Formation of colloidal suspension of hydrophobic compounds with an amphiphilic self-assembling peptide

Colloids and Surfaces B: Biointerfaces 55 (2007) 200–211

Formation of colloidal suspension of hydrophobic compounds with an amphiphilic self-assembling peptide S.Y. Fung, H. Yang, P. Chen ∗ Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada Received 22 August 2006; received in revised form 29 November 2006; accepted 1 December 2006 Available online 14 December 2006

Abstract The amphiphilic self-assembling peptide EAK16-II was found to be able to stabilize hydrophobic compounds in aqueous solution. Micro/nanocrystals of a hydrophobic compound, pyrene, and a hydrophobic anticancer agent, ellipticine, were stabilized by EAK16-II to form colloidal suspensions in water. Initial evidence of the association between EAK16-II and hydrophobic compounds was the observation of a clouding phenomenon and a difference in fluorescence spectra of the solution. A further investigation on the interaction between EAK16-II and pyrene was carried out using fluorescence spectroscopy and scanning electron microscopy (SEM). It was found that the pyrene–peptide complex formation required mechanical stirring, and the freshly prepared peptide solution (containing peptide monomers and/or peptide protofibrils) was more effective at stabilizing pyrene than the mature fibrils in aged peptide solutions. The time duration over which the complex formed was about 22 h. The data on the complexation of pyrene and EAK16-II at various concentrations suggested that the maximum amount of stabilized pyrene was concentration dependent. SEM images showed that peptide concentration did not significantly affect the size of the complexes/suspensions but altered the structures of the peptide coating on the surface of the complex. Atomic force microscopy (AFM) was conducted to study the interaction of EAK16-II with a model hydrophobic surface, which provided some detailed information of how peptide adsorbed onto the hydrophobic compounds and stabilize them. This study shows the potential of self-assembling peptides for encapsulation of hydrophobic compounds. © 2006 Elsevier B.V. All rights reserved. Keywords: Colloidal suspension; Hydrophobic compounds; Peptides; Self-assembly; Ellipticine; Pyrene; Fluorescence spectroscopy; Scanning electron microscopy (SEM); Atomic force microscopy (AFM); Encapsulation

1. Introduction A special class of self-assembling peptides, originally discovered from a yeast z-DNA binding protein, has shown great potential in a wide range of applications, from tissue scaffolding, biological surface patterning to controlled drug delivery [1,2]. These peptides contain unique sequences of alternating hydrophobic and hydrophilic residues, and can self-assemble into ␤-sheets and further form nanofibrils and macroscopic membranes. The self-assembled nano/microstructures have been successfully used as scaffoldings for tissue repair [3–5] and neurite outgrowth [6,7], as well as a carrier for controlled delivery of an endothelium cell growth factor in vivo [8]. Our early publication demonstrated the capability of a self-assembling peptide, EAK16-II, carrying a hydrophobic compound and

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delivering it to a cell membrane mimic [9]. In this paper, we report detailed studies on how EAK16-II interacts with a model hydrophobic compound as well as a hydrophobic anticancer agent to form colloidal suspensions in aqueous solution. The self-assembling peptide EAK16-II consists of 16 amino acids in sequence with alternating charged (E and K) and hydrophobic (A) residues; the charged residues are arranged in a type II fashion “− − + + − − + +”. This unique arrangement of the amino acids provides an amphiphilic property for EAK16-II molecules, with hydrophobic and charged residues lying in the opposing directions (Fig. 1a). It has been found that EAK16II can self-assemble into ␤-sheet riched nanofibrils through hydrogen bonding, ionic complementarity and hydrophobic interaction [2,10,11]. These ␤-sheet structures are very stable at extreme pHs (1.5 and 11) and in the presence of various proteases (trypsin, ␣-chymotrypsin and pronase) and denaturation agents (sodium dodecyl sulfate and urea) [12]. In addition, EAK16-II has shown no immune responses when being introduced into mice, rabbits and goats [2,13]. These features make EAK16-II

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Fig. 1. (a) Molecular structure of EAK16-II. The shaded red and blue regions indicate the charged and hydrophobic residues, respectively, on the opposite side of the peptide backbone. (b) Pyrene crystals in water with and without EAK16-II before mechanical stirring. (c) Formation of colloidal suspensions of pyrene microcrystals in EAK16-II solutions after 12 h stirring. The control sample without EAK16-II remained transparent with pyrene crystals afloat on top or precipitating at the bottom. The pyrene concentration was 0.50 mg/mL, and the EAK16-II concentrations ranged from 0.024 to 0.20 mg/mL (left to right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

a good candidate as a carrier for hydrophobic compounds, such as anticancer drugs. It is expected that the hydrophobic region of EAK16-II can interact with hydrophobic compounds while the charged residues stabilize the complex in aqueous solution. Solubilization and stabilization of hydrophobic compounds in aqueous systems is crucial in pharmaceutical industry. Many drugs with very low water solubility have limited or no activity in clinical usage due to the difficulty of transferring them in aqueous physiological systems. Thus, these hydrophobic drugs often require amphiphilic molecules to either provide hydrophobic microdomains or form stable colloidal dispersions for solubilization. Micelles are widely studied and

applied to hydrophobic drug delivery [14]. Similar systems include polymeric vesicles [15–19], microemulsions [20–22], liposomes [23–25] and nanoparticles [26–29]. All of these involve self-assembly of various amphiphilic molecules to provide hydrophobic microdomains to contain hydrophobic drugs. Comparing to these well-characterized delivery systems, selfassembling peptides are emerging biomaterials with a potential to outperform the current systems for hydrophobic drug delivery [9]. This paper studies the ability of EAK16-II interacting with hydrophobic compounds in forming colloidal suspensions in aqueous solution. Our goal is to investigate how EAK16-II

