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Peptide-based spherulitic films—formation and properties
Journal of Colloid and Interface Science 343 (2010) 387–391
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Short Communication
Peptide-based spherulitic films—formation and properties Noam Sidelman a,b, Yuri Rosenberg c, Shachar Richter a,b,* a
The Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel-Aviv, Israel The Center for NanoScience and NanoTechnology, Tel-Aviv University, Tel-Aviv, Israel c The Wolfson Applied Materials Research Center, Tel-Aviv University, Tel-Aviv, Israel b
a r t i c l e
i n f o
Article history: Received 24 September 2009 Accepted 10 November 2009 Available online 18 November 2009 Keywords: Spherulites Films Peptide nanotubes
a b s t r a c t Peptide nanotube-based spherulitic films are a recently discovered phenomenon, which was demonstrated in the case of the self-assembled diphenylalanine peptide nanotubes. Here we show that the film-formation method can be implemented with other peptides. We also demonstrate that a critical physical parameter, an elevated level of environmental hydration, is required for film growth. A possible formation mechanism is suggested. The optical, morphological and mechanical properties of these films are characterized and are found to be substantially different from those of non-spherulitic deposits. Ó 2009 Elsevier Inc. All rights reserved.
Spherulites are poly-crystalline entities comprised of a nucleation center from which multiple lamellas span out, maintaining a space-filling shape [1,2]. Although spherulitic growth has been recognized since the early days of crystal growth research, its mechanism is not yet completely understood [1,2]. Nevertheless, spherulitic moieties can be found in various types of materials— inorganic [3–5], organic [6–10] biologic [11–20], and even in the element selenium [21]. In particular, spherulites are encountered in polymers [1]. The significance of their existence in soft matter stems from the fact that spherulitic composition has been shown to alter the polymers’ physical properties, such as optical and mechanical [6,22–25]. It was previously shown [26] that the peptide diphenylalanine (FF) can self-assemble to form peptide nanotubes (PNTs). The formation of PNTs has been previously discussed in view of its self assembly properties [26]. Self assembly [27,28] is an important phenomenon with great importance in nature and in nano-sciences since it offers a way to construct complex nanostructures. Interestingly, self assembly can be seen in various scales, ranging from molecular and up to micro-sized objects [28]. We have previously shown [29] that self-assembly capability of FF can be utilized to form spherulitic films on solid surfaces. These films are an excellent example of multi scale self assembly, that is ranging from the molecular self assembly, and up to the spherulitic structure. Here we demonstrate that the spherulitic film growth phenomenon is governed by several physical factors, and not by the chemical composition of the peptide molecules. We show that
* Corresponding author. Address: The Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel-Aviv, Israel. Fax: +972 3 6405612. E-mail address: [email protected] (S. Richter). 0021-9797/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2009.11.028
environmental humidity level is a critical factor in the peptide’s spherulitic growth. This is demonstrated by the formation of spherulitic films from another PNT-forming peptide—dileucine (LL). We also illustrate that the mechanical properties of the peptide spherulitic films differ from those of their non-spherulitic counterparts. Our method of forming peptide spherulitic films [29] consists of depositing an aqueous peptide solution, diluted with the additive N-methyl pyrrolidone (NMP), onto a heated flat solid substrate. The solution is subsequently allowed to completely evaporate. The conditions for such spherulitic growth are not as harsh as in the case of some polymer spherulites: similarly shaped and packed spherulites can be found in bulk polymers, which are crystallized from the melt at temperatures exceeding 100 °C, such as nylon [7] and n-alkanes [10]. The mild conditions required for peptide spherulitic film formation suggest that their formation from other peptides should not interfere with peptides’ functions; thus, various peptide films with enhanced mechanical properties can be formed which still retain their function. Moreover, although spherulites have been previously shown to be formed by peptides [13–15], such structures were three-dimensional in nature and were spread sporadically in the matrix in which they were formed. Our films are comprised of two-dimensional closely arranged spherulites, similar to those found in bulk polymers [2,6], and therefore have potential uses in biologic coating technology. We have recently shown that the beta-amyloid derived diphenylalanine [26] (FF, Fig. 1a) can be induced to form films on flat solid surfaces [29]. These films are comprised of multiple closely arranged two-dimensional spherulites, each exhibiting a nucleation center and multiple lamellas. These lamellas grow from the nucleation center until they encounter the growth fronts of neighboring spherulites, thus forming a grain boundary. Optical, atomic
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force (AFM) and scanning electron (SEM) microscopy of the films are shown in Fig. 1. We found that FF spherulitic films form only when a specific physical parameter exists. Specifically, a high level of environmental relative humidity (RH) (RT, RH > 75%). The high humidity was found to be a critical factor: without it, no spherulites formed. To test whether spherulitic films can also form from other peptides, and whether our film-formation procedure can be applied to various peptides or proteins, the spherulitic film-formation procedure found suitable for FF was applied to a different dipeptide— dileucine (L-Leu–L-Leu; LL, Fig. 2a). It was also of interest to note whether the dependency on humidity levels would still exist. LL, like FF, is a dipeptide with hydrophobic side chains, but it differs from FF by its lack of aromatic moieties. LL has been previously found to be a PNT-forming peptide [30], as it is comprised of channels that form due to its crystalline structure, like FF. The channels in LL are smaller than the 10 Å channels found in FF, and are rectangular, with dimensions of 2.5 Å by 6.0 Å [30]. When the FF-based film-formation procedure was applied to LL, spherulitic films were also successfully formed. The LL films were similarly comprised of closely arranged spherulites, exhibiting distinct nucleation centers and grain boundaries. Optical, AFM and SEM analyses of the film are shown in Fig. 2. As in the case of the FF films, the same humidity dependency was found with the LL spherulitic films, which formed only at elevated levels of environmental humidity.
