Time-Lapse Atomic Force Microscopy Observations of the Morphology

Dec 17, 2009 - Time-lapse atomic force microscopy is used in this contribution to directly watch the growth of nanofibers of a lipidated peptide on a ...
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J. Phys. Chem. B 2010, 114, 620–625

Time-Lapse Atomic Force Microscopy Observations of the Morphology, Growth Rate, and Spontaneous Alignment of Nanofibers Containing a Peptide-Amphiphile from the Hepatitis G Virus (NS3 Protein) Konrad J. Weron´ski,*,† Pilar Cea,‡ Ismael Diez-Pere´z,§,| Maria Antonia Busquets,† Josefina Prat,† and Victoria Girona† Department of Physical Chemistry, Faculty of Pharmacy, UniVersity of Barcelona, AV. Joan XXII s/n, 08028 Barcelona, Spain, Department of Organic and Physical Chemistry (Faculty of Science) and Institute of Nanoscience of Aragon (INA), UniVersity of Zaragoza, Plaza San Francisco s/n, 50009, Zaragoza, Spain, and Laboratory of Electrochemistry and Materials (LCTEM), Department of Physical Chemistry, Faculty of Chemistry, UniVersity of Barcelona, Martı´ I Franque`s 1, 08028 Barcelona, Spain ReceiVed: September 13, 2009; ReVised Manuscript ReceiVed: October 24, 2009

Time-lapse atomic force microscopy is used in this contribution to directly watch the growth of nanofibers of a lipidated peptide on a mica surface. Specifically, the studied lipopeptide is the palmitoyl derivative of the fragment 505-514 of NS3 protein from the hepatitis G virus, abbreviated as Palmitoyl-NS3 (505-514). Data on the morphology, growth rate, and orientation of these peptide-amphiphile nanofibers have been obtained. From these data, it can be concluded that this synthetic lipopeptide forms two types of fiber-like aggregates: (i) half-spherical fibrous aggregates with lengths of hundreds of nanometers and (ii) spherical fibrous aggregates with lengths of several micrometers. In addition, when a fresh lipopeptide aqueous solution is deposited onto a mica surface, the aggregates spontaneously orient parallel to each other, yielding wellaligned nanofibers on large areas of the mica surface. A significant growth in both the length and the number of the fibers was observed during the first minutes after the solution deposition. Elongation of the fibrous aggregates from one end is more frequent, though elongation from both ends also occurs, with growth rates in the 4-5 nm/s range. The effects of dilution, mechanical perturbation, and pH on the aggregation behavior of Palmitoyl-NS3 (505-514) are also detailed in this paper. 1. Introduction In recent years, synthetic lipopeptides have attracted a great deal of attention in the multidisciplinary and emerging field of nanobiomedicine.1-13 This is due to several reasons including their ability to self-assemble into well-defined nanostructures, the chemical diversity which can be tolerated within these nanostructures, their ease of synthesis, their high purity, lot to lot consistency, their high specificity in eliciting immune responses, and their low production costs.14,15 Thus, these types of biological materials are promising candidates to become peptide vaccines5,12,13,16-23 because they can generate immune responses without the use of adjuvants, which are often toxic. Especially remarkable is the demonstration of lipopeptides as components in drug and vaccine delivery systems to desired biological sites.24 In addition, several groups are intensively working on the fabrication of solid scaffolds that incorporate peptide sequences known to promote cell adhesion or differentiation and to form by self-assembly from aqueous solutions of peptide amphiphiles.25-36 These studies are still in their infancy, but interest in the area is intense because an ability to engineer or design water-soluble self-assembly systems offers * To whom correspondence should be addressed. E-mail: weronski@ ub.edu. Tel.: +34 93 402 4556. Fax: +34 93 403 5987. † Faculty of Pharmacy, University of Barcelona. ‡ University of Zaragoza. § Faculty of Chemistry, University of Barcelona. | Present affiliation: Bielectronics and Biosensors, Biodesign Institute, Arizona State University, AZ85287.