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interacts with hydrophobic compounds and characterize the properties of the peptide–hydrophobic compound colloids. Two hydrophobic compounds are selected in this work. Pyrene is used as a model hydrophobic compound for its low water solubility (∼ 6 × 10−7 M) [30,31] and well-characterized fluorescence properties [31–33]. The hydrophobic anticancer agent ellipticine is chosen to demonstrate the potential of EAK16-II as a carrier for stabilizing and delivering hydrophobic drugs. Initial investigation indicates that EAK16-II interacts with hydrophobic pyrene to form colloid suspensions in aqueous solution. The effect of EAK16-II and pyrene concentrations on their colloid formation is studied with three techniques: optical visualization, spectroscopy and microscopy. Steady-state fluorescence spectroscopy is a primary experimental method used to obtain information about the interaction of EAK16-II with hydrophobic compounds. Scanning electron microscopy (SEM) is used to visualize the peptide–hydrophobic compound colloids and to estimate their size distributions. The detailed structural information on the EAK16-II coating on the surface of the hydrophobic compounds is investigated by atomic force microscopy (AFM) on a model hydrophobic surface, highly ordered pyrolytic graphite (HOPG). Results on the interaction of EAK16-II with ellipticine are presented. The information obtained by the combination of these techniques provides a rather comprehensive collection of knowledge on interactions between hydrophobic compounds and the self-assembling peptide EAK16-II, leading to future development of self-assembling peptide based carriers for hydrophobic drug delivery. 2. Experimental 2.1. Materials The peptide, EAK16-II (1657 g/mol, crude), was purchased from Invitrogen (Burlington, Canada) and CanPeptide Inc. (Montr´eal, Canada), and used without further purification. The N- and C-termini were protected with acetyl and amide groups, respectively, to avoid end-to-end interaction. Pyrene and the anticancer agent ellipticine (99.8% pure) were obtained from Sigma–Aldrich (Oakville, Canada). Pyrene was recrystallized three times in ethanol to eliminate any impurities prior to use while ellipticine was used without further purification. Tetrahydrofuran (THF, distilled in glass 99.9%) and ethanol (HPLC, 99.8%) were purchased from Caledon Laboratories Ltd. (Georgetown, Canada). Highly ordered pyrolytic graphite (HOPG, ZYB grade) was bought from SPI Supplies (West Chester, USA). 2.2. Formation of colloidal suspensions Amounts of pyrene (0.50 mg/mL) or ellipticine crystals (0.20 mg/mL) were added into freshly prepared EAK16-II solutions with various peptide concentrations ranging from 0.01 to 0.20 mg/mL. Solutions of pyrene and ellipticine with appropriate concentrations were prepared in pure water as controls. A magnetic stir bar was put in each sample vial, and all samples were continuously stirred at 900 rpm on a stir plate (Fisher Scientific

Inc., Canada) at room temperature; while stirring, each sample vial was wrapped with a thin parafilm on the cap to avoid possible evaporation, and covered with aluminum foil to avoid light degradation on the compounds. The samples were photographed with a digital camera (Cannon PowerShot A95) initially, 12 h after and 6 days after. After 1 week of stirring, aliquots of the solutions were taken from the vials to prepare specimens for SEM imaging; the fluorescence spectra of the solutions were also acquired. 2.3. Factors influencing the formation of colloidal suspensions It is known that EAK16-II can self-assemble into nanofibrils over time [10]; thus, one can expect that, at a given time, peptide monomers and their assemblies (protofibrils and mature fibrils) coexist in the solution. The amount of each peptide (assembly) state is related to the age of the peptide solution; peptide monomers/protofibrils are dominant species in the solution initially, and they gradually turn into mature fibrils. To better understand if the solution age affects the formation of colloidal suspensions, peptide solutions (0.10 mg/mL) were prepared in three different manners (related to the solution age) before mixing with pyrene. The first peptide solution was freshly prepared to assure that peptide monomers and protofibrils are dominant. The second one was made 2 weeks prior to the mixing to allow the formation of mature fibrils. The third one was diluted from a 0.40 mg/mL peptide stock solution, which was stored at 4 ◦ C for about 1 month. The high concentration and 1-month storage were expected to drive peptide self-assembly toward mature fibrils. The solutions were then transferred to 7 mL sample vials containing a thin film of solid pyrene at the bottom, to have a final pyrene concentration of 0.50 mg/mL. The thin film of pyrene was prepared in the following procedure: pyrene crystals were dissolved in THF to make the stock solution. One milliliter of the pyrene–THF stock solution was transferred into a glass vial and dried with nitrogen gas for 1 h; a thin film of solid pyrene was formed at the bottom of the vial. This procedure is referred to as the “THF film method” throughout the paper. This method was used to prepare solutions that have very low pyrene concentrations, which could not be achieved by directly weighing the pyrene solid crystals. The pyrene–peptide samples were stirred for 2 days, and then monitored with a spectrofluorometer. One may suspect that the way of pyrene–peptide mixing also affects the formation of the colloidal suspensions. To elucidate this effect, one sample containing 0.40 mg/mL peptide and 0.10 mg/mL pyrene was tested using three different mixing methods: volumetric shaking, bath sonicating and magnetic stirring. Each method has a different mixing mechanism: volumetric shaking provides the mildest disturbance in solution, mainly through shear flow, among all methods; bath sonicating generates molecular vibration as medium disturbance in solution; magnetic stirring is expected to cause the largest solution disturbance by directly applying mechanical forces to the solution. The sample was first put into a bench top incubated shaker (Model 3527-5, Lab-Line Instruments, Inc., Melrose Park, IL, USA) at a speed of 250 rpm for 70 h. It was then sonicated in an ultrasonic

