Colloids and Surfaces B: Biointerfaces 125 (2015) 104–109
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Enzyme-functionalized gold-coated magnetite nanoparticles as novel hybrid nanomaterials: Synthesis, purification and control of enzyme function by low-frequency magnetic field Alexander Majouga a,d,∗ , Marina Sokolsky-Papkov b , Artem Kuznetsov a , Dmitry Lebedev a , Maria Efremova a , Elena Beloglazkina a , Polina Rudakovskaya a , Maxim Veselov a , Nikolay Zyk a , Yuri Golovin a,c , Natalia Klyachko a,b , Alexander Kabanov a,b a
Laboratory of Chemical Design of Bionanomaterials, Chemistry Department, M.V. Lomonosov Moscow State University, Russian Federation Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA c R.G. Derzhavin Tambov State University, Russian Federation d National University of Science and Technology MISiS, Leninsky Ave, 4, 119049 Moscow, Russian Federation b
a r t i c l e
i n f o
Article history: Received 6 July 2014 Received in revised form 1 November 2014 Accepted 11 November 2014 Available online 20 November 2014 Keywords: Gold-coated magnetite nanoparticles Enzyme immobilization Purification Super low-frequency non-heating magnetic field Enzyme catalytic activity inhibition
a b s t r a c t The possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears attractive. From a practical point of view, this effect opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. Gold-coated magnetite nanoparticles are perfect candidates for such application. Herein, we have successfully synthesized core–shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized ChT. We show that the enzyme activity decreases upon exposure of the NPs to the field. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The integration of nanotechnology into biology and medicine is expected to produce major advances in molecular diagnostics, therapeutics, molecular biology and bioengineering. Success in the synthesis of nanoparticles (NPs) has led to the development of functional NPs able to react to an applied external stimulus. These NPs can be covalently linked to biological molecules such as peptides, proteins and nucleic acids. Due to their size-dependent properties and dimensional similarities to biomacromolecules, NPs offer exciting new opportunities for many biotechnological applications. In this frame, the possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears particularly attractive. For example, a gold nanocrystal has been covalently
∗ Corresponding author at: Laboratory of Chemical Design of Bionanomaterials, Chemistry Department, M.V. Lomonosov Moscow State University, Russian Federation. Tel.: +7 9258581024. E-mail address: [email protected] (A. Majouga). http://dx.doi.org/10.1016/j.colsurfb.2014.11.012 0927-7765/© 2014 Elsevier B.V. All rights reserved.
attached to a double-helical DNA; upon exposition to an oscillating radio frequency (RF) magnetic field, the DNA double helix opened up and closed back, i.e., denaturated and renaturated [1]. In a similar way, it has been reported that it is possible to switch on and off an enzyme in vitro by attaching to it a gold nanocrystal and modulating an applied RF field [2]. Recently, we have demonstrated that super low-frequency non-heating magnetic field can alter the kinetics of chemical reactions catalyzed by the enzymes ␣-chymotrypsin (ChT) and -galactosidase (-GaL) immobilized on nanoscale magnetic NP aggregates and core–shell magnetic NPs [3]. The observation is unprecedented and suggests the significance of magneto-mechanochemical effects induced by realignment of MNP magnetic moments in an alternating current (AC) magnetic field rather than traditional heating. Such low frequency and amplitude fields are safe and are not expected to cause any damage to biological tissues [4]. Thus, from a practical point of view, this investigation opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. However, to fully benefit from these potential opportunities there is a need to advance the NP surface chemistry approaches allowing
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functionalization of magnetite NPs with various biological molecules. It is well known that gold NPs offer excellent opportunities for surface chemical modification and facile attachment of broad range of small molecules as well as synthetic biological macromolecules. In the present report, we demonstrate a facile and simple aqueous-based method to fabricate Fe3 O4 /Au bifunctional hybrid materials based on reduction of AuCl4 − on the surface of magnetite NPs [5]. Herein, we first develop a procedure for the purification of synthesized gold-coated magnetic NPs using flash chromatography on a Sepharose gel. Resulting NPs were consistently modified by thiolated carboxylic acids with different chain lengths (to form covalent bond Au–S) and ChT using the carbodiimide method. To investigate the influence of the spacer nature on the kinetics of chemical reaction catalyzed by the ChT, the resulting hybrid nanomaterials were investigated under the influence of non-heating magnetic fields.
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Fe3 O4 @Au NPs was suspended in 50 ml of an aqueous ligand solution (0.103 mol of ligand) and stirred overnight. After decantation of the NP solution from Sephadex and dialysis in DI water (3 times in 1 L), the NP solution was purified from any Sephadex traces using 0.22 m hydrophilic PES filters.
2.5. Immobilization of ChT on Fe3 O4 @Au core–shell NPs The 0.75 ml of as-purified NP solution (freshly sonicated for 20 min) was mixed with 0.25 ml of citrate buffer (20 mM, pH 5.9). Then, the mixture was supplemented first by 0.7 mg of 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.2 mg of N-hydroxysulphosuccinimide (Sulpho-NHS) (both used as 10 mg/ml aqueous stock solutions) and second by 600 l of 10 mg/ml ChT aqueous solution. The final solution was shaken for 2 h at r.t. The last step was the removal of any non-bound enzyme by multiple centrifugal filtrations (1800 × g, rotary 100 kDa filters).
