Anion-Specific Effects on the Assembly of Collagen Layers Mediated

Dec 26, 2013 - ... Kieran Fuller , Naledi Shologu , Michael Keeney , Manus J. Biggs , Abhay Pandit , Dimitrios I. Zeugolis. Advanced Healthcare Materi...
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Anion-Specific Effects on the Assembly of Collagen Layers Mediated by Magnesium Ion on Mica Surface Li Wang,* Yan Guo, Pengcheng Li, and Yonghai Song* Key Laboratory of Functional Small Organic Molecule, Ministry of Education, College of Chemistry and Chemical Engineering, Jiangxi Normal University, 99 Ziyang Road, Nanchang 330022, China ABSTRACT: The well-ordered assembly of collagen molecules on mica surface has attracted extensive attention because it has great potential applications or can be served as a model system for study on the assembly process. Although the epitaxially guided collagen assembly mediated by potassium ion on mica surface has been reported several times over these years, specific effects of anions in this field has never been surveyed and discussed before now. In this work, atomic force microscopy was employed to visually follow the assembly of collagen on mica surface mediated by three kinds of Mg2+ salts with different anions, including MgAc2, MgSO4, and MgCl2. It was found that at high ionic concentration anions could critically affect the interaction between collagen microfibrils and mica surface and accordingly resulted in different structures. Almost parallelly aligned collagen fibrils in one direction were achieved for acetates, sparse and small fibrils in two main directions rotated by 120° were acquired for sulfate, while flat film with some defects was obtained for chloride, respectively. The Hofmeister series and Collins’ model were used to interpret the results. This study would be useful for controlling the morphologies of assembled collagen on a surface.

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INTRODUCTION Collagen has been widely investigated not only for exploring the structure component of mammalian connective tissues1,2 but also for its important industrial and biomedical applications.3,4 Collagen is a fibrous protein and can form hierarchical fibers after a complex self-organizing process.5,6 The formation of collagen fibrils presents a fascinating case for the study on hierarchical structure, assembly, and design of novel biomaterials.7−9 Better understanding and controlling of the collagen self-assembled process has both a fundamental and a practical importance. In the past 2 decades, abundant information about the selfassembly of collagen molecules on flat substrates has been obtained.10−15 For example, Sun et al. proved that the direction of collagen alignment on mica was actually dependent on interactions between collagen and the mica lattice.16 Loo et al. found that potassium ion was responsible for creating a single domain array with aligned bundles of intertwined microfibrils.17 Leow et al. observed bidirectional growth of collagen fibrils on phlogopite mica and parallel alignment of collagen fibrils on muscovite mica and discussed the formation mechanism of collagen fibrils.18 It was found that the growth direction of collagen fibrils was determined by the nucleation step. Recently, He et al. confirmed that the dehydration of collagen by Cr3+ was more significant than that by Al3+, resulting in the aggregation of collagen fibrils.19 Thanks to the insightful observations and detailed discussion, people have realized the importance of metal ions especially K+ and the interaction among metal cations, collagen molecules and substrate when collagen was assembled on a surface. Unfortunately, very little attention has been focused on the effect of anions on the © 2013 American Chemical Society

assembly of collagen molecules, which might be very important due to the Hofmeister effect and Collins’ model. Hofmeister effect, which is known as specific ion effect, has commonly been used to describe the interactions among ions, water and macromolecules in bulk solution and at interfaces.20−23 According to the ability of salts to alter the hydrogen bonding network of water and precipitate certain proteins from an aqueous solution, the classical Hofmeister anion series could be arranged as SO42− > CH3COO− > OH− > F− > Cl− > Br− ≈ NO3− > I− > ClO4− > SCN− (Figure 1). The species to the left

Figure 1. Typical ordering of anions in the classical Hofmeister series.

of Cl− are referred to as kosmotropes (water structure maker), which are strongly hydrated and have stabilizing and salting-out effects on proteins and macromolecules. Meanwhile, those species to the right of Cl− are called chaotropes (water structure breaker), which are weakly hydrated and known as Received: May 22, 2013 Revised: December 18, 2013 Published: December 26, 2013 511

