- Email: [email protected]
Interaction of magnetic nanoparticles with lysozyme amyloid fibrils
Author’s Accepted Manuscript Interaction of magnetic lysozyme amyloid fibrils
nanoparticles
with
Veronika Gdovinová, Natália Tomašovičová, Ivan Batko, Marianna Batková, Lucia Balejčíková, Vasyl M. Garamus, Viktor. I. Petrenko, Mikhail V. Avdeev, Peter Kopčanský www.elsevier.com/locate/jmmm
PII: DOI: Reference:
S0304-8853(16)32074-1 http://dx.doi.org/10.1016/j.jmmm.2016.09.035 MAGMA61816
To appear in: Journal of Magnetism and Magnetic Materials Received date: 30 June 2016 Revised date: 17 August 2016 Accepted date: 5 September 2016 Cite this article as: Veronika Gdovinová, Natália Tomašovičová, Ivan Batko, Marianna Batková, Lucia Balejčíková, Vasyl M. Garamus, Viktor. I. Petrenko, Mikhail V. Avdeev and Peter Kopčanský, Interaction of magnetic nanoparticles with lysozyme amyloid fibrils, Journal of Magnetism and Magnetic Materials, http://dx.doi.org/10.1016/j.jmmm.2016.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interaction of magnetic nanoparticles with lysozyme amyloid fibrils Veronika Gdovinov´aa,b, Nat´ alia Tomaˇsoviˇcov´aa,b,∗, Ivan Batkoa , Marianna a Batkov´a , Lucia Balejˇc´ıkov´aa, Vasyl M. Garamusc , Viktor. I. Petrenkob,d, Mikhail V. Avdeevd , Peter Kopˇcansk´ ya a Institute
of Experimental Physics SAS, Watsonova 47, 040 01 Koˇ sice, Slovakia Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region, Russian Federation c Helmholtz-Zentrum Geesthacht: Zentrum fr Material- und Kstenforschung GmbH Max-Plank-Strae 1, Geesthacht, 216502 Germany d Physics Department, Taras Shevchenko Kyiv National University, Volodymyrska Street 64, 01601 Kyiv, Ukraine b Frank
Abstract This work is devoted to the structural study of complex solutions of magnetic nanoparticles with lysozyme amyloid fibrils due to possible ordering of such system by applying the external magnetic field. The interaction of magnetic nanoparticles with amyloid fibrils has been followed by atomic force microscopy and small-angle X-ray scattering. It has been observed that magnetic nanoparticles (MNPs) adsorb to lyzozyme amyloid fibrils. It was found that MNPs alter amyloids structures, namely the diameter of lysozyme amyloid fibrils is increased whereas the length of fibrils is decreased. In same time MNPs do not change the helical pitch significantly. Keywords: magnetic nanoparticles, lysozyme fibrils, SAXS, AFM
1. Introduction Amyloid fibrils have an important role in nanotechnology and biomaterials applications due to their unique physical and mechanical properties [1, 2]. Also amyloid fibrils, highly ordered nanoscale assemblies of protein protofibrils ∗ Corresponding
author Email address: [email protected] (Nat´ alia Tomaˇsoviˇcov´ a)
Preprint submitted to Journal of LATEX Templates
September 6, 2016
5
with characteristic cross- stacking perpendicular to the long axis of the fiber [3] are associated with various neurodegenerative diseases. Therefore the detail study of amyloids is of great interest. The most used and well characterised model protein for in vitro study of amyloid fibrillation is Hen egg white lysozyme (HEWL) that represents a structural homologue of human lysozyme
10
with globular shape. While native lysozyme is a relatively stable enzyme under physiological conditions and does not easily form amyloids, mutant versions are implicated in some forms of hereditary systemic amyloidosis in humans [4]. Therefore forming amyloids from HEWL in vitro requires the use of specific environmental conditions, as high concentrations in solutions under acidic pH,
15
constant stirring, high temperature and ionic strength or presence of various salts [5]. It was shown, that the kinetics of fibrils formation under these conditions is concentration dependent [6]. Fibrils may show several polymorphic states such as twisted ribbons, helical ribbons or nanotubes [7]. The coexistence of twisted and helical ribbons has been reported in the case of egg lysozyme after
20
long incubation time [8]. It was observed that there exist some time- dependent transformations of twisted ribbon to nanotubes thought the helical ribbon intermediated [9]. Except the study of amyloid fibrils, nowadays there is increasing interest of studing of magnetic nanoparticles (MNPs) as novel materials very suitable
25