2. Materials and methods 2.1. Materials
2.6. Enzyme activity study
Iron chloride (II) tetrahydrate (FeCl2 ·4H2 O, 98%), hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 ·3H2 O, 98%), iron (III) chloride (FeCl3 , anhydrous, 97%), ammonium solution (NH3 ·H2 O, 29%), nitric acid (HNO3 , 69%), hydrochloric acid (HCl, 37%) and 11-mercaptoundecanoic acid (11-MUA, 98%) were obtained from Sigma–Aldrich. Perchloric acid (HClO4 , 70%) and l-cysteine (99%) were purchased from Acros Organics, sodium citrate from Reachem (Russia), HS-PEG-COOH (5 kDa) and HS-PEG-OMe (5 kDa) from Nanocs Inc. All water used in experiments was deionized (18.2 M cm−1 , Millipore Milli-Q Academic System). All vessels were washed with hot solution of aqua regia and then rinsed with DI water before conducting syntheses. 2.2. Synthesis of Fe3 O4 NPs
Activity of free or conjugated ChT was determined by UV–vis spectrometry by measuring the rate of enzymatic hydrolysis of a specific substrate, N-benzoyl-l-tyrosine p-nitroanilide (BTNA). Briefly, 1–2 l of BTNA solution (17 mg/ml) in a dioxane–acetonitrile mixture (1:1 v:v) was added to 1 ml of 20 mM Tris–HCl buffer, pH 8.2 and then mixed with 2 l of the free or immobilized enzyme solutions. Formation of the product (pnitroaniline) was recorded at 380 nm over time. The immobilized enzyme activity was calculated and compared with a standard curve of free enzyme activity. The enzymatic hydrolysis measurements followed Michaelis–Menten kinetics, showing a linear dependence on enzyme concentration and hyperbolic dependence on substrate concentration. Substrate concentrations were adjusted to maintain constant hydrolysis rates for time periods required for AC field exposures (approx. 1 h).
Fe3 O4 NPs were prepared by co-precipitation of Fe(II) and Fe(III) salts according to the procedure [5].
2.7. Immobilized enzyme quantity
2.3. Synthesis of gold-coated Fe3 O4 NPs Gold-coated magnetite NPs were synthesized by a modified protocol [5,6]. Briefly, 120 ml of HAuCl4 water solution (35 mg of HAuCl4 ·3H2 O) under vigorous stirring was heated under reflux to boiling-state, and 5 ml of freshly prepared Fe3 O4 NP dispersion was quickly added to HAuCl4 solution. After 10 min, 5 ml of sodium citrate (80 mM) was rapidly added to the reaction mixture. This mixture was boiled under reflux with vigorous stirring for 5 min; the heating was then turned off, and the mixture was cooled to room temperature (r.t.). 2.4. Purification and modification of Fe3 O4 @Au core–shell NPs by organic ligands The 0.15 g of Sephadex G-100 was mixed with an excess of water and kept overnight at 5 ◦ C and then placed into the empty 12 g chromatography column cartridge (Interchim PF-50SIHP) with a downside filter. All chromatography procedures were made under gravity flow conditions. Sephadex was washed with 50 ml of citrate buffer solution (pH 5.0), and 15 ml of as-prepared Fe3 O4 @Au core–shell freshly sonicated (30 min) NP dispersion was added to the column. After all of the dispersion entered into the Sephadex, the column was washed with 20 ml of citrate buffer to remove the uncovered Fe3 O4 NPs. Then, the Sephadex gel with purified
Total immobilized ChT quantity was determined using a Micro BCA protein assay kit. 150 l of reagents were added to 150 l samples, which contained ChT (the measurements were conducted in microplates). The mixture was intensively stirred in the shaker for 1 min and then thermostated at 37 ◦ C for 2 h. After that, the microplate was cooled to r.t. and solution absorbance at 562 nm was measured. The quantity of immobilized enzyme was determined from the linear (in the range of 2–40 g of enzyme/ml) calibration graph for standard samples. The studied samples were several times diluted.
2.8. Number of free amino groups Free amino groups on the surface of NPs were determined using 2,4,6-trinitrobenzenesulphonic acid (TNBSA) as described in [7]. 25 l of 0.1 M sodium tetraborate Na2 B4 O7 in 0.1 M sodium hydroxide NaOH and then 1 l of 1 M TNBSA solution were added to 25 l of studied/standard samples (in microplates). The mixture was incubated at 23 ◦ C for exactly 5 min, and then 100 l of 0.1 M sodium sulphite Na2 SO3 solution and 0.1 M sodium dihydrogen phosphate NaH2 PO4 mixture (1:65 v:v) were added to it. After that, the absorbance at 420 nm was measured. The quantity of free amino groups was determined from the linear calibration graph for standard samples.
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Scheme 1. Synthesis, purification and modification of core–shell magnetite–gold nanoparticles.
2.9. Characterization of NPs Transmission electron microscopy (TEM), JEM-2010(HC), 200 kV) was used to examine the morphology of the core–shell NPs. TEM specimens were prepared by aspirating samples onto a copper TEM grid. The average diameter of the magnetic NPs was calculated from TEM images by analysing at least 300 NPs for each sample using ImageJ software (National Institutes of Health, USA). The hydrodynamic size and zeta potential of the magnetic NPs were measured by dynamic light scattering (DLS) using a NanoS Zeta Sizer (Malvern Instruments) just after sonication for 10 s. The average particle sizes with error ranges were obtained from three measurements of each sample. Optical absorption spectroscopy measurements were performed on HITACHI U-2900 spectrophotometer using plastic cuvettes. Spectra were collected within a range of 300–800 nm. NP tracking analysis (NTA) measurements were performed on NanoSight NS500. 2.10. Experiments in the AC magnetic field (a) Unit description: The unit contained a sinusoid current generator with variable power (up to 1.5 kW), frequency (in the range from 30 to 3000 Hz) and amplitude. The unit was equipped with a water-cooled inductor with a ferromagnetic core and temperature-controlled active volume of 30 mm × 30 mm × 60 mm and a controller, which allowed generating required duration of exposition to magnetic field intervals and pauses between them. Magnetic field amplitude could be varied in the range from several mT to 250 mT. Magnetic field heterogeneity in the range of standard spectrophotometer cell active volume (10 mm × 10 mm × 25 mm) did not exceed 0.3% of maximum field value (that eliminated pullin forces effect on magnetic NPs in suspension). The experiments were conducted at 50 Hz frequency, and magnetic field intensity was varied from 15 to 220 kA/m. (b) Procedure description: The
100–400 l of samples and 6 l of BTNA solution (0.04 mM) in a dioxane–acetonitrile mixture (1:1 v:v) were added to 2 ml of 20 mM Tris–HCl buffer, pH 8.2. The mixture was then intensively stirred and divided between two uniform translucent cells. One cell served as a control and was placed into the spectrophotometer initially; meanwhile, another cell was exposed to the magnetic field. After 5.5 min of field exposition (twice for 2.5 min in field and 0.5 min pause), this cell was placed into the spectrophotometer and the control cell was thermostated without the magnetic field. This procedure was repeated several times. Field effect was evaluated as a ratio between ChT activity in field exposed cell and ChT activity in the control cell.
