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Quantification and visualization of HER2 protein using nanocrystals conjugated with single-domain antibodies

Name
Sergey
Surname
Glukhov
Scientific organization
Moscow Engineering Physics Institute
Academic degree
PhD
Position
Research Engineer
Scientific discipline
Life Sciences & Medicine
Topic
Quantification and visualization of HER2 protein using nanocrystals conjugated with single-domain antibodies
Abstract
At present, three classes of fluorescent labels are used for biomedical applications. These are heterocyclic molecules, fluorescent proteins, and photoluminescent (PL) semiconductor nanocrystals or quantum dots (QD). The main advantages of QDs are an extremely high resistance to photobleaching, high PL quantum yield, and continuous range of emission spectra from the ultraviolet to the infrared region depending on the QDs' size. In this study, HER2 protein was visualized and its content was estimated using single-domain antibodies conjugated in a site-specific manner with QDs.
Keywords
Quantum dots, QD; near infrared, NIR; single-domain antibody, sdAb; HER2 protein
Summary

Sergey Glukhov,1 Mikhail Berestovoy,1 Patrick Chames,2 Daniel Baty,2 Fernanda Ramos Gomes3, Frauke Alves3, Igor Nabiev,1,4 Alyona Sukhanova1,4

1Laboratory of Nano-Bioengineering, National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Moscow, Russian Federation, 2INSERM U1068 and CNRS UMR7258, Centre de Recherche en Cancérologie de Marseille, Institut Paoli-Calmettes and Université Aix-Marseille, Marseille, France, 3 Max-Planck Institute for Experimental Medicine, Goettingen, Germany, 4Laboratoire de Recherche en Nanosciences, LRN - EA4682, Université de Reims Champagne-Ardenne, Reims, France

 

Summary

Development of new fluorescent-based visualization is increasing in geometric progression, since fluorescent labeling of macromolecules has become one of the most widespread methods of imaging in modern biology and biotechnology (Fili and Toseland, 2014). The most common fluorophores for biolabeling are low-molecular-weight heterocyclic organic molecules. This type of fluorophores is characterized by a wide range of emission spectra, but their fluorescence is susceptible to photobleaching. Fluorescent proteins, such the widely used green fluorescent protein (GFP), have a higher photostability than heterocyclic molecules, and, in addition, intriguing properties of photoinduced shift of fluorescent emission spectra (Ando et al., 2002) and generation of reactive oxygen species (Wojtovich and Foster, 2014). Nevertheless, some difficulties with producing proteins with infrared fluorescence  pose obstacles to their wider applications.

            The third type of fluorescent labels includes photoluminescent (PL) semiconductor nanocrystals or quantum dots (QDs). QDs are synthesized from elements of groups IV–VI, such as Cdor Te. Absorbing UV/vis light, QDs emit fluorescence in the form of a narrow peak at wavelengths directly proportional to the QD size (Bruchez Jr., 1998). Due to the continuous variation of the emission maximum, it is possible to obtain nanocrystals with desired emission characteristics. Another useful feature of QDs is their extremely high resistance to photobleaching (Hardzei et al., 2012). The high stability of fluorescence makes QDs very interesting for labeling macromolecules in studying bioprocesses in vivo (Lim et al., 2015; Samanta and Medintz, 2016; Tu et al., 2016).

            Near-infrared light (NIR) is very weakly absorbed by tissues of living organisms in comparison with UV or visual electromagnetic waves; that is why this region of the optical spectrum is the most promising for bio-imaging. The second attractive feature of IR radiation is its harmlessness for living systems. That is why the use of fluorescence in the IR region of the optical spectrum is considered a very promising approach to biomedical imaging (Song et al., 2016).

  In the present study, we have prepared a PL nanoprobe for imaging of human epidermal growth factor receptor 2 (HER2), a very important prognostic marker for cancer diagnostic and treatment (Hadi, 2015; Harris et al., 2007). For the detection of HER2 protein, we have conjugated QDs with a maximum of emission spectra at 570 nm, as well as NIR-light-emitting QDs, with single-domain antibodies (sdAbs ) against HER2 protein.

We have developed ultraminiature nanoprobes consisting of highly affine sdAbs or "nanobodies", which are the smallest possible functionally active antibodies (13 kDa), conjugated with QDs in a strictly oriented manner. The highly ordered orientation of sdAbs on the QD surface is ensured by site-specific conjugation via an additional cysteine residue attached to the C-terminal region of the sdAb amino acid sequence. Each nanoprobe contains four sdAb molecules attached to a QD in the same orientation relative to its surface and has a hydrodynamic diameter smaller than 12 nm.

We present here our results of ELISA quantification of HER2 protein with anti-HER2-QD conjugates and in situ visualization of HER2 protein on the surface of HER2-positive cells.

When further validating the flow cytometric detection of cancer markers using QD–sdAb conjugates, we experimentally demonstrated that all other QD–sdAb conjugates obtained were highly specific and could be used to differentially detect very small relative numbers of cancer cells (Figure 1). Experiments with mixtures of human breast cancer cells expressing HER2 (the SK-BR-3 cell line) stained and not stained with conjugates of QDs with anti-HER2 sdAbs, as well as human epidermoid carcinoma cells (the A431 cell line) stained and not stained with conjugates of QDs with anti-EGFR sdAbs, in different ratios showed strong correlation between the numbers of cells identified as HER2- and EGFR-positive and the actual numbers of the respective labeled cells in the mixtures. It proved possible to detect as small proportions of labeled cells as 0.5 and 0.25% among SK-BR-3 and A431 cells, respectively.

 

Figure 1. Testing of conjugates of quantum dots (QDs) with single-domain antibodies (sdAbs) against HER2 and EGFR. SK-BR-3 cells stained and not stained with conjugates of QDs and anti-HER2 sdAbs (the upper row) and A431 cells stained and not stained with conjugates of QDs and anti-EGFR sdAbs (the lower row) were mixed in specified ratios. 105 cells were incubated in the presence of conjugates containing QD570 (30 μg/ml) for 1.5 h at 4°C in a final volume of 50 μl. Immediately after the last washing stage, the ratio between stained and unstained cells was determined using flow cytometry. The numbers above the plots (black) show the actual, prespecified percentages of stained cells; the numbers in the upper right corners of the plots (pink), the estimated percentages of stained cells.

 

The results of our research allow us to propose a method for fabricating QD-based nanoprobes with advanced functional characteristics, including an enhanced sensitivity. Thus, the ultraminiature QD–sdAb nanoprobes with highly ordered orientation of the Ab molecules relative to the QD surface developed in our studies have numerous implications for advanced integrated diagnostics.

 

Acknowledgments

The authors would like to thank Dr. Tina Van den Broeck, Line De Kimpe, and Dr. Frans Nauwelaers (BD Biosciences, Erembodegem, Belgium) for the assistance with flow cytometry experiments. This study was supported by the Federal Target Program for Research and Developments of the Ministry of Education and Science of the Russian Federation (grant no. 14.584.21.0012, contract no. RFMEFI58415X0012).

 

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