for biomedical uses such as hyperthermia, magnetic resonance imaging, drug delivery etc. Zaman et al. suggested existence of critical concentration at which amyloid fibrils are ordered to pure biological liquid crystal (LC) [10]. Our expectation is that magnetite nanoparticles will interact with lysozyme amyloid fibrils (LAF). Main aim is to study the fibrils-magnetic nanoparticles complexes if they
30
may be oriented by applying the external magnetic field in order to obtain a biological liquid crystal. In our recent work the adsorption of MNPs on amyloid fibrils of HEWL in 2 mg/ml acidic solutions has been detected for the MNPs concentration range of 0.01-0.1 vol% [11]. It has been observed that adsorption is determined by the MNPs content and aggregates of the MNPs follow the rod-
35
like structure of fibrils at high MNP concentrations. However, the mechanism of 2
such nanoparticle effect on protein amyloid aggregation has not been explained yet. Several researches show that different size, surface and concentration of nanoparticles affect protein aggregation in different way [12, 13]. Therefore, the main goal of present research is the detail study and understanding the inter40
action between MNPs and solution of LAF with protein concentration of 10 mg/ml that is much higher as it was used in previous experiments with LAF. This finding about the interaction of LAF with MNPs can open a new field of research related to study of fibrils-magnetic nanoparticles complexes if they may be oriented by applying the external
45
magnetic field in order to obtain a biological liquid crystal. It should be mentioned that slightly different procedure for amyloids formation was used in this work. The inner structural organization of initial fibrillized amyloid solution and its mixtures with MNPs were analyzed by means small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM).
50
2. Materials and Methods Hen egg white lysozyme (lyophilized powder, lot number L6876, 50.000 units mg−1 protein) was obtained from Sigma-Aldrich Chemical Company (St Louis, MO). All other chemicals were obtained from Sigma or Fluka and were of analytical reagent grade. Lysozyme amyloid fibrils were prepared by dissolving
55
of lysozyme powder to a final concentration of 10 mg/ml in 0.2 M glycine-HCl buffer with pH=2.4 and 80 mMNaCl. Prepared solution in enclosed bottle was heated for 2 hours at 65◦ C and constant stirring (250 rpm). The magnetite nanoparticles were prepared by co-precipitation method [14]. Electrostatically stabilized magnetic nanoparticles with HClO4 were dispersed in water to obtain
60
magnetic fluid with mean diameter of nanoparticles d = 26 nm. The size of MNPs was determined by dynamic light scattering (Zetasizer). Such magnetic fluids were used for preparation of mixtures with solution of LAF (sLAF). Magnetic fluid (MF) with concentration of MNPs of 45 mg/ml was added to the initial solution of LAF with concentration of 10 mg/ml to achieve three differ-
3
65
ent ratios between volumes of sLAF and MNPs solutions (VsLAF :VMF ) in the mixture: 1ml: 0.0023ml, 1ml: 0.011ml, 1ml: 0.023 ml to obtain final mixture with mass ratio 10:0.1, 10:0.5 and 10:1, respectively. The SAXS experiments were conducted on a Nanostar (Bruker AXS GmbH, Karlsruhe, Germany) laboratory device with the following characteristic parameters: microfocus X- ray
70
souce IμS, CuKα wavelength of 1.54 ˚ A detector VANTEC-2000 (14 x 14 cm in size, resolution of 2048 x 2048 pixels). The samples were placed into glass capillary vessels with a diameter of 2 mm. The measurements were performed at 25±1◦ C. The primary processing of data with taking into account the scattering from the solvent and capillary, detector efficiency, and background signal,
75
as well as the recalculation to the absolute cross section was performed with the SuperSAXS (Jan Pedersen&Cristiano Oliveira) code. Atomic force microscopy measurements were performed using Agilent 5500 AFM system equipped by PicoView 1.14.3 control software. The topography images were acquired in the tapping mode with a standard silicon cantilevers (Olympus, model OMCL-AC
80
160TS) with resonant frequency 300 kHz (typ.), and spring constant 26 N/m (typ.). All measurements were carried out at ambient temperature in air, while relative humidity was in the range of 30-40%. Images were processed using freely available software from Gwyddion (http://gwyddion.net). Samples for AFM observations were prepared by dropping the sample on freshly cleaved
