Bioconjugate Chem. 2007, 18, 1095−1100
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Biodelivery of a Fullerene Derivative Bogdan Belgorodsky, Ludmila Fadeev, Jenny Kolsenik, and Michael Gozin* School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel . Received November 21, 2006; Revised Manuscript Received March 29, 2007
Most current nanotoxicology research is focused on examining the influence of nanomaterials at the tissue and cellular levels. To explore these interactions on the molecular level, new carboxyfullerenes interact with transport proteins at the molecular level. The carboxyfullerenes exhibited an unusual mode of binding outside the calyx of β-lactoglobulin (a typical representative of lipocalin family of barrier liquid proteins). The complexes were studied by various techniques, including mass spectrometry, UV/vis and circular dichroism spectroscopy, chromatographic methods, gel electrophoresis, and dynamic light scattering. The fullerene ligands were transferred from β-lactoglobulin to human serum albumin (a representative of a blood transport protein), thus providing a model of how fullerene-based nanomaterials interact with biomolecules and are transported in biological systems.
INTRODUCTION During the past decade, research related to carbonaceous nanomaterials has increased, because these materials have potential in applications ranging from biosensor components (1, 2) to therapeutics (3) to diagnostic agents (4). In this context, fullerenes and their derivatives have great potential because of their unique structural (5, 6), physical (7, 8), and chemical properties (9-12). Bioactivity of fullerenes has been demonstrated in antibacterial (13), neuroprotection (14), DNA cleavage (15, 16), and apoptosis (17) assays. Taking into account that fullerene industrial production approaches the scale of tens of tons per year (and may reach hundreds of tons in the future), human and environmental exposure to carbonaceous nanomaterials will undoubtedly increase. In a light of this prospect, studies have begun to appear in the literature addressing the toxicity of nanomaterials. Specifically, acute toxicity studies of nanocolloidal suspensions of C60 fullerene clusters (nanoC60) in water have been performed on several environmentally relevant species, such as freshwater crustaceans and fish (14). Hughes and co-workers found that growth of prokaryotic cells is inhibited upon exposure to low concentrations of nanoC60 (18). Cytotoxicity of fullerene has been shown for both normal (19-22) and carcinogenic cells (20-22). West and co-workers reported that nanoC60 suspensions disrupt normal cellular function through lipid peroxidation (21). All these studies indicate the potential risk of uncontrolled exposure to our environment to fullerene-based nanomaterials. Current knowledge of the toxicology of carbonaceous nanomaterials suggests that these materials may have adverse effects at their portals of entry into living organisms, particularly the respiratory and digestive systems and the skin (23). Most nanotoxicology research is focused on examining the influence of nanomaterials at the tissue and cellular levels, while interactions of carbon nanomaterials with native proteins, especially transport proteins, remain mostly unexplored. Only a few complexes of fullerene derivatives with proteins have been reported. This includes enzyme inhibition by fullerene-based compounds (24, 25) and interactions with some other proteins such as a fullerene-specific antibody (26, 27), lysozyme (28), cytochrome C (29), bovine and human serum albumins (30* Corresponding author: Telephone: +972-3-6405878. Fax: +9723-6405879. E-mail:
[email protected].
32), and apomyoglobin (33, 34). However, in most cases, the reported information provides only partial data on the structure and properties of these fullerene-protein complexes. Our recent studies have been aimed at preparing novel complexes of fullerenes with transport proteins in an attempt to elucidate the structure and properties of these materials. These studies should lead to better understanding of the biodelivery of carbon nanomaterials. In the present study, we focused on lipocalins, a family of transport proteins present in serum, lymph, and barrier liquids, such as saliva and mucus. The lipocalins are a family of small secreted proteins, characterized by similarity in their tertiary structures and molecular recognition properties. These proteins bind to hydrophobic molecules (such as retinol) and to specific cell-surface receptors (to promote endocytosis) (35, 36). Bovine β-lactoglobulin (β-LG) is a typical representative of the lipocalin family of proteins and was selected as a model compound for the preparation of lipocalin-fullerene complexes. Here, we report preparation of complexes formed by interaction of β-LG with carboxyfullerene (CF) and structural characterization of these compounds. We then showed how a fullerene ligand could be transported to the human serum albumin (HSA) to model carbonaceous nanomaterial delivery from an epithelial liquid barrier protein to a major blood transport protein.