For both FF and LL, the dependence on humidity was found to outlive the film-deposition process itself. At RH > 75% (RT), films were observed immediately after deposition. In contrast, filmdeposition attempts carried out at lower environmental humidity (e.g. RT, RH 60%) did not initially yield spherulitic films. However, when the samples were exposed to elevated levels of humidity, spherulitic films appeared on the substrate. An example of this effect can be seen in Fig. 3. Fig. 3a shows the result of attempted FF film deposition at a humidity level below 70% (RT). No spherulitic film is formed, and the only observable feature is a small fibrous branched shape. Similar behavior was observed for LL, as can be seen in Fig. 3c, which shows only small fibrous features appearing shortly after deposition. Exposure of the samples to elevated levels of environmental humidity resulted in the formation of a spherulitic film (Fig. 3b and d, FF and LL, respectively). The fiber seen in Fig. 3a became the nucleation center of the spherulites, and is an example of a nucleation ‘‘sheaf”, which represents a stage of homogeneous nucleation [1,31]. It should be noted that spherulites comprised of small organic molecules are rarely encountered [2]. Nevertheless, the few small organic molecules that have been previously described [1] have a common property—they are all aromatic hydrocarbons. LL is therefore unique in the sense that it is a small non-aromatic organic molecule that forms spherulites. Based on our observations, and on what is known about spherulitic composition in polymers, we determined the effect of
Fig. 1. (a) Scheme of L-Phe–L-Phe (FF). (b) An optical image of an FF spherulitic film. Bar = 50 lm. (c) An SEM image of a homogenous nucleation site of a FF spherulite, bar = 10 lm. (d) An AFM phase image of a spherulite’s homogenous nucleation site. Bar = 9.3 lm. (e) An SEM image of an FF spherulitic film. Multiple closely arranged spherulites can be seen. Bar = 100 lm. (f) An AFM image showing the lamellas comprising an FF spherulite. Bar = 1.6 lm. (g) and (h), Optical images of a homogenous nucleation site of FF spherulite (bar = 20 lm) and a heterogeneous one (bar = 50 lm). Films were prepared as follows: the peptide was dissolved in water, at a conc. of 2 mg/ ml. N-methyl-2-pyrrolidone was added at a ratio of 10% v/v and the solution was drop-casted and dried on the substrate at 60 °C. The resulting films are 700–1500 nm thick.
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humidity on peptide spherulitic growth to be as follows: polymer spherulites form when the polymer is grown from the melt. It has been previously suggested that the viscosity of the melt is important for spherulite formation, and it was noted that large viscosities seem to be required [2,6,32]. This has also been observed in the case of the rarely encountered spherulitic growth of small organic molecules [2]. In the case of our peptide spherulites, the spherulites grow from a saturated solution which contains the additive NMP. Following deposition of the solution onto the heated flat solid substrate and its evaporation, the substance remaining on the substrate appears to be completely solid, but is in fact a gel, and is comprised of the peptide molecules, NMP and a minute amount of water (most of it having evaporated). The NMP, without which no film forms, has a relatively low vapor pressure, and thus remains in sufficient quantity on the substrate. In the case of our peptide spherulitic films, as the substrate is exposed to elevated levels of humidity, the deposited substance absorbs water molecules from the environment and turns into a solid gel-like substance. The adsorbance of water from the environment regulates the viscosity of the gel-like substance, making it slightly less solid, i.e. highly viscous. When a level of viscosity suitable for peptide spherulitic growth is reached, the growth process is initiated. This correlates to viscosity dependence in the growth of spherulitic polymers, and this finding is also in agreement with a previous claim [33] that spherulitic growth is a crystallization process that occurs far from equilibrium: it is diffusion-dependent and therefore viscosity-dependent as well.
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It should be emphasized that although the FF and LL dipeptides behave, to some extent, similarly to polymeric spherulites, neither FF nor LL undergo polymerization, as verified by X-ray powder diffraction (XRD) for FF, and mass spectrometry for LL (not shown). This means that no chemical reaction has taken place before or during the spherulitic film’s growth process. In general, spherulite recognition and characterization are based on their optical behavior—polymer spherulites are recognized by the appearance of a Maltese cross extinction pattern under a polarized light microscope [1,2]. We observed this optical pattern in FF and LL spherulites as well. Images of FF spherulites viewed by polarized light microscopy can be seen in Fig. 4a–d. The images clearly show multiple spherulites packed together, each exhibiting a typical Maltese cross pattern, due to the twodimensional nature of the peptide spherulitic films. In polymers, the Maltese cross pattern is attributed to the chains being perpendicular the to the radius of the spherulite, and to a difference in the index of refraction between the direction parallel to the chain and the direction normal to the chain. This birefringence accompanied by spherical geometry is the reason for the extinction along the axis of the filter in the microscope [34]. In small peptide molecules like FF and LL, the reason for this pattern is attributed to the molecular orientation in the lamellar crystals, analogous to the long chain orientation effect in polymers. The spherulitic nature of the films has a marked effect on their mechanical properties. PNTs which were deposited on the substrate and resulted in a non-spherulitic mesh were not cleavable.
