Adsorption and Disruption of Lipid Bilayers by Nanoscale Protein

Spectroscopic and microscopic studies of vesicle rupture by AgNPs attack to screen the cytotoxicity of nanomaterials. Ha Nee Umh , Younghun Kim. Journ...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/Langmuir

Adsorption and Disruption of Lipid Bilayers by Nanoscale Protein Aggregates Atsushi Hirano,† Hiroki Yoshikawa,‡ Shuhei Matsushita,‡ Yoichi Yamada,‡ and Kentaro Shiraki*,‡ †

Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8562, Japan ‡ Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan ABSTRACT: Nanoparticles taken into biological systems can have biological impacts through their interactions with cell membranes, accompanied by protein adsorption onto the nanoparticle surfaces, forming a so-called protein corona. Our current research aims to demonstrate that nanoscale protein aggregates behave like such nanoparticles with regard to the interaction with lipid membranes. In this study, the adsorption and disruption of the lipid membranes by protein aggregates were investigated using amyloid fibrils and nanoscale thermal aggregates of lysozyme. Both types of protein aggregates had disruptive effects on the negatively charged liposomes, similar to polycationic nanoparticles. Interestingly, adsorption of liposomes on the amyloid fibrils preceding disruption occurred even if the net charge of the liposome was zero, suggesting the importance of hydrophobic interactions in addition to electrostatic interactions. The results of the present study provide new insights into the biological impacts of nanoparticles in vivo.

■

and disruption31 of lipid membranes. It is also noteworthy that nanoparticles in biological systems may indirectly cause cytotoxicity through the amyloid fibril formation affected by them.32,33 Thus, the interactions of lipid membranes with artificial nanoparticles and amyloid aggregates have been studied in connection with cytotoxicity in research fields, such as nanotoxicology and biology. Our current research aims to demonstrate the similarity between nanoscale protein aggregates and artificial nanoparticles with regard to their interactions with lipid membranes. Our previous study indicated that both protein-adsorbed carbon nanotubes and amyloid fibrils showed identical effects on lipid membranes in physiological saline.34 Although such common effects on lipid membranes may be ascribed to their nanoscale size, little is known about the details of their interactions. In the present study, to clarify the effects of nanoscale protein aggregates on lipid membranes, amyloid fibrils of lysozyme at each phase of the fibrillation process and start aggregates of lysozyme were used. Lysozyme is often used to study amyloid fibril formation.35,36 Particularly, hen egg white lysozyme is one of the best-characterized proteins,37−40 and therefore lysozyme was used as a model protein in the present study. Disruption of the lipid membranes by the amyloid aggregates increased with growth of amyloid fibrils accompanied by increases in their number, which can be accounted for by their irreversible

INTRODUCTION The increases in both basic and applied research into nanoparticles for nanotechnology and biotechnology have raised concerns regarding the possible environmental and health issues related to nanoparticles. It has been reported that uptake of nanoparticles into biological systems can cause cytotoxicity.1−7 Accordingly, it is a key challenge to control the balance between the advantages and disadvantages of nanoparticles.8 One of the most important factors affecting the balance is the interaction between nanoparticles and cell membranes, which is dependent on the electrostatic interaction, shape, size, surface area, flexibility, surface chemical properties, and amphipathic character of the nanoparticles.9−17 When the nanoparticles are charged, electrostatic interactions mainly determine their interplay with lipid membranes.9,12 In addition to artificial materials, biopolymers such as proteins and peptides can also form nanoscale structures. Amyloid aggregates are among the most well-known nanoscale protein aggregates. The deposition of insoluble amyloid fibrils in specific tissues and organs causes several diseases, such as type II diabetes, Alzheimer’s disease, and Parkinson’s disease.18−20 Although amyloid fibrils, which share a common cross β-sheet structure,21 were initially considered to be the cytotoxic species involved in amyloid diseases, many recent reports have focused on soluble oligomers as the primary cytotoxic species.22−26 However, several recent reports have again drawn attention to the cytotoxicity of amyloid fibrils.27,28 A possible mechanism of the cytotoxic action of mature amyloid fibrils involves their impact on cell membranes, which was suggested by the observations that amyloid aggregates induce adsorption,29 aggregation,30 © 2012 American Chemical Society

Received: November 30, 2011 Revised: January 13, 2012 Published: January 25, 2012 3887