EXPERIMENTAL SECTION Preparation and Separation of β-LG/CF Complexes. Carboxyfullerene was synthesized according to previously reported methods (37-39). Bovine β-lactoglobulin, fine chemicals, and solvents were purchased from Sigma-Aldrich. A solution of 4.0 mg of β-LG (1 equiv) in 2 mL of sodium phosphate buffer (20 mM, pH 7.2) and 4, 10, 20, or 40 µL of 100 mM CF in DMSO (2, 5, or 10 equiv, respectively) was incubated for 2 h at 37 °C and then overnight at 10 °C. Additional incubations were prepared in phosphate buffer (20 mM) at pH 6.2 and pH 8.2 at the same concentrations. The complexes were separated from noncomplexed CF on a Sephadex G-25 gel permeation column (Pharmacia Biotech) with sodium phosphate buffer (20 mM, pH 7.2). No differences in the extent of complexation were found among incubations with β-LG A, β-LG B, or a mixture of these proteins; therefore, the latter was used in our further studies. The buffer concentration was reduced by reloading the complex-containing fraction on a second Sephadex G-25 column
10.1021/bc060363+ CCC: $37.00 © 2007 American Chemical Society Published on Web 06/09/2007
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and eluting the complex with sodium phosphate buffer (1 mM, pH 7.2). The resulting solution was lyophilized for storage and further experiments. After reconstitution in water, the concentration of complex in solution was determined by UV-vis spectroscopy and a BioRad protein assay (BioRad). Liquid Chromatography. All HPLC analyses were performed on an Agilent 1100 chromatograph equipped with diodearray detector (Agilent). Size-exclusion liquid chromatography of β-LG, CF, and β-LG/CF complexes was performed on a 4.6 mm × 3.0 cm TSK-gel Super SW2000 column (Tosoh Bioscience) using a sodium phosphate/sodium chloride buffer (100 mM sodium phosphate, pH 7.2, and 100 mM sodium chloride) as the isocratic eluent at a flow rate of 0.2 mL/min. Ion exchange chromatography analyses of starting materials and incubated mixtures were performed on an SP Sepharose column (HiTrap SP FF, Amersham Biosciences) using sodium phosphate/citric acid (20 mM, pH 4.2) as the starting buffer and sodium phosphate/citric acid/sodium chloride (100 mM sodium phosphate, pH 7.2, and 400 mM sodium chloride) as the elution buffer. A total of 72 fractions (200 µL each) were collected on a Costar UV 96-well plate (Corning), and absorption of each fraction was analyzed by a microplate reader (SpectraMax M2, Molecular Devices) at 280 and 485 nm. Mass Spectrometry. Mass spectrometric analyses were performed on a Finnigan LCQ Classic ion-trap mass spectrometer (Finnigan) equipped with a standard ESI source. The ESI source was operated at 3.5 kV, and the capillary was set to 150 °C and 50 V. Raw data were processed using ZNoVa ProMass version 1.4 software (Novatia). Dynamic Light Scattering. Particle sizing was performed at 25 °C using a high-performance particle size analyzer ALVNIBS/HPPS (ALV-Laser Vertriebsgesellschaft). Freshly prepared solutions of β-LG, CF, and β-LG/CF complex in sodium phosphate buffer (20 mM, pH 7.2) were filtered through a 0.2 µm filter. In all experiments, a final concentration of 100 µM protein was used. The particle size was calculated by the autocorrelation function of the ALV sizer software. Circular Dichroism. Circular dichroism measurements were performed on a spectrophotometer equipped with a temperaturecontrolled cell holder (202 Model, Aviv Biomedical). Spectra of protein samples (2 µM) dissolved in a sodium phosphate buffer (20 mM, pH 7.2) were recorded at room temperature in the 190-400 nm spectral range (in 1 nm steps); each spectrum (averaged over 10 scans) was corrected according to the appropriate blank. Docking Studies. The three-dimensional structure of the β-LG protein was obtained from Protein Data Bank (1BEB). A three-dimensional structure of CF was obtained from HarteeFock energy-minimization calculations (Gaussian 03, Gaussian.com). Molecular docking calculations were performed using the PatchDock algorithm (40, 41). In order to estimate the CF binding site on β-LG, the PatchDock algorithm was applied without any a priori data to indicate putative CF binding sites.