3. Results and discussion 3.1. Synthesis and purification of NPs The properties and behaviour of Fe3 O4 NPs in biomedical applications are expected to strongly depend on the NP surface properties and stability in physiological solutions [8]. Unfortunately, Fe3 O4 NPs often undergo a spontaneous surface oxidation, which can hinder their use. Chemical methods to predictably tune the surface functionality of Fe3 O4 NPs also need further advancement. Therefore, Au-coated Fe3 O4 NPs were recently synthesized to reduce surface oxidation and expand possibilities for surface functionalization [9]. The magnetic NPs, especially high moment metallic magnetic NPs, can be stabilized with such an Au coating under corrosive biological conditions (another great advantage of magnetite coated with gold layer is absolutely passivity under bio conditions, especially in acidic media, where pure magnetite nanoparticles dissolves with a production of toxic species) and readily functionalized through well-developed Au–S chemistry. Moreover, the coating also renders the magnetic NPs
Fig. 1. TEM images of (A) the as-prepared uncoated Fe3 O4 NPs; (B) the as-prepared core–shell Fe3 O4 -Au NPs, mean diameter 27 ± 8 nm; (C) the purified core–shell Fe3 O4 -Au NPs, mean diameter 30 nm ± 7 nm.
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Table 1 Structure of attached ligands. Sample
Attached ligands
Structure
H2N 1
COOH
l-Cysteine
SH COOH 2
3-Mercaptopropionic acid
HS COOH 3
d,l-Lipoic acid
4
11-Mercaptoundecanoic acid (MUA)
HS
5
HS-PEG-COOH (MW 5000)
HS
with plasmonic properties [10]. Such Fe3 O4 @Au core–shell NPs can combine properties of the Au nanoshells and regular magnetite NPs. The former can display valuable optical properties suitable for combined imaging and therapy,[11] as they can simultaneously provide both scattering and absorption properties at specific frequencies. On the latter, these NPs can be remotely actuated by an external magnetic field or even transported by the field within proximity of targeted tissue in the body [12]. Composite materials that have both optical properties of Au nanoshells and magnetic properties of superparamagnetic NPs have already been reported [13–15]. However, the available methods for preparation of Fe3 O4 @Au core–shell NPs often result in an uncontrollable formation of a pure magnetite and core–shell NP mixture [16]. Therefore, for further integration of magnetic NPs into smart electronic devices, drug delivery systems and others, a reliable methodology for the preparation of pure Fe3 O4 @Au core–shell NPs must be developed. Synthesis of magnetite NP in aqueous media could be facile and easy for subsequent conjugation of biomolecules to the particles. Herein, we used a simple two-step reaction to synthesize water-soluble, Fe3 O4 @Au core–shell NPs based on a well-known procedure [5,17,18]. Iron oxide NPs were synthesized in aqueous media using a standard co-precipitation method, starting from Fe2+ and Fe3+ salts. This was followed by the reduction of a HAuCl4 to add a layer of gold at the iron oxide NP surface (Scheme 1). It can be seen from the TEM images in Fig. 1 that both uncoated Fe3 O4 NPs and Fe3 O4 @Au core–shell NPs are roughly spherical, particle size distribution is also displayed. The TEM images suggest that the as-prepared core–shell Fe3 O4 Au NPs were rather heterogeneous (Fig. 1B). Attempts to separate uncoated and Au coated magnetite NPs using a magnetic field were reported previously by Ning et al. [19]. Herein, we developed a new procedure for the purification of the synthesized NPs based on affinity chromatography. In this approach a sepharose gel (Sephadex) was used to separate the uncovered Fe3 O4 NPs from the desired core–shell Fe3 O4 -Au NPs. The solution of non-purified NPs was passed through the column, and uncovered NPs were eluted with
S S
COOH O
nO
COOH
10 mM citrate buffer, whereas the core–shell Fe3 O4 -Au NPs were retained in the column, presumably due to interaction of the sugar hydroxyl groups of the sepharose gel with the Au surface of the NPs. The next step included washout of the core–shell NPs from the sepharose gel using a thiolated ligands, a well-known Au–S chemistry that provides modification of the Au shell of the NPs. Several ligands presented in Table 1 were explored that allow for subsequent covalent attachment of ChT to NPs through the carbodiimide chemistry. To remove the excess unbound ligands, multiple steps of dialysis against water were applied. The core–shell Fe3 O4 -Au NPs became more uniform after the purification as seen in the TEM image (Fig. 2C). Compared to the initial uncoated Fe3 O4 NPs, these purified coated NPs appeared to have much more contrast on the TEM, suggesting their higher electron
Fig. 2. Magnetic field effect on the enzymatic activity of ChT immobilized at the core–shell Fe3 O4 -Au NPs using different linkers. The data represent the mean of five measurements, and the error bars are the standard deviation.
