Full length articleConformational control of human transferrin covalently anchored to carbon-coated iron nanoparticles in presence of a magnetic field
Graphical abstract
Introduction
The unique properties of nanoparticles have generated strong current interest due to their potential for application in a broad range of areas. The prospective application of nanoparticles include medicine [1], [2], natural and technical sciences [3], [4] and various nanotechnology approaches, for instance: magnetooptic devices for optical information processing [5], nanosensors [6], [7], new efficient recoverable catalysts [8], [9], [10], [11] and substrates for surface-enhanced Raman scattering (SERS) measurements [12], [13], [14]. Among the variety of promising nanoparticles developed so far, magnetic nanoparticles (M Nps) offer many opportunities, especially as bio-nanomaterials dedicated to biomedicine and nano-biointerface studies [15], [16], [17]. The main advantages of magnetic nanoparticles are those that they can be visualized (superparamagnetic Nps) and guided or held in selected locations by using a magnetic field [18]. Small ferromagnetic and ferrimagnetic nanoparticles (below a critical size of ∼3–50 nm, depending on the used material) belong to the group of superparamagnetic particles [19], [20]. One of the most widespread elements in this group are the superparamagnetic iron oxide nanoparticles (SPIONs) [21], [22], [23]. The unique characteristics of M Nps, such as high surface-to-volume ratio and size-dependent optical and magnetic properties are completely different compared to their bulk materials. On the other hand, their small size and large surface area can cause the aggregation of these nanoparticles and, in consequence, limit their potential applications. In fact, only nanoparticles with the appropriate size and surface chemistry are responsible for effective linkage with other materials/compounds, as well as for catalytic properties and activity with respect to biomolecules.
Due to the high tendency to an aggregation process and to corrosion of the core metal, which leads to the growth of the toxicity of these nanosystems, the simple magnetic nanoparticles such as Fe and Co oxides cannot be used in biochemical investigations. Of particular importance here is the process of coating of magnetic nanoparticles, which leads to the formation of “core-shell” heterogeneous systems. Such systems may have superior corrosion resistance [24], [25]. Among all materials used in the coating process, carbon is the best candidate, due to the ease of its further functionalization and stability in acidic and basic media as well as in many inorganic and organic solvents [26], [27], [28]. Moreover, the carbon shell protects the metal core against oxidation and, in consequence, preserves its specific properties and significantly limits the interactions between the nanoparticles. A major benefit of the carbon shell is that it is biocompatible and therefore can be widely applied in medical applications. The introduction of carbon coating into M Nps also improves their magnetic properties. For example, the maximum achievable magnetization of SPIONs is between 80 and 90 emu·g−1. In contrast, in the case of coating of Co or Fe it is possible to obtain the nanomaterial with increased magnetization to more than 150 emu·g−1. This property significantly increases their potential applications as a ferromagnetic electrode modifier for efficient enzyme immobilization in its active form [29].
Any realistic biomedical application of magnetic nanoparticles requires that they maintain a specific ability to recognize and bind the selected molecular targets (e.g. tumor receptors, pathogens, antigens and proteins) without losing their biological activities. SPIONs are widely used in biomedicine especially as drug delivery systems. The key point in such approaches is targeting them to specific cancerous cells. It is known that tumor cells have a large number of transferrin (Tf) receptors at the membrane surface (several times higher than in the normal cells) [30], [31]. Thus, transferrin can be successfully used in the delivery of drug-M Nps conjugates to specific cells through the blood brain barrier. Unfortunately, the direct contact of transferrin with SPIONs leads to irreversible changes in the protein structure and finally to the release of iron ions [32] so transferrin becomes useless in the drug delivery system. The coating of the SPIONs surface with 3-aminopropyltriethoxysilane layer that couples transferrin by amide bonds leads to good magnetic properties and high stability of such nanoconjugates, which provides possibilities for successful biomedical applications [33]. To avoid the necessity of adding an extra layer and in consequence preserving the properties of nanoparticles it is better to apply the carbon-coated iron nanoparticles. Additionally such nanoparticles eliminate the problem with unwanted iron release, which is shown in this paper. Our investigations were focused on the influence of magnetic field on the conformational changes of human transferrin due to its interaction with carbon-coated iron magnetic nanoparticles functionalized with carboxylic groups (Fe@C-COOH Nps). In consequence the electroactivity of the studied protein was preserved. To the best of our knowledge, this is the first report describing the dependence of the orientation and packing density of transferrin in the layer on the direct electron transfer between the protein and the electrode surface. To characterize the interactions between human transferrin and Fe@C-COOH nanoparticles in the presence and the absence of a magnet various techniques were applied: cyclic voltammetry (CV), quartz crystal microbalance with dissipation (QCM-D), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), circular dichroism (CD) and UV–vis spectroscopy.
Section snippets
Materials
NaH2PO4, Na2HPO4, Na2SO4 (all from POCH, Poland), cysteamine (CSH), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), N-hydroxy-succinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and human transferrin (Tf) (all from Sigma-Aldrich) were of the highest purity available and used as received. Carbon-coated iron nanoparticles functionalized with carboxylic groups (Fe@C-COOH Nps) were synthesized according to the procedure described in reference [34].
FTIR spectroscopy
The attachment of Tf molecules to the Fe@C-COOH nanoparticles via amide bonds was confirmed by FTIR spectroscopy. Typical FTIR spectra of human transferrin (Tf), Fe@C-COOH nanoparticles and Fe@C-COOH/Tf conjugate synthesized in the absence and presence of magnetic field are presented in Fig. 1. For the unmodified nanoparticles (Fe@C-COOH Nps), the characteristic stretching vibrations: of CO in carboxylic groups at ca. 1700 cm−1, CC in the hexagonal graphite lattice at 1580 and 1530 cm−1, and CO
Conclusions
We have demonstrated that the combination of external magnetic field and ferromagnetic electrode modifier (Fe@C-COOH Nps), during the formation of a transferrin layer on the support matrix, is necessary to successful covalent immobilization of the enzyme in its electroactive form. The QCM-D and TGA data have showed that the protein layer formed under such conditions consists of more than one monolayer. Transferrin molecules attached directly to ferromagnetic nanoparticles are tightly packed and
Acknowledgments
This work was supported by the National Science Centre of Poland [No. 2014/15/D/ST4/02989] and Ministry of Science and Higher Education of Poland [DS-6002-4693-14/Na/12]. The synthesis of carbon-coated iron nanoparticles was supported by the National Centre for Research and Development [LIDER/021/527/L-4/12/NCBR/2013].
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These authors contributed equally.