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Figure 2. AFM topography images of collagen fibrils assembled on mica surface at different concentrations of collagen in the presence of 500 mM MgSO4: (a) 5, (b) 10, (c) 25, and (d) 50 μg/mL. Scale bar = 500 nm. Vertical scale: 2.0 (a, b), 3.0 (c), and 3.5 nm (d).

protein denaturants, increasing protein solubility.24,25 It clearly indicates that the anion plays an important role in stabilizing the protein structure, which would affect the protein assembly on a solid surface accordingly. Moreover, the interactions between water molecules and ions or charged groups of protein can also be interpreted with Collins’ concept of matching water affinities.26 Ions are approximately considered as a sphere with just a point charge in the center. When the ions are small, the surrounding water molecules are tightly bound (the ions are hard or kosmotropic), whereas when the ions are big, the hydration shell is only loosely bound (the ions are soft or chaotropic). When two strongly hydrated small ions with opposite charge come together, very strong attraction occurred between them. Consequently, they can form direct ion pairs and extrude the hydration spheres between them. While for two weakly hydrated soft big ions, the hydration spheres are so loosely bound that they can also form direct ion pairs and extrude the hydration water. The interaction between one hard ion and one oppositely charged soft ion is not strong enough to form direct ion pairs or extrude the hydration shells. A most striking success of this concept is the qualitatively correct prediction of the reversal of the Hofmeister series after changing the counterion.27−29 It also indicated that the anion played an important role in protein assembly on a solid surface.

According to Hofmeister series and Collins’ concept, anions have stronger interactions with water and collagen molecules relative to cations. Thus, in this work we try to use atomic force microscopy (AFM) to investigate the effect of different anions including sulfate, acetate and chloride on the Mg2+-mediated assembly of collagen on mica surfaces.

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MATERIALS AND METHODS

Materials. Collagen (type I, pI = 7.830) from calf skin was purchased from Sigma-Aldrich. Magnesium sulfate anhydrous, magnesium chloride hexahydrate, magnesium acetate tetrahydrate and acetic acid (36%) were purchased from Sinopharm Group Chemical Reagent Co. Ltd. (Shanghai, China). Other reagents were purchased from Beijing Chemical Reagent Factory (Beijing, China). All reagents (analytical grade) were used as received without further purification. All solutions were prepared with ultrapure water purified by a Millipore-Q System (18.2 MΩ cm). The muscovite mica substrate [KAl2(AlSi3)O10(OH)2] were purchased from Linhe Street Commodity Marketplace (Changchun, China). Samples. A 0.2 M acetic acid (pH 2.7) solution was prepared by diluting original acetic acid (36%) with ultrapure water, and used as the solvent to prepare collagen solutions and to dissolve various metal salts. Then 1.0 mg/mL collagen solution was prepared by dissolving collagen in 0.2 M acetic acid solution. Other collagen solutions were obtained by further diluting the above solution. All of these collagen solutions were stored at 4 °C for further use. Original concentrations of these magnesium salt solutions were 1.0 M. Magnesium salt 512