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Table 2 Characteristics of the purified core–shell Fe3 O4 -Au NPs (DLS, each value is an average of five independent measurements ± standard deviation measured as RMS (mean square) of each SD value; NTA, the data is reported as the mode with one standard deviation of three runs on the same sample). Sample
Concentration, particles per ml
Size by DLS, nm
1 2 3 4 5
1.5 × 1010 1.0 × 1010 3.1 × 109 1.1 × 1010 5.0 × 1010
93 69 65 65 70
± ± ± ± ±
Size by NTA, nm
34 33 33 25 35
71 47 46 47 48
density due to the presence of the Au shell. Their average increased to 30 ± 7 nm vs. 8 ± 2 nm for the uncoated Fe3 O4 NPs. This suggests that an Au layer with an average thickness of 11 ± 4 nm was added to the surface of the NPs, resulting in an increase of the particle diameter. The aqueous dispersion of the core–shell Fe3 O4 -Au NPs exhibited an absorption band at 515–535 nm, which is typical for surface plasmon (SP) resonance of Au NPs larger than 2 nm; this is a trait of the unique optical properties of the gold nanostructures (Supplementary material) [20]. The particles sizes and size distribution in dispersion were also determined using the DLS and NTA (Table 2). These data demonstrate generally good reproducibility of the NP characteristics for different batches, each obtained using ligand moiety as presented in Table 1. Notably, as expected, the DLS diameter representing the z-average value exceeded the NTA diameter, which is a number average value. Both the DLS and NTA sizes were larger than the value obtained in TEM, which is also typical, as TEM does not account for the hydration of the NPs. Table 2 also presents the -potential values determined by DLS. It should be noted that in all cases the obtained samples were stable, as initial NP concentration (measured by NTA) remained unchanged even after storage at r.t. for two weeks. The last step of the present study involved conjugation of ChT to the synthesized NPs (samples 1–3) using EDC and Sulpho-NHS to couple the amino groups of the protein to the carboxylic groups displayed at the NPs surface according to a published procedure [3]. A standard TNBSA test for the number of the free primary amino groups was used to quantify the number of enzymes molecules attached to the NPs. It is known that native ChT molecule contains 15 free amino groups [21], and after conjugation, the enzyme retained only five to seven free amino groups per protein molecule. Together with known data on the ChT structure and our observation, we propose that several (1/2 or 1/3 of total) groups are involved in formation of the links to the NP surface. The activity of the NPs immobilized ChT in comparison with the free enzyme in solution was determined by the reaction of the hydrolysis of a specific substrate of ChT. It was found that the reaction kinetics complied with the standard Michaelis–Menten equation and the reaction rate linearly depended on the enzyme concentration. The Michaelis–Menten kinetic parameters (KM , kkat ) for the immobilized ChT calculated using the linearization of the rate vs. substrate concentration dependence in double
Table 3 NP sizes for different samples before and after conjugation of the enzyme (DLS, each value is an average of five independent measurements ± standard deviation measured as RMS (mean square) of each SD value; NTA, the data is reported as the mode with one standard deviation of three runs on the same sample). Sample
NP diameter by NTA, nm Before conjugation
After conjugation
1 2 3
71 ± 33 47 ± 32 46 ± 35
81 ± 36 64 ± 33 65 ± 33
± ± ± ± ±
33 32 35 32 34
-potential, mV
Concentration of NPs in solution after 14 days, particles per ml
−8.9 −19.1 −5.0 −20.4 −7.7
0.4 × 1010 0.2 × 1010 3.1 × 109 1.0 × 1010 4.3 × 1010
reciprocal coordinates (Supplementary material) were similar to those for free enzyme in solution [22]. To demonstrate the effect of the spacer between NPs and ChT on the kinetics of the enzyme reaction, we conducted a series of experiments with synthesized materials under an AC magnetic field. Field exposures (50 Hz, 110 kA/m) resulted in considerable decreases of the enzymatic reaction rates compared to the reference sample, albeit no effect on the free enzyme or on the non-conjugated enzyme–NP mixture was observed for samples 1–3. In all cases, the reaction mixture was treated three times in magnetic field for 5.5 min (twice for 2.5 min in field and 0.5 min pause). Residual activity after exposure vs. control sample without exposure to the magnetic field was used as a criterion to evaluate the effect. As seen from Fig. 2, in all cases exposure of the immobilized ChT to the field resulted in a decrease in the enzyme activity, which is consistent with the previously observed effect [3,23]. As proposed earlier, such an AC magnetic field effect on the activity of the immobilized enzyme can be achieved in the case of enzymes conjugated between two different NPs. For samples 1–3, we used NTA data to explore whether the enzyme immobilization increases the particle size (Table 3). Indeed, for all samples, the size distribution maxima were shifted to larger values. An increase of the size distribution by 1.5 times indicates the formation of mostly dimer associates as was mentioned previously [24]. Recently, we performed numerical evaluations of the forces, stresses, and strains of enzymes attached to core–shell magnetic nanoparticles [25]. It was shown that a nanomechanical device consisting of two magnetic nanoparticles makes it possible to perform four types of deformation of the MMs attached to their surface: tension, compression, shear, and torsion. In the case of pair of magnetic nanoparticles connected by developed in present study “linkerenzyme-linker” chain, the particles roll over the surface of each other. The theoretical observations suggests that the forced applied to the enzyme (up to several hundreds of piconewtons) induce in the enzyme structure stress that is sufficient to change the topology of active centres, as well as the secondary/tertiary structure.
4. Conclusions We have successfully synthesized core–shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized enzyme. We show that the enzyme activity decreases upon exposure of the NPs to the field. The present result suggests that activity of selected enzymes can be efficiently manipulated by a remote magnetic field using the proposed enzymes-superparamagnetic NP hybrid structures, which may be useful in various biomedical and nanotechnology applications. Supporting information
A. Majouga et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 104–109
