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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Stable and Biocompatible Monodispersion of C60 in Water by Peptides Matteo Di Giosia,† Federica Nicolini,† Lucia Ferrazzano,† Alice Solda,̀ † Francesco Valle,‡ Andrea Cantelli,† Tainah Dorina Marforio,† Andrea Bottoni,† Francesco Zerbetto,† Marco Montalti,† Stefania Rapino,† Alessandra Tolomelli,*,† and Matteo Calvaresi*,† †
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Dipartimento di Chimica “Giacomo Ciamician”, Alma Mater Studiorum − Università di Bologna, Via Francesco Selmi, 2 − 40126 Bologna, Italy ‡ Istituto per lo Studio dei Materiali Nanostrutturati, ISMN-CNR, via Gobetti 101, 40129 Bologna, Italy S Supporting Information *
ABSTRACT: The lack of solubility in water and the formation of aggregates hamper many opportunities for technological exploitation of C60. Here, different peptides were designed and synthesized with the aim of monomolecular dispersion of C60 in water. Phenylalanines were used as recognizing moieties, able to interact with C60 through π−π stacking, while a varying number of glycines were used as spacers, to connect the two terminal phenylalanines. The best performance in the dispersion of C60 was obtained with the FGGGF peptidic nanotweezer at a pH of 12. A full characterization of this adduct was carried out. The peptides disperse C60 in water with high efficiency, and the solutions are stable for months both in pure water and in physiological environments. NMR measurements demonstrated the ability of the peptides to interact with C60. AFM measurements showed that C60 is monodispersed. Electrospray ionization mass spectrometry determined a stoichiometry of C60@(FGGGF)4. Molecular dynamics simulations showed that the peptides assemble around the C60 cage, like a candy in its paper wrapper, creating a supramolecular host able to accept C60 in the cavity. The peptide-wrapped C60 is fully biocompatible and the C60 “dark toxicity” is eliminated. C60@(FGGGF)4 shows visible light-induced reactive oxygen species (ROS) generation at physiological saline concentrations and reduction of the HeLa cell viability in response to visible light irradiation.
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INTRODUCTION Dispersion of fullerenes in water is an essential step for the technological exploitation of C60 in materials science1−11 and nanomedicine.12−18 The unique chemical and physical properties of C60 will not be fully delivered until the lack of solubility of C60 in water is entirely overcome. Four strategies are commonly used to deal with the strong hydrophobicity of C60: (i) mechanical dispersion−stabilization of C60 (metastable dispersions of fullerenes are obtained, but mechanochemical treatment may often determine the surface chemical modification of C6019); (ii) chemical derivatization of the fullerene, by introduction of water-soluble substituents on the C60 cage20 (soft derivatization processes maintain the tendency of these amphiphilic C60 derivatives to aggregate, while multiple functionalizations efficiently increases C60 solubility in water, but at the same time affects C 60 peculiar properties21); (iii) use of dispersants22,23 such as surfactants, block copolymers, amphiphilic polymers, micelles, and liposomes (large quantities of C60 in water are dispersed, but the resulting solutions are polydispersions of fullerene aggregates of different sizes23,24); (iv) fullerene solvation using a supramolecular host25 (this approach produces dispersion of C60 by the synergistic interplay of hydrophobic, CH−π, n−π, and π−π interactions between host and C60). Different hosts can disperse C60 in water, but the resulting complexes are © XXXX American Chemical Society
usually metastable and subsequent aggregation of the inclusion complex is common,26 especially in physiological environment. In addition, for biological and medical applications of C60, some of the hosts may be toxic and the use of fully biocompatible molecules is highly desirable. Analysis of the C60 hosts shows that two basic design principles are followed; the first is the inclusion of the molecule in a well-suited lipophilic cavity,25 the second is the construction of bivalent, tweezer-like receptors27 with two units that recognize C60, which are connected through a spacer. The binding constants found for tweezer-like receptors are often comparable to those of the structurally more elaborate macrocyclic-type receptors, thus indicating that the molecular tweezers design is an optimal strategy to disperse fullerene.27 Stable dispersions of C60 in water have been recently obtained using proteins.28−53 The interactions between aromatic residues and carbon nanomaterials are crucial for recognition.28,30,53−60 On this basis, we applied a reductionist approach to design peptidic nanotweezeers (PNTs) able to disperse C60 in water. Additional advantages of peptides over traditional supramolecular hosts reside in the fact that peptides are (i) fully Received: December 21, 2018 Revised: January 7, 2019 Published: January 7, 2019 A
DOI: 10.1021/acs.bioconjchem.8b00916 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry biocompatible, (ii) already used in nanotechnology as supramolecular building blocks, and (iii) synthesizable by simple and well-established chemistry. Aromatic molecules interact with C60 through π−π stacking and the phenyl group is an ideal moiety for chemical recognition, able to interact with C60.