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Figure 3. AFM topography images of collagen fibrils assembled on mica surface at 25 μg/mL collagen in the presence of different concentration of MgSO4: (a) 100, (b) 200, (c) 300, (d) 400, (e) 450, (f) 500, (g) 550, (h) 600, and (i) 750 mM. Inset (bottom) in part g showed its two-dimensional (2D) Fourier transform. Insets (upper) in parts g−i were the corresponding section analysis. Scale bare = 1250 nm. Vertical scale: 2.0 (a, c, g, h), 3.0 (b, d−f), and 1.3 nm (i). The pink arrows in the images were used to point the starfish-like collagen assemblies. solutions with various concentrations were obtained by diluting the original solutions and used for our experiment. The muscovite mica was cut into about 1.2 × 1.2 cm2 square pieces as substrates. Both sides of the mica were freshly cleaved before use. A 10 μL aliquot of the mixture of collagen and magnesium salt was deposited onto the freshly cleaved mica surface, incubated at room temperature for 10 min, then rinsed with water and dried in a sealed desiccator at room temperature. After the sample was dried absolutely, the mica sheet was then affixed on a metal disk for AFM imaging. AFM Imaging. For morphological characterization, an AFM (AJIII microscopy, Shanghai Aijian Nanotechnology) was employed in tapping mode. Standard silicon cantilevers (spring constant of 0.6−6 nN/m, radius of curvature less than 10 nm) were purchased from MikroMasch Inc. (Model of NSC11, made in Estonia) and used close to their resonance frequencies (typically, 60−150 kHz). All AFM images were obtained at room temperature under ambient conditions. The signal-to-noise ratio was maintained higher than 10. The scanning frequency was 1 Hz for small-area scans ( Ac− > Cl−. Only Cl− is referred to chaotrope among them, which tends to act as protein denaturant and increase protein solubility. While SO42‑ and Ac− are kosmotropes which have stabilizing and salting-out effects on proteins and macromolecules. Apparently, in this work, Cl− actually enhanced solubility of collagen and resulted in the formation of flat film on mica surface as revealed by AFM images. In the presence of SO42‑ and Ac −, the collagen layer on mica surface was preferentially guided by Mg2+. The formation mechanism of this assembled pattern was similar to K+-guided assembly of collagen layers.17,18 Collagen molecules adsorbed on mica surface were growing along substrate direction and rotated energetically to the most favorable direction as the same as the layer near mica. Some delicate morphology difference always existed under the same concentration of both magnesium salts. Hence, it could be concluded that the Mg2+ ion is actually regulating the assembly of collagen by neutralizing negative charge on mica surface, enhancing surface diffusion and providing a favorable ambient for collagen assembly. For SO42‑ and Ac −, although they are both kosmotropes, the characteristics shown in AFM images are different due to their different hydratability. In fact, the former it ranks in the Hofmeister series, the stronger hydratability it has. According to Collins’ concept of matching water affinities, Mg2+ has a big and loosely bounded hydration shell, which can not form ion pairs with SO42‑. Thus, free Mg2+ is abundant in a solution, and most of them would be adsorbed rapidly onto mica surface or collagen backbone by electrostatic force.32 Meanwhile, the hydrogen bonds in the polypeptide chains are reduced and weaken. This assumption could also be confirmed by small height of collagen fibrils shown in Figure 3g−i. As a consequence, the collagen fibrils assembled on mica surface along mica lattice to form two main directions which were 120° apart. Nevertheless, in the case of Ac−, Mg2+ could combine with Ac− to form weak but effective direct ion pairs because the sizes of their hydration shells were approximately equal.

of collagen pattern on mica surface might be due to the effect of the different anions in Mg2+ salts. To further illustrate that anions could have effects on the behaviors of Mg2+-mediated assembly of collagen on mica surface, MgCl2 was employed as another control experiment. Since KCl was extensively used to study the effects of K+ on collagen microfibril assembly on mica surface, the same anion of Cl−, was employed in this experiment for comparison. Images of collagen assemblies on mica surface under two MgCl2 concentrations are shown in Figure 5. Disordered collagen assemblies on mica surface were always observed at 100 mM MgCl2 (Figure 5a), which was similar to the assemblies obtained at 100 mM MgSO4 or MgAc2. It suggested that Mg2+ played a leading role in mediating the assembly of collagen at low concentration of Mg2+ salts. While such disordered assembly of collagen has also been reported as 200 mM NaCl was used in previous work which led to a conclusion that K+ was responsible for creating an ordered pattern of collagen.17 In our case, further increasing the concentration of MgCl2 to 300 mM, as shown in Figure 5b, a large scale of flat film with 1.02 nm thickness (disclosed by the inset of Figure 5b) and many defects was observed on mica surface. Such structure was hardly observed in previous studies, suggesting anions did play a crucial role in the Mg2+-mediated assembly of collagen on mica surface at high concentration of Mg2+ salts. It must be pointed out that there were some disadvantages in our ex situ AFM observation. For example, the observed morphologies of air-dried collagen samples might not completely represent the actual structure of the adsorbed phase under liquid due to some distortion of the protein arrangement by the strong directional drying forces. It was impossible to capture the very initial stages of the pattern formation and follow specific regions of the surface as they evolved, too.31,32 However, a lot of information on the formation process of the monolayers can be still revealed by ex situ AFM studies.33−36 It has been fortunately pointed out that air-dried collagen sample showed the same alignment as that under liquid in previous work.16 In addition, changing AFM scan direction with respect to mica substrate orientation did not affect the arrangement either, excluding the possibility of tip-induced arrangement. 516