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

characteristic relaxation time τc can be found using the correlation function of the fluctuations; it is linked to the diffusion coefficient of nanoparticles D (protein aggregation in this case) by the following expression: D = 1/2τck2, where k is the wave vector of scattered light. Then, the hydrodynamic radius of the particles, Rh, can be calculated using the Stokes−Einstein equation: D = kBT/6πηRh, where kB is the Boltzmann constant, T is temperature, and η is solvent viscosity. Preparation of Liposomes. Unilamellar liposomes composed of DOPG/DOPC (1:1 molar ratio) and DOPC were prepared using the extrusion method with 200 nm pore size polycarbonate membranes. The appropriate amounts of lipids in chloroform were mixed; subsequently, the solvent was completely removed in a vacuum desiccator connected to a rotary vacuum pump for 12 h. An appropriate amount of 70 mM calcein (pH 7.4) was added to this dry lipid film, and then the suspension was vortexed for several seconds at room temperature. The solution was then extruded through 200 nm pore size polycarbonate membranes (Avanti Mini-Extruder; Avanti Polar Lipids, Inc., Alabaster, AL). Subsequently, to remove the untrapped calcein, the liposome suspension was eluted through a Sephadex G-75 column with 140 mM sodium chloride. The concentrations of phospholipids in the samples were determined on the basis of the Fiske−Subbarow method and the Bartlett method.45,46 Liposome Leakage Assay for Amyloid Fibrils. Calcein leakage from the liposomes induced by the amyloid aggregates was detected using a spectrofluorometer (FP6500; Japan Spectroscopic). Aliquots of 10 μL of 400 μM liposome suspensions prepared as described above were mixed with 1460 μL of 10 mM sodium phosphate buffer or 3.3 mM sodium citrate buffer (pH 7.4) containing 140 mM sodium chloride. The amyloid fibrils thus prepared were diluted 1.4-fold with water, 30 μL of which was added to the liposome solutions. The fluorescence intensities of the solution in the absence and presence of 6 mM sodium dodecyl sulfate (SDS) were designated as 0% and 100% leakage, respectively. Liposome Leakage Assay for Start Aggregates. Similar to amyloid fibrils, calcein leakage by the start aggregates was also detected using a spectrofluorometer. Aliquots of 10 μL of the 400 μM liposome suspensions prepared as described above were mixed with 740 μL of 20 mM sodium phosphate buffer (pH 7.4) containing 280 mM sodium chloride; subsequently, 750 μL of the start aggregates prepared as described above was added to the liposome solution. The fluorescence intensities of the solution in the absence and presence of 6 mM sodium dodecyl sulfate (SDS) were designated as 0% and 100% leakage, respectively. Measurement of Adsorbed Amounts of Lipids on Amyloid Fibrils. The amounts of adsorbed lipids on the amyloid fibrils were determined as follows. Aliquots of 26 μM liposome suspensions mixed with 0−40 μM amyloid fibrils, 140 mM NaCl, and 3.3 mM sodium citrate (pH 7.4) were incubated for 5 min or 5 h at 25 °C, and the mixed solutions were centrifuged at 9000 × g for 1 min. Sodium citrate buffer was used in place of sodium phosphate buffer because phosphate interferes with the detection of phospholipids by the Fiske−Subbarow method and the Bartlett method. The lipid concentrations in the supernatants were defined as the unadsorbed amounts determined by the Fiske−Subbarow method and the Bartlett method. Thus, adsorbed amounts of the lipids were determined by subtracting the unadsorbed amounts from the initial amounts.

adsorption onto the fibrils due to electrostatic and hydrophobic interactions. Interestingly, start aggregates being nonfibrillar nanoscale aggregates, which are generated at the initial stage of heating,41−44 also disrupted the lipid membrane. These results indicated that the interaction of nanoscale protein aggregates with lipid membranes is similar to that of artificial polyelectrolyte nanoparticles. Such interaction should be a universal characteristic of nanoscale protein aggregates, and it will also be valid in the protein corona.

■

EXPERIMENTAL SECTION

Chemicals. Chicken egg white lysozyme, sodium metabisulfite, sodium sulfite, ammonium molybdate tetrahydrate, and Sephadex-G75 were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium chloride, potassium chloride, hydrochloric acid, sodium hydroxide, hydrogen peroxide, 1-amino-2-naphthol-4-sulfonic acid, thioflavin T (ThT), sodium dihydrogenphosphate, calcein, sodium dodecyl sulfate (SDS), glycine, and citric acid were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) were obtained from NOF Corp. (Tokyo, Japan). All chemicals used were of reagent grade and used as received. Preparation of Amyloid Fibrils. Amyloid fibrils of lysozyme were formed as follows. A stock solution containing 2.0 mg/mL (140 μM) lysozyme, 136.7 mM sodium chloride, and 1.34 mM potassium chloride was adjusted to pH 2.0 by addition of HCl. Subsequently, the samples were incubated for 0−3 h at 50 °C with continuous agitation using a stirrer to form amyloid fibrils. The amyloid fibrils at each time point were determined by spectroscopic and microscopic methods as described below. Thioflavin T Fluorescence Assay. ThT was used to determine the amount of amyloid fibrils. Aliquots of 20 μL of the samples prepared as described above were mixed with 1980 μL of the solution containing 5 μM ThT and 50 mM glycine (pH 8.5). Fluorescence of ThT excited at 440 nm was measured at 480 nm using a Jasco spectrofluorometer (FP-6500; Japan Spectroscopic, Co., Ltd., Tokyo, Japan), equipped with a Peltier thermal control system. Imaging of Amyloid Fibrils by Atomic Force Microscopy. Atomic force microscopy (AFM) was used to image the amyloid fibrils. The samples incubated at each time point as described above were diluted 100-fold with pure water. Subsequently, 1 μL of the diluted samples was placed on freshly cleaved mica and dried in air for 10 min. AFM images were obtained using S-image (SII NanoTechnology Inc., Chiba, Japan) operating in taping mode. Preparation and Determination of Start Aggregates. Start aggregates of lysozyme were formed as follows. Stock solutions containing 0.4 mg/mL (28 μM) lysozyme and 25 mM sodium phosphate (pH 7.4) were incubated for 0−10 min at 80 °C using a temperature control system (PC-880; Astec, Fukuoka, Japan). The subsequent studies were performed using the samples diluted 10-fold with water. To investigate the structural changes in lysozyme, circular dichroism (CD) spectra of the samples were measured with a Jasco spectropolarimeter (model J-820 W; Japan Spectroscopic). In addition, the residual activity of the lysozyme was determined as follows. A 100 μL portion of the protein solution was mixed with 1400 μL of 0.4 mg/mL Micrococcus lysodeikticus (Wako Pure Chemical Industries Ltd.) in 50 mM sodium phosphate buffer (pH 7.4). Subsequently, the decrease in turbidity of the solution was determined by monitoring its absorbance at 600 nm with a spectrophotometer (V-630; Japan Spectroscopic). The residual activity was determined from the initial decrement in turbidity. Light scattering intensity and hydrodynamic radius of the start aggregates in the sample solutions were determined by dynamic light scattering using a light-scattering spectrometer (Zetasizer Nano ZA; Malvern Instruments, Worcestershire, U.K.). This method allows determination of the sizes of the aggregates based on measurements of time-dependent fluctuations in their scattering intensities. The

