Intercalation of Bovine Serum Albumin Coated Gold Clusters Between

Article pubs.acs.org/JPCB

Intercalation of Bovine Serum Albumin Coated Gold Clusters Between Phospholipid Bilayers: Temperature-Dependent Behavior of Lipid-AuQC@BSA Assemblies with Red Emission and Superlattice Structure Balázs Söptei, Judith Mihály, Júlia Visy, András Wacha, and Attila Bóta* Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 2 Magyar tudósok blvd, Budapest 1117, Hungary S Supporting Information *

ABSTRACT: A method has been developed to encapsulate bovine serum albumin (BSA)-coated gold quantum clusters (AuQC@BSA) in a multilamellar system of dipalmitoylphosphatidylcholine (DPPC). Results have shown that intercalation of AuQC@BSA particles into lipid bilayers occurs in the presence of CaCl2. Intense red photoluminescence emission was observed after encapsulation of the clusters. A well-defined structure was found with periodic distances drastically larger than that in the pure DPPC/water system. Although Ca2+ ions can change the dipole characteristics of the lipid bilayer surface, leading to unbinding between the bilayers of multilamellar DPPC/water system, the repulsion is shielded in the presence of AuQC@BSA particles. A coherent superlattice structure evolves due to mixed Ca2+-DPPC and Ca2+-AuQC@BSA interactions. Studies at different temperatures have suggested a correlation between the luminescence properties of the clusters and phase transition of the lipid layers. The temperature-dependent behavior assumes the connection between the coating and the lipid bilayer surface. Temperature-dependent features of lipid intercalated Au clusters provide new opportunities in their application.

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INTRODUCTION Protein-coated gold quantum clusters (AuQCs) represent a novel type of luminescent nanomaterials. Having unique emission characteristics, AuQCs are promising materials for a variety of application fields.1 It has been shown that AuQCs can be used for the analysis of metal ions (Hg2+, Cu2+), hydrogen peroxide, glucose, or glutathione in different matrices.2−6 Moreover, protein-coated gold clusters are excellent imaging tools due to their biocompatibility and photostability.7−9 Although protein-coated AuQCs are highly stable, they can react with several substances resulting in unwanted changes of photoluminescence properties of the clusters. It has been demonstrated that the emission intensity of gold clusters is highly dependent on the chemical environment, which is relevant in biological media: the presence of amino acids, small peptides, or sulfide ions, for example, can significantly decrease photoluminescence emission even at low concentration.6,10 Changes in the pH of the medium can also have a negative effect on the emission intensity.11 Synthesis and optical characterization of a great number of different types of AuQCs are described.12−15 The detailed investigation of their behavior in different matrices, however, is still in its infancy. In this study, we focus on the physicochemical behavior of these clusters, connected to their biological application. Multilamellar vesicles (MLV) of © 2014 American Chemical Society

dipalmitoylphosphatidylcholine (DPPC) were applied as a model membrane system to follow the self-organization of protein-coated gold clusters with lipids. MLVs are frequently used as model membrane systems, in which the interactions with organic molecules or nanoparticles can be studied directly.16−22 Moreover, vesicles are commonly applied as diagnostic tools and drug delivery vehicles because of their favorable properties such as biocompatibility, hydrophilic− hydrophobic character, and their ability to protect the incorporated molecules or particles from the external environment.23 Here, we report that bovine serum albumin-coated gold clusters (AuQC@BSA) can be embedded between phospholipid bilayers in an ordered way, forming a super layer-lattice with large periodicity.

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EXPERIMENTAL METHODS Gold(III) chloride hydrate (99.999% trace metal basis) was purchased from Aldrich (USA). BSA (lyophilized powder, ≥ 98%, essentially fatty acid free, essentially globulin free) was obtained from Sigma (USA). DPPC (>99%) was purchased from Avanti Polar Lipids (AL, USA). Sodium hydroxide Received: December 19, 2013 Revised: February 14, 2014 Published: March 14, 2014 3887