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Consequently, the interaction between Mg2+ and negatively charged mica surface or collagen chains under Ac− would be weaker than that under SO42‑. Then those collagen fibrils formed with MgAc2 could freely diffuse on mica surface to form parallel alignment in one direction as a result of equilibrium of all interactions among collagen, MgAc2 and mica surface. Such an arrangement decreased interactions between collagen chains to a minimum value and produced the most stable configuration. The height of collagen fibrils with 750 mM MgAc2 shown in Figure 4f is also about 1.7 nm, which is equal to the height of fibrils with 550 mM MgSO4 in Figure 3g. Thus, we can assume that when 500 mM MgSO4 or 750 mM MgAc2 is in the solution of 25 μg/mL collagen, the interaction among cations, anions, collagen molecules, and mica surface is balanced, and accordingly collagen molecules are able to form oriented fibrils on mica substrate based on the appropriate interaction.

(2) Friedrichs, J.; Taubenberger, A.; Franz, C. M.; Müller, D. J. Cellular Remodelling of Individual Collagen Fibrils Visualized by Time-lapse AFM. J. Mol. Biol. 2007, 372, 594−607. (3) Wu, B.; Mu, C. D.; Zhang, G. Z.; Lin, W. Effects of Cr3+ on the Structure of Collagen Fiber. Langmuir 2009, 25, 11905−11910. (4) He, L. R.; Mu, C. D.; Shi, J. B.; Zhang, Q.; Shi, B.; Lin, W. Modification of Collagen with a Natural Cross-Linker, Procyanidin. Int. J. Biol. Macromol. 2011, 48, 354−359. (5) Hulmes, D. J. S.; Miller, A. Quasi-hexagonal Molecular Packing in Collagen Fibrils. Nature 1979, 282, 878−880. (6) Kadler, K. E.; Holmes, D. F.; Trotter, J. A.; Chapman, J. A. Collagen Fibril Formation. Biochem. J. 1996, 316, 1−11. (7) Ramshaw, J. A. M.; Werkmeister, J. A.; Glattauer, V. Collagenbased Biomaterial. Biotechnol. Genet. Eng. Rev. 1996, 13, 335−382. (8) Monroe, M. R.; Li, Y.; Ajinkya, S. B.; Gower, L. B.; Douglas, E. P. Directed Collagen Patterning on Gold-Coated Silicon Substrates via Micro-Contact Printing. Mater. Sci. Eng., C 2009, 29, 2365−2369. (9) Salhi, B.; Vaurette, F.; Grandidier, B.; Stievenard, D.; Melnyk, O.; Coffnier, Y.; Boukherroub, R. The Collagen Assisted Self-Assembly of Silicon Nanowires. Nanotechnology 2009, 20, 235601−235607. (10) Chernoff, E. A. G.; Chernoff, D. A. Atomic Force Microscope Images of Collagen Fibers. J. Vac. Sci. Technol. A 1992, 10, 596−599. (11) Gale, M.; Pollanen, M.; Markiewicz, P.; Goh, M. Sequential Assembly of Collagen Revealed by Atomic Force Microscopy. Biophys. J. 1995, 68, 2124−2128. (12) Dong, M.; Xu, S.; Bünger, M. H.; Brikedal, H.; Besenbacher, F. Temporal Assembly of Collagen Type II Studied by Atomic Force Microscopy. Adv. Eng. Mater. 