Fig. 2. (a) Scheme of L-Leu–L-Leu (FF). (b) An optical image of an LL spherulitic film. Bar = 50 lm. (c) An SEM image of a grain boundary where lamellas of two spherulites meet. Bar = 1 lm. (d) An SEM image showing a nucleation site of an LL spherulite. Bar = 10 lm. (e) An SEM image of an LL spherulitic film. Multiple closely arranged spherulites can be seen. Bar = 100 lm. (f) An AFM image showing the lamellas comprising an LL spherulite. Bar = 3 lm. (g) and (h) Optical images of LL spherulites. The spacefilling tendency can be observed. Bar = 50 lm. Films were prepared as follows: the peptide was dissolved in water, at a conc. of 2 mg/ml. N-methyl-2-pyrrolidone was added at a ratio of 10% v/v and the solution was drop-casted and dried on the substrate at 60 °C. The resulting films are 700–1500 nm thick.
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Fig. 3. Optical images showing the results of FF (3a) and LL (3c) film depositions conducted at low environmental RH level. Following exposure to elevated levels of environmental humidity, spherulitic films formed on the substrates (FF – 3b, LL – 3d). All bars = 50 lm. 3e – the fracture line of a cleaved FF film. The brittleness can be clearly observed.
All cleavage attempts of such samples were unsuccessful. However, the spherulitic samples were successfully cleaved. The fracture lines of the spherulitic films indicate that they are somewhat brittle. Fig. 3e shows the fracture line of a cleaved FF film. The brittleness can be attributed to a change in the degree of crystallinity between spherulitic and non-spherulitic samples. The crystal structure characterization of various LL samples was performed by powder XRD. The normalized XRD patterns obtained with Cu Ka radiation on the theta–theta powder diffractometer ‘‘Scintag”, equipped with liquid nitrogen cooled Ge solid-state detector, are shown in Fig. 5. The lowest red pattern of the untreated LL powder fits well the orthorhombic structure with lattice parameters 5.35 Å, 16.76 Å and 33.31 Å, determined by single crystal XRD [35]. It is evident that the diffraction pattern of surface-deposited LL PNTs (blue) differs completely from that of the original powder. It was verified by mass-spectrometry that no chemical change in the molecules comprising the crystal occurred during solvent addition and heating. Therefore we can conclude that differing from the FF case [36], the polymorphic transition comes about during LL peptide nanotubes formation. Moreover, the surface-deposited LL spherulitic film comprises both LL polymorphic modifications as its XRD pattern (green) demonstrates. Although the unambiguous indexing of the surface-deposited LL XRD pattern was not achieved, it is intriguing that most of the indexing results tend to ‘‘concentrate around” the hexagonal unit cell with in-plane lattice parameters of about 23.40 Å and out-ofplane lattice parameter ranging from 3.9 Å, up to 5.1 Å, depending on the specific space group chosen. These values are quite close to the parameters found for FF [35]. When comparing LL and FF PNT diffraction patterns (two upper curves in Fig. 5) one can see that, indeed, some similarity exists. Further studies are needed to determine whether this likeness is completely casual or reflects any important mechanisms of PNT formation from dipeptides. An intriguing similarity has been hinted at [13–15,37,38], between spherulites and the protein deposits (plaques) observed in amyloidogenic diseases such as Alzheimer’s and CreutzfeldtJakob. It is also intriguing that many of the known peptide spherulites are amyloid-related [38–43] and that plaques are recognized by their Maltese cross pattern [44,45]. Our findings, which suggest that the environment’s hydration level has a profound effect on peptide spherulitic growth, and the fact that FF is derived from
Fig. 4. (a)–(d) The Maltese cross pattern observed in FF spherulites when viewed using polarized light microscopy. 4a bar = 200 lm. 4b–d bar = 100 lm. Amyloid plaques exhibiting similar optical behavior can be seen in Ref. [39].
N. Sidelman et al. / Journal of Colloid and Interface Science 343 (2010) 387–391
Fig. 5. The X-ray powder diffraction patterns of untreated LL powder (red), surfacedeposited LL spherulitic film (green), surface-deposited LL PNTs (blue), and the diffraction pattern of FF surface deposited PNTs (magenta). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
plaque-forming beta-amyloids, further highlight the importance of the largely overlooked similarity between plaques and spherulites, which might explain some observations in amyloidogenic diseases. In conclusion, we show that two-dimensional peptide spherulitic films can be formed by a simple, mild process. We also show that optically, these films behave similarly to polymer spherulites, and that there are mechanical differences between the spherulitic films and their non-spherulitic counterparts. We believe that our film-formation procedure can be applied to other peptides and therefore has potential use in bio-coating technologies. References [1] P.J. Phillips, in: D.T.J. Hurle (Ed.), Handbook of Crystal Growth, vol. 2b, Elsevier, Amsterdam, 1994, pp. 1167–1216. [2] J.H. Magill, Journal of Materials Science 36 (2001) 3143. [3] R.C. Ewing, Science 184 (1974) 561. [4] V. Hangloo, S. Pandita, K.K. Bamzai, P.N. Kotru, N. Sahni, Journal of Materials Science 39 (2004) 1743. [5] C. Sun, C. Kuan, F.J. Kao, Y.M. Wang, J.C. Chen, C.C. Chang, P. Shen, Materials Science and Engineering A 379 (2004) 327. [6] D.C. Bassett, Journal of Macromolecular Science; Physics B42 (2003) 227. [7] C.R. Lindegren, Journal of Polymer Science 50 (1961) 181. [8] J.A. Creek, G.R. Ziegler, J. Runt, Biomacromolecules 7 (2006) 761. [9] G.R. Ziegler, J.A. Creek, J. Runt, Biomacromolecules 6 (2005) 1547.
