix bundle structure, are a promising class of disease-specific ligands with high affinity [2], [3], [4]. Different from antibodies, the key features of affibody molecules are their much smaller size, faster tumor targeting ability, and more well-defined structure which could potentially be site-specifically modified. The simple, robust structure of affibody molecules in combination with their low molecular weight (7 kDa) make them suitable for a wide variety of applications, especially in tumor-targeted imaging. For example, radiolabeled (e.g., 18F, 64Cu, 68Ga, 111In, 125I, and 177Lu) affibody molecules have shown great promise for tumor positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging and radiotherapy [5], [6], [7], [8], [9], [10], [11], [12]. The near-infrared (NIR) dyes labeled epidermal growth factor receptor (EGFR)-specific affibody molecules have also exhibited excellent properties for in vivo optical imaging of EGFR-overexpressing tumors [13], [14].

Nanobiotechnology, the combination of nanotechnology and biomedicine, has become a flourishing research area because of their great potential to offer abundant opportunities and tools for discovering and understanding new materials, processes, and phenomena in biology and medicine. The basic rationale is that the metal, metal oxide, semiconductor, or self-assembled molecular nanostrucuters have novel properties and functions that are not available from bulk counterparts or individual molecules. Among the well-established nanomaterials, both QDs and IO nanoparticles have found notable and successful applications in biomedicine: the former one are receiving increased acceptance as fluorescent probes for visualizing biological processes in vitro and in vivo [15], [16], [17], [18], while the latter one have served as magnetic resonance imaging (MRI) contrast agents for the clinical diagnosis of many diseases, including cancers [19], [20], [21]. Recently, nanoplatform-based molecular imaging has attracted more and more attentions because of the unique properties and multifunctionality in nanoplatforms [22], [23], [24], [25]. Besides the QDs as optical probes and IO nanoparticles as MRI contrast agents, there are many other novel nanomaterials developed as excellent molecular imaging agents. For example, Rabin et al. reported the polymer-coated Bi2S3 nanoparticles as an injectable computed tomography (CT) contrast agent [26]. De la Zerda et al. demonstrated that carbon nanotubes can be used as targeted photoacoustic molecular imaging agents after conjugated with cyclic Arg-Gly-Asp (RGD) peptides [27].

The integration of affibody with nanoprobes as targeted molecular probes may be of great importance in the field of molecular imaging and cancer diagnosis. Herein, using two kinds of well-established nanomaterials (QDs with emission wavelength at about 800 nm, denoted as QD800, and IO nanoparticles) as representative examples, we conjugated an anti–HER2 affibody molecule (ZHER2:342) and demonstrated the high specificity of affibody-based nanoprobes for HER2-expressing cell and tumor imaging. HER2 is a well-established tumor target overexpressed in a wide variety of cancers, including breast, ovarian, lung, and gastric cancers. In this study, we designed and chemically synthesized the anti–HER2 affibody molecules (ZHER2:342) with adding a cysteine residue at the N-terminus of the protein, then precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates (Scheme 1). Comparing with radiolabeled affibody molecules, the multivalent binding effect of nanoparticle-affibody conjugates may potentially enhance the targeting ability because the collective binding in a multivalent interaction is much stronger than that of the monovalent binding [28], [29], [30]. The in vitro and in vivo study further showed that the nanoparticle-affibody conjugates were highly specific to target HER2-expressing cell and tumor (e.g., SKOV3). The fluorescence imaging results indicated that QD800-affibody displayed strong fluorescent signal in SKOV3 tumor with high specificity, cellular MRI data revealed the significant MR contrast signal in IO-affibody treated SKOV3 cancer cells over IO-PEG treated SKOV3 cancer cells.

Section snippets

Materials

InAs/InP/ZnSe core/shell/shell QDs (∼5 nm in diameter, denoted as QD800) [31] and magnetite nanoparticles (∼15 nm in diameter, denoted as IO nanoparticles) [32] were provided by Ocean Nanotech LLC (Fayetteville, Arkansas). Affibody (ZHER2:342 with the amino acid sequence of VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPPK) with the N-terminus cysteine residue (Cys-ZHER2:342) and acetylation was produced by conventional solid phase peptide synthesis using a peptide synthesizer

Characterization of nanoparticle-affibody conjugates

The as-produced QD800 and IO nanoparticles only dispersed in non-polar organic solvents, such as hexane and chloroform. Poly(ethylene glycol) phosphatidylethanolamine (e.g., DSPE-PEG2000 amine) is one kind of micelle-forming hydrophilic polymer-grafted lipids [33], [36], [37]. This well-established micelle strategy indeed provides a general route to accomplish the transformation and modification of hydrophobic nanoparticles [34]. We thus used DSPE-PEG2000 amine to coat the nanoparticles by

Conclusion

In summary, the small, simple, precise, and robust structure of affibody molecules with high affinity and specificity provide promising advantages as targeting entities. The general procedure of bioconjugation and the negligible influence on the size of nanoparticles after conjugation warrant the ability of affibody-based nanoprobes for highly specific targeting and imaging. We demonstrate the high specificity of nanoparticle-affibody to HER2-overexpressing cancer cells using QD800 and IO

Acknowledgments

This work was partially supported by NCI/NIH R21 CA121842 (to Z.C.) and NCI of Cancer Nanotechnology Excellence Grant U54 CA119367 (to S.S.G.).

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