■

RESULTS Elongation of Amyloid Fibrils. Lysozyme forms amyloid fibrils on incubation in salt solution at acidic pH.34,47,48 In this study, lysozyme was incubated in the presence of 136.7 mM sodium chloride and 1.34 mM potassium chloride at pH 2.0 to reproducibly prepare amyloid fibril samples. To monitor the appearance and growth of fibrils, fluorescence measurement was performed using ThT (Figure 1). The fluorescence intensity showed a typical sigmoidal curve with incubation time of lysozyme. There was lag time for ca. 1 h prior to the increase in intensity. This phase corresponded to the nucleation 3888

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

Disruption of Lipid Membranes by Amyloid Fibrils. Liposome leakage assay was performed to investigate the effects of amyloid fibrils prepared as described above on disruption of lipid membranes. As demonstrated in our previous study, the disruption of calcein-containing liposomes can be detected by measuring the fluorescence changes of calcein leaked from the liposomes.34 Thus, DOPG/DOPC liposomes, which are negatively charged, containing calcein were used to assess the effects of incubation with amyloid fibrils for various periods on the disruption of lipid membranes. The leakage of calcein after mixing the liposomes with the amyloid fibrils increased with time in physiological saline, i.e., in the presence of 10 mM sodium phosphate and 140 mM sodium chloride at pH 7.4 (Figure 3), indicating disruption of the liposomes by the

Figure 1. Fibrillation of lysozyme detected by ThT fluorescence.

process of lysozyme aggregates toward the amyloid fibrils.49 From 1.5 to 2.5 h, the fluorescence intensity increased sharply, indicating fibril elongation due to interaction of amyloid fibrils with monomers. The fibrillation was considered to be completed by ca. 3.0 h as fluorescence intensity of ThT reached a plateau. Although fluorescence measurement using ThT is useful to evaluate fibrillation, information on their morphology cannot be obtained. To examine the morphology of the amyloid fibrils, AFM images were taken during the fibrillation process (Figure 2). As expected from the fluorescence measurements, few amyloid fibrils were formed by 1 h. After incubation for more than 1 h, the number of fibrils increased markedly, which was consistent with the fluorescence measurements (Figure 1). Importantly, the incubation of lysozyme under these conditions resulted in an increase in number of amyloid fibrils rather than in their length. The observed changes in ThT fluorescence intensity with incubation time were therefore considered to reflect the number of amyloid fibrils.

Figure 3. Representative time-dependent changes of calcein leakage from 2.6 μM 1:1 DOPG/DOPC liposomes in the presence of 2.0 μM lysozyme incubated for various periods.

interaction with amyloid fibrils. The amount of leakage at each time point tended to increase with increasing incubation time

Figure 2. AFM images of lysozyme incubated for 0.5 h (A), 0.75 h (B), 1 h (C), 1.25 h (D), 1.5 h (E), 1.75 h (F), 2 h (G), and 2.25 h (H). Scale bars, 3 μm. 3889

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

at that time point. The maximum leakage was constant from 0 to 0.5 h, i.e., ca. 10%, indicating that monomeric or nonfibrillar lysozyme as shown in Figure 2 can disrupt few liposomes. The distribution can be mainly ascribed to electrostatic attraction between the positively charged lysozyme and the negatively charged liposomes. 50−53 From 0.5 to 2.0 h, the leakage increased with incubation time, indicating that membrane disruption was attributable to the amount of amyloid fibrils. Adsorption of Liposomes onto Amyloid Fibrils. As mentioned above, the liposome is considered to be irreversibly adsorbed on the amyloid fibrils; therefore, it is meaningful to investigate the adsorbed amount of lipids onto the amyloid fibrils. Figure 5A shows the adsorbed lipid concentrations after incubation of 26 μM 1:1 DOPG/DOPC liposome solutions with 0−40 μM amyloid fibrils for 5 min and 5 h in the presence of 3.3 mM sodium citrate and 140 mM sodium chloride at pH 7.4, where the adsorbed amounts of the lipids were determined by subtracting the unadsorbed amounts from the initial amounts. The adsorbed lipid concentrations increased up to more than 50% by addition of 1 μM amyloid fibrils. Addition of 10 μM amyloid fibrils resulted in complete adsorption of the lipid. Importantly, the adsorbed lipid concentrations were almost independent of the mixing time. On the basis of the adsorbed lipid concentration after mixing for 5 h at 1 μM amyloid fibrils, the molar ratio of the bound lipid to the amyloid fibrils was roughly estimated to be ca. 17. Consequently, the lipid membrane disruption shown in Figures 3 and 4 was attributable to irreversible adsorption onto the amyloid fibrils. The adsorption of amyloid fibrils of lysozyme may be associated with their electrostatic interaction. To elucidate the role of electrostatic interaction, the adsorption experiment was performed using DOPC liposomes, the net charge of which is zero (Figure 5B). Interestingly, DOPC liposomes were also adsorbed onto the amyloid fibrils. It should be noted that the adsorption rates of DOPC liposomes onto the amyloid fibrils were apparently dependent on the incubation time in contrast to DOPG/DOPC liposomes (Figure 5A), suggesting that the adsorption of DOPC liposomes occurred slower than that of DOPG/DOPC liposomes. In addition, DOPC liposomes showed less adsorption than DOPG/DOPC liposomes; i.e., the adsorbed lipid concentrations after mixing for 5 h were