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[10] I.L. Hosier, D.C. Bassett, Polymer 41 (2000) 8801. [11] K. Monar, P.J. Phillips, Journal of Polymer Science Part B: Polymer Physics 35 (1997) (1843). [12] K. Monar, P.J. Phillips, Macromolecules 32 (1999) 5852. [13] M.R.H. Krebs, C.E. MacPhee, A.F. Miller, L.E. Dunlop, C.M. Dobson, A.M. Donald, Proceedings of National Academy Science USA 101 (2004) 14420. [14] M.R.H. Krebs, E.H.C. Bromley, S.S. Rogers, A.M. Donald, Biophysical Journal 88 (2005) 2013. [15] S.S. Rogers, M.R.H. Krebs, E.H.C. Bromley, E. van der Linden, A.M. Donald, Biophysical Journal 90 (2006) 1043. [16] S.B. Murray, A.C. Neville, International Journal of Biological Macromolecules 22 (1998) 137. [17] J. Sakamoto, J. Sugiyama, S. Kimura, T. Imai, T. Itoh, T. Watanabe, S. Kobayashi, Macromolecules 33 (2000) 4155. [18] T. Tanaka, J. Magoshi, Y. Magoshi, B. Lotz, S.I. Inoue, M. Kobayashi, H. Tsuda, M.A. Becker, Z. Han, S. Nakamura, Journal of Thermal Analysis and Calorimetry 64 (2001) 645. [19] S.J. Hamodrakas, A. Hoenger, V.A. Iconomidou, Journal of Structural Biology 145 (2004) 226. [20] R. Martin, L. Waldmann, D.L. Kaplan, Biopolymers 70 (2003) 435. [21] J. Bisault, G. Ryschenkow, G. Faivre, Journal of Crystal Growth 110 (1991) 889. [22] H.W. Starkweather Jr., R.E. Brooks, Journal of Applied Polymer Science 1 (1959) 236. [23] V.I. Pavlov, Materials Science 4 (1971) 438. [24] D. Krishnegowda, S.R. Kumaraswamy, M.S. Madhava, R. Somashekar, D. Revannasiddaiah, Molecular Crystals and Liquid Crystals Science and Technology Section A: Molecular Crystals and Liquid Crystals 250 (1994) 21. [25] J.J. Point, Polymer 47 (2006) 3186. [26] M. Reches, E. Gazit, Science 300 (2003) 625. [27] K. Ariga, J.P. Hill, M.V. Lee, A. Vinu, R. Charvet, S. Acharya, Science and Technology of Advanced Materials 9 (2008) 96. [28] G.M. Whitesides, B. Grzybowski, Science 295 (2002) 2418. [29] N. Hendler, N. Sidelman, M. Reches, E. Gazit, Y. Rosenberg, S. Richter, Advanced Materials 19 (2007) 1485. [30] Carl Henrik Görbitz, Chemistry – A European Journal 13 (2007) 1022. [31] L. Granasy, T. Pusztai, G. Tegze, J.A. Warren, J.F. Douglas, Physical Review E 72 (2005). [32] J.L. Hutter, J. Bechhoefer, Journal of Crystal Growth 217 (2000) 332. [33] H. Imai, Topics in Current Chemistry 270 (2007) 43. [34] P.C. Hiemenz, Polymer Chemistry, the Basic Concepts, Marcel Dekker, New York, 1984. [35] C.H. Görbitz, Chemistry—A European Journal 7 (2001) 5153. [36] C.H. Görbitz, Chemical Communications (2006) 2332. [37] G.P. Gellermann, K. Ullrich, A. Tannert, C. Unger, G. Habicht, S.R.N. Sauter, P. Hortschansky, U. Horn, U. Mollmann, M. Decker, J. Lehmann, M. Fandrich, Journal of Molecular Biology 360 (2006) 251. [38] T. Ban, K. Morigaki, H. Yagi, T. Kawasaki, A. Kobayashi, S. Yuba, H. Naiki, Y. Goto, Journal of Biological Chemistry 281 (2006) 33677. [39] G.G. Glenner, New England Journal of Medicine 302 (1980) 1283. [40] G.G. Glenner, New England Journal of Medicine 302 (1980) 1333. [41] P.T. Lansbury, Biochemistry 31 (1992) 6865. [42] P.T. Lansbury, P.R. Costa, J.M. Griffiths, E.J. Simon, M. Auger, K.J. Halverson, D.A. Kocisko, Z.S. Hendsch, T.T. Ashburn, R.G.S. Spencer, B. Tidor, R.G. Griffin, Nature Structural Biology 2 (1995) 990. [43] C.L. Masters, G. Simms, N.A. Weinman, G. Multhaup, B.L. McDonald, K. Beyreuther, Proceedings of National Academy Science USA 82 (1985) 4245. [44] L. Manuelidis, W. Fritch, Y.G. Xi, Science 277 (1997) 94. [45] C. Tranchant, L. Geranton, C. Guiraud-Chaumeil, M. Mohr, J.M. Warter, Neurology 52 (1999) 1244.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Short Communication
Peptide-based spherulitic films—formation and properties Noam Sidelman a,b, Yuri Rosenberg c, Shachar Richter a,b,* a
The Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel-Aviv, Israel The Center for NanoScience and NanoTechnology, Tel-Aviv University, Tel-Aviv, Israel c The Wolfson Applied Materials Research Center, Tel-Aviv University, Tel-Aviv, Israel b
a r t i c l e
i n f o
Article history: Received 24 September 2009 Accepted 10 November 2009 Available online 18 November 2009 Keywords: Spherulites Films Peptide nanotubes
a b s t r a c t Peptide nanotube-based spherulitic films are a recently discovered phenomenon, which was demonstrated in the case of the self-assembled diphenylalanine peptide nanotubes. Here we show that the film-formation method can be implemented with other peptides. We also demonstrate that a critical physical parameter, an elevated level of environmental hydration, is required for film growth. A possible formation mechanism is suggested. The optical, morphological and mechanical properties of these films are characterized and are found to be substantially different from those of non-spherulitic deposits. Ó 2009 Elsevier Inc. All rights reserved.