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cleaner (Model 2510, Branson Untrasonics Corp., Danbury, CT, USA) at 55 ◦ C for 17 h. After that, the sample was further stirred using a magnetic stir bar for 2 days. The fluorescence spectra of pyrene were collected right after each mixing method was applied. 2.4. Time dependence of complex/colloid formation Two concentrations of EAK16-II, 0.01 and 0.50 mg/mL, were used in this test. Pyrene crystals were put into the EAK16-II solutions to have a pyrene concentration of 0.10 mg/mL (0.5 mM). All solutions were continuously stirred for 1 week at room temperature, and the change in their fluorescence spectra was monitored continuously on a spectrofluorometer. The testing frequency was once an hour initially and gradually decreased to once a day after 30 h. The total experimental period was 5 days. 2.5. Concentration dependence The colloidal suspensions were prepared with different EAK16-II and pyrene concentrations using the THF film method. For each set of samples, peptide concentrations ranged from 0.00001 to 0.40 mg/mL with a fixed pyrene concentration. Pyrene concentrations were 0.025, 0.05 and 0.50 mg/mL for three sets of samples, respectively. All samples were stirred for 2 days at room temperature before being monitored with a spectrofluorometer. 2.6. EAK16-II self-assembly on HOPG surface The self-assembly of EAK16-II on hydrophobic HOPG surface was investigated to reveal the nature of interaction between the peptide and the hydrophobic compounds. The sample was prepared in the following procedure: a freshly cleaved HOPG surface was affixed on an AFM liquid sample plate using a double-sided tape. The liquid cell was set up on the sample plate and filled with 500 ␮L pure water. One hundred microliters of EAK16-II stock solutions (0.022 mg/mL) was injected into the liquid cell using a 100 ␮L syringe. The sample was incubated inside the AFM environmental chamber for 1.5 and 9 h to allow adhesion and self-assembly of the peptide on the HOPG surface, followed by liquid AFM imaging (see below).

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ing ellipticine, they were excited at 294 nm while the emission was acquired at wavelengths from 320 to 580 nm. The fluorescence intensities of pyrene were averaged from 500 to 530 nm to give the intensity value of the excimer for each sample. The fluorescence spectrum of pyrene crystals was collected using a triangular cell while that of ellipticine was acquired using a solid sample holder on the type QM4-SE spectrofluorometer. A standard was used in each run to correct for lamp intensity variations. One solution with 0.10 mg/mL EAK16-II and 1.0 mg/mL pyrene was monitored 10 times for error analysis. The error within 95% confidence level was found to be ±0.0009 a.u. 2.8. Scanning electron microscopy LEO model 1530 field emission SEM (GmbH, Oberkochen, Germany) was employed to study the morphology and dimension of the pyrene–peptide and ellipticine–peptide colloids. The SEM samples were prepared by depositing 10 ␮L of the colloidal suspensions on a freshly cleaved mica surface. The mica was affixed on an SEM stub using a conductive carbon tape. The sample was settled under a Petridish-cover for 10 min to allow the complexes adhering to the mica surface. It was then washed twice with a total of 200 ␮L pure water and air-dried in a desicator overnight. All samples were coated with a 20 nm thick gold layer prior to the imaging; the images were acquired using the secondary electron (SE2) mode at 5 kV. 2.9. Atomic force microscopy The liquid AFM imaging was performed on a PicoScanTM AFM (Molecular Imaging, Phoenix, AZ) using tapping mode. A scanner with the maximum scan size of 6 ␮m × 6 ␮m was used with a silicon nitride tip, which has a nominal spring constant of 0.58 N/m (DNP-S, Digital Instruments) and a typical tip radius of 10 nm. For the best imaging, the typical tapping frequency was set between 16 and 18 kHz and the scan rates were controlled between 0.8 and 1 Hz. To avoid evaporation of the peptide solution, all liquid imaging was performed in an environmental control chamber at room temperature. All images were taken with a resolution of 256 × 256 pixels. 3. Results and discussion 3.1. Interaction of peptides with a model hydrophobic compound—pyrene

2.7. Steady-state fluorescence All fluorescence spectra were acquired on a spectrofluorometer with either type LS-100 or type QM-4SE (Photon Technology International, London, Ont., Canada). The light source used for type LS-100 was a pulsed xenon flash lamp while that used for type QM-4SE was a continuous xenon lamp. For each sample, approximately 3 mL of solution were transferred from a vial into a square quartz cell (1 cm × 1 cm) through a pasteur glass pipette. For samples containing pyrene, they were irradiated at 336 nm, and the emission fluorescence spectra were collected at the wavelengths ranging from 350 to 600 nm; for those contain-