2007, 9, 1129−1133. (13) Cisneros, D. A.; Friedrichs, J.; Taubenberger, A.; Franz, C. M.; Müller, D. J. Creating Ultrathin Nanoscopic Collagen Matrices for Biological and Biotechnological Applications. Small 2007, 3, 956−963. (14) Jiang, F.; Horber, H.; Howard, J.; Müller, D. J. Assembly of Collagen into Microribbons: Effects of pH and Electrolytes. J. Struct. Biol. 2004, 148, 268−278. (15) Li, Y.; Asadi, A.; Monrroe, M. R.; Douglas, E. P. pH Effects on Collagen Fibrillogenesis in vitro: Electrostatic Interactions and Phosphate Binding. Mater. Sci. Eng., C 2009, 29, 1643−1649. (16) Sun, M.; Stetco, A.; Merschrod, S. E. F. Surface-Templated Formation of Protein Microfibril Arrays. Langmuir 2008, 24, 5418− 5421. (17) Loo, R. W.; Goh, M. C. Potassium Ion Mediated Collagen Microfibril Assembly on Mica. Langmiur 2008, 24, 13276−13278. (18) Leow, W. W.; Hwang, W. Epitaxially Guided Assembly of Collagen Layers on Mica Surfaces. Langmiur 2011, 27, 10907−10913. (19) He, L. R.; Cai, S. M.; Wu, B.; Mu, C. D.; Zhang, G. Z.; Lin, W. Trivalent Chromium and Aluminum Affect the Thermostability and Conformation of Collagen very Differently. J. Inorg. Biochem. 2012, 117, 124−130. (20) Hofmeister, F. About the Science of the Effect of Salts. Arch. Exp. Pathol. Pharmakol 1888, 24, 247−260. (21) Kunz, W. Specific Ion Effects in Colloidal and Biological Systems. Curr. Opin. Colloid Interface Sci. 2010, 15, 34−39. (22) Kunz, W.; Henle, J.; Ninham, B. W. ‘Zur Lehre von der Wirkung der Salze’ (about the science of the effect of salts): Franz Hofmeister’s Historical Papers. Curr. Opin. Colloid Interface Sci. 2004, 9, 19−37. (23) Nostro, P. L.; Ninham, B. W. Hofmeister Phenomena: An Update on Ion Specificity in Biology. Chem. Rev. 2012, 112, 2286− 2322. (24) Zhang, Y.; Cremer, P. S. Interactions between Macromolecules and Ions: the Hofmeister Series. Curr. Opin. Chem. Biol. 2006, 10, 658−663. (25) Chen, X.; Flores, S. C.; Lim, S. M.; Zhang, Y.; Yang, T.; Kherb, J.; Cremer, P. S. Specific Anion Effects on Water Structure Adjacent to Protein Monolayers. Langmuir 2010, 26, 16447−16454. (26) Vlachy, N.; Jagoda-Cwiklik, B.; Vacha, R.; Touraud, D.; Jungwirth, P.; Kunz, W. Hofmeister Series and Specific Interactions of Charged Headgroups with Aqueous Ions. Adv. Colloid Interface Sci. 2009, 146, 42−47.