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made for geometrical distortions and self-absorption, and the intensities were calibrated into absolute units using a precalibrated glassy carbon specimen. Radial scattering curves were obtained from the fully corrected and calibrated images via azimuthal averaging. For direct visualization of the liposome samples, freezefracture procedure and electron microscopy (FF-TEM) were used. Gold sample holders were incubated at 24 °C, aliquots of 1 μL of the sample were pipetted onto the sample holders and frozen in partially solidified Freon and stored in liquid nitrogen. Fracturing was performed at −100 °C with a Balzers freezefracture device (Balzers BAF 400D, Balzers AG, Vaduz, Liechtenstein). A platinum−carbon shadowing was applied. The replicas were placed on 200-mesh copper grids. A MORGAGNI 268D (FEI, USA) transmission electron microscope was used for the visualization. A Varian FTS-2000 FTIR Spectrometer (Varian Inc., USA) with a Golden Gate single reflection diamond ATR (SPECAC Ltd., UK) accessory was used for the collection of FTIR spectra in attenuated total reflection (ATR) mode. A custom liquid cell was applied permitting temperature-controlled measurements in the range of 20 to 50 °C. Aliquots (approximately 5 μL) were spread on the top of the ATR crystal. Spectra were collected by acquisition of 128 scans at a resolution of 2 cm−1 using an incubation time of 5 min at each temperature. Before evaluation, ATR correction and water background extraction were performed. Peak frequencies of the complex IR bands were determined by peak fitting with Lorentzian curves (Grams32 software package, Galactic Inc., USA). Dynamic light scattering (DLS) measurements were carried out at 25 °C using an AvidNano W130i DLS instrument (λlaser = 660 nm). Samples were filtered through an Acrodisc polyvinylidene fluoride syringe filter with a cutoff of 0.2 μm.

solution (1 M, analytical grade) was ordered from Carlo Erba (France). Deionized water of Millipore purity (18.2 MΩ·cm) was used in all experiments. BSA-protected gold clusters were prepared via a slightly modified version of a method described by Xie et al.24 Briefly, a 25 mg.mL−1 solution of BSA was mixed with equal volume of 10−2 M HAuCl4. After 10 min of stirring, a solution of 1 M NaOH (5% of the protein−gold mixture in volume) was added. After reacting for 16 h at 40 °C, the brown solution of clusters was washed by ultrafiltration using Amicon Ultra Centrifugal Filters (Ultracel-10 k Membrane) with 3 × 1 mL ultrapure water (18.2 MΩ·cm Purelab Classic UV, ELGA Labwater, UK). Aliquots of AuQC@BSA samples were mixed with a solution of 5 mM CaCl2. Protein concentration was adjusted to 4.2 mg· mL−1. The mixture was added to dry DPPC to achieve a final lipid concentration of 5%. Suspensions were then homogenized by heating/cooling in five cycles. In a single cycle, samples were kept at 40 °C for 10 min and at 5 °C for further 10 min. In order to confirm the role of Ca2+ ions in the encapsulation process, control samples were prepared in the absence of CaCl2 by the same method (referred as AuQC@BSA/DPPC/water). For photoluminescence (PL) spectroscopy measuremenets, AuQC@BSA samples were diluted 10 times of their original volume in 5 mM CaCl2. Lipid containing samples were centrifuged at 10 000 rpm for 15 min, the supernatant and the lipid phase were separated. In order to minimize the light scattering of the samples, diluted MLV systems were investigated. Approximately 1 μL of the sediment was suspended in 50 μL of a solution of 5 mM CaCl2. Temperature-dependent photoluminescence properties of the lipid encapsulated AuQC@BSA particles were investigated in the range of 20 to 50 °C. Measurements were carried out using a Shimadzu RFPC 3501 luminescence spectrometer. Samples were excited at 350 nm, emission and excitation slits of 10 nm were used. Thermotropic behavior of the samples (hydrated, containing 5% DPPC) were examined with a Setaram μDSC3 evo differential scanning calorimetry (DSC) apparatus. All samples were scanned at least three times in heating/cooling cycles in the temperature interval of 20 to 70 °C. Scanning rate was 1 °C/min first time, then 0.5 °C/min during heating period, and 1 °C/min during cooling period. An empty sample holder was used as a reference. About 10 mg of the undiluted MLV suspensions were used for the measurements. Small-angle X-ray scattering (SAXS) was measured by a recently built laboratory instrument (details are to be published in J. Appl. Cryst.). Monochromatic Cu Kα X radiation was produced by a GeniX3D Cu ULD beam delivery system (Xenocs Ltd., Sassenage, France), which consists of a 30W microfocus X-ray tube and a parabolic multilayer mirror. The X-ray beam exiting the mirror was collimated by three pinholes, two 0.7 and one 0.8 mm diameter. For measurement, samples were filled into 1 mm thick quartz capillaries (average wall thickness 0.01 mm). These were inserted into an aluminum capillary holder block, which enabled the temperature control via water circulation. Small-angle scattering was measured with a Pilatus 300k two-dimensional position sensitive detector (Dectris, Baden, Switzerland), with 392 mm sample-to-detector distance. The capillaries were under atmospheric pressure during the measurement, and the flight path after the samples was evacuated to 0.03 mbar. Scattering from X-ray windows (Kapton foils) and air around the sample was subtracted from all scattering patterns, corrections were