Hydrophobic compounds are difficult to dissolve in aqueous systems due to their poor water solubility. The solubility of pyrene and ellipticine in water was found to be ∼6 × 10−7 M [30,31] and 6.2 × 10−7 M [34], respectively. In order to solubilize such hydrophobic compounds in aqueous systems, amphiphilic molecules are often required [14,19]. The selfassembling peptide EAK16-II has an amphiphilic property with hydrophobic residues (A) on one side and charged residues (E and K) on the other side (Fig. 1a). This peptide is expected to interact with hydrophobic compounds and stabilize them in aqueous solution.

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Pyrene was used as a model hydrophobic compound in this study because of its well-characterized fluorescence properties [30,32,35,36]. When pyrene crystals were added to EAK16-II solutions, the initially clear solutions (Fig. 1b) became turbid (Fig. 1c) after 12 h stirring; in the meanwhile, the control sample without EAK16-II remained clear. The pyrene crystals in the control sample vial either stayed afloat on top of the solution or deposited at the bottom. The clouding phenomenon seemed to be independent on the peptide concentration (0.01–0.20 mg/mL) as no significant difference in turbidity was observed among samples in Fig. 1c. It should be noted that, however, the detailed information of colloid formation cannot be examined by visualization of the samples’ turbidity; hence, fluorescence spectroscopy was employed to characterize the pyrene–peptide complex formation (see the following sections). These results indicate that EAK16-II is capable of stabilizing the pyrene crystals in water by forming colloidal pyrene–peptide complexes. The formation of pyrene–peptide complexes can also be shown from the fluorescence spectra of pyrene in Fig. 2. The fluorescence spectrum of the colloidal suspensions with 0.20 mg/mL EAK16-II and 0.50 mg/mL pyrene exhibited a broad peak with a peak maximum at ∼470 nm. The rising of such peak indicates the formation of pyrene excimers. Pyrene excimers can be formed either through diffusional encounters between two pyrene molecules or from the direct excitation of preassociated pyrene dimers [36]. In order to know which pathway is attributed to the observed excimer peak from the colloidal suspensions, the fluorescence spectrum of solid pyrene crystals was acquired, and it looked very similar to that of the colloidal suspensions. Our early publication also proved that pyrene was in a crystalline state in these stabilized colloids by time-resolved fluorescence measurements [9]. On the other hand, the control sample (pyrene in water) exhibited a spectrum with relatively low fluorescence intensity and several spikes between 370 and 400 nm, which is the characteristic of minute soluble pyrene monomers in water; no trace of pyrene excimers was observed since the concentration of soluble pyrene in water was too low

to form excimers, and the pyrene crystals were not stable in water without the peptide. As a result, the presence of EAK16II certainly helps stabilize pyrene crystals by forming colloidal pyrene–peptide complexes in water.

The peptide EAK16-II has been found to be able to selfassemble over time into fibrils or membranes [2,10]. The self-assembly process takes several days to a few weeks to approach equilibrium [10]. The question arises as to whether the single peptide molecules or the peptide fibrils are more effective to interact with pyrene. To answer this question, a series of experiments were performed using the steady-state fluorescence technique. Fig. 3 shows the fluorescence spectra obtained from three colloidal suspensions, which were prepared in three different ways as described in Section 2. All three samples contained the same amount of pyrene (0.50 mg/mL) and EAK16-II (0.10 mg/mL). The freshly prepared solution had the highest excimer peak, while the solution diluted from a high concentration peptide solution (0.40 mg/mL) prepared a month earlier had the lowest excimer intensity. The middle spectrum was obtained from the solution prepared 2 weeks prior to the measurement. The fluorescence excimer intensities infer the amount of pyrene crystals that are stabilized in the solution: the higher the fluorescence excimer intensity is, the more the pyrene crystals may be solubilized in the solution. The observed fluorescence excimer intensities varied in the three samples with different solution preparation methods may be related to the amounts of peptide fibrils present in the solutions. Since the self-assembly of EAK16-II occurs over time, all solutions should have some peptide assemblies—mainly peptide fibrils. However, more peptide fibrils would be expected to form in an old peptide solution than in a freshly prepared one. If the “mature” peptide fibrils are more effective to stabilize the pyrene crystals, higher fluorescence excimer intensity will be expected. However, as shown in Fig. 3, the fluorescence excimer inten-

Fig. 2. Fluorescence spectra of pyrene crystals in solid state, water and EAK16II solution. The pyrene and EAK16-II concentrations were 0.50 and 0.20 mg/mL, respectively. The fluorescence intensities of pyrene solid crystals and pyrene in peptide solution were normalized according to their peak maxima, while that of pyrene in water was normalized according to the fluorescence signal of pyrene in peptide solution for the comparison.