routes to novel biomaterials with potential applications in regenerative medicine, drug delivery, biological surface engineering, etc. From the chemical point of view, a lipidated peptide is an amphiphilic molecule composed of a hydrophobic aliphatic tail of a variable length and a hydrophilic peptide sequence attached to that tail through an amide bond. The tendency of lipopeptides to aggregate in aqueous solutions above the critical micelle concentration is driven by the aliphatic tail, while the peptide portion displays the active functional groups for particular applications. The dimensions and the shape of these aggregated structures depend on different factors, such as the geometry of the polar group, the shape of each molecule, complementary shape among the individual components, and the interactions among them (van der Waals, electrostatic, and hydrogen bonding).37 In this respect, several amphiphilic peptide structures have been described, including spheroid, nanotapes, ribbons, twisted ribbons, double walled nanotubes, nanofibers, and nanovesicles.38-46 In addition, the characteristics of lipopeptide aggregates in aqueous solutions can be affected by many factors including molecular concentration, pH, and temperature changes.41,45 The way in which lipopeptides self-assemble and especially disassemble is of interest from both the theoretical interest in surface chemistry and their potential use in particular medical applications.8 The possibility of direct observation of the assembly and disassembly processes as well as the understanding of the driving forces which control these processes are also a matter of considerable current interest in the design, construc-

10.1021/jp9088436  2010 American Chemical Society Published on Web 12/17/2009

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Figure 1. Upper figure shows the chemical structure of Palmitoyl-NS3 (505-514). The lower illustration shows the space-filling molecular model.

tion, placement, and orientation of nanometer-scale biomaterials for future medical applications.35,47-49 After the hepatitis G virus (GBV-C/HGV) was discovered in 1995,50,51 a significant decrease in the mortality of patients coinfected with GBV-C/HGV and HIV was reported in Japan and later in the USA, Germany, and France.52 However, despite the obvious advances in vaccines and treatments of hepatitis, the confinement of proteins of the hepatitis G virus into welldefined one-dimensional supramolecular structures may become important in future biomedical applications for either in vitro or in vivo cell therapies. Prompted by this picture of the current landscape, we have sought to investigate the self-assembly and disassembly behavior of a lipopeptide consisting of the hydrophilic peptide of the NS3 protein of the hepatitis G virus (fragment 505-514), whose chemical structure is depicted in Figure 1. The chosen sequence, SAELSMQRRG, has a net positive charge, with the whole molecule being amphiphilic thanks to the combination of the hydrophobic character of the long alkyl tail of palmitic acid with the hydrophilic peptide region. The amphiphilic character together with the electrostatic interactions among the peptide groups and the molecule conical shape, in which the hydrophilic headgroup is somewhat bulkier than its narrow hydrophobic tail, will determine the shape and aggregation behavior of this material. This paper is intended to demonstrate the Palmitoyl-NS3 (505-514) of GBV-C/HGV tendency to self-assemble into autoaligned fibrous aggregates when exposed to an aqueous media. In addition, the effects of dilution, mechanical perturbation, and pH on the aggregation behavior of Palmitoyl-NS3 (505-514) are also described. These investigations were carried out by time-lapse atomic force microscopy (AFM). In biology, the distinct advantage of AFM over SEM (scanning electron microscopy) or TEM (transmission electron microscopy) is its ability to in situ monitor changes in the aggregation state of supramolecular structures and to study dynamic aspects of molecular interactions in their physiological buffer solution.

Thus, the biological material can be maintained “alive” in the aqueous environment, while a particular sample area can be repeatedly imaged with high spatial resolution. 2. Experimental Section The lipopeptide Palmitoyl-NS3 (505-514) was synthesized in solid phase and then lyophilized by the literature methods.53 The author’s laboratory has recently reported that this lipopeptide self-assembles in a 0.77 mM water solution,11 with aggregates showing a diameter of ca. 8 ( 1 nm and lengths between a few dozen of nanometers and several micrometers. AFM experiments were conducted at room temperature using an extended multimode AFM with a Nanoscope IIIa controller (Digital Instruments Veeco Metrology Group, Santa Barbara, CA, USA). All images were recorded under aqueous solution in Tapping Mode AFM using working resonant frequencies between 6 and 9 kHz and low set point amplitudes of around 0.5 V (∼5 nm). A 15 µm2 piezoelectric scanner (d-scanner) was employed. Triangular Si3N4 tips, with a nominal force constant of 0.1 N/m (Olympus, Japan), were used, and the forces were minimized during the scans. Prior to measurements, the AFM liquid cell was washed with ethanol and Milli Pore Milli-Q water and allowed to dry in an N2 stream. Green Muscovite Mica discs (Metafix, France) were cleaved with Scotch-tape and glued onto a Teflon disk using a water-insoluble epoxy. These Teflon discs were glued onto a steel disk and then set onto the piezoelectric scanner. Previous to sample imaging, the tip was stabilized in the working buffer solution for 10-15 min. Digital data were analyzed using the 5.12r3 version of the Nanoscope III software. 3. Results and Discussion 3.1. Self-Assembly Process and Influence of the Solution Age. A lipopeptide aqueous solution (38.5 µM) was prepared just before the deposition onto freshly cleaved mica. AFM