of the amyloid fibrils. This result indicated that monomeric lysozyme molecules did not disrupt the membrane, whereas the amyloid fibrils did, implying the importance of lysozyme aggregate morphology in the disruption. More importantly, irrespective of the samples, the leakage reached the respective maximum by 4 h (data not shown), indicating that the amyloid fibrils interacted with a limited number of liposomes. This result suggested that the lipid membranes were irreversibly adsorbed on the amyloid fibrils. If the lipid membranes were reversibly adsorbed on the amyloid fibrils, then the liposomes would be completely disrupted. The vertical axis intercept of the curves for the leakage by amyloid fibrils incubated for a long time was not zero because their strong disruptive effect made it difficult to detect the initial leakage. Figure 4 shows the maximum of liposome leakage by incubation with amyloid fibrils for each period. As the

Figure 4. Maximum leakage amounts of calcein from 2.6 μM 1:1 DOPG/DOPC liposomes after mixing with 2.0 μM lysozyme incubated for various periods.

leakage reached the respective maximum by 4 h, the maximum leakage was defined as the percentage of leakage

Figure 5. Concentrations of adsorbed lipids on the amyloid fibrils after mixing 26 μM 1:1 DOPG/DOPC liposomes (A) and 26 μM DOPC liposomes (B) with various concentrations of amyloid fibrils for 5 min (●) and 5 h (○). 3890

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

increased up to ca. 50% by addition of 5 μM amyloid fibrils, which was ca. 5 times higher than the concentration for DOPG/DOPC (Figure 5A). However, the addition of 40 μM amyloid fibrils resulted in complete adsorption of the lipids. These results suggest that adsorption of liposomes onto amyloid fibrils is due not only to electrostatic interactions, but also to hydrophobic interactions. The adsorbability of amyloid fibrils onto the lipid membranes is considered to affect their disruption. Thus, liposome leakage assay was performed using DOPG/DOPC and DOPC liposomes in the presence of 3.3 mM sodium citrate and 140 mM sodium chloride at pH 7.4, where 4 μM amyloid fibrils formed by incubation for 3 h and 2.6 μM liposomes were used. Figure 6 shows the maximum leakage of calcein after mixing the

adsorbability of liposomes onto the amyloid fibrils is related to the disruptive effect. Disruption of Lipid Membranes by Start Aggregates. As described above, formation of the positively charged amyloid fibrils of lysozyme resulted in disruption of the negatively charged lipid membranes. This raised the question of whether the disruption is unique to the amyloid fibrils or common to nanoscale protein aggregates. Experiments using another nanoscale protein aggregate should be useful to address this question. Generally, thermal aggregates of proteins are formed through their denaturation by heating. It was reported previously that nanoscale thermal aggregates, so-called start aggregates,41 are generated at the initial stage of heating.41−44 Thus, the start aggregate of lysozyme was used to test the generality of disruption by nanoscale protein aggregates. The thermal aggregates of lysozyme were formed by heating at 80 °C. With heating time, the secondary structure gradually changed, which was accompanied by inactivation of the antibacterial action of lysozyme (Figure 7). To detect lysozyme aggregation, the intensity of the protein solution was measured by dynamic light scattering. The intensity increased monotonously with heating time at 80 °C, indicating thermal aggregate formation (Figure 8A). The hydrodynamic radius of the aggregates changed with heating time. The hydrodynamic radius of native lysozyme was ca. 4 nm (Figure 8B). Heating for 3 min resulted in marginal changes of the radius; specifically, the intensity from the thermal aggregates, even if detectable, was insignificant. After heating for 5 min, the intensity corresponding to the aggregates of ca. 80 nm increased, indicating the formation of start aggregates. However, intensity corresponding to the monomeric molecules of lysozyme remained even after 5 min, indicating that the monomeric molecules coexisted with the start aggregates. After heating for 10 min, intensity corresponding to monomeric molecules disappeared and the radius of the aggregates increased, suggesting subsequent association of the start aggregates. The liposome leakage assay was performed using the native monomer and the start aggregates of lysozyme (Figure 8C). Similar to amyloid fibrils, the disruption of DOPG/DOPC liposomes was induced by the start aggregates, whereas no such effect was observed on DOPC liposomes. Despite the intensity

Figure 6. Maximum amount of calcein leakage from 2.6 μM 1:1 DOPG/DOPC and DOPC liposomes in the presence of 4.0 μM amyloid fibrils.

samples for 4 h. As expected, the amount of leakage for DOPG/DOPC liposomes was greater than that for DOPC liposomes, indicating that electrostatic interactions between amyloid fibrils and lipid membranes affect disruption of the lipid membranes. Consequently, it was clarified that the

Figure 7. Circular dichroism (A) and residual activity (B) of lysozyme incubated for 0, 3, 5, and 10 min at 80 °C. 3891

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

Figure 8. Intensity of light scattering (A) and size distribution (B) of the aggregates formed by incubation for different periods at 80 °C. (C) Maximum amounts of calcein leakage from 1:1 DOPG/DOPC and DOPC liposomes in the presence of the aggregates.