85
pieces of mica (PELCO Mica Sheets Grade V5, 15 X 15 mm2 ). After the deposition, the drop was left to incubate for 5-10 minutes, and then removed by rinsing the surface of mica substrate by ultra-pure water. Samples were dried in air at ambient temperature (typically for 1 hour). 3. Experimental results
90
Fig. 1 and Fig. 2 show AFM images of pure lysozyme fibrils and those doped with magnetic fluid, respectively. The doped sample for AFM was prepared with ratio of sLAF volume to magnetic fluid volume (sLAF/MF= 1:0.023). The doped sample was deposed on mica 1 hour after preparation. As the vol-
4
ume concentration of magnetic particles is high, it is seen that some fibrils are 95
completely covered by magnetic particles (see e.g. Profile 4 in Fig. 2), however it is clearly seen, that in sample are presented also fibrils covered only with few particles as well as without particles (as reveals Profile 3 in Fig. 2). If compare Fig. 1 and Fig. 2 it is clearly shown that after doping with MF the length of fibrils is shorter. AFM measurements also reveal presence of a helical structure
100
but as it can be concluded based on Profiles 1-4 in Fig. 1 the pitch of the helical structure is different for different fibrils (see text in the caption of Fig. 1). Also, comparisons of mutual distance between Profiles 1 and 2 in Fig. 2 (system with MNPs) with the mutual distances between pairs of Profiles in Fig. 1 yields values for the pitch
105
of the helical structure of the same order, what might indicate that adding of MNPs to the system (using above described technology process) does not change this pitch significantly. The obtained diameter of the fibrils in initial solution is 7-9 nm. In the doped sample with MNPs the diameter is 9-10nm.
110
Experimental SAXS curves for the mixtures of amyloid aggregates with MNPs (Fig. 3) were analyzed by applying the Indirect Fourier Transform (IFT) method developed by Glatter [15] in the version of Pedersen [16]. Taking into account elongated shape of the particles, the cross-section of pair distance distribution (PDD) functions, pcs (r), have been obtained in Fig. 4 that indicates the
115
helical structure of lysozyme fibrils which is similar to previous works [17, 18]. The power- law type scattering at small q values (linear parts in the graph) with power exponent close to -1 indicates the presence of elongated particles. Model of cylinder-like shape is an approximation of the real shape of the amyloid aggregates. Fibrils have shown some periodic structure most probably helical. Radius
120
of primary component is ∼ 1.4-2 nm and diameter of whole helixes slightly less than 7 nm. By adding magnetic fluid to lysozyme fibrils, the structure of the lysozyme was changed. It leads to disappearing of second maxima (helical structure) and some shift of first maximum (primary protein component). The shift of maximum for primary component points to adsorption of MF and increasing 5
28 26
Profile 1 Profile 2 Profile 3 Profile 4
z (nm)
24 22 20 18 16 14 0
20
40
60
80
100
120
distance (nm)
Figure 1: AFM height image and their representative profile cross section for pure lysozyme fibrils. Distances between profiles 1-2 (∼ 60 nm) and 3-4 represent ( 100 nm) approximately the quarter of the period of the helical structure.
125
of effective cross section (Fig. 4. 4. Conclusion The interaction between lysozyme amyloid fibrils and MNPs was investigated by SAXS and AFM measurements. Our results have shown that lysozyme fibrils have helical structure with a diameter of 7 nm approximately. By adding a small
130
amount of MNPs to lysozyme amyloid fibrils caused that the helical structure of
6
28 26
Profile 1 Profile 2
z (nm)
24 22 20 18 16 0
5
10
15
20
distance (nm) 45
Profile 3 Profile 4
40
z (nm)
35 30 25 20 15 10 0
100
200
300
distance (nm)
Figure 2: AFM height image and their representative profile cross section for lysozyme fibrils doped with magnetic nanoparticles after 1 hour incubation. The distance between profiles 1 and 2 (∼ 90 nm) is approximately the quarter of the period of the helical structure.
7
Figure 3: Experimental SAXS curves for sLAF and mixtures of sLAF and various concentrations of MNPs.