Spherulites are poly-crystalline entities comprised of a nucleation center from which multiple lamellas span out, maintaining a space-filling shape [1,2]. Although spherulitic growth has been recognized since the early days of crystal growth research, its mechanism is not yet completely understood [1,2]. Nevertheless, spherulitic moieties can be found in various types of materials— inorganic [3–5], organic [6–10] biologic [11–20], and even in the element selenium [21]. In particular, spherulites are encountered in polymers [1]. The significance of their existence in soft matter stems from the fact that spherulitic composition has been shown to alter the polymers’ physical properties, such as optical and mechanical [6,22–25]. It was previously shown [26] that the peptide diphenylalanine (FF) can self-assemble to form peptide nanotubes (PNTs). The formation of PNTs has been previously discussed in view of its self assembly properties [26]. Self assembly [27,28] is an important phenomenon with great importance in nature and in nano-sciences since it offers a way to construct complex nanostructures. Interestingly, self assembly can be seen in various scales, ranging from molecular and up to micro-sized objects [28]. We have previously shown [29] that self-assembly capability of FF can be utilized to form spherulitic films on solid surfaces. These films are an excellent example of multi scale self assembly, that is ranging from the molecular self assembly, and up to the spherulitic structure. Here we demonstrate that the spherulitic film growth phenomenon is governed by several physical factors, and not by the chemical composition of the peptide molecules. We show that
* Corresponding author. Address: The Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel-Aviv, Israel. Fax: +972 3 6405612. E-mail address: [email protected] (S. Richter). 0021-9797/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2009.11.028
environmental humidity level is a critical factor in the peptide’s spherulitic growth. This is demonstrated by the formation of spherulitic films from another PNT-forming peptide—dileucine (LL). We also illustrate that the mechanical properties of the peptide spherulitic films differ from those of their non-spherulitic counterparts. Our method of forming peptide spherulitic films [29] consists of depositing an aqueous peptide solution, diluted with the additive N-methyl pyrrolidone (NMP), onto a heated flat solid substrate. The solution is subsequently allowed to completely evaporate. The conditions for such spherulitic growth are not as harsh as in the case of some polymer spherulites: similarly shaped and packed spherulites can be found in bulk polymers, which are crystallized from the melt at temperatures exceeding 100 °C, such as nylon [7] and n-alkanes [10]. The mild conditions required for peptide spherulitic film formation suggest that their formation from other peptides should not interfere with peptides’ functions; thus, various peptide films with enhanced mechanical properties can be formed which still retain their function. Moreover, although spherulites have been previously shown to be formed by peptides [13–15], such structures were three-dimensional in nature and were spread sporadically in the matrix in which they were formed. Our films are comprised of two-dimensional closely arranged spherulites, similar to those found in bulk polymers [2,6], and therefore have potential uses in biologic coating technology. We have recently shown that the beta-amyloid derived diphenylalanine [26] (FF, Fig. 1a) can be induced to form films on flat solid surfaces [29]. These films are comprised of multiple closely arranged two-dimensional spherulites, each exhibiting a nucleation center and multiple lamellas. These lamellas grow from the nucleation center until they encounter the growth fronts of neighboring spherulites, thus forming a grain boundary. Optical, atomic
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force (AFM) and scanning electron (SEM) microscopy of the films are shown in Fig. 1. We found that FF spherulitic films form only when a specific physical parameter exists. Specifically, a high level of environmental relative humidity (RH) (RT, RH > 75%). The high humidity was found to be a critical factor: without it, no spherulites formed. To test whether spherulitic films can also form from other peptides, and whether our film-formation procedure can be applied to various peptides or proteins, the spherulitic film-formation procedure found suitable for FF was applied to a different dipeptide— dileucine (L-Leu–L-Leu; LL, Fig. 2a). It was also of interest to note whether the dependency on humidity levels would still exist. LL, like FF, is a dipeptide with hydrophobic side chains, but it differs from FF by its lack of aromatic moieties. LL has been previously found to be a PNT-forming peptide [30], as it is comprised of channels that form due to its crystalline structure, like FF. The channels in LL are smaller than the 10 Å channels found in FF, and are rectangular, with dimensions of 2.5 Å by 6.0 Å [30]. When the FF-based film-formation procedure was applied to LL, spherulitic films were also successfully formed. The LL films were similarly comprised of closely arranged spherulites, exhibiting distinct nucleation centers and grain boundaries. Optical, AFM and SEM analyses of the film are shown in Fig. 2. As in the case of the FF films, the same humidity dependency was found with the LL spherulitic films, which formed only at elevated levels of environmental humidity.