Acknowledgment The authors gratefully acknowledge the financial support of the Ministry of Education and Science of the Russian Federation (No. 11.G34.31.0004, K1-2014-022 (MISIS)) and RSF-14-13-00731. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.11.012. References [1] K. Hamad-Schifferli, J.J. Schwartz, A.T. Santos, S. Zhang, J.M. Jacobson, Nature 415 (2002) 152–155. [2] P. Ball, Nat. Nanotechnol. 3 (2008) 139–143. [3] N.L. Klyachko, M. Sokolsky-Papkov, N. Pothayee, M.V. Efremova, D.A. Gulin, N. Pothayee, A.A. Kuznetsov, A.G. Majouga, J.S. Riffle, Y.I. Golovin, A.V. Kabanov, Angew. Chem. Int. Ed. 51 (2012) 1–5. [4] F. Gazeau, M. Levy, C. Wilhelm, Nanomedicine 3 (2008) 831–844. [5] C.K. Lo, D. Xiao, M.M.F. Choi, J. Mater. Chem. 17 (2007) 2418–2427. [6] P.G. Rudakovskaya, E.K. Beloglazkina, A.G. Majouga, N.V. Zyk, Mend. Commun. 20 (2010) 158–160. [7] R. Fields, Methods Enzymol. 25 (1972) 464–469. [8] L.H. Reddy, J.L. Arias, J. Nicolas, P. Couvreur, Chem. Rev. 112 (2012) 5818–5878. [9] S. Wei, Q. Wang, J. Zhu, L. Sun, H. Line, Z. Guo, Nanoscale 3 (2011) 4474–4502.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Enzyme-functionalized gold-coated magnetite nanoparticles as novel hybrid nanomaterials: Synthesis, purification and control of enzyme function by low-frequency magnetic field Alexander Majouga a,d,∗ , Marina Sokolsky-Papkov b , Artem Kuznetsov a , Dmitry Lebedev a , Maria Efremova a , Elena Beloglazkina a , Polina Rudakovskaya a , Maxim Veselov a , Nikolay Zyk a , Yuri Golovin a,c , Natalia Klyachko a,b , Alexander Kabanov a,b a
Laboratory of Chemical Design of Bionanomaterials, Chemistry Department, M.V. Lomonosov Moscow State University, Russian Federation Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA c R.G. Derzhavin Tambov State University, Russian Federation d National University of Science and Technology MISiS, Leninsky Ave, 4, 119049 Moscow, Russian Federation b
a r t i c l e
i n f o
Article history: Received 6 July 2014 Received in revised form 1 November 2014 Accepted 11 November 2014 Available online 20 November 2014 Keywords: Gold-coated magnetite nanoparticles Enzyme immobilization Purification Super low-frequency non-heating magnetic field Enzyme catalytic activity inhibition
a b s t r a c t The possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears attractive. From a practical point of view, this effect opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. Gold-coated magnetite nanoparticles are perfect candidates for such application. Herein, we have successfully synthesized core–shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized ChT. We show that the enzyme activity decreases upon exposure of the NPs to the field. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The integration of nanotechnology into biology and medicine is expected to produce major advances in molecular diagnostics, therapeutics, molecular biology and bioengineering. Success in the synthesis of nanoparticles (NPs) has led to the development of functional NPs able to react to an applied external stimulus. These NPs can be covalently linked to biological molecules such as peptides, proteins and nucleic acids. Due to their size-dependent properties and dimensional similarities to biomacromolecules, NPs offer exciting new opportunities for many biotechnological applications. In this frame, the possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears particularly attractive. For example, a gold nanocrystal has been covalently
∗ Corresponding author at: Laboratory of Chemical Design of Bionanomaterials, Chemistry Department, M.V. Lomonosov Moscow State University, Russian Federation. Tel.: +7 9258581024. E-mail address: [email protected] (A. Majouga). http://dx.doi.org/10.1016/j.colsurfb.2014.11.012 0927-7765/© 2014 Elsevier B.V. All rights reserved.
attached to a double-helical DNA; upon exposition to an oscillating radio frequency (RF) magnetic field, the DNA double helix opened up and closed back, i.e., denaturated and renaturated [1]. In a similar way, it has been reported that it is possible to switch on and off an enzyme in vitro by attaching to it a gold nanocrystal and modulating an applied RF field [2]. Recently, we have demonstrated that super low-frequency non-heating magnetic field can alter the kinetics of chemical reactions catalyzed by the enzymes ␣-chymotrypsin (ChT) and -galactosidase (-GaL) immobilized on nanoscale magnetic NP aggregates and core–shell magnetic NPs [3]. The observation is unprecedented and suggests the significance of magneto-mechanochemical effects induced by realignment of MNP magnetic moments in an alternating current (AC) magnetic field rather than traditional heating. Such low frequency and amplitude fields are safe and are not expected to cause any damage to biological tissues [4]. Thus, from a practical point of view, this investigation opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. However, to fully benefit from these potential opportunities there is a need to advance the NP surface chemistry approaches allowing
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functionalization of magnetite NPs with various biological molecules. It is well known that gold NPs offer excellent opportunities for surface chemical modification and facile attachment of broad range of small molecules as well as synthetic biological macromolecules. In the present report, we demonstrate a facile and simple aqueous-based method to fabricate Fe3 O4 /Au bifunctional hybrid materials based on reduction of AuCl4 − on the surface of magnetite NPs [5]. Herein, we first develop a procedure for the purification of synthesized gold-coated magnetic NPs using flash chromatography on a Sepharose gel. Resulting NPs were consistently modified by thiolated carboxylic acids with different chain lengths (to form covalent bond Au–S) and ChT using the carbodiimide method. To investigate the influence of the spacer nature on the kinetics of chemical reaction catalyzed by the ChT, the resulting hybrid nanomaterials were investigated under the influence of non-heating magnetic fields.
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Fe3 O4 @Au NPs was suspended in 50 ml of an aqueous ligand solution (0.103 mol of ligand) and stirred overnight. After decantation of the NP solution from Sephadex and dialysis in DI water (3 times in 1 L), the NP solution was purified from any Sephadex traces using 0.22 m hydrophilic PES filters.
2.5. Immobilization of ChT on Fe3 O4 @Au core–shell NPs The 0.75 ml of as-purified NP solution (freshly sonicated for 20 min) was mixed with 0.25 ml of citrate buffer (20 mM, pH 5.9). Then, the mixture was supplemented first by 0.7 mg of 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.2 mg of N-hydroxysulphosuccinimide (Sulpho-NHS) (both used as 10 mg/ml aqueous stock solutions) and second by 600 l of 10 mg/ml ChT aqueous solution. The final solution was shaken for 2 h at r.t. The last step was the removal of any non-bound enzyme by multiple centrifugal filtrations (1800 × g, rotary 100 kDa filters).