Figure 4: The cross-section of PDD functions as a result of the IFT procedure for scattering from mixture of sLAF and various concentrations of MNPs.
amyloid fibrils was partly lost. Diameter of the mixture of LAF and MNPs was increased that provide an experimental evidence of interaction of MNPs with LAF. Diameter of LAF after doping with MNPs was increased that
8
provide an experimental evidence of their mutual interaction. 135
This
should be the first step to order such complex systems by applying magnetic field. Acknowledgements This work was supported by project VEGA 2/0045/13], the Slovak Research and Development Agency under the contract No. APVV- 015-0453, Ministry of
140
Education Agency for Structural Funds of EU in frame of project 26220120033, EU FP7 M-era.Net MACOSYS. [1] I. Cherny, E. Gazit. Angew. Chem. Int. Ed. Engl. 47 (2008) 4062. DOI: 10.1002/anie.200703133 [2] T.P. Knowles, T. W. Oppenheim, A. K. Buell, D. Y. Chirgadze, M. E.
145
Welland. Nat. Nanotechnol. 5 (2010) 204. DOI: 10.1038/nnano.2010.26 [3] C. M. Dobson. Nature 426 (2003) 884. DOI: 10.1038/nature02261 [4] M. B. Pepys, P. N. Hawkins, D. R. Booth, D. M. Vigushin, G. A. Tennent, A. K. Soutar, N. Totty, O. Nguyen, C. C. F. Blake, C. J. Terry, T. G. Feest, A. M. Zalin and J. J. Hsuan, Nature 362(1993) 553. DOI: 10.1038/362553a0
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[5] M. R. Krebs, D. K. Wilkins, E. W. Chung, M. C. Pitkeathly, A. K. Chamberlain, J. Zurdo, C. V. Robinson, C. M. Dobson. J. Mol. Biol. 300 (2000) 541. DOI: 10.1006/jmbi.2000.3862 [6] L. N. Arnaudov, R. de Vries. Biophys. J. 88 (2005) 515. DOI: 10.1529/biophysj.104.048819
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[7] V. Castelletto, I. W. Hamley, C. Cenker, U. Olsson. J. Phys. Chem. 114 (2010) 8002. DOI: 10.1021/jp102744g [8] C. Lara, J. Adamcik, S. Jordens, R. Mezzenga, Biomacromolecules 12 (2011) 1868. DOI: 10.1021/bm200216u
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[9] E. T. Pashuck, S. I. Stupp. J. Am. Chem. Soc. 132 (2010) 8819. DOI: 160
10.1021/ja100613w [10] M. Zaman, E. Ahmad, A. Quadeer, G. Rabbani, R. H. Khan. International Journal of Nanomedicine, 9 (2014), 899. DOI: 10.2147/IJN.S54171 [11] J. Majorosova, V. I. Petrenko, K. Siposova, M. Timko, N. Tomasovicova, V. M. Garamus, M. Koralewski, M. V. Avdeev, B. Leszczynski, S. Jurga,
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Z. Gazova, S. Hayryan, Ch.-K. Hu, P. Kopcansky. submited in Colloids and Surfaces B, DOI: 10.1016/j.colsurfb.2016.07.024 [12] S. Rocha, Mhwald,
A. F. Thnemann, G. Brezesinski,
C. Pereira Mdo,
M. Coelho,
H.
Biophys. Chem. 137 (2008) 35. DOI:
10.1016/j.bpc.2008.06.010. 170
[13] L. Xiao, D. Zhao, W.-H. Chan, M.M.F. Choi, H.-W. Li, Biomaterials 31 (2010) 91. DOI: 10.1016/j.biomaterials.2009.09.014 [14] R.
Massart,
IEEE
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(1981)
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DOI:
10.1109/TMAG.1981.1061188 [15] O. J. Glatter, Appl. Crystalogr. 10 (1977) 415. 175
[16] J. S. Pedersen, Adv. Coll. Inter. Sci. 70 (1997) 171. [17] M.V.Avdeev, V. L. Aksenov, Z. Gazova, L. Almasy, V. I. Petrenko, H. Gojzewski, A. V. Feoktystov, K. Sipovova, A. Antosova, M. Timko, P. Kopcansky. J. Appl. Cryst. 46 (2013) 224. DOI: 10.1107/S0021889812050042 [18] V.I.Petrenko, M. V. Avdeev, V. M. Garamus, M. Kubovcikova, Z. Gazova,
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K. Siposova, L. A. Bulavin, L. Almasy, V. L. Aksenov, P. Kopcansky. Phys. Solid State 56(1) (2014) 129.