electrostatic interactions were enhanced by increasing the charge density through aggregate formation.54 In addition, both lipid membranes were adsorbed on the amyloid fibrils irrespective of the presence of a net charge (Figure 5). This result suggested the occurrence of hydrophobic interactions, probably due to the enhanced hydrophobicity of lysozyme through fibrillation.27,30,59 However, hydrophobic interactions only play an auxiliary role in the disruption of lipid membranes, whereas electrostatic interactions are crucial. Therefore, nanoscale protein aggregates are considered to behave like polyelectrolytes with regard to interaction with lipid membranes. The information about protein aggregates described above will be useful to understand the biological impacts of nanoparticles. Recently, it was suggested that cytotoxicity of nanoparticles cannot be understood without considering protein adsorption onto their surfaces in biological systems. Such proteins on nanoparticles form a “protein corona.”60,61 Our previous study showed that carbon nanotubes with lysozyme have disruptive effects on lipid membranes,34 which was ascribed to the presence of the positively charged protein on the carbon nanotubes. Importantly, the disruptive effect was similar to those of amyloid fibrils and start aggregates (Figures 6 and 8). These previous results and the findings of the present study yielded a novel insight into the nanoparticles; i.e., the nanoparticles taken into biological systems are identical to nanoscale protein aggregates. As the surfaces of the nanoparticles tend to form protein corona in biological systems,60−64 the physical attributes of the surfaces will be comparable to those of nanoscale protein aggregates, at least in terms of their interactions with cell membranes. Thus, knowledge regarding nanoscale protein aggregates will be useful to understand the cytotoxicity of not only protein aggregates but also of nanoparticles in biological systems. The observed interactions of amyloid fibrils with liposomes also provide important perspectives about the biological impacts of amyloid fibrils themselves, because the solutions used for liposome leakage assay in this study contained moderate concentrations of neutral salts at pH 7.4, which mimicked biological systems. Despite reports indicating that amyloid oligomers act as cytotoxic species, several more recent studies have demonstrated the cytotoxicity of amyloid fibrils.27,28 One possible cause of the cytotoxicity of amyloid fibrils is leakage of toxic oligomers from the unstable fibrils.65 Although amyloid fibrils commonly possess stable cross-β structures, polymorphism of the fibrils can result in leakage of the oligomers from the fibrils. Another possible cause is specific

increase by heating (Figure 8A), the leakage by the start aggregates formed by heating for 5 and 10 min was almost comparable. Although the detailed mechanism is not yet clear, the decrease in number of start aggregates due to their association may be responsible for the reduction of the disruptive effect. Taken together, the findings of the present study suggest that the disruption of lipid membranes by the protein aggregates is due to their nanoscale size. The increase in charge density of their surface by aggregation is probably responsible for the disruption, as discussed below.

■

DISCUSSION Our previous study showed that amyloid fibrils of lysozyme disrupt negatively charged liposomes.34 The disruption was attributed to the electrostatic attraction between the amyloid fibrils and the liposomes because the leakage of liposome contents was significantly reduced at pH near the isoelectric point of lysozyme. Despite our previous study, little is known about the effects of the elongation of amyloid fibrils on adsorption and disruption. Therefore, this was one of the focuses of the present study. The disruption of DOPG/DOPC liposomes was induced by lysozyme fibrillation (Figure 4). On the other hand, the monomeric or nonfibrillar state at the initial stage of incubation hardly disrupted the liposomes. These results indicated that the disruptive effect is due to the fibrillation. Interestingly, the start aggregate also disrupted the lipid membranes (Figure 8). As our previous study showed that suspension of amorphous aggregates prepared by heating at 98 °C and subsequent ultrasonication induced marginal disruption,34 the protein appeared to impact the lipid membrane through the formation of nanoscale aggregates, such as amyloid fibrils and start aggregates. Such disruption of the lipid membranes by these nanoscale aggregates is probably a universal characteristic of charged proteins, in a manner similar to the interaction of amyloid fibrils with polyelectrolytes54 and soluble proteins.55 Similar observations were also reported for the interplay between polycationic polymers and lipid membranes.9,12,14,56−58 The polycationic polymers were reported to affect lipid membranes due to electrostatic interaction.12 Disruption of the lipid membranes by polycationic polymers can be derived from their nanoscale size.58 On the basis of these studies, it was suggested that proteins develop polyelectrolyte-like impacts on lipid membranes through the formation of nanoscale aggregates. Indeed, in this study, the nanoscale aggregates of lysozyme disrupted DOPG/DOPC liposomes to a greater extent than the native monomer (Figure 6), indicating that 3892