(267 and 341 nm) together with an absorption band in the range of 400−550 nm due to the charge transfer from an electron-donor moiety (Phe residue) to C60 with formation of weak donor−acceptor complexes. The appearance of this band is typical of C60 dispersion by proteins/peptides28,29,33 and is due to the charge transfer to C60 from aromatic residues (Phe, Trp, Tyr) that compose the C60 supramolecular host. The 13C NMR (Figure S1) of the water solution of C60@FGGGF revealed the typical chemical shift of fullerene aromatic carbon at 146.1. In Figure 1, the results obtained by using different PNTs at various pH’s are reported. The formation of peptide-C60 supramolecular structures does not result from a single intermolecular noncovalent interactions, but pertains to the synergistic effect of various terms, including hydrogen bonding, π−π stacking, electrostatic, hydrophobic, and van der Waals interactions.61,62 The host−guest interaction is mainly driven by thermodynamics, but kinetics has a critical role in the structural modulation and function integration.61,62 In addition, host−guest interaction is regulated also by host− host and guest−guest tendency to self-assembly, in particular, when the interactions involve peptides and C60, that show a high tendency to self-assemble. Many factors regulate the formation of C60-peptide adducts. In this paper we deeply investigated the role of the pH of the solution that affects (i) the global charge of the peptide, and (ii) the formation of hydrogen-bonds, that regulate the peptide−peptide process of self-assembly. Other factors such as temperature, that strongly affects hydrogen-bonding and hydrophobic interactions, or ionic strength, that has a great impact on electrostatic interactions, can play important roles. A high pH plays a crucial role for the effective dispersion of C60. At low pH, positively charged peptides tend to disperse large aggregations of C60. The surface of C60 aggregates is negatively charged63 and positively charged peptides tend to absorb onto the C60 aggregate. At neutral pH, peptides are zwitterionic and have a natural tendency to self-assemble (in particular, phenylalanine based peptides). For these pH’s, peptide−peptide interactions are favored over peptide−C60 interactions. At high pH, negatively charged peptides are able to disaggregate the C60 clusters, that are negatively charged,63 favoring monodispersion of C60 and their stability in water. The best performance in the dispersion of C60 was obtained with the FGGGF peptidic nanotweezer at a pH of 12. A full characterization of this adduct was carried out. Stability of C60@FGGGF Hybrids. The analysis of the stability of the C60@FGGGF complex at different pH’s shows that the solution is perfectly stable from pH 6.5 to pH 12, while is metastable at pH lower than 4, where aggregation occurs on the time scale of a week (Figure 2a). More importantly, the C60@FGGGF complex is stable at physiological saline concentrations (Figure 2b), while usually C60 dispersed by γ-cyclodextrins or nC60 rapidly precipitate.26 The C60@FGGGF solutions are stable for months both in water and in PBS. AFM Imaging of C60@FGGGF Hybrids. Dispersion of C60 in water is a necessary but not sufficient condition for C60 applications because the photophysical/photochemical properties of C6064−66 and its toxicity66−68 depend strictly on the nature of the fullerene dispersion. To study the aggregation state of C60@FGGGF, direct observation of the complex was conducted using atomic force microscopy (AFM) (Figure 3). The AFM images of a solution of C60@FGGGF drop-cast on a
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RESULTS AND DISCUSSION The choice as a recognition moiety fell on the phenylalanine (F) amino acidic residue while glycine (G) oligomers, Glyn (n = 0−3), were selected as suitable spacers for holding the two recognizing units at the appropriate distance to bind C60 (Scheme 1). We synthesized FF, FGF, FGGF, and FGGGF Scheme 1. Peptidic Nanotweezeer (PNT) Structures