For both FF and LL, the dependence on humidity was found to outlive the film-deposition process itself. At RH > 75% (RT), films were observed immediately after deposition. In contrast, filmdeposition attempts carried out at lower environmental humidity (e.g. RT, RH 60%) did not initially yield spherulitic films. However, when the samples were exposed to elevated levels of humidity, spherulitic films appeared on the substrate. An example of this effect can be seen in Fig. 3. Fig. 3a shows the result of attempted FF film deposition at a humidity level below 70% (RT). No spherulitic film is formed, and the only observable feature is a small fibrous branched shape. Similar behavior was observed for LL, as can be seen in Fig. 3c, which shows only small fibrous features appearing shortly after deposition. Exposure of the samples to elevated levels of environmental humidity resulted in the formation of a spherulitic film (Fig. 3b and d, FF and LL, respectively). The fiber seen in Fig. 3a became the nucleation center of the spherulites, and is an example of a nucleation ‘‘sheaf”, which represents a stage of homogeneous nucleation [1,31]. It should be noted that spherulites comprised of small organic molecules are rarely encountered [2]. Nevertheless, the few small organic molecules that have been previously described [1] have a common property—they are all aromatic hydrocarbons. LL is therefore unique in the sense that it is a small non-aromatic organic molecule that forms spherulites. Based on our observations, and on what is known about spherulitic composition in polymers, we determined the effect of
Fig. 1. (a) Scheme of L-Phe–L-Phe (FF). (b) An optical image of an FF spherulitic film. Bar = 50 lm. (c) An SEM image of a homogenous nucleation site of a FF spherulite, bar = 10 lm. (d) An AFM phase image of a spherulite’s homogenous nucleation site. Bar = 9.3 lm. (e) An SEM image of an FF spherulitic film. Multiple closely arranged spherulites can be seen. Bar = 100 lm. (f) An AFM image showing the lamellas comprising an FF spherulite. Bar = 1.6 lm. (g) and (h), Optical images of a homogenous nucleation site of FF spherulite (bar = 20 lm) and a heterogeneous one (bar = 50 lm). Films were prepared as follows: the peptide was dissolved in water, at a conc. of 2 mg/ ml. N-methyl-2-pyrrolidone was added at a ratio of 10% v/v and the solution was drop-casted and dried on the substrate at 60 °C. The resulting films are 700–1500 nm thick.
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humidity on peptide spherulitic growth to be as follows: polymer spherulites form when the polymer is grown from the melt. It has been previously suggested that the viscosity of the melt is important for spherulite formation, and it was noted that large viscosities seem to be required [2,6,32]. This has also been observed in the case of the rarely encountered spherulitic growth of small organic molecules [2]. In the case of our peptide spherulites, the spherulites grow from a saturated solution which contains the additive NMP. Following deposition of the solution onto the heated flat solid substrate and its evaporation, the substance remaining on the substrate appears to be completely solid, but is in fact a gel, and is comprised of the peptide molecules, NMP and a minute amount of water (most of it having evaporated). The NMP, without which no film forms, has a relatively low vapor pressure, and thus remains in sufficient quantity on the substrate. In the case of our peptide spherulitic films, as the substrate is exposed to elevated levels of humidity, the deposited substance absorbs water molecules from the environment and turns into a solid gel-like substance. The adsorbance of water from the environment regulates the viscosity of the gel-like substance, making it slightly less solid, i.e. highly viscous. When a level of viscosity suitable for peptide spherulitic growth is reached, the growth process is initiated. This correlates to viscosity dependence in the growth of spherulitic polymers, and this finding is also in agreement with a previous claim [33] that spherulitic growth is a crystallization process that occurs far from equilibrium: it is diffusion-dependent and therefore viscosity-dependent as well.
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It should be emphasized that although the FF and LL dipeptides behave, to some extent, similarly to polymeric spherulites, neither FF nor LL undergo polymerization, as verified by X-ray powder diffraction (XRD) for FF, and mass spectrometry for LL (not shown). This means that no chemical reaction has taken place before or during the spherulitic film’s growth process. In general, spherulite recognition and characterization are based on their optical behavior—polymer spherulites are recognized by the appearance of a Maltese cross extinction pattern under a polarized light microscope [1,2]. We observed this optical pattern in FF and LL spherulites as well. Images of FF spherulites viewed by polarized light microscopy can be seen in Fig. 4a–d. The images clearly show multiple spherulites packed together, each exhibiting a typical Maltese cross pattern, due to the twodimensional nature of the peptide spherulitic films. In polymers, the Maltese cross pattern is attributed to the chains being perpendicular the to the radius of the spherulite, and to a difference in the index of refraction between the direction parallel to the chain and the direction normal to the chain. This birefringence accompanied by spherical geometry is the reason for the extinction along the axis of the filter in the microscope [34]. In small peptide molecules like FF and LL, the reason for this pattern is attributed to the molecular orientation in the lamellar crystals, analogous to the long chain orientation effect in polymers. The spherulitic nature of the films has a marked effect on their mechanical properties. PNTs which were deposited on the substrate and resulted in a non-spherulitic mesh were not cleavable.