Fig. 3. Effect of peptide self-assemblies on the formation of pyrene–peptide complexes. All the samples have a pyrene concentration of 0.50 mg/mL and an EAK16-II concentration of 0.10 mg/mL. The samples were prepared freshly (blue line), 2 weeks prior to the measurement (red line), and by diluting a concentrated solution with 0.40 mg/mL EAK16-II (green line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. Important factors affecting complex formation

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sities from the two solutions containing more EAK16-II fibrils were lower than that from the freshly prepared solution. This indicates that mature peptide fibrils, which were formed after a longer period in solution, are less effective at stabilizing pyrene crystals than the peptide monomers, which should be abundant in the freshly prepared peptide solution. This conclusion may be because peptide molecules and small peptide aggregates, such as protofibrils, in young peptide samples are more surface active and tend to associate with hydrophobic pyrene. In fact, in other peptide self-assembly systems, it has been reported that peptide protofibrils and their precursors are more active binding to cell membranes and cause cell death [37,38]. In addition to the peptide (assembly) states (i.e., monomers, protofibrils and mature fibrils), the mechanical stirring of the solution was found to be another crucial factor affecting the formation of pyrene–peptide colloids. Three mixing methods, stirring, shaking and sonicating, were studied and compared. The fluorescence spectra of the solutions with the three mixing methods are shown in Fig. 4. For shaken and sonicated solutions, the fluorescence spectra revealed typical features of pyrene monomer in water, with no trace of an excimer peak, and the solutions remained transparent. This indicates that the aqueous solutions were saturated with pyrene monomers, within the limit of its solubility. The stirred solution, on the other hand, became turbid and exhibited a large excimer peak. This implies that the pyrene–peptide solution requires a specific type of mechanical disturbance for the complex formation. Possible explanations for this unexpected result include: (i) the ability of the magnetic stir bar to break up pyrene crystals into micro/nanocrystals which can be more easily stabilized by the peptide molecules and (ii) the formation of excessive active peptide protofibrils by the mechanical stirring. Further experiments need to be carried out to examine these possibilities. 3.3. Complex formation kinetics The formation of pyrene–peptide complexes was monitored over time with fluorescence techniques to study the complexation kinetics. The excimer intensities were plotted as a function

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Fig. 5. Time-dependent pyrene–peptide complex formation. Two EAK16-II concentrations, 0.01 and 0.50 mg/mL, were used with a fixed pyrene concentration of 0.10 mg/mL. The time for pyrene uptake is about 20 h for both EAK16-II concentrations.

of time in Fig. 5. Two EAK16-II concentrations, either above (0.50 mg/mL) or below (0.01 mg/mL) the critical aggregation concentration (CAC, ∼0.1 mg/mL) [10], were used with a pyrene concentration set at 0.10 mg/mL. It was found that, for both EAK16-II concentrations, the fluorescence excimer intensities increased with time and reached a plateau after ∼20 h. These plateaus represent the maximum amount of pyrene crystals that can interact with the peptide and be stabilized in solution. Thus, the different plateau values show that at a given pyrene concentration, more pyrene can be stabilized in solution if a higher peptide concentration is used. Similar results have been found in the concentration-dependent studies, which will be discussed later (see Section 3.4). The two profiles can be simply fitted with an exponential equation, to determine the time scale of the complex formation: I = A(1 − e−kt )

(1)

where I is the fluorescence excimer intensity, and A is a constant representing the plateau value in Fig. 5. Normally, 1/k indicates the risetime, and the plateau is reached after approximately five times the risetime. Thus, 5(1/k) represents the time required for the completion of the complex formation. It is worth noting that Eq. (1) is purely mathematic, not representing a mechanism of the complex formation. The kinetic parameters for the complex formation fitted with Eq. (1) were listed in Table 1. The results showed that the complex formation was complete after about 22 ± 2 h for both EAK16-II concentrations. It seems that the peptide concentration has little influence on the rate of formation of Table 1 Fitting parameters of complex formation kinetics, Eq. (1) Fitting parameters

Fig. 4. Effect of solution disturbance on the formation of colloidal suspensions. All solutions contain 0.10 mg/mL pyrene and 0.50 mg/mL EAK16-II. The pyrene–peptide solution with mechanical stirring exhibits an excimer peak (red line); on the other hand, shaking and sonicating methods lead to a pyrene monomer spectrum (black line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

A k (h−1 ) 5(1/k) (h)

EAK16-II concentration (mg/mL) 0.01

0.50

0.039 ± 0.004 0.228 ± 0.025 21.9 ± 2.4

0.054 ± 0.004 0.232 ± 0.022 21.6 ± 2.0

A: plateau value; k: rate constant of the formation of stabilized pyrene crystals in solution (complex formation); 5(1/k): approximate time required for complete complex formation. All errors are calculated at the 95% confidence level.