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Figure 2. Formation of NS3 (505-514) GBV-C/HGV fibers onto freshly cleaved mica observed by time-lapse AFM. (A) Selected area on the mica revealing the presence of a large fibrous aggregate, indicated by the white arrow, as well as shorter and thinner fibrous aggregates which are distributed all over the mica substrate. (B) The same area scanned 260 s after. A new thick and large aggregate together with many thin ones appear on the mica. (C) The same area 600 s after. The elongation of thick fibers is evident. (D) The same area 771 s after. The arrows point to the thick aggregates whose elongation with time can be observed in the gallery of images. The appearance of new fibers is also observed. (E) Image recorded 1157 s after. The thick aggregate elongation continues and is accompanied by the appearance of new fibers. (F) The same area 1327 s after. The thick aggregate pointed to by the arrow on the left of the image ceases its growing. The growth of other thick aggregates is observed.

images were collected a few minutes after the sample deposition (time necessary for the stabilization of thermal and mechanical drift), and the same area of the mica was consecutively scanned to analyze the evolution of the aggregation process with time. The resulting images of this experiment are shown in Figure 2(A-F). The white arrow in Figure 2A points to a long fibrillike structure, more than one micrometer in length, and many shorter fibrous aggregates (a few hundred of nanometers in length) which were observed just a few minutes after the sample deposition. In addition, all these fibers were spontaneously oriented in the same direction and nearly parallel to each other, which may be triggered by the charged mica surface. It is worth noting that all the short fibers have a similar height, which is slightly higher than half of the height of the large aggregates (see the section analysis in Figure 3). The precise height of both types of aggregates depends slightly on the initial concentration of the aqueous solution although we have always found a height ratio of ca. 1:2. The order of height of the short aggregates is in good agreement with that reported before for hemimicelles (characteristic half-spherical fibrous aggregates formed at the water-solid interface), while the height of the large fibers could be explained by a spherical aggregate.40,41 Half-spherical and spherical aggregates of Palmitoyl-NS3 (505514) are schematically represented in Figure 4. A comparison of the length of the Palmitoyl-NS3 (505-514) molecule, estimated from molecular models (5.8 nm), and the diameter