10
nanoparticles
with
Veronika Gdovinová, Natália Tomašovičová, Ivan Batko, Marianna Batková, Lucia Balejčíková, Vasyl M. Garamus, Viktor. I. Petrenko, Mikhail V. Avdeev, Peter Kopčanský www.elsevier.com/locate/jmmm
PII: DOI: Reference:
S0304-8853(16)32074-1 http://dx.doi.org/10.1016/j.jmmm.2016.09.035 MAGMA61816
To appear in: Journal of Magnetism and Magnetic Materials Received date: 30 June 2016 Revised date: 17 August 2016 Accepted date: 5 September 2016 Cite this article as: Veronika Gdovinová, Natália Tomašovičová, Ivan Batko, Marianna Batková, Lucia Balejčíková, Vasyl M. Garamus, Viktor. I. Petrenko, Mikhail V. Avdeev and Peter Kopčanský, Interaction of magnetic nanoparticles with lysozyme amyloid fibrils, Journal of Magnetism and Magnetic Materials, http://dx.doi.org/10.1016/j.jmmm.2016.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interaction of magnetic nanoparticles with lysozyme amyloid fibrils Veronika Gdovinov´aa,b, Nat´ alia Tomaˇsoviˇcov´aa,b,∗, Ivan Batkoa , Marianna a Batkov´a , Lucia Balejˇc´ıkov´aa, Vasyl M. Garamusc , Viktor. I. Petrenkob,d, Mikhail V. Avdeevd , Peter Kopˇcansk´ ya a Institute
of Experimental Physics SAS, Watsonova 47, 040 01 Koˇ sice, Slovakia Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region, Russian Federation c Helmholtz-Zentrum Geesthacht: Zentrum fr Material- und Kstenforschung GmbH Max-Plank-Strae 1, Geesthacht, 216502 Germany d Physics Department, Taras Shevchenko Kyiv National University, Volodymyrska Street 64, 01601 Kyiv, Ukraine b Frank
Abstract This work is devoted to the structural study of complex solutions of magnetic nanoparticles with lysozyme amyloid fibrils due to possible ordering of such system by applying the external magnetic field. The interaction of magnetic nanoparticles with amyloid fibrils has been followed by atomic force microscopy and small-angle X-ray scattering. It has been observed that magnetic nanoparticles (MNPs) adsorb to lyzozyme amyloid fibrils. It was found that MNPs alter amyloids structures, namely the diameter of lysozyme amyloid fibrils is increased whereas the length of fibrils is decreased. In same time MNPs do not change the helical pitch significantly. Keywords: magnetic nanoparticles, lysozyme fibrils, SAXS, AFM
1. Introduction Amyloid fibrils have an important role in nanotechnology and biomaterials applications due to their unique physical and mechanical properties [1, 2]. Also amyloid fibrils, highly ordered nanoscale assemblies of protein protofibrils ∗ Corresponding
author Email address: [email protected] (Nat´ alia Tomaˇsoviˇcov´ a)
Preprint submitted to Journal of LATEX Templates
September 6, 2016
5
with characteristic cross- stacking perpendicular to the long axis of the fiber [3] are associated with various neurodegenerative diseases. Therefore the detail study of amyloids is of great interest. The most used and well characterised model protein for in vitro study of amyloid fibrillation is Hen egg white lysozyme (HEWL) that represents a structural homologue of human lysozyme
10
with globular shape. While native lysozyme is a relatively stable enzyme under physiological conditions and does not easily form amyloids, mutant versions are implicated in some forms of hereditary systemic amyloidosis in humans [4]. Therefore forming amyloids from HEWL in vitro requires the use of specific environmental conditions, as high concentrations in solutions under acidic pH,
15
constant stirring, high temperature and ionic strength or presence of various salts [5]. It was shown, that the kinetics of fibrils formation under these conditions is concentration dependent [6]. Fibrils may show several polymorphic states such as twisted ribbons, helical ribbons or nanotubes [7]. The coexistence of twisted and helical ribbons has been reported in the case of egg lysozyme after
20
long incubation time [8]. It was observed that there exist some time- dependent transformations of twisted ribbon to nanotubes thought the helical ribbon intermediated [9]. Except the study of amyloid fibrils, nowadays there is increasing interest of studing of magnetic nanoparticles (MNPs) as novel materials very suitable
25
for biomedical uses such as hyperthermia, magnetic resonance imaging, drug delivery etc. Zaman et al. suggested existence of critical concentration at which amyloid fibrils are ordered to pure biological liquid crystal (LC) [10]. Our expectation is that magnetite nanoparticles will interact with lysozyme amyloid fibrils (LAF). Main aim is to study the fibrils-magnetic nanoparticles complexes if they