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pyrene–peptide complexes. Such an observation was unexpected as more peptides were thought to be more effective at interacting with pyrene. One possible reason could be that the factor of mechanical stirring overwhelms other factors such as peptide concentration. The rate limiting step may be the mechanical stirring to break up the pyrene crystals into microcrystals, which are subsequently stabilized by the peptide. If this speculation is true, it may result in a similar size distribution of pyrene microcrystals in the complexes (see below). However, only mechanical stirring without the presence of peptides cannot induce the formation of colloidal suspensions as shown in Fig. 1b and c (controls). Therefore, it should be emphasized that both mechanical stirring and amphiphilic peptides are necessary to stabilize hydrophobic compounds and form colloidal suspensions in water. 3.4. Concentration dependence of complex formation A wide range of peptide concentrations was prepared with three pyrene concentrations to study the concentration dependence of the complex formation. The excimer intensities (after 2 days stirring) were plotted as a function of peptide concentrations (0.00001–0.40 mg/mL) in Fig. 6. Each profile has a fixed pyrene concentration: 0.025 mg/mL (open triangles), 0.05 mg/mL (crosses) and 0.50 mg/mL (open squares). For each curve, the excimer intensity first increases with increasing peptide concentration and then reaches a plateau. The data points on each profile reflect the amount of pyrene that could be stabilized by the peptide in water. When the peptide concentration is too low, only a limited amount of pyrene crystals can be stabilized to form colloidal suspensions. When the peptide concentration is high enough, almost all pyrene crystals are stabilized in the solution, resulting in a plateau region in each profile. As a result, the breaking point between the rise region and the plateau may represent the minimum amount of peptide that is required to stabilize all pyrene at a given pyrene concentration. The position of the breaking point can be estimated by fitting the data on each region with a straight line, one sloping up for the rise region and one horizontal for the plateau. The intersection of the two fitting lines is considered to be the breaking point. The

Fig. 6. The effects of pyrene and EAK16-II concentration on the complex formation. The excimer intensities are plotted as a function of EAK16-II concentration (0.00001–0.40 mg/mL) at three different pyrene concentrations (0.025, 0.05 and 0.50 mg/mL). Each curve has a sharp increase and a plateau region. Between the two regions is a breaking point.

Table 2 Pyrene loading capacity at different pyrene concentrations Pyrene concentration (mg/mL) Rising slope Pyrene loading capacity (mol/mol)

0.025 0.19 ± 0.02 2.91 ± 0.68

0.05 0.45 ± 0.08 10.3 ± 2.6

0.50 1.39 ± 0.14 78.8 ± 9.8

The large errors are possibly due to that only a few data points were fitted for the two curves in Fig. 6 to generate the breaking point. The pyrene loading capacity increases with the increase of pyrene concentration, indicating that the pyrene loading capacity is highly related to the pyrene concentration.

ratio of the amount of pyrene at the breaking point to the corresponding EAK16-II concentration may be treated as the “loading capacity” of pyrene. This definition is different from that of a typical drug loading capacity, which is defined as the maximum amount of a drug that can be loaded by a given amount of a carrier. The pyrene loading capacities with different pyrene concentrations were listed in Table 2. It was unusual that the loading capacity was not a constant, and increased with increasing pyrene concentration. The loading capacity was 2.9 ± 0.7 mol pyrene/mol peptide at a pyrene concentration of 0.025 mg/mL, and increased to 79 ± 10 mol pyrene/mol peptide at a pyrene concentration of 0.50 mg/mL. The difference between these values indicates that the pyrene loading capacity could be a function of pyrene concentration. Normally, the loading capacity of a hydrophobic compound in a colloidal system, e.g., micelles, is independent of its concentration; the loading capacity is simply based on the partition coefficient of the hydrophobic compounds between the stabilizer and the aqueous phase [16,18,30,39,40]. The observation that EAK16-II could stabilize more pyrene in water at a higher pyrene concentration may be rationalized as follows. First, the stabilized pyrene in the pyrene–peptide complexes was in crystalline form (Fig. 2 and ref. [9]) in contrast to the monomerically solubilized pyrene in polymeric and surfactant micelles. In these systems, the hydrophobic compounds are incorporated inside the hydrophobic interior of the micelles. In this work, however, the peptide molecules seem to adsorb onto the surface of the hydrophobic pyrene crystals and stabilize them in solution. When the pyrene crystal is larger, the surface area of the crystal is relatively smaller; hence, fewer peptide molecules are required. In order to stabilize the pyrene crystals at high pyrene concentrations by a certain number of peptide molecules, one way is to enlarge the crystal size so that the total surface area of crystals can be reduced. Thus, the different values of pyrene loading capacity may imply that the surface area per volume of the pyrene crystals decreases with increasing pyrene concentration, i.e., the sizes are larger. Second, the binding of the peptide to the pyrene crystals might exhibit a cooperative relationship, where the previously bound pyrene crystals could possibly affect the binding of subsequent ones. The cooperative property is well known in molecular binding systems such as the binding of O2 to hemoglobin [41,42]. Cooperative peptide binding onto the pyrene crystals could be one reason why the pyrene loading capacity changes at different pyrene concentrations.

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Fig. 7. Size distribution of the pyrene–peptide complexes with different EAK16II concentrations (0.024, 0.05, 0.10 and 0.20 mg/mL). The pyrene concentration is 0.50 mg/mL. The size distribution was generated by analyzing the area of the complexes from SEM images using ImageJ software. Peptide concentration does not have significant influence on the size distribution of the complexes.