RESULTS AND DISCUSSION β-LG/CF Complexes Preparation. A series of incubations of native β-LG A, β-LGB, or a mixture of these proteins was performed with different amounts of CF ligand (under various pH conditions and salt concentrations). Using the analytical methods described below, all these experiments produced very similar results and showed the formation of stable β-LG/CF complexes. Size Exclusion Chromatography. Analyses of the β-LG/ CF complexes were performed by high-performance, size exclusion liquid chromatography (SEC-HPLC) coupled to a diode array UV-vis detector. Figure 1 presents chromatograms of the CF ligand, the β-LG protein, and the β-LG/CF complex.
Belgorodsky et al.
Figure 1. (A) Size exclusion chromatograms (monitored at 280 nm): blue, β-LG protein; red, β-LG/CF complex; black, unbound CF ligand. (B) Corresponding UV-vis spectra obtained using a diode array detector: blue, β-LG protein; red, β-LG/CF complex; black, unbound CF ligand. Table 1. CF Content (equivalents) in β-LG/CF Complexes ratio of [CF]:[β-LG] in β-LG/CF complex
incubation ratio [β-LG]:[CF]
pH 7.2
pH 6.2
pH 8.2
1:2 1:5 1:10
1:1.0 1:1.9 1:2.6
1:1.7 1:2.9 1:3.2
1:1.0 1:2.0 1:2.8
These size exclusion chromatograms clearly show that under our elution conditions the retention time of the proposed β-LG/ CF complex (RT ) 15.3 min) closely matched that of the free β-LG host (RT ) 15.1 min), unambiguously showing very similar sizes of both molecules. Determination of CF Ligand Binding Stoichiometry. Different host-to-ligand ratios were present in the β-LG/CF complexes. Protein-to-ligand ratios were obtained from analyses of purified complexes prepared under various experimental conditions by comparing the amount of the CF ligand in a certain complex (determined at λmax ) 485 nm, ) 4800 M-1 cm-1) to the amount of β-LG protein in the same complex (determined by the BioRad protein assay). Our quantitative analyses of complexes showed that these complexes contained up to three bound CF ligands per a single β-LG host, depending on the amount of the CF ligand used in complexes preparation (Table 1). These unusually high binding ratios between the β-LG host and the CF ligand are in contrast to the typical binding mode of this protein, in which a calix hydrophobic cavity functions
Biodelivery of Fullerene Derivative
Figure 2. Ion-exchange chromatograms (monitored at 280 nm): blue, β-LG protein; red, β-LG/CF complex; black, unbound CF ligand.
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Figure 4. Native polyacrylamide gel electrophoresis of β-LG/CF complexes. Lanes contain (1) β-LG A; (2) β-LG B; (3) β-LG A/β-LG B mixture; β-LG/CF at ratios of (4) 1:2, (5) 1:5, (6) 1:10, and (7) 1:20.
Figure 5. Deconvoluted ESI-mass spectra of β-LG/CF complex.
Figure 6. Circular dichroism spectra of β-LG protein (blue) and β-LG/ CF mixtures in ratios of 1:5 (red) and 1:20 (green).