peptides where the terminal phenylalanine residues act as functional ligands while the glycine sequence acts as a spacer to regulate the distances between the two recognition moieties. Standard Fmoc solid-phase peptide synthesis protocols were used to synthesize the four PNTs (see SI). The structures were purified by HPLC and confirmed by NMR and MS. The C60@ PNT complexes were prepared using the same approach previously described for proteins.28−31 Briefly, C60 powder was sonicated, using a probe tip sonicator, in a solution of PNT. After sonication the solution was centrifuged, and the supernatant collected. The experiment was performed at different pH values (2, 7, 12) because the PNTs offer the additional advantage, with respect to the nonpeptidic hosts, of a tuning of their charge state as a function of pH (PNTs are positively charged at pH 2, zwitterionic at pH 7, and negatively charged at pH 12). The presence of C60 in the water solutions was demonstrated by UV−vis spectroscopy and 13C NMR. The UV−visible spectrum (Figure 1) of the water solution of C60@FGGGF revealed the distinctive absorption of C60
Figure 1. Left: UV−visible spectra of FGGGF (solid line) and C60@ FGGGF (dotted line). Right: Quantitative dispersion of C60 by PNTs at the different pH’s (white histograms pH 2; gray histograms pH 7; black histograms pH 12) measuring the absorbance at 341 nm. B
DOI: 10.1021/acs.bioconjchem.8b00916 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
fleshly cleaved mica surface show dot-like structure. From the height distribution analysis of the AFM images (Figure 3) a monodisperse state of C60, wrapped by peptides is visible. Control experiments were carried out using solutions of peptides at the same concentrations, that gave barely visible topographic signal. The AFM analysis was also repeated in the hydrated state (Figure S2) and confirms the results on dried mica, even if with a lower resolution due to the slight drift of the objects in the hydrated state. NMR Characterization of C60@FGGGF Hybrids. Direct proof for the formation of C60 complexes is obtained through NMR spectroscopy. We compared experiments on samples of the free peptide before and after complexation with C60. 1H NMR provides evidence of the presence of supramolecular noncovalent interactions by chemical shift perturbation. Diffusion-ordered NMR Spectroscopy, DOSY, provides evidence of complexation by variation of the translational diffusion coefficient of the peptide. Superimposition of the 1H NMR spectra, reported in Figure 4, shows a significant perturbation of the signals of the N-terminal phenylalanine residue. The α-proton chemical shift is deshielded going from
4.19 to 4.26 ppm (Δδ = 0.07 ppm), and the β-proton chemical shift moves from 3.17 to 3.21 ppm (Δδ = 0.04 ppm). At basic conditions, the C-terminal phenylalanine is negatively charged, so that it points toward the water solution, while the Nterminal phenylalanine is neutral and interacts strongly with the C60 cage. This structural arrangement is crucial to explain the data in Figure 2. In fact, the presence of a negatively charged carboxylate is critical for the stability of the C60@ FGGGF hybrids. When the carboxylate group is negatively charged, it provides the necessary electrostatic repulsion to prevent aggregation/flocculation of the C60@FGGGF hybrids, while when it is neutral (pH below 4.7 for a carboxylate group) the aggregation process starts. Upon C60 binding, DOSY experiments showed an increase of 0.54 × 10−10 m2 s−1 of the translational diffusion coefficient of the FGGGF peptide (from 3.38 × 10−10 m2 s−1 to 3.92 × 10−10 m2 s−1). Such a change agrees with those reported for small molecule-C60 complexes.69,70 Determination of the Stoichiometry of C60@FGGGF Hybrids. Based on the absorption spectra of FGGGF and C60 in water, a 4:1 stoichiometry is estimated (see SI for details). The stoichiometry of the C 60 @FGGGF complex was confirmed by electrospray ionization (ESI) mass spectrometry. ESI-MS analyses were carried out on FGGGF and C60@ FGGGF solutions at a pH value of 12. The ESI-MS spectra of C60@FGGGF solutions (see Figure S3) shows a peak at m/z + 4 = 664.0, under positive ionization conditions, corresponding to a complex of four FGGGF