2. Materials and methods 2.1. Materials
2.6. Enzyme activity study
Iron chloride (II) tetrahydrate (FeCl2 ·4H2 O, 98%), hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 ·3H2 O, 98%), iron (III) chloride (FeCl3 , anhydrous, 97%), ammonium solution (NH3 ·H2 O, 29%), nitric acid (HNO3 , 69%), hydrochloric acid (HCl, 37%) and 11-mercaptoundecanoic acid (11-MUA, 98%) were obtained from Sigma–Aldrich. Perchloric acid (HClO4 , 70%) and l-cysteine (99%) were purchased from Acros Organics, sodium citrate from Reachem (Russia), HS-PEG-COOH (5 kDa) and HS-PEG-OMe (5 kDa) from Nanocs Inc. All water used in experiments was deionized (18.2 M cm−1 , Millipore Milli-Q Academic System). All vessels were washed with hot solution of aqua regia and then rinsed with DI water before conducting syntheses. 2.2. Synthesis of Fe3 O4 NPs
Activity of free or conjugated ChT was determined by UV–vis spectrometry by measuring the rate of enzymatic hydrolysis of a specific substrate, N-benzoyl-l-tyrosine p-nitroanilide (BTNA). Briefly, 1–2 l of BTNA solution (17 mg/ml) in a dioxane–acetonitrile mixture (1:1 v:v) was added to 1 ml of 20 mM Tris–HCl buffer, pH 8.2 and then mixed with 2 l of the free or immobilized enzyme solutions. Formation of the product (pnitroaniline) was recorded at 380 nm over time. The immobilized enzyme activity was calculated and compared with a standard curve of free enzyme activity. The enzymatic hydrolysis measurements followed Michaelis–Menten kinetics, showing a linear dependence on enzyme concentration and hyperbolic dependence on substrate concentration. Substrate concentrations were adjusted to maintain constant hydrolysis rates for time periods required for AC field exposures (approx. 1 h).
Fe3 O4 NPs were prepared by co-precipitation of Fe(II) and Fe(III) salts according to the procedure [5].
2.7. Immobilized enzyme quantity
2.3. Synthesis of gold-coated Fe3 O4 NPs Gold-coated magnetite NPs were synthesized by a modified protocol [5,6]. Briefly, 120 ml of HAuCl4 water solution (35 mg of HAuCl4 ·3H2 O) under vigorous stirring was heated under reflux to boiling-state, and 5 ml of freshly prepared Fe3 O4 NP dispersion was quickly added to HAuCl4 solution. After 10 min, 5 ml of sodium citrate (80 mM) was rapidly added to the reaction mixture. This mixture was boiled under reflux with vigorous stirring for 5 min; the heating was then turned off, and the mixture was cooled to room temperature (r.t.). 2.4. Purification and modification of Fe3 O4 @Au core–shell NPs by organic ligands The 0.15 g of Sephadex G-100 was mixed with an excess of water and kept overnight at 5 ◦ C and then placed into the empty 12 g chromatography column cartridge (Interchim PF-50SIHP) with a downside filter. All chromatography procedures were made under gravity flow conditions. Sephadex was washed with 50 ml of citrate buffer solution (pH 5.0), and 15 ml of as-prepared Fe3 O4 @Au core–shell freshly sonicated (30 min) NP dispersion was added to the column. After all of the dispersion entered into the Sephadex, the column was washed with 20 ml of citrate buffer to remove the uncovered Fe3 O4 NPs. Then, the Sephadex gel with purified
Total immobilized ChT quantity was determined using a Micro BCA protein assay kit. 150 l of reagents were added to 150 l samples, which contained ChT (the measurements were conducted in microplates). The mixture was intensively stirred in the shaker for 1 min and then thermostated at 37 ◦ C for 2 h. After that, the microplate was cooled to r.t. and solution absorbance at 562 nm was measured. The quantity of immobilized enzyme was determined from the linear (in the range of 2–40 g of enzyme/ml) calibration graph for standard samples. The studied samples were several times diluted.
2.8. Number of free amino groups Free amino groups on the surface of NPs were determined using 2,4,6-trinitrobenzenesulphonic acid (TNBSA) as described in [7]. 25 l of 0.1 M sodium tetraborate Na2 B4 O7 in 0.1 M sodium hydroxide NaOH and then 1 l of 1 M TNBSA solution were added to 25 l of studied/standard samples (in microplates). The mixture was incubated at 23 ◦ C for exactly 5 min, and then 100 l of 0.1 M sodium sulphite Na2 SO3 solution and 0.1 M sodium dihydrogen phosphate NaH2 PO4 mixture (1:65 v:v) were added to it. After that, the absorbance at 420 nm was measured. The quantity of free amino groups was determined from the linear calibration graph for standard samples.
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Scheme 1. Synthesis, purification and modification of core–shell magnetite–gold nanoparticles.
2.9. Characterization of NPs Transmission electron microscopy (TEM), JEM-2010(HC), 200 kV) was used to examine the morphology of the core–shell NPs. TEM specimens were prepared by aspirating samples onto a copper TEM grid. The average diameter of the magnetic NPs was calculated from TEM images by analysing at least 300 NPs for each sample using ImageJ software (National Institutes of Health, USA). The hydrodynamic size and zeta potential of the magnetic NPs were measured by dynamic light scattering (DLS) using a NanoS Zeta Sizer (Malvern Instruments) just after sonication for 10 s. The average particle sizes with error ranges were obtained from three measurements of each sample. Optical absorption spectroscopy measurements were performed on HITACHI U-2900 spectrophotometer using plastic cuvettes. Spectra were collected within a range of 300–800 nm. NP tracking analysis (NTA) measurements were performed on NanoSight NS500. 2.10. Experiments in the AC magnetic field (a) Unit description: The unit contained a sinusoid current generator with variable power (up to 1.5 kW), frequency (in the range from 30 to 3000 Hz) and amplitude. The unit was equipped with a water-cooled inductor with a ferromagnetic core and temperature-controlled active volume of 30 mm × 30 mm × 60 mm and a controller, which allowed generating required duration of exposition to magnetic field intervals and pauses between them. Magnetic field amplitude could be varied in the range from several mT to 250 mT. Magnetic field heterogeneity in the range of standard spectrophotometer cell active volume (10 mm × 10 mm × 25 mm) did not exceed 0.3% of maximum field value (that eliminated pullin forces effect on magnetic NPs in suspension). The experiments were conducted at 50 Hz frequency, and magnetic field intensity was varied from 15 to 220 kA/m. (b) Procedure description: The
100–400 l of samples and 6 l of BTNA solution (0.04 mM) in a dioxane–acetonitrile mixture (1:1 v:v) were added to 2 ml of 20 mM Tris–HCl buffer, pH 8.2. The mixture was then intensively stirred and divided between two uniform translucent cells. One cell served as a control and was placed into the spectrophotometer initially; meanwhile, another cell was exposed to the magnetic field. After 5.5 min of field exposition (twice for 2.5 min in field and 0.5 min pause), this cell was placed into the spectrophotometer and the control cell was thermostated without the magnetic field. This procedure was repeated several times. Field effect was evaluated as a ratio between ChT activity in field exposed cell and ChT activity in the control cell.