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RESULTS AND DISCUSSION PL spectra, presented in Figure 1a, show the red emission of AuQC@BSA clusters dispersed in water or encapsulated in the MLVs (referred as AuQC@BSA/DPPC/CaCl2aq). The supernatant of the centrifuged AuQC@BSA/DPPC/CaCl2aq system did not show emission in the red interval. The decrease in the

Figure 1. (a) Photoluminescence emission spectra of native AuQC@ BSA clusters (green), AuQC@BSA/DPPC/CaCl2aq sediment (black), AuQC@BSA/DPPC/CaCl2aq supernatant (gray), DPPC/CaCl2 sediment (red), and the AuQC@BSA/DPPC/water sediment (magenta) excited at 350 nm (absolute concentration of AuQCs in AuQC@BSA/ DPPC/CaCl2aq was lower than that of the native AuQC@BSA samples); (b) Photograph of DPPC/CaCl2 (left) and AuQC@BSA/ DPPC/CaCl2aq (right) under UV lamp; samples were prepared in 5 w/w% lipid/water forms. 3888

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headgroup region of the bilayers. It is worth noting that the SAXS pattern of AuQC@BSA/DPPC/water system exhibited the characteristic periodicity of DPPC MLVs (data not shown), pointing out that MLVs of pure DPPC form in the absence of Ca2+ ions. The freeze fractured surface morphology of the systems provides visual information on the significant differences in the SAXS pattern of the AuQC@BSA/DPPC/CaCl2aq and DPPC/CaCl2aq systems. In the presence of Au clusters, the typical morphology of the DPPC vesicles has changed. Instead of nearly spherical vesicles, laterally extended stacks of closely packed layers were observed (Figure 3a).26 The sheets consisting of several layers are not planar, but rather crumpled. Moreover, the surface of the stacks and vesicles is patchy (Figure 3b). In spite of the grainy surface, layers are closely packed showing a peculiar surface characteristic of the AuQC@ BSA/DPPC/CaCl2aq system (see inset in Figure 3b). In the hydrated DPPC/CaCl2aq system, only vesicles with smooth surface are present. Inside the huge vesicles, which are entirely broken through, smaller ones are embedded. The number of layers in vesicles is reduced, multilamellar forms appear occasionally (Figure 3c). The significant loss in multilayer correlation is typical in micrographs giving a visual explanation for the disappearance of the Bragg reflections in the SAXS patterns of the multilamellar vesicles (Figure 3d). The calorimetric (DSC) curves of these lipid systems are similar and only the biologically relevant part, i.e., the thermotropic features of hydrated MLVs show differences. The fully hydrated pure DPPC system undergoes two phase transitions in the temperature range between 30 and 50 °C. Characteristic changes in enthalpy and the phase transition temperatures altered in the systems with CaCl2 and AuQC@ BSA with respect to that of the pure lipid system. The pretransition (between the gel and rippled gel phases) shifted to lower temperatures (from 35.1 down to 34.4 °C) and exhibited a reduced change in phase transition enthalpy (ΔH = 2.5 instead of 7.0 kJ/mol, Table S1). The main transition of the AuQC@BSA/DPPC/CaCl2aq and DPPC/CaCl2aq systems (shown in Figure 4) occurred at about 0.5 °C higher temperature and exhibited reduced ΔH values compared to the pure DPPC system. First of all, these noticeable, but not drastic changes in the thermograms indicate that the characteristic phospholipid-bilayers are still present, but perturbed. Similarities in the phase behavior of the AuQC@BSA/DPPC/ CaCl2aq and DPPC/CaCl2aq systems point out that the effect of CaCl2 remains dominant, even in the presence of Au clusters. The thermotrophic behavior of DPPC seems to affect photoluminescence properties of the embedded AuQC@BSA clusters. The emission wavelength shows a local minimum around the pretransition temperature, which implies a conjunction between the lipid and the clusters (Figure 5a). The effect of the temperature can also be observed on the emission intensity (Figure 5b): increase of the temperature leads to the decrease of PL intensity. Molecular interactions in the AuQC@BSA/DPPC/CaCl2aq system were followed by FTIR-ATR. Lipid spectral features of AuQC@BSA loaded and pure DPPC/CaCl2aq systems resembled, indicating that the incorporation of AuQC@BSA does not perturb the lipid−lipid interactions in the bilayers. The abrupt increase in the temperature-dependence of the CH2 stretching frequency, due to the change from all-trans to gauche conformers of acyl chains during the main endothermic phase transition, is in correspondence with the DSC results. Both the