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

(3) Colvin, V. L. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 2003, 21, 1166−1170. (4) Donaldson, K.; Aitken, R.; Tran, L.; Stone, V.; Duffin, R.; Forrest, G.; Alexander, A. Carbon nanotubes: A review of their properties in relation to pulmonary toxicology and workplace safety. Toxicol. Sci. 2006, 92, 5−22. (5) Hardman, R. A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. Environ. Health Perspect. 2006, 114, 165−172. (6) Nel, A.; Xia, T.; Madler, L.; Li, N. Toxic potential of materials at the nanolevel. Science 2006, 311, 622−627. (7) Lewinski, N.; Colvin, V.; Drezek, R. Cytotoxicity of nanoparticles. Small 2008, 4, 26−49. (8) Leroueil, P. R.; Hong, S.; Mecke, A.; Baker, J. R.; Orr, B. G.; Banaszak Holl, M. M. Nanoparticle interaction with biological membranes: Does nanotechnology present a Janus face? Acc. Chem. Res. 2007, 40, 335−342. (9) Hong, S.; Leroueil, P. R.; Janus, E. K.; Peters, J. L.; Kober, M. M.; Islam, M. T.; Orr, B. G.; Baker, J. R. Jr.; Banaszak Holl, M. M. Interaction of polycationic polymers with supported lipid bilayers and cells: Nanoscale hole formation and enhanced membrane permeability. Bioconjugate Chem. 2006, 17, 728−734. (10) Ginzburg, V. V.; Balijepalli, S. Modeling the thermodynamics of the interaction of nanoparticles with cell membranes. Nano Lett. 2007, 7, 3716−3722. (11) Qiao, R.; Roberts, A. P.; Mount, A. S.; Klaine, S. J.; Ke, P. C. Translocation of C60 and its derivatives across a lipid bilayer. Nano Lett. 2007, 7, 614−619. (12) Lee, H.; Larson, R. G. Lipid bilayer curvature and pore formation induced by charged linear polymers and dendrimers: The effect of molecular shape. J. Phys. Chem. B 2008, 112, 12279−12285. (13) Roiter, Y.; Ornatska, M.; Rammohan, A. R.; Balakrishnan, J.; Heine, D. R.; Minko, S. Interaction of nanoparticles with lipid membrane. Nano Lett. 2008, 8, 941−944. (14) Leroueil, P. R.; Berry, S. A.; Duthie, K.; Han, G.; Rotello, V. M.; McNerny, D. Q.; Baker, J. R. Jr.; Orr, B. G.; Holl, M. M. Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers. Nano Lett. 2008, 8, 420−424. (15) Roiter, Y.; Ornatska, M.; Rammohan, A. R.; Balakrishnan, J.; Heine, D. R.; Minko, S. Interaction of lipid membrane with nanostructured surfaces. Langmuir 2009, 25, 6287−6299. (16) Verma, A.; Stellacci, F. Effect of surface properties on nanoparticle-cell interactions. Small 2010, 6, 12−21. (17) Kelly, C. V.; Leroueil, P. R.; Nett, E. K.; Wereszczynski, J. M.; Baker, J. R. Jr.; Orr, B. G.; Banaszak Holl, M. M.; Andricioaei, I. Poly(amidoamine) dendrimers on lipid bilayers I: Free energy and conformation of binding. J. Phys. Chem. B 2008, 112, 9337−9345. (18) Stefani, M.; Dobson, C. M. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J. Mol. Med. 2003, 81, 678−699. (19) Chiti, F.; Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 2006, 75, 333−366. (20) Stefani, M. Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world. Biochim. Biophys. Acta 2004, 1739, 5−25. (21) Sunde, M.; Blake, C. The structure of amyloid fibrils by electron microscopy and X-ray diffraction. Adv. Protein Chem. 1997, 50, 123− 159. (22) Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 2003, 300, 486−489. (23) Reixach, N.; Deechongkit, S.; Jiang, X.; Kelly, J. W.; Buxbaum, J. N. Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2817−2822. (24) Shankar, G. M.; Li, S.; Mehta, T. H.; Garcia-Munoz, A.; Shepardson, N. E.; Smith, I.; Brett, F. M.; Farrell, M. A.; Rowan, M. J.; Lemere, C. A.; Regan, C. M.; Walsh, D. M.; Sabatini, B. L.; Selkoe,

cytotoxicity of amyloid fibrils themselves. For example, even amyloid fibrils of hen egg white lysozyme (a non-diseaseassociated protein) induce apoptosis-like cell death pathway in contrast to the amyloid oligomers inducing necrosis-like cell death.27 Another study indicated that the amyloid fibrils formed from hen egg white lysozyme have the ability to disrupt lipid membranes and to reduce cell viability.31 In addition, it has been shown that amyloid fibrils formed under different conditions have different degrees of cytotoxicity.66,67 Taken together with the present results shown in Figures 5 and 6, it is suggested that the irreversible adsorption of lipid membranes on the amyloid fibrils of lysozyme is directly associated with their cytotoxicity, which is induced by electrostatic and probably also hydrophobic interactions, as discussed above. Thus, an important insight into the cytotoxicity of amyloid fibrils was obtained; specifically, positively charged amyloid fibrils, such as those of lysozyme, are different from amyloid oligomers or negatively charged amyloid fibrils, such as those of amyloid β (apart from the local positively charged surfaces),68 in their interaction with lipid membranes. The net charge of amyloid fibrils is considered to be one of their crucial physical attributes involved in the cytotoxicity. A previous study indicated no disruption of POPC/POPG/ cholesterol liposomes by amyloid fibrils of hen egg white lysozyme,30 the result of which seems to be contradictory to our results. The lipid components may account for susceptibility of the lipid bilayer to the amyloid fibrils. Moreover, it should be noted that the liposome content did not leak completely from the liposomes even after 4 h when the leakage reached the maximum (Figure 6). These results suggest that the broken lipid membranes on the amyloid fibrils undergo restructuring. Such structural changes in the lipid membranes may be associated with the cytotoxicity. In conclusion, the results of the present study indicated that the nanoscale protein aggregates have universal disruptive effects on lipid membranes due to enhancement of electrostatic and hydrophobic interactions by aggregation. This effect was found in both amyloid fibrils and start aggregates of lysozyme. These findings can be utilized as models of the biological impacts of nanoparticles with protein corona. In addition, the information regarding the interactions between amyloid fibrils and lipid membranes revealed in the present study will yield important perspectives regarding their cytotoxicity.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-29-853-5306. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful to Dr. Shunsuke Tomita for valuable discussion. This work was supported in part by a Grant-in-Aid for Scientific Research (C), No. 23550189, and Scientific Research (A), No. 23246063, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

■

REFERENCES

(1) Oberdorster, G. Pulmonary effects of inhaled ultrafine particles. Int. Arch. Occup. Environ. Health 2001, 74, 1−8. (2) Oberdorster, G.; Oberdorster, E.; Oberdorster, J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 2005, 113, 823−839. 3893