To verify whether the size of stabilized pyrene crystals is dependent on the peptide concentration, SEM was conducted to study the size of the pyrene–peptide complexes. Over 2000 complexes, observed on at least six SEM images (1000× magnification) for each peptide concentration (0.024–0.20 mg/mL) with a fixed pyrene concentration of 0.50 mg/mL, were analyzed

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by ImageJ 1.34s (free software, National Institute of Health, USA) to get the surface area of each particle. The surface area was than converted to an equivalent diameter by assuming that the particle was spherical. The frequency percentage of the complexes with different equivalent diameters at a given peptide concentration was plotted in Fig. 7. No significant difference in the size distribution was observed between different peptide concentrations as over 90% of the complexes have an equivalent diameter below 5 ␮m for each peptide concentration. Detailed examinations on the size distribution showed that there seemed to be some minor differences in frequency percentage: more complexes with the size smaller than 0.5 ␮m were observed at high peptide concentrations (0.10 and 0.20 mg/mL) while the most abundant population of the complexes at 0.024 and 0.05 mg/mL peptide concentrations were 1–5 and 0.5–1 ␮m, respectively. The SEM results showed that the size of the pyrene–peptide complexes was not strongly dependent on the peptide concentrations. This seems to support our previous speculation that the mechanical stirring may be more important for the complex formation than the peptide concentration (see Section 3.3). All samples were experienced the same amount of mechanical stirring, which may result in a similar size distribution of the pyrene–peptide complexes regardless of peptide concentration.

Fig. 8. SEM images of the pyrene–peptide complexes with different EAK16-II concentrations: 0.024 mg/mL (a), 0.05 mg/mL (b), 0.10 mg/mL (c) and 0.20 mg/mL (d). The pyrene concentration is 0.50 mg/mL. The peptide coatings on the complexes have different morphology depending on the EAK16-II concentration.

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This result indicates that different pyrene loading capacities cannot be rationalized by the size of the stabilized pyrene crystals. Further experiments are needed to verify whether the cooperative binding between peptides and pyrene or other factors would cause the different pyrene loading capacities mentioned above. Although peptide concentration did not have much influence on the size of the pyrene–peptide complexes, it affected the peptide coating on the pyrene micro/nanocrystals. As shown in Fig. 8, the morphology of the peptide coating changed according to the peptide concentration. A low peptide concentration (0.024 mg/mL) resulted in porous coating while a high peptide concentration (0.20 mg/mL) had solid coating; both porous and solid coatings existed when the peptide concentrations were in between these two concentrations. This demonstrates that the peptide coating on complexes can be controlled. One implication could be in controlled release of the hydrophobic compound into the environment. The early work showed that the release rate of pyrene from a porous coated pyrene–peptide complex was faster than that from a solid coated one [9].

3.5. Interaction of EAK16-II with a hydrophobic HOPG surface The SEM images show the peptide coating on the pyrene micro/nanocrystals; however, the detailed features of the peptide coating cannot be seen, leading to a question how EAK16II adheres onto the surface of the hydrophobic compounds to form the peptide coating. To answer this question, liquid AFM imaging was performed on HOPG surfaces, which served as a model hydrophobic surface for peptide coating. The atomic flat HOPG surface provides a perfect imaging platform to directly observe the peptide coating process and the nanoscale peptide assemblies; AFM visualizes the surface morphology in a liquid environment, which may reveal a more realistic circumstance of how peptides interact with hydrophobic compounds in solution. Fig. 9 shows the AFM images of peptide nanofibrils on HOPG in water. The images were collected after 1.5 h (Fig. 9a) and 9 h (Fig. 9b) incubation on HOPG. It is clearly seen that the amount of peptide nanofibrils on HOPG increased with a longer

Fig. 9. AFM images of peptide self-assembly on the hydrophobic HOPG surface in water at different incubation time: 1.5 h (a) and 9 h (b). The left images represent the topography images while the right ones are phase images. The surface is covered by the EAK16-II nanofibrils, and the surface coverage increases with time from 19.2 to 76.8%. The scale bar represents 200 nm.

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incubation time. The surface coverage of the peptide nanofibrils was obtained using ImageJ software; phase images (right) were used for the image analysis because they provided better contrast and resolution. The surface coverage increased from 19 to 77% as the incubation time increased from 1.5 to 9 h. These images clearly shows that the peptide coating on the hydrophobic HOPG surface is made of many parallel patterned nanofibrils with dimensions of ∼7 nm in width and 0.5–1 nm in height. Comparing the dimension of peptide nanofibrils with that of a single, ␤-stranded EAK16-II (∼6 nm in length and ∼0.7 nm in width), it may be concluded that the nanofibrils are made of a single layer of EAK16-II ␤-sheets. In this case, the hydrophobic side of EAK16-II is expected to interact with the hydrophobic HOPG surface, leaving the charged residues facing the liquid phase; hence, the hydrophobicity of the HOPG surface can be reduced. Recently, Yang et al. [43] found that the EAK16-II modified HOPG surface had a reduced water contact angle, indicating the modified surface became relatively hydrophilic. This may explain why the peptide coating can stabilize hydrophobic compounds in aqueous solution. It should be noted that the dimension of these peptide nanofibrils are so small that they could not be observed from the SEM images. The formation of the patterned peptide nanofibrils may be due to the way of peptide self-assembly on HOPG surfaces. More details of EAK16-II self-assembly on HOPG are currently under investigation. These results demonstrate that EAK16-II is capable of interacting with hydrophobic surfaces and forming nanofibrils to coat the surface; surface coverage of the peptide coating increases with time. 3.6. Formation of colloidal ellipticine–peptide complexes Ellipticine was selected as a model hydrophobic drug for the following reasons. First, it is extremely hydrophobic with a water solubility of 6.2 × 10−7 M [34] similar to that of pyrene; hence, amphiphilic molecules are required to stabilize them in aqueous solution. Second, ellipticine is capable of being fluorescent, and its photophysical properties make it easy to be monitored [44]. Third, ellipticine has potential anticancer activity [45]; a successful stabilization of ellipticine in aqueous systems would be the first step toward developing a delivery system for ellipticine and further for other hydrophobic anticancer agents. Similar to pyrene, ellipticine can be stabilized by EAK16-II to form colloidal suspensions in water (Fig. 10a). The solutions with the presence of EAK16-II became turbid with light yellow color after 6 days stirring while the control sample remained clear. The fluorescence spectrum of the colloidal suspensions had a major peak with the peak maximum around 470 nm (Fig. 10b). This spectrum is very different from that of molecularly solubilized ellipticine, which exhibits a peak ∼430 nm representing its neutral form and a peak ∼520 nm as its protonated form [44,46]. Although such a new fluorescence spectrum has not yet been reported in the literature, we suspected that it may come from the ellipticine crystals. The fluorescence spectrum of solid ellipticine crystals was then acquired to confirm this speculation, and the spectrum was very similar to that of the colloidal suspensions with a sharp peak around 470 nm. This