30
may be oriented by applying the external magnetic field in order to obtain a biological liquid crystal. In our recent work the adsorption of MNPs on amyloid fibrils of HEWL in 2 mg/ml acidic solutions has been detected for the MNPs concentration range of 0.01-0.1 vol% [11]. It has been observed that adsorption is determined by the MNPs content and aggregates of the MNPs follow the rod-
35
like structure of fibrils at high MNP concentrations. However, the mechanism of 2
such nanoparticle effect on protein amyloid aggregation has not been explained yet. Several researches show that different size, surface and concentration of nanoparticles affect protein aggregation in different way [12, 13]. Therefore, the main goal of present research is the detail study and understanding the inter40
action between MNPs and solution of LAF with protein concentration of 10 mg/ml that is much higher as it was used in previous experiments with LAF. This finding about the interaction of LAF with MNPs can open a new field of research related to study of fibrils-magnetic nanoparticles complexes if they may be oriented by applying the external
45
magnetic field in order to obtain a biological liquid crystal. It should be mentioned that slightly different procedure for amyloids formation was used in this work. The inner structural organization of initial fibrillized amyloid solution and its mixtures with MNPs were analyzed by means small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM).
50
2. Materials and Methods Hen egg white lysozyme (lyophilized powder, lot number L6876, 50.000 units mg−1 protein) was obtained from Sigma-Aldrich Chemical Company (St Louis, MO). All other chemicals were obtained from Sigma or Fluka and were of analytical reagent grade. Lysozyme amyloid fibrils were prepared by dissolving
55
of lysozyme powder to a final concentration of 10 mg/ml in 0.2 M glycine-HCl buffer with pH=2.4 and 80 mMNaCl. Prepared solution in enclosed bottle was heated for 2 hours at 65◦ C and constant stirring (250 rpm). The magnetite nanoparticles were prepared by co-precipitation method [14]. Electrostatically stabilized magnetic nanoparticles with HClO4 were dispersed in water to obtain
60
magnetic fluid with mean diameter of nanoparticles d = 26 nm. The size of MNPs was determined by dynamic light scattering (Zetasizer). Such magnetic fluids were used for preparation of mixtures with solution of LAF (sLAF). Magnetic fluid (MF) with concentration of MNPs of 45 mg/ml was added to the initial solution of LAF with concentration of 10 mg/ml to achieve three differ-
3
65
ent ratios between volumes of sLAF and MNPs solutions (VsLAF :VMF ) in the mixture: 1ml: 0.0023ml, 1ml: 0.011ml, 1ml: 0.023 ml to obtain final mixture with mass ratio 10:0.1, 10:0.5 and 10:1, respectively. The SAXS experiments were conducted on a Nanostar (Bruker AXS GmbH, Karlsruhe, Germany) laboratory device with the following characteristic parameters: microfocus X- ray
70
souce IμS, CuKα wavelength of 1.54 ˚ A detector VANTEC-2000 (14 x 14 cm in size, resolution of 2048 x 2048 pixels). The samples were placed into glass capillary vessels with a diameter of 2 mm. The measurements were performed at 25±1◦ C. The primary processing of data with taking into account the scattering from the solvent and capillary, detector efficiency, and background signal,
75
as well as the recalculation to the absolute cross section was performed with the SuperSAXS (Jan Pedersen&Cristiano Oliveira) code. Atomic force microscopy measurements were performed using Agilent 5500 AFM system equipped by PicoView 1.14.3 control software. The topography images were acquired in the tapping mode with a standard silicon cantilevers (Olympus, model OMCL-AC
80
160TS) with resonant frequency 300 kHz (typ.), and spring constant 26 N/m (typ.). All measurements were carried out at ambient temperature in air, while relative humidity was in the range of 30-40%. Images were processed using freely available software from Gwyddion (http://gwyddion.net). Samples for AFM observations were prepared by dropping the sample on freshly cleaved
85
pieces of mica (PELCO Mica Sheets Grade V5, 15 X 15 mm2 ). After the deposition, the drop was left to incubate for 5-10 minutes, and then removed by rinsing the surface of mica substrate by ultra-pure water. Samples were dried in air at ambient temperature (typically for 1 hour). 3. Experimental results
90