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CONCLUSION In summary, a series of magnesium salts with different anion including SO42‑, Ac−, and Cl− mediating the assembly of collagen on mica surfaces were carried out to study the anionic effects on collagen assembly for the first time. It was found that different kinds and concentrations of magnesium salts could lead to completely different collagen fibrils pattern on mica surface. Under 25 μg/mL collagen, 500 mM MgSO4 led to oriented thick and close fibrils pattern with two main directions which were 120° apart, 750 mM MgAc2 resulted in parallelly arranged compact collagen fibrils in one direction, while 300 mM Cl− produced flat collagen film with many defects. Hofmeister series related to different anions’ sequence were used to explain the different interactions between these charged groups and Collins’ concept were used to explain the affinities of water toward Mg2+ and different anions, respectively. In other words, oriented collagen fibrils covered macroscale substrates could be obtained with appropriate sequence of anions, choice of cations, and concentration ratio of protein-tosalts. This study can provide some information about the ionmediated assembly of molecules on a solid surface.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone/Fax: +86-791-88120861. E-mail: lwanggroup@ aliyun.com (L.W.). *E-mail: [email protected] (Y.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (21065005, 21165010), Young Scientist Foundation of Jiangxi Province (20112BCB23006 and 20122BCB23011), the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201310), and the Open Project Program of Key Laboratory of Functional Small organic molecule, Ministry of Education, Jiangxi Normal University (No. KLFS-KF-201214; KLFS-KF-201218).

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REFERENCES

(1) Thomas, A. C.; Campbell, G. R.; Campbell, J. H. Advances in Vascular Tissue Engineering. Cardiovasc. Pathol. 2003, 12, 271−276. 517

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(27) Collins, K. D. Ions from the Hofmeister Series and Osmolytes: Effects on Proteins in Solution and in the Crystallization Process. Methods 2004, 34, 300−311. (28) Mason, P. E.; Neilson, G. W.; Enderby, J. E.; Saboungi, M. L.; Dempsey, C. E.; MacKerell, A. D.; Brady, J. W. The Structure of Aqueous Guanidinium Chloride Solutions. J. Am. Chem. Soc. 2004, 126, 11462−11470. (29) Mason, Ph. E.; Dempsey, Ch. E.; Vrbka, L.; Heyda, J.; Brady, J. W.; Jungwirth, P. Specificity of Ion−Protein Interactions: Complementary and Competitive Effects of Tetrapropylammonium, Guanidinium, Sulfate, and Chloride Ions. J. Phys. Chem. B 2009, 113, 3227− 3234. (30) Highberger, J. H. The Isoelectric Point of Collagen. J. Am. Chem. Soc. 1939, 61 (9), 2302−2303. (31) Wang, L.; Ye, S. H.; Yuan, H. Z.; Song, Y. H.; Zhu, H. Z.; Hou, H. Q.; Li, P. C. α, ω-Dihexylsexithiophene Self-Assembled Nanostructures on Mica: Atomic Force Microscopy Study. Microsc. Microanal. 2012, 18, 844−851. (32) Song, Y. H.; Guo, C. L.; Sun, L. L.; Wei, G.; Peng, C. Y.; Wang, L.; Sun, Y. J.; Li, Z. Effects of Bridge Ions, DNA Species, and Developing Temperature on Flat-Lying DNA Monolayers. J. Phys. Chem. B 2007, 111, 461−468. (33) Wang, L.; Song, Y. H.; Han, X. J.; Zhang, B. L.; Wang, E. K. Growth of Cationic Lipid toward Bilayer Lipid Membrane by Solution Spreading: Scanning Probe Microscopy Study. Chem. Phys. Lipids 2003, 123, 177−185. (34) Wang, L.; Jiang, J. G.; Song, Y. H.; Zhang, B. L.; Wang, E. K. Self-Assembled Monolayer Growth of Octanol on Mica: Atomic Force Microscopy and Fourier-Transform Infrared Spectroscopy Studies. Langmuir 2003, 19, 4953−4957. (35) Bierbaum, K.; Grunze, M.; Baski, A. A.; Chi, L. F.; Schrepp, W.; Fuchs, H. Growth of Self-Assembled n-Alkyltrichlorosilane Films on Si(100) Investigated by Atomic Force Microscopy. Langmuir 1995, 11, 2143−2150. (36) Song, Y. H.; Li, Zh.; Liu, Zh. G.; Wei, G.; Wang, L.; Sun, L. L.; Guo, C. L.; Sun, Y. J.; Yang, T. A Novel Strategy to Construct a FlatLying DNA Monolayer on a Mica Surface. J. Phys. Chem. B 2006, 110, 10792−10798.

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