photoluminescence intensity of AuQC@BSA/DPPC/CaCl2aq can be related to the absorption and light scattering of the MLVs. A considerable blue shift of the red emission peak was observed in the spectra of AuQC@BSA/DPPC/CaCl2aq, indicating changes in the chemical environment of the clusters. In the case of AuQC@BSA/DPPC/water samples, PL measurements pointed out that AuQC@BSA particles were not encapsulated in the lipid phase. We assume that the intercalation of AuQC@BSA clusters between DPPC bilayers was assured by the presence of Ca2+ ions. This effect of Ca2+ ions is in correspondence with the observation that a strong interaction occurs between BSA and DPPC in 5 mM CaCl2 in a wide pH range.25 Photograph of the red emitting AuQC@BSA/DPPC/CaCl2aq system and the hydrated DPPC system in 5 mM CaCl2 (DPPC/CaCl2aq) is shown in Figure 1b. Significant structural differences were revealed in the lipid matrices by the SAXS method (Figure 2). SAXS measurements

Figure 2. SAXS patterns of AuQC@BSA/DPPC/CaCl2aq (black), DPPC/CaCl2aq (red), and DPPC/water (blue) systems at different temperatures.

were carried out at 28, 38, and 46 °C related to the characteristic temperature domains of the three biologically relevant phases of DPPC/water system. The SAXS patterns of the pure hydrated DPPC system exhibit the characteristic Bragg reflections in several orders corresponding to the well-defined and temperature-dependent periodic distances of a regular multilamellar arrangement (64 Å at 28 °C, 71 Å at 38 °C, 67 Å at 46 °C). If Ca2+ ions are present, lamellae are uncorrelated, as it can be clearly seen in the SAXS profiles in all the three temperature domains; that is, the form factor of the single lipid bilayer is not more than a broad hump. Features of the SAXS patterns have provided strong evidence about the intercalation of protein-coated Au clusters between the periodically arranged DPPC layers. Bragg reflections, observed in three orders, have proven the existence of large layer distances: 105 Å at 28 °C, 106 Å at 38 °C, 130 Å at 46 °C. The new periodicity does not follow the tendency known in the pure lipid system, that is, i.e., at 46 °C the repeating distance is far longer than in the other two states. Ratios of the peak intensities are quite different from those of the DPPC/water system, reflecting the significant change in the structural unit. In the same temperature domain, the mean diameter of AuQC@BSA clusters in water increases monotonously from 8 up to 9 nm (determined by DLS, Figure S1). We conclude that AuQC@BSA clusters must be flattened in gel phases and swollen in the liquid crystalline phase. The enormous relative change in the periodicity of the AuQC@ BSA/DPPC/CaCl2aq system clearly shows that conformational changes in the protein shell of AuQC@BSA clusters must have occurred, induced by the interaction with the interfacial 3889

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Figure 3. FF-TEM images of MLV systems of AuQC@BSA/DPPC/CaCl2aq (a,b); and DPPC/CaCl aq (c,d).