Fig. 1 and Fig. 2 show AFM images of pure lysozyme fibrils and those doped with magnetic fluid, respectively. The doped sample for AFM was prepared with ratio of sLAF volume to magnetic fluid volume (sLAF/MF= 1:0.023). The doped sample was deposed on mica 1 hour after preparation. As the vol-
4
ume concentration of magnetic particles is high, it is seen that some fibrils are 95
completely covered by magnetic particles (see e.g. Profile 4 in Fig. 2), however it is clearly seen, that in sample are presented also fibrils covered only with few particles as well as without particles (as reveals Profile 3 in Fig. 2). If compare Fig. 1 and Fig. 2 it is clearly shown that after doping with MF the length of fibrils is shorter. AFM measurements also reveal presence of a helical structure
100
but as it can be concluded based on Profiles 1-4 in Fig. 1 the pitch of the helical structure is different for different fibrils (see text in the caption of Fig. 1). Also, comparisons of mutual distance between Profiles 1 and 2 in Fig. 2 (system with MNPs) with the mutual distances between pairs of Profiles in Fig. 1 yields values for the pitch
105
of the helical structure of the same order, what might indicate that adding of MNPs to the system (using above described technology process) does not change this pitch significantly. The obtained diameter of the fibrils in initial solution is 7-9 nm. In the doped sample with MNPs the diameter is 9-10nm.
110
Experimental SAXS curves for the mixtures of amyloid aggregates with MNPs (Fig. 3) were analyzed by applying the Indirect Fourier Transform (IFT) method developed by Glatter [15] in the version of Pedersen [16]. Taking into account elongated shape of the particles, the cross-section of pair distance distribution (PDD) functions, pcs (r), have been obtained in Fig. 4 that indicates the
115
helical structure of lysozyme fibrils which is similar to previous works [17, 18]. The power- law type scattering at small q values (linear parts in the graph) with power exponent close to -1 indicates the presence of elongated particles. Model of cylinder-like shape is an approximation of the real shape of the amyloid aggregates. Fibrils have shown some periodic structure most probably helical. Radius
120
of primary component is ∼ 1.4-2 nm and diameter of whole helixes slightly less than 7 nm. By adding magnetic fluid to lysozyme fibrils, the structure of the lysozyme was changed. It leads to disappearing of second maxima (helical structure) and some shift of first maximum (primary protein component). The shift of maximum for primary component points to adsorption of MF and increasing 5
28 26
Profile 1 Profile 2 Profile 3 Profile 4
z (nm)
24 22 20 18 16 14 0
20
40
60
80
100
120
distance (nm)
Figure 1: AFM height image and their representative profile cross section for pure lysozyme fibrils. Distances between profiles 1-2 (∼ 60 nm) and 3-4 represent ( 100 nm) approximately the quarter of the period of the helical structure.
125
of effective cross section (Fig. 4. 4. Conclusion The interaction between lysozyme amyloid fibrils and MNPs was investigated by SAXS and AFM measurements. Our results have shown that lysozyme fibrils have helical structure with a diameter of 7 nm approximately. By adding a small
130
amount of MNPs to lysozyme amyloid fibrils caused that the helical structure of
6
28 26
Profile 1 Profile 2
z (nm)
24 22 20 18 16 0
5
10
15
20
distance (nm) 45
Profile 3 Profile 4
40
z (nm)
35 30 25 20 15 10 0
100
200
300
distance (nm)
Figure 2: AFM height image and their representative profile cross section for lysozyme fibrils doped with magnetic nanoparticles after 1 hour incubation. The distance between profiles 1 and 2 (∼ 90 nm) is approximately the quarter of the period of the helical structure.
7
Figure 3: Experimental SAXS curves for sLAF and mixtures of sLAF and various concentrations of MNPs.
Figure 4: The cross-section of PDD functions as a result of the IFT procedure for scattering from mixture of sLAF and various concentrations of MNPs.
amyloid fibrils was partly lost. Diameter of the mixture of LAF and MNPs was increased that provide an experimental evidence of interaction of MNPs with LAF. Diameter of LAF after doping with MNPs was increased that
8
provide an experimental evidence of their mutual interaction. 135
This
should be the first step to order such complex systems by applying magnetic field. Acknowledgements This work was supported by project VEGA 2/0045/13], the Slovak Research and Development Agency under the contract No. APVV- 015-0453, Ministry of
140
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