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

D. J. Amyloid-β protein dimers isolated directly from Alzheimer’s brains impair synaptic plasticity and memory. Nat. Med. 2008, 14, 837−842. (25) Silveira, J. R.; Raymond, G. J.; Hughson, A. G.; Race, R. E.; Sim, V. L.; Hayes, S. F.; Caughey, B. The most infectious prion protein particles. Nature 2005, 437, 257−261. (26) Campioni, S.; Mannini, B.; Zampagni, M.; Pensalfini, A.; Parrini, C.; Evangelisti, E.; Relini, A.; Stefani, M.; Dobson, C. M.; Cecchi, C.; Chiti, F. A causative link between the structure of aberrant protein oligomers and their toxicity. Nat. Chem. Biol. 2010, 6, 140−147. (27) Gharibyan, A. L.; Zamotin, V.; Yanamandra, K.; Moskaleva, O. S.; Margulis, B. A.; Kostanyan, I. A.; Morozova-Roche, L. A. Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways. J. Mol. Biol. 2007, 365, 1337−1349. (28) Pieri, L.; Bucciantini, M.; Nosi, D.; Formigli, L.; Savistchenko, J.; Melki, R.; Stefani, M. The yeast prion Ure2p native-like assemblies are toxic to mammalian cells regardless of their aggregation state. J. Biol. Chem. 2006, 281, 15337−15344. (29) Sasahara, K.; Hall, D.; Hamada, D. Effect of lipid type on the binding of lipid vesicles to islet amyloid polypeptide amyloid fibrils. Biochemistry 2010, 49, 3040−3048. (30) Chaudhary, N.; Nagaraj, R. Hen lysozyme amyloid fibrils induce aggregation of erythrocytes and lipid vesicles. Mol. Cell. Biochem. 2009, 328, 209−215. (31) Xue, W. F.; Hellewell, A. L.; Gosal, W. S.; Homans, S. W.; Hewitt, E. W.; Radford, S. E. Fibril fragmentation enhances amyloid cytotoxicity. J. Biol. Chem. 2009, 284, 34272−34282. (32) Linse, S.; Cabaleiro-Lago, C.; Xue, W. F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S. E.; Dawson, K. A. Nucleation of protein fibrillation by nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8691−8696. (33) Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Lindman, S.; Minogue, A. M.; Thulin, E.; Walsh, D. M.; Dawson, K. A.; Linse, S. Inhibition of amyloid β protein fibrillation by polymeric nanoparticles. J. Am. Chem. Soc. 2008, 130, 15437−15443. (34) Hirano, A.; Uda, K.; Maeda, Y.; Akasaka, T.; Shiraki, K. Onedimensional protein-based nanoparticles induce lipid bilayer disruption: carbon nanotube conjugates and amyloid fibrils. Langmuir 2010, 26, 17256−17259. (35) Booth, D. R.; Sunde, M.; Bellotti, V.; Robinson, C. V.; Hutchinson, W. L.; Fraser, P. E.; Hawkins, P. N.; Dobson, C. M.; Radford, S. E.; Blake, C. C.; Pepys, M. B. Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 1997, 385, 787−793. (36) Canet, D.; Sunde, M.; Last, A. M.; Miranker, A.; Spencer, A.; Robinson, C. V.; Dobson, C. M. Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants. Biochemistry 1999, 38, 6419−6427. (37) Krebs, M. R.; Wilkins, D. K.; Chung, E. W.; Pitkeathly, M. C.; Chamberlain, A. K.; Zurdo, J.; Robinson, C. V.; Dobson, C. M. Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the β-domain. J. Mol. Biol. 2000, 300, 541−549. (38) Yonezawa, Y.; Tanaka, S.; Kubota, T.; Wakabayashi, K.; Yutani, K.; Fujiwara, S. An insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle Xray and neutron scattering study. J. Mol. Biol. 2002, 323, 237−251. (39) Frare, E.; Mossuto, M. F.; Polverino de Laureto, P.; Dumoulin, M.; Dobson, C. M.; Fontana, A. Identification of the core structure of lysozyme amyloid fibrils by proteolysis. J. Mol. Biol. 2006, 361, 551− 561. (40) Frare, E.; Polverino De Laureto, P.; Zurdo, J.; Dobson, C. M.; Fontana, A. A highly amyloidogenic region of hen lysozyme. J. Mol. Biol. 2004, 340, 1153−1165. (41) Markossian, K. A.; Yudin, I. K.; Kurganov, B. I. Mechanism of suppression of protein aggregation by α-crystallin. Int. J. Mol. Sci. 2009, 10, 1314−1345. (42) Khanova, H. A.; Markossian, K. A.; Kurganov, B. I.; Samoilov, A. M.; Kleimenov, S. Y.; Levitsky, D. I.; Yudin, I. K.; Timofeeva, A. C.;