cm−1, due to the hydrogen bonded CO groups.27 The presence of divalent Ca2+ ions elevates the relative amount of H-bonded CO band, implying a hydration in the lipid interface region. Since no changes were observed in the region of acyl chain vibrations, we assume that the secondary hydration shell of the lipid increases, which might change the dipole potential of the lipid membrane surface.28 This is reflected in the formation of lipid structures with uncorrelated layers observed in SAXS curves of DPPC/CaCl2aq system. The phenomenon is suppressed by the presence of AuQC@BSA clusters. It seems plausible that both the lipid headgroups (ester carbonyl region) and the protein surface of AuQC@BSA are able to interact with Ca2+ ions. The conjunction between AuQC@BSA and lipid headgroup region was confirmed also by analyzing the Amide I vibrational band region of the albumin peptide backbone, frequently used for characterization of protein secondary structure.11 Temperature-induced spectral pattern of Amide I band of the native AuQC@BSA complex shows a rather monotonic decrease. The total shift of the Amide I band envelope (Figure 5d) can be interpreted as the relative increase of band components characteristic of exposed α-helix and turn structures, compared to the one of buried α-helix.29 As in the case of the AuQC@ BSA/DPPC/CaCl2aq system, the maximum wavenumber values of the Amide I band envelope show a local minimum around 36 °C, followed by a recovering period up to 40 °C, than a further decrease. Cooling experiments (Figure S4) have evidenced that most of the changes in the physicochemical parameters are reversible, which suggests that degradation and denaturation processes do

Figure 4. DSC thermograms of AuQC@BSA/DPPC/CaCl2aq (black), DPPC/CaCl2 (red) and DPPC/water (blue); inset shows the pretransition of the corresponding samples.

presence of AuQC@BSA and CaCl2 cause a broadening of the curve and a shift of the phase transition toward higher temperatures (Figure 5c). More rigorous analysis of the CO stretching band contour arising from the ester carbonyls reveals subtle changes (Figure S2). In highly hydrated bilayers, this band splits into two overlapping components: a high wavenumber band at approximately 1742 cm−1 of the non-hydrogen bonded C O groups and a low wavenumber band at approximately 1728 3890

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Figure 5. Temperature-dependent behavior of the AuQC@BSA-lipid systems: (a) Emission wavelength of AuQC@BSA/DPPC/CaCl2aq. (b) Emission intensity of AuQC@BSA/DPPC/CaCl2aq. (c) Wavemember of symmetric CH2 stretching of the DPPC acyl chains in AuQC@BSA/ DPPC/CaCl2aq (black), DPPC/CaCl2 (green) and DPPC/water (blue). d) Wavenumber of the Amide I bands of AuQC@BSAaq (light green) and AuQC@BSA/DPPC/CaCl2aq (black).

Author Contributions

not take place within the investigated temperature interval. Considering the thermotropic behavior of the lipid system, the above results imply that temperature-dependent conformational changes of the albumin outer layer of AuQC@BSA are strongly affected by the phase transition in the lipid bilayers.

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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CONCLUSIONS

ACKNOWLEDGMENTS The authors thank Teréz Kiss for the FF-TEM investigations. This work was supported by the Hungarian Scientific Research Fund (OTKA, Hungary) and the National Innovation Office (NIH, Hungary) under Grant Agreement CNK-81056.

In summary, we have shown the interactions between BSA coated Au clusters and cell membrane-mimetic phospholipid bilayers. The attraction between the two species is assured by Ca2+ ions, which are typically present in biological matrices. The intercalation of clusters between the lipid layers assumes an ability for specific localization, even in local compartment of tissues. We can conclude that the coherent structure is a result of mixed Ca2+-DPPC and Ca2+-AuQC@BSA interactions. Measurements carried out at different temperatures suggests a correlation between photoluminescence properties of the clusters and the layer structure. Moreover, investigation of the Au@BSA/DPPC/CaCl2aq has shown that the temperaturedependent behavior of the system is determined by both the phase transition of the lipid bilayers and the conformational changes of the protein. Changes in emission characteristics of the embedded Au clusters offer a way to develop ‘local sensor quantum clusters’ to indicate the alterations in the physicalchemical behavior of surrounding medium.

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ABBREVIATIONS ATR, attenuated total reflection; AuQC, gold quantum cluster; BSA, bovine serum albumin; DLS, dynamic light scattering; DPPC, dipalmitoylphosphatidylcholine; DSC, differential scanning calorimetry; FF-TEM, freeze-fracture transmission electron microscopy; MLV, multilamellar vesicle; PL, photoluminescence; SAXS, small-angle X-ray scattering

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ASSOCIATED CONTENT

S Supporting Information *

Contents: results of DLS measurements, deconvolution of the lipid CO FTIR bands, and DSC results. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: 003613826427. 3891

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