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Fig. 10. Interaction of EAK16-II with the anticancer agent ellipticine. (a) The formation of colloidal suspensions of ellipticine–peptide solutions after 12 h stirring. The control sample without EAK16-II remains clear. The ellipticine concentration is 0.20 mg/mL, and the EAK16-II concentrations vary from 0.024 to 0.20 mg/mL. (b) Fluorescence spectra of ellipticine crystals in solid state (orange), water (blue) and 0.20 mg/mL EAK16-II solution (red). The fluorescence intensities of ellipticine solid crystals and in peptide solution were normalized according to their peak maxima, while that of ellipticine in water was normalized according to of ellipticine in peptide solution for the comparison. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

indicates that the stabilized ellipticine in the ellipticine–peptide complexes is also in crystalline form, just like stabilized pyrene micro/nanocrystals. The control sample of ellipticine in water without EAK16-II had a similar fluorescence spectrum but with much less intensity. This may be due to the tiny amounts of ellipticine crystals suspended in water after mechanical stirring. A shoulder at ∼520 nm of the spectrum from the ellipticine in water indicates the presence of some protonated ellipticine. Another evidence of the formation of colloidal ellipticine–peptide complexes was shown in the SEM images (Fig. 11). The large ellipticine crystals were broken down into micro/nanocrystals with the presence of EAK16-II, in combination of mechanical stirring. There are several advantages using self-assembling peptides in stabilizing hydrophobic compounds in aqueous systems, particularly for drug delivery. First, these peptides are able to stabilize large amounts of hydrophobic compounds in the form of micro/nanocrystals, and form colloidal suspensions. It is more efficient than using micelles, which usually stabilize monomeric hydrophobic molecules. The large capacity of these peptides stabilizing hydrophobic compounds makes them beneficial in hydrophobic drug delivery, where less delivery vehicles would

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the mature peptide fibrils, and the formation of the colloidal suspensions need both peptides and mechanical stirring. The complex formation took about 22 ± 2 h to complete. The peptide concentration did not have a significant influence on the size of the complexes, but affected the peptide coating on the surface of the complexes. The pyrene “loading capacity” of EAK16-II was found to be highly related to the pyrene concentration. These results demonstrate the ability of the self-assembling peptide EAK16-II stabilizing hydrophobic compounds in aqueous solution, which sets up the future work toward developing peptide carriers for hydrophobic drugs. Acknowledgments This research was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Research Chairs (CRC) program and the Canada Foundation for Innovation (CFI). We are grateful to Jeremy Bezaire and Christine Keyes-Baig for providing help in initial fluorescence experiments. We also thank Dr. Mohammand Elias Biswas for the general information on molecular binding, and Dr. Jean Duhamel and Dr. Yooseong Hong for helpful discussions. References

Fig. 11. SEM images of ellipticine crystals (a) and ellipticine–peptide complexes (b). Both the ellipticine and EAK16-II concentrations are 0.20 mg/mL. The presence of EAK16-II with mechanical stirring breaks up the ellipticine crystals and stabilizes them in water.

be required. Second, self-assembly of these peptides could generate a protective layer coated on the surface of hydrophobic compounds to control their release and to protect them from being attacked by degradation agents. In addition, the peptide protective layer may reduce the chance of exposing the drug to normal tissues after administration, rendering less side effects. Third, the peptide sequence can be designed to incorporate certain biological functions, such as cell targeting [47,48] and membrane penetration [49,50]. This study has set up a beginning toward developing future delivery systems of hydrophobic compounds using self-assembling peptides. 4. Conclusions The self-assembling peptide EAK16-II was shown to stabilize a model hydrophobic compound pyrene and the hydrophobic anticancer agent ellipticine in water. The mixing of the hydrophobic compounds with EAK16-II in solution involving mechanical stirring resulted in the formation of colloidal suspensions. The stabilized pyrene and ellipticine in sub-micron complexes were in crystalline form as the peptide assemblies coated on the surface of the micro/nanocrystals. Detailed studies were done using the model hydrophobic compound pyrene and the results lead to the following: the peptide monomers and protofibrils were more effective to stabilize pyrene crystals than

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