Figure 3. DLS analyses. (A) Unbound CF ligand. (B) Dimer of β-LG protein. (C) β-LG/CF complex.
as a single binding site for ligand (35). This observation suggests that there are alternative sites for ligand binding or aggregates formation. Ion Exchange Chromatography. We first explored the possibility that binding between CF and β-LG is stabilized through ionic interactions by analyzing the complexes by ion
exchange chromatography (IEC); IEC should disrupt most ionic interactions. On IEC, the free β-LG protein and β-LG/CF complex were observed, unambiguously showing that the interactions between the CF ligand and the β-LG host are mostly hydrophobic. There was only a small difference in retention time between complex and free protein. This could be explained by small differences in the overall charge of the free host versus the complex, as the latter contains more carboxylic groups (Figure 2). Dynamic Light Scattering. Our next step was to exclude the possibility of nonspecific binding of β-LG protein to clusters formed by CF molecules (42). We performed dynamic light scattering (DLS) experiments, examining aqueous solutions of the CF ligand, the free β-LG protein, and the purified β-LG/ CF complex. In aqueous solution, we observed two populations
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Figure 7. Representative, high-score docking model of the β-LG/CF complex; the β-LG dimer is shown as a ribbon model (gray) and the four CF ligands as space-filling models (red).
of CF particles with average sizes of 1 and 1200 nm (broadly distributed), corresponding to single CF molecules and large CF clusters, respectively (Figure 3A). In solutions of the free β-LG protein and the purified β-LG/CF complex, only one type of CF species was observed with an average size of 3 nm (Figure 3B,C). The latter results are in excellent agreement with the approximately 3.5 nm size structure reported for β-LG dimer (PDB: 1BEB) (43). The DLS experiment results suggest that a process of β-LG-CF complex formation proceeds through CF clusters dissolution by β-LG protein and may represent a mechanism by which fullerene nanoclusters undergo an uptake into biological systems. Gel Electrophoresis. Native polyacrylamide gel electrophoresis (PAGE) was used as an additional technique to examine the size, the overall charge, and the stability of the β-LG/CF complexes. The PAGE results showed progressive disappearance of bands of the free protein and appearance of diffuse bands with that migrated more rapidly through the gel than free protein, corresponding to complexes, as the concentration of CF increased (Figure 4). The faster mobility of β-LG/CF complexes probably results from a larger number of the incorporated CF ligands, producing compounds with higher overall negative charge, but similar size to native proteins, as shown in the DLS experiments. Mass Spectrometry. Electrospray mass spectrometry (ESIMS) analysis of a representative purified β-LG/CF complex was performed. The deconvoluted ESI-MS spectrum of this material (Figure 5) revealed the presence of a β-LG dimer bound to up to four CF ligands and additional peaks corresponding to β-LG/ CF complexes with 1:3 binding stoichiometry. These β-LG/CF complexes had remarkable stability, as most noncovalent protein complexes do not remain intact under these mass spectrometry conditions. Notably, these results are in agreement with the data obtained by UV-vis spectroscopy and the stoichiometry determination previously described (Table 1), which showed 1:2.8 protein-to-ligand ratio for the same complex. Circular Dichroism. To assess the structural features of these complexes, we explored whether the β-LG protein remains folded close to its native conformation in the β-LG/CF complex. For these purposes, circular dichroism (CD) spectroscopy studies of β-LG/CF complexes were performed. All the measured CD spectra of these complexes exhibited a typical negative peak