Fig. 2. (a) Scheme of L-Leu–L-Leu (FF). (b) An optical image of an LL spherulitic film. Bar = 50 lm. (c) An SEM image of a grain boundary where lamellas of two spherulites meet. Bar = 1 lm. (d) An SEM image showing a nucleation site of an LL spherulite. Bar = 10 lm. (e) An SEM image of an LL spherulitic film. Multiple closely arranged spherulites can be seen. Bar = 100 lm. (f) An AFM image showing the lamellas comprising an LL spherulite. Bar = 3 lm. (g) and (h) Optical images of LL spherulites. The spacefilling tendency can be observed. Bar = 50 lm. Films were prepared as follows: the peptide was dissolved in water, at a conc. of 2 mg/ml. N-methyl-2-pyrrolidone was added at a ratio of 10% v/v and the solution was drop-casted and dried on the substrate at 60 °C. The resulting films are 700–1500 nm thick.
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Fig. 3. Optical images showing the results of FF (3a) and LL (3c) film depositions conducted at low environmental RH level. Following exposure to elevated levels of environmental humidity, spherulitic films formed on the substrates (FF – 3b, LL – 3d). All bars = 50 lm. 3e – the fracture line of a cleaved FF film. The brittleness can be clearly observed.
All cleavage attempts of such samples were unsuccessful. However, the spherulitic samples were successfully cleaved. The fracture lines of the spherulitic films indicate that they are somewhat brittle. Fig. 3e shows the fracture line of a cleaved FF film. The brittleness can be attributed to a change in the degree of crystallinity between spherulitic and non-spherulitic samples. The crystal structure characterization of various LL samples was performed by powder XRD. The normalized XRD patterns obtained with Cu Ka radiation on the theta–theta powder diffractometer ‘‘Scintag”, equipped with liquid nitrogen cooled Ge solid-state detector, are shown in Fig. 5. The lowest red pattern of the untreated LL powder fits well the orthorhombic structure with lattice parameters 5.35 Å, 16.76 Å and 33.31 Å, determined by single crystal XRD [35]. It is evident that the diffraction pattern of surface-deposited LL PNTs (blue) differs completely from that of the original powder. It was verified by mass-spectrometry that no chemical change in the molecules comprising the crystal occurred during solvent addition and heating. Therefore we can conclude that differing from the FF case [36], the polymorphic transition comes about during LL peptide nanotubes formation. Moreover, the surface-deposited LL spherulitic film comprises both LL polymorphic modifications as its XRD pattern (green) demonstrates. Although the unambiguous indexing of the surface-deposited LL XRD pattern was not achieved, it is intriguing that most of the indexing results tend to ‘‘concentrate around” the hexagonal unit cell with in-plane lattice parameters of about 23.40 Å and out-ofplane lattice parameter ranging from 3.9 Å, up to 5.1 Å, depending on the specific space group chosen. These values are quite close to the parameters found for FF [35]. When comparing LL and FF PNT diffraction patterns (two upper curves in Fig. 5) one can see that, indeed, some similarity exists. Further studies are needed to determine whether this likeness is completely casual or reflects any important mechanisms of PNT formation from dipeptides. An intriguing similarity has been hinted at [13–15,37,38], between spherulites and the protein deposits (plaques) observed in amyloidogenic diseases such as Alzheimer’s and CreutzfeldtJakob. It is also intriguing that many of the known peptide spherulites are amyloid-related [38–43] and that plaques are recognized by their Maltese cross pattern [44,45]. Our findings, which suggest that the environment’s hydration level has a profound effect on peptide spherulitic growth, and the fact that FF is derived from
Fig. 4. (a)–(d) The Maltese cross pattern observed in FF spherulites when viewed using polarized light microscopy. 4a bar = 200 lm. 4b–d bar = 100 lm. Amyloid plaques exhibiting similar optical behavior can be seen in Ref. [39].
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Fig. 5. The X-ray powder diffraction patterns of untreated LL powder (red), surfacedeposited LL spherulitic film (green), surface-deposited LL PNTs (blue), and the diffraction pattern of FF surface deposited PNTs (magenta). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
plaque-forming beta-amyloids, further highlight the importance of the largely overlooked similarity between plaques and spherulites, which might explain some observations in amyloidogenic diseases. In conclusion, we show that two-dimensional peptide spherulitic films can be formed by a simple, mild process. We also show that optically, these films behave similarly to polymer spherulites, and that there are mechanical differences between the spherulitic films and their non-spherulitic counterparts. We believe that our film-formation procedure can be applied to other peptides and therefore has potential use in bio-coating technologies. References [1] P.J. Phillips, in: D.T.J. Hurle (Ed.), Handbook of Crystal Growth, vol. 2b, Elsevier, Amsterdam, 1994, pp. 1167–1216. [2] J.H. Magill, Journal of Materials Science 36 (2001) 3143. [3] R.C. Ewing, Science 184 (1974) 561. [4] V. Hangloo, S. Pandita, K.K. Bamzai, P.N. Kotru, N. Sahni, Journal of Materials Science 39 (2004) 1743. [5] C. Sun, C. Kuan, F.J. Kao, Y.M. Wang, J.C. Chen, C.C. Chang, P. Shen, Materials Science and Engineering A 379 (2004) 327. [6] D.C. Bassett, Journal of Macromolecular Science; Physics B42 (2003) 227. [7] C.R. Lindegren, Journal of Polymer Science 50 (1961) 181. [8] J.A. Creek, G.R. Ziegler, J. Runt, Biomacromolecules 7 (2006) 761. [9] G.R. Ziegler, J.A. Creek, J. Runt, Biomacromolecules 6 (2005) 1547.