Weron´ski et al. of the spherical (ca. 7 nm) and half-spherical aggregates (ca. 4.3 nm) are a clear indication of the alkyl chains not being fully extended but in a fluid-like distribution, in an attempt to increase the mean distance between the charged head groups to minimize the repulsive interactions between them and thus lower the interaction energy per molecule (relative to the energy of a uniform distribution), which is in good agreement with previous observations.37 Under these experimental conditions (a fresh 38.5 µM aqueous solution), successive scans of the same area (Figure 2B-F) reveal the growth of many of the short fibers observed on the previous scans as well as the appearance of new long aggregates. It is also noteworthy that the elongation of the fibrous aggregates from one end is more frequent, though the elongation from both ends also occurs. Figure 5 illustrates the growth rate for the two fibrous aggregates indicated by arrows in Figure 2. Both fibers increased their lengths by ∼1 µm per scan (every ∼200 s). These observations are consistent with Palmitoyl-NS3 (505-514) being deposited onto the mica surface after partial assembly that may occur in the bulk solution above the mica. The negatively charged mica surface may attract and orient the positively charged polar head of Palmitoyl-NS3 (505514) molecules, which results in the formation of new fibrous aggregates and the growth of others previously deposited onto the mica surface. However, the ceasing of the aggregate elongation was observed after a few hours. This phenomenon may be explained in terms of the formation of a “closedterminated” aggregate (Figure 6) which precludes the incorporation of more molecules into the fibrous aggregate. In addition, the elongation of fibers from only one end may be due to the early formation of a closed-terminated aggregate on one end of the fiber, which has been related before to the conical shape of the molecule37,41 as well as to the hydrophobic effect of the alkyl tail in self-assembly processes in water.41,54 Another point worth mentioning is that the increase in the aggregate length and the formation of new fibrous aggregates were only observed when the lipopeptide solution had been prepared immediately before the sample deposition onto the mica. On the contrary, when the lipopeptide solution was prepared 24 h before the AFM visualization, neither changes in the aggregate length nor the formation of new aggregates was observed, which is consistent with the aggregate growth and deposition of new fibers being related to the formation of new aggregates in the fresh solution above the mica. In addition, the aggregates formed from an aged solution were loosely placed without the parallel orientation observed in freshly prepared solutions, and many aggregates were also coiled to each other. 3.2. Effect of a Mechanical Perturbation on Aggregate Elongation. The next stage in these investigations was to explore the capability of the aggregates to elongate when a mechanical perturbation was applied through the AFM tip in an equilibrated system. The experimental conditions were slightly varied with respect to the foregoing experiments with a view to ensuring that the analyzed area was clear enough, without too many fibers which could make the observations rather difficult. Thus, a fresh solution was prepared just before conducting the AFM study. The concentration of the lipopeptide was reduced to a value of 19.25 µM to decrease the number of deposited aggregates onto the mica. The system was in equilibrium ca. 2 h after the solution deposition; i.e., no new aggregates were deposited onto the mica, and no elongation of any of the existing fibers took place. Under these equilibrium conditions, a fragment from a long fiber was cut and removed by applying forces >50 nN using the AFM tip (Figure 7A, where

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Figure 3. 2D AFM image of Palmitoyl-NS3 (505-514) fibers (left) and section analysis (right).

Figure 4. Schematic model for half-spherical fibrous aggregates (left) and spherical fibrous aggregates (right).

Figure 6. AFM view of closed-terminated aggregates and schematic representation in the inset cartoon. Figure 5. Variation of the length of the fibrous aggregates, indicated in Figure 2 by arrows, with time. The slope of the dotted lines (linear fit of the experimental data) is a good indication of the fiber growth rate.

the cutoff region is marked with arrows). After the breakage of the fiber, the next scan revealed a growth of the fiber at one end accompanied by a growth and a bifurcation at the other end (indicated by arrows in Figure 7B). A further growth of the fibrous aggregate until it was completely regenerated was observed in the next scans (Figure 7C and D). The elongation of the cutoff aggregate is believed to follow the same mechanism as the conventional growth of the fibers after the solution deposition. Thus, the growth of a fiber can be explained by the presence of free lipopeptide molecules in the solution and an “open” fiber end. The bifurcation observed in the above-described experiment could be due to an irregularity in the fiber structure produced on account of a not exactly perpendicular cut of the fibrous aggregate by the AFM tip. Note that the cutoff fragment was ideally reconstructed.

3.3. Dilution and pH Effects on the Fiber Disaggregation. The effect of the concentration of the peptide solution on the fiber’s growth or diminution was also investigated by analyzing the deposition of aggregates from a more diluted solution. Thus, a 10.0 µM lipopeptide aqueous solution was deposited onto the freshly cleaved mica, and AFM scans were recorded after the achievement of the equilibrium conditions, ca. 1 h. With the purpose of studying the possible disaggregation of PalmitoylNS3 (505-514) already assembled in fibers, the solution above the mica surface was removed and replaced by the same volume of water. This procedure was repeated two more times, but no changes were observed in the AFM images. A similar procedure was followed for a fiber which was mechanically removed by the AFM tip with no reconstruction of the fiber. The solution above the mica was also changed three times, with no modification in the aggregate length (60 min of observation), which is indicative of strong electrostatic interactions between the aggregates and the mica. Thus, neither the mechanical perturbation, which involves the removal of a part of the fiber to ensure

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Figure 9. (A) Pristine sample showing fiber-like aggregates (a mica region with few aggregates was chosen for clarifying purposes). (B) The same sample scanned 60 min after the HCl addition to the solution above the mica. The inset figures show a more detailed picture of the aggregates.