3. Results and discussion 3.1. Synthesis and purification of NPs The properties and behaviour of Fe3 O4 NPs in biomedical applications are expected to strongly depend on the NP surface properties and stability in physiological solutions [8]. Unfortunately, Fe3 O4 NPs often undergo a spontaneous surface oxidation, which can hinder their use. Chemical methods to predictably tune the surface functionality of Fe3 O4 NPs also need further advancement. Therefore, Au-coated Fe3 O4 NPs were recently synthesized to reduce surface oxidation and expand possibilities for surface functionalization [9]. The magnetic NPs, especially high moment metallic magnetic NPs, can be stabilized with such an Au coating under corrosive biological conditions (another great advantage of magnetite coated with gold layer is absolutely passivity under bio conditions, especially in acidic media, where pure magnetite nanoparticles dissolves with a production of toxic species) and readily functionalized through well-developed Au–S chemistry. Moreover, the coating also renders the magnetic NPs
Fig. 1. TEM images of (A) the as-prepared uncoated Fe3 O4 NPs; (B) the as-prepared core–shell Fe3 O4 -Au NPs, mean diameter 27 ± 8 nm; (C) the purified core–shell Fe3 O4 -Au NPs, mean diameter 30 nm ± 7 nm.
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Table 1 Structure of attached ligands. Sample
Attached ligands
Structure
H2N 1
COOH
l-Cysteine
SH COOH 2
3-Mercaptopropionic acid
HS COOH 3
d,l-Lipoic acid
4
11-Mercaptoundecanoic acid (MUA)
HS
5
HS-PEG-COOH (MW 5000)
HS
with plasmonic properties [10]. Such Fe3 O4 @Au core–shell NPs can combine properties of the Au nanoshells and regular magnetite NPs. The former can display valuable optical properties suitable for combined imaging and therapy,[11] as they can simultaneously provide both scattering and absorption properties at specific frequencies. On the latter, these NPs can be remotely actuated by an external magnetic field or even transported by the field within proximity of targeted tissue in the body [12]. Composite materials that have both optical properties of Au nanoshells and magnetic properties of superparamagnetic NPs have already been reported [13–15]. However, the available methods for preparation of Fe3 O4 @Au core–shell NPs often result in an uncontrollable formation of a pure magnetite and core–shell NP mixture [16]. Therefore, for further integration of magnetic NPs into smart electronic devices, drug delivery systems and others, a reliable methodology for the preparation of pure Fe3 O4 @Au core–shell NPs must be developed. Synthesis of magnetite NP in aqueous media could be facile and easy for subsequent conjugation of biomolecules to the particles. Herein, we used a simple two-step reaction to synthesize water-soluble, Fe3 O4 @Au core–shell NPs based on a well-known procedure [5,17,18]. Iron oxide NPs were synthesized in aqueous media using a standard co-precipitation method, starting from Fe2+ and Fe3+ salts. This was followed by the reduction of a HAuCl4 to add a layer of gold at the iron oxide NP surface (Scheme 1). It can be seen from the TEM images in Fig. 1 that both uncoated Fe3 O4 NPs and Fe3 O4 @Au core–shell NPs are roughly spherical, particle size distribution is also displayed. The TEM images suggest that the as-prepared core–shell Fe3 O4 Au NPs were rather heterogeneous (Fig. 1B). Attempts to separate uncoated and Au coated magnetite NPs using a magnetic field were reported previously by Ning et al. [19]. Herein, we developed a new procedure for the purification of the synthesized NPs based on affinity chromatography. In this approach a sepharose gel (Sephadex) was used to separate the uncovered Fe3 O4 NPs from the desired core–shell Fe3 O4 -Au NPs. The solution of non-purified NPs was passed through the column, and uncovered NPs were eluted with
S S
COOH O
nO
COOH
10 mM citrate buffer, whereas the core–shell Fe3 O4 -Au NPs were retained in the column, presumably due to interaction of the sugar hydroxyl groups of the sepharose gel with the Au surface of the NPs. The next step included washout of the core–shell NPs from the sepharose gel using a thiolated ligands, a well-known Au–S chemistry that provides modification of the Au shell of the NPs. Several ligands presented in Table 1 were explored that allow for subsequent covalent attachment of ChT to NPs through the carbodiimide chemistry. To remove the excess unbound ligands, multiple steps of dialysis against water were applied. The core–shell Fe3 O4 -Au NPs became more uniform after the purification as seen in the TEM image (Fig. 2C). Compared to the initial uncoated Fe3 O4 NPs, these purified coated NPs appeared to have much more contrast on the TEM, suggesting their higher electron
Fig. 2. Magnetic field effect on the enzymatic activity of ChT immobilized at the core–shell Fe3 O4 -Au NPs using different linkers. The data represent the mean of five measurements, and the error bars are the standard deviation.