391
[10] I.L. Hosier, D.C. Bassett, Polymer 41 (2000) 8801. [11] K. Monar, P.J. Phillips, Journal of Polymer Science Part B: Polymer Physics 35 (1997) (1843). [12] K. Monar, P.J. Phillips, Macromolecules 32 (1999) 5852. [13] M.R.H. Krebs, C.E. MacPhee, A.F. Miller, L.E. Dunlop, C.M. Dobson, A.M. Donald, Proceedings of National Academy Science USA 101 (2004) 14420. [14] M.R.H. Krebs, E.H.C. Bromley, S.S. Rogers, A.M. Donald, Biophysical Journal 88 (2005) 2013. [15] S.S. Rogers, M.R.H. Krebs, E.H.C. Bromley, E. van der Linden, A.M. Donald, Biophysical Journal 90 (2006) 1043. [16] S.B. Murray, A.C. Neville, International Journal of Biological Macromolecules 22 (1998) 137. [17] J. Sakamoto, J. Sugiyama, S. Kimura, T. Imai, T. Itoh, T. Watanabe, S. Kobayashi, Macromolecules 33 (2000) 4155. [18] T. Tanaka, J. Magoshi, Y. Magoshi, B. Lotz, S.I. Inoue, M. Kobayashi, H. Tsuda, M.A. Becker, Z. Han, S. Nakamura, Journal of Thermal Analysis and Calorimetry 64 (2001) 645. [19] S.J. Hamodrakas, A. Hoenger, V.A. Iconomidou, Journal of Structural Biology 145 (2004) 226. [20] R. Martin, L. Waldmann, D.L. Kaplan, Biopolymers 70 (2003) 435. [21] J. Bisault, G. Ryschenkow, G. Faivre, Journal of Crystal Growth 110 (1991) 889. [22] H.W. Starkweather Jr., R.E. Brooks, Journal of Applied Polymer Science 1 (1959) 236. [23] V.I. Pavlov, Materials Science 4 (1971) 438. [24] D. Krishnegowda, S.R. Kumaraswamy, M.S. Madhava, R. Somashekar, D. Revannasiddaiah, Molecular Crystals and Liquid Crystals Science and Technology Section A: Molecular Crystals and Liquid Crystals 250 (1994) 21. [25] J.J. Point, Polymer 47 (2006) 3186. [26] M. Reches, E. Gazit, Science 300 (2003) 625. [27] K. Ariga, J.P. Hill, M.V. Lee, A. Vinu, R. Charvet, S. Acharya, Science and Technology of Advanced Materials 9 (2008) 96. [28] G.M. Whitesides, B. Grzybowski, Science 295 (2002) 2418. [29] N. Hendler, N. Sidelman, M. Reches, E. Gazit, Y. Rosenberg, S. Richter, Advanced Materials 19 (2007) 1485. [30] Carl Henrik Görbitz, Chemistry – A European Journal 13 (2007) 1022. [31] L. Granasy, T. Pusztai, G. Tegze, J.A. Warren, J.F. Douglas, Physical Review E 72 (2005). [32] J.L. Hutter, J. Bechhoefer, Journal of Crystal Growth 217 (2000) 332. [33] H. Imai, Topics in Current Chemistry 270 (2007) 43. [34] P.C. Hiemenz, Polymer Chemistry, the Basic Concepts, Marcel Dekker, New York, 1984. [35] C.H. Görbitz, Chemistry—A European Journal 7 (2001) 5153. [36] C.H. Görbitz, Chemical Communications (2006) 2332. [37] G.P. Gellermann, K. Ullrich, A. Tannert, C. Unger, G. Habicht, S.R.N. Sauter, P. Hortschansky, U. Horn, U. Mollmann, M. Decker, J. Lehmann, M. Fandrich, Journal of Molecular Biology 360 (2006) 251. [38] T. Ban, K. Morigaki, H. Yagi, T. Kawasaki, A. Kobayashi, S. Yuba, H. Naiki, Y. Goto, Journal of Biological Chemistry 281 (2006) 33677. [39] G.G. Glenner, New England Journal of Medicine 302 (1980) 1283. [40] G.G. Glenner, New England Journal of Medicine 302 (1980) 1333. [41] P.T. Lansbury, Biochemistry 31 (1992) 6865. [42] P.T. Lansbury, P.R. Costa, J.M. Griffiths, E.J. Simon, M. Auger, K.J. Halverson, D.A. Kocisko, Z.S. Hendsch, T.T. Ashburn, R.G.S. Spencer, B. Tidor, R.G. Griffin, Nature Structural Biology 2 (1995) 990. [43] C.L. Masters, G. Simms, N.A. Weinman, G. Multhaup, B.L. McDonald, K. Beyreuther, Proceedings of National Academy Science USA 82 (1985) 4245. [44] L. Manuelidis, W. Fritch, Y.G. Xi, Science 277 (1997) 94. [45] C. Tranchant, L. Geranton, C. Guiraud-Chaumeil, M. Mohr, J.M. Warter, Neurology 52 (1999) 1244.