Figure 7. Watching Palmitoyl-NS3 (505-514) fiber bifurcation and reconstruction. (A) Area before the experiment. The two arrows point to the area to be cut off by the AFM tip. (B) The same area 790 s after the removal of a fraction of the fiber. Bifurcation of the upper end and the absence of the fiber fragment are evident. (C) The same area 1300 s after. (D) The aggregate is “ideally” reconstructed.

Figure 8. (A) Pristine sample showing fiber-like aggregates. (B) The same sample scanned 46 min after the NaOH addition to the solution above the mica. The arrows point to the disassembled regions of the aggregates.

the existence of an open end, nor the change of the solution caused any disassembly of the fibers or a decrease in length. The pH of the solution was found before8 to trigger the assembly or disassembly processes of synthetic peptides. Further insight into the Palmitoyl-NS3 (505-514) tendency to aggregation was obtained by changing the pH of the aqueous solution. After formation of the fibrous aggregates onto a mica substrate (Figure 8A), the lipopeptide solution above the mica surface was removed, and a 0.01 M NaOH aqueous solution was added. The exposure of the aggregates to the NaOH solution leads to a decrease in the aggregate length as evidenced by Figure 8B, which was taken 46 min after the change of the solution and the NaOH addition. The observed disaggregation phenomenon in a high pH medium may be explained in terms of a reduction in the electrostatic interactions between the lipopeptide and the mica as a consequence of the neutralizing effect that the basic media has on the net positive peptide charge. In these conditions, a decrease in the peptide concentration results in a disaggregation effect. The speeds at which the decrease of the fiber lengths occur are gathered in Table 1. The lengths of the fibers were decreased to certain values (reported in Table 1), and subsequent dilutions with NaOH and water did not lead to any further changes in the aggregate lengths. The cease in the disassembly process is probably a consequence of the close of the fiber ends. An experiment similar to the foregoing described was performed, but this time the solution above the mica was removed and replaced by a 0.1 M HCl aqueous solution. Figure 9A and 9B show the fibers formed before and after the HCl

TABLE 1: Decrease in the Length of the Fibrous Aggregates of Figure 8 time (min) 0 28 46 difference 0-46 min decreasing speed

length of fiber number 1

length of fiber number 2

271 nm 166 nm 105 nm 166 nm 0.06 nm/s

459 nm 392 nm 293 nm 166 nm 0.06 nm/s

addition, respectively. From these figures, it can be concluded that a low pH in the media surrounding the mica triggers the formation of aggregates. In addition, long aggregates are preferentially formed in the acid media with the molecules forming these large fibers coming from the hydrolysis of short fibers previously formed in the neutral media. 4. Conclusions We have thoroughly studied the aggregation behavior of a lipidated peptide, the palmitoyl derivative of the fragment 505514 of NS3 protein of GBV-C/HGV virus, in aqueous media concluding that fiber-like aggregates are deposited from the aqueous solution onto freshly cleaved mica. Two different types of nanofibers are formed: (i) half-spherical fibrous aggregates with lengths of hundreds of nanometers and a height consistent with the length of one Palmitoyl-NS3 (505-514) molecule and (ii) spherical fibrous aggregates with lengths of several micrometers and whose heights are approximately twice the value of the half-spherical fibers. Moreover, these aggregates, when deposited from a fresh solution, are aligned with each other over large areas of the mica substrate forming a well-defined structure consisting of parallel nanofibers. To our knowledge, an autoaligned deposition of amphiphilic peptide fibers over large substrate areas has not been reported before and could be of interest in many of the technological applications envisioned for supramolecular self-assembly. Synthetic lipopeptides have so far been investigated to a small extent by time-lapse atomic force microscopy despite the wealth of opportunities offered by this method for conveniently analyzing the morphology, growth rate, and directionality of growth of aggregate nanostructures. Throughout this paper, timelapse atomic force microscopy has shown itself as a powerful tool for the in situ characterization of the assembly and disassembly processes of lipopeptide aggregates and has allowed us to analyze changes in the length of individual fibers as they were appearing and growing. From a quantitative point of view, the growth rate of these nanofibers has been determined, with values in the 4 to 5 nm/s range. In addition, the effect of a

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