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Table 2 Characteristics of the purified core–shell Fe3 O4 -Au NPs (DLS, each value is an average of five independent measurements ± standard deviation measured as RMS (mean square) of each SD value; NTA, the data is reported as the mode with one standard deviation of three runs on the same sample). Sample
Concentration, particles per ml
Size by DLS, nm
1 2 3 4 5
1.5 × 1010 1.0 × 1010 3.1 × 109 1.1 × 1010 5.0 × 1010
93 69 65 65 70
± ± ± ± ±
Size by NTA, nm
34 33 33 25 35
71 47 46 47 48
density due to the presence of the Au shell. Their average increased to 30 ± 7 nm vs. 8 ± 2 nm for the uncoated Fe3 O4 NPs. This suggests that an Au layer with an average thickness of 11 ± 4 nm was added to the surface of the NPs, resulting in an increase of the particle diameter. The aqueous dispersion of the core–shell Fe3 O4 -Au NPs exhibited an absorption band at 515–535 nm, which is typical for surface plasmon (SP) resonance of Au NPs larger than 2 nm; this is a trait of the unique optical properties of the gold nanostructures (Supplementary material) [20]. The particles sizes and size distribution in dispersion were also determined using the DLS and NTA (Table 2). These data demonstrate generally good reproducibility of the NP characteristics for different batches, each obtained using ligand moiety as presented in Table 1. Notably, as expected, the DLS diameter representing the z-average value exceeded the NTA diameter, which is a number average value. Both the DLS and NTA sizes were larger than the value obtained in TEM, which is also typical, as TEM does not account for the hydration of the NPs. Table 2 also presents the -potential values determined by DLS. It should be noted that in all cases the obtained samples were stable, as initial NP concentration (measured by NTA) remained unchanged even after storage at r.t. for two weeks. The last step of the present study involved conjugation of ChT to the synthesized NPs (samples 1–3) using EDC and Sulpho-NHS to couple the amino groups of the protein to the carboxylic groups displayed at the NPs surface according to a published procedure [3]. A standard TNBSA test for the number of the free primary amino groups was used to quantify the number of enzymes molecules attached to the NPs. It is known that native ChT molecule contains 15 free amino groups [21], and after conjugation, the enzyme retained only five to seven free amino groups per protein molecule. Together with known data on the ChT structure and our observation, we propose that several (1/2 or 1/3 of total) groups are involved in formation of the links to the NP surface. The activity of the NPs immobilized ChT in comparison with the free enzyme in solution was determined by the reaction of the hydrolysis of a specific substrate of ChT. It was found that the reaction kinetics complied with the standard Michaelis–Menten equation and the reaction rate linearly depended on the enzyme concentration. The Michaelis–Menten kinetic parameters (KM , kkat ) for the immobilized ChT calculated using the linearization of the rate vs. substrate concentration dependence in double
Table 3 NP sizes for different samples before and after conjugation of the enzyme (DLS, each value is an average of five independent measurements ± standard deviation measured as RMS (mean square) of each SD value; NTA, the data is reported as the mode with one standard deviation of three runs on the same sample). Sample
NP diameter by NTA, nm Before conjugation
After conjugation
1 2 3
71 ± 33 47 ± 32 46 ± 35
81 ± 36 64 ± 33 65 ± 33
± ± ± ± ±
33 32 35 32 34
-potential, mV
Concentration of NPs in solution after 14 days, particles per ml
−8.9 −19.1 −5.0 −20.4 −7.7
0.4 × 1010 0.2 × 1010 3.1 × 109 1.0 × 1010 4.3 × 1010
reciprocal coordinates (Supplementary material) were similar to those for free enzyme in solution [22]. To demonstrate the effect of the spacer between NPs and ChT on the kinetics of the enzyme reaction, we conducted a series of experiments with synthesized materials under an AC magnetic field. Field exposures (50 Hz, 110 kA/m) resulted in considerable decreases of the enzymatic reaction rates compared to the reference sample, albeit no effect on the free enzyme or on the non-conjugated enzyme–NP mixture was observed for samples 1–3. In all cases, the reaction mixture was treated three times in magnetic field for 5.5 min (twice for 2.5 min in field and 0.5 min pause). Residual activity after exposure vs. control sample without exposure to the magnetic field was used as a criterion to evaluate the effect. As seen from Fig. 2, in all cases exposure of the immobilized ChT to the field resulted in a decrease in the enzyme activity, which is consistent with the previously observed effect [3,23]. As proposed earlier, such an AC magnetic field effect on the activity of the immobilized enzyme can be achieved in the case of enzymes conjugated between two different NPs. For samples 1–3, we used NTA data to explore whether the enzyme immobilization increases the particle size (Table 3). Indeed, for all samples, the size distribution maxima were shifted to larger values. An increase of the size distribution by 1.5 times indicates the formation of mostly dimer associates as was mentioned previously [24]. Recently, we performed numerical evaluations of the forces, stresses, and strains of enzymes attached to core–shell magnetic nanoparticles [25]. It was shown that a nanomechanical device consisting of two magnetic nanoparticles makes it possible to perform four types of deformation of the MMs attached to their surface: tension, compression, shear, and torsion. In the case of pair of magnetic nanoparticles connected by developed in present study “linkerenzyme-linker” chain, the particles roll over the surface of each other. The theoretical observations suggests that the forced applied to the enzyme (up to several hundreds of piconewtons) induce in the enzyme structure stress that is sufficient to change the topology of active centres, as well as the secondary/tertiary structure.
4. Conclusions We have successfully synthesized core–shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized enzyme. We show that the enzyme activity decreases upon exposure of the NPs to the field. The present result suggests that activity of selected enzymes can be efficiently manipulated by a remote magnetic field using the proposed enzymes-superparamagnetic NP hybrid structures, which may be useful in various biomedical and nanotechnology applications. Supporting information
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Acknowledgment The authors gratefully acknowledge the financial support of the Ministry of Education and Science of the Russian Federation (No. 11.G34.31.0004, K1-2014-022 (MISIS)) and RSF-14-13-00731. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.11.012. References [1] K. Hamad-Schifferli, J.J. Schwartz, A.T. Santos, S. Zhang, J.M. Jacobson, Nature 415 (2002) 152–155. [2] P. Ball, Nat. Nanotechnol. 3 (2008) 139–143. [3] N.L. Klyachko, M. Sokolsky-Papkov, N. Pothayee, M.V. Efremova, D.A. Gulin, N. Pothayee, A.A. Kuznetsov, A.G. Majouga, J.S. Riffle, Y.I. Golovin, A.V. Kabanov, Angew. Chem. Int. Ed. 51 (2012) 1–5. [4] F. Gazeau, M. Levy, C. Wilhelm, Nanomedicine 3 (2008) 831–844. [5] C.K. Lo, D. Xiao, M.M.F. Choi, J. Mater. Chem. 17 (2007) 2418–2427. [6] P.G. Rudakovskaya, E.K. Beloglazkina, A.G. Majouga, N.V. Zyk, Mend. Commun. 20 (2010) 158–160. [7] R. Fields, Methods Enzymol. 25 (1972) 464–469. [8] L.H. Reddy, J.L. Arias, J. Nicolas, P. Couvreur, Chem. Rev. 112 (2012) 5818–5878. [9] S. Wei, Q. Wang, J. Zhu, L. Sun, H. Line, Z. Guo, Nanoscale 3 (2011) 4474–4502.
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