with a minimum around 215 nm, as previously reported for β-LG (44). A series of complexes in which the ratio between β-LG and CF was progressively increased were evaluated. The ellipticity of the complex noticeably decreased upon addition of 20 excess equivalents of the CF ligand (Figure 6). These results strongly suggest that the native structure of the β-LG protein is only slightly affected (mostly in R-helix regions) by binding of CF, even when multiple CF ligands are bound. Docking Studies. To explore possible CF-binding sites on the β-LG dimer interface, docking calculations were performed using the dimer (PDB: 1BEB) structure of β-LG (40, 41). In our models, the first CF unit was located in the interface formed between β-LG monomers, near the R-helix (Figure 7). The resulting structure was used as a basis for further docking experiments as more CF units were added in each round of calculations, producing a model in which four ligands bound near the dimerization site (Figure 7). These docking results are strongly supported by experimental observations; we found that CF binding by β-LG was unaffected by changes in pH conditions and therefore nonrelated to the Tanford transition (45), as the Tanford transition involves pH-dependent movement of EF loop, controlling the entry to β-LG’s calyx binding site. Moreover, in good correlation with CD results, docking studies showed interactions between CF units and the main R-helices of β-LG dimer, located near the dimerization site. Delivery of CF from the β-LG/CF Complex to HSA. Nanomaterials can enter the human body through different ports, including lungs and intestines, and then undergo rapid translocation through the bloodstream to other vital organs (23). The pharmacokinetic behavior of different types of nanoparticles requires detailed investigation, starting at the molecular biochemical level. For the first time, we were able to monitor the transport of nanomaterial between two biochemical systems: CF dissociated from the β-LG/CF complex, where β-LG represented a barrier liquid protein, and bound to a blood plasma protein (represented by HSA). Specifically, 25 µM β-LG/CF complex (at a 1:3 ratio) was incubated with 25 µM HSA protein in phosphate buffer (20 mM, pH 7.2). Transport of CF ligand from the β-LG/CF complex to HSA was monitored by SECHPLC (Figure 8), using the previously described HSA-CF complex as a reference standard (31). At 485 nm, the noncomplexed proteins exhibited almost no absorbance (dark blue
Biodelivery of Fullerene Derivative
Figure 8. Delivery of CF ligand from β-LG/CF complex to HSA, monitored by size exclusion chromatography (at 485 nm). Dark blue, HSA and β-LG proteins without CF; red, after incubation of 0.1 min; black, 2 min; green, 120 min; light blue, 1200 min. Inset: timedependent changes in relative fullerene content in HSA-CF complexes during CF delivery process.
chromatogram; Figure 8), thus allowing us to detect only CFcontaining compounds. As expected, the larger-size HSA-CF complex eluted with a shorter retention time than β-LG/CF (13.6 and 15.3 min, respectively). CF uptake by HSA was clearly observed even at the beginning of the process (after only 0.1 min incubation, red chromatogram; Figure 8). Over the time course, the intensity of the peak at 13.6 min gradually increased, while the peak at 15.3 min decreased (insert, Figure 8). The HSA-CF complex in these experiments was spectroscopically and chromatographically identical to the previously reported HSA-CF (1:1 protein-to-ligand) complex (31). The observed process of CF transfer from β-LG/CF to HSA is very rapid, which could be explained by significant differences in CF affinity toward these proteins. The HSA-CF complex has a relatively high binding constant of 1.2 × 107 M-1 as determined by fluorescence titration (31). A binding constant for CF to β-LG could not be precisely determined due to the presence of multiple competing binding sites (see Supporting Information). After 60 min of incubation, CF uptake by HSA reached a maximum. However, it is expected that, in the real world, the amount of serum albumin protein will be far greater than the amount of lipocalin, thus leading to more complete ligand depletion in β-LG/CF complex.
CONCLUSIONS The characterization of the complex formed by CF and β-LG reported here provides insight into how fullerene-based nanomaterials interact with native liquid barrier proteins. Capable of multiple carbonaceous ligand binding, a representative lipocalin protein, β-lactoglobulin, bound efficiently to carboxyfullerene. Moreover, we provide the first example of transfer of CF from one protein, β-lactoglobulin, to another, albumin, in a model of the delivery process of nanomaterials in biological systems.
ACKNOWLEDGMENT The authors thank the Israeli Science Foundation for their generous financial support. Supporting Information Available: Addtional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.
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