Muranov, K. O.; Ostrovsky, M. A. Mechanism of chaperone-like activity. Suppression of thermal aggregation of βL-Crystallin by αCrystallin. Biochemistry 2005, 44, 15480−15487. (43) Golub, N.; Meremyanin, A.; Markossian, K.; Eronina, T.; Chebotareva, N.; Asryants, R.; Muronets, V.; Kurganov, B. Evidence for the formation of start aggregates as an initial stage of protein aggregation. FEBS Lett. 2007, 581, 4223−4227. (44) Tomita, S.; Yoshikawa, H.; Shiraki, K. Arginine controls heatinduced cluster-cluster aggregation of lysozyme at around the isoelectric point. Biopolymers 2011, 95, 695−701. (45) Fiske, C. H.; Subbarow, Y. The colorimetric determination of phosphorus. J. Biol. Chem. 1925, 66, 375−400. (46) Bartlett, G. R. Phosphorus assay in column chromatography. J. Biol. Chem. 1959, 234, 466−468. (47) Wang, S. S.; Liu, K. N.; Wu, C. H.; Lai, J. K. Investigating the influences of redox buffer compositions on the amyloid fibrillogenesis of hen egg-white lysozyme. Biochim. Biophys. Acta 2009, 1794, 1663− 1672. (48) Sarkar, N.; Kumar, M.; Dubey, V. K. Effect of sodium tetrathionate on amyloid fibril: Insight into the role of disulfide bond in amyloid progression. Biochimie 2011, 93, 962−968. (49) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry 2001, 40, 6036−6046. (50) Bergers, J. J.; Vingerhoeds, M. H.; van Bloois, L.; Herron, J. N.; Janssen, L. H.; Fischer, M. J.; Crommelin, D. J. The role of protein charge in protein-lipid interactions. pH-dependent changes of the electrophoretic mobility of liposomes through adsorption of watersoluble, globular proteins. Biochemistry 1993, 32, 4641−4649. (51) Posse, E.; De Arcuri, B. F.; Morero, R. D. Lysozyme interactions with phospholipid vesicles: Relationships with fusion and release of aqueous content. Biochim. Biophys. Acta 1994, 1193, 101−106. (52) Witoonsaridsilp, W.; Panyarachun, B.; Sarisuta, N.; MullerGoymann, C. C. Influence of microenvironment and liposomal formulation on secondary structure and bilayer interaction of lysozyme. Colloids Surf. B 2010, 75, 501−509. (53) Zschornig, O.; Paasche, G.; Thieme, C.; Korb, N.; Arnold, K. Modulation of lysozyme charge influences interaction with phospholipid vesicles. Colloids Surf. B 2005, 42, 69−78. (54) Calamai, M.; Kumita, J. R.; Mifsud, J.; Parrini, C.; Ramazzotti, M.; Ramponi, G.; Taddei, N.; Chiti, F.; Dobson, C. M. Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes. Biochemistry 2006, 45, 12806−12815. (55) Konno, T. Amyloid-induced aggregation and precipitation of soluble proteins: An electrostatic contribution of the Alzheimer’s β(25−35) amyloid fibril. Biochemistry 2001, 40, 2148−2154. (56) Mecke, A.; Lee, D. K.; Ramamoorthy, A.; Orr, B. G.; Holl, M. M. Synthetic and natural polycationic polymer nanoparticles interact selectively with fluid-phase domains of DMPC lipid bilayers. Langmuir 2005, 21, 8588−8590. (57) Mecke, A.; Uppuluri, S.; Sassanella, T. M.; Lee, D. K.; Ramamoorthy, A.; Baker, J. R. Jr.; Orr, B. G.; Banaszak Holl, M. M. Direct observation of lipid bilayer disruption by poly(amidoamine) dendrimers. Chem. Phys. Lipids 2004, 132, 3−14. (58) Mecke, A.; Majoros, I. J.; Patri, A. K.; Baker, J. R. Jr.; Holl, M. M.; Orr, B. G. Lipid bilayer disruption by polycationic polymers: the roles of size and chemical functional group. Langmuir 2005, 21, 10348−10354. (59) Huang, B.; He, J.; Ren, J.; Yan, X. Y.; Zeng, C. M. Cellular membrane disruption by amyloid fibrils involved intermolecular disulfide cross-linking. Biochemistry 2009, 48, 5794−5800. (60) Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the nanoparticleprotein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2050−2055. (61) Lynch, I.; Cedervall, T.; Lundqvist, M.; Cabaleiro-Lago, C.; Linse, S.; Dawson, K. A. The nanoparticle-protein complex as a 3894

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895

Langmuir

Article

biological entity; a complex fluids and surface science challenge for the 21st century. Adv. Colloid Interface Sci. 2007, 134−135, 167−174. (62) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14265−14270. (63) Casals, E.; Pfaller, T.; Duschl, A.; Oostingh, G. J.; Puntes, V. Time evolution of the nanoparticle protein corona. ACS Nano 2010, 4, 3623−3632. (64) Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F. B.; Dawson, K. A. Physical-chemical aspects of protein corona: Relevance to in vitro and in vivo biological impacts of nanoparticles. J. Am. Chem. Soc. 2011, 133, 2525−2534. (65) Stefani, M. Biochemical and biophysical features of both oligomer/fibril and cell membrane in amyloid cytotoxicity. FEBS J. 2010, 277, 4602−4613. (66) Mossuto, M. F.; Dhulesia, A.; Devlin, G.; Frare, E.; Kumita, J. R.; de Laureto, P. P.; Dumoulin, M.; Fontana, A.; Dobson, C. M.; Salvatella, X. The non-core regions of human lysozyme amyloid fibrils influence cytotoxicity. J. Mol. Biol. 2010, 402, 783−796. (67) Petkova, A. T.; Leapman, R. D.; Guo, Z.; Yau, W. M.; Mattson, M. P.; Tycko, R. Self-propagating, molecular-level polymorphism in Alzheimer’s β-amyloid fibrils. Science 2005, 307, 262−265. (68) Yoshiike, Y.; Akagi, T.; Takashima, A. Surface structure of amyloid-β fibrils contributes to cytotoxicity. Biochemistry 2007, 46, 9805−9812.

3895

dx.doi.org/10.1021/la204717c | Langmuir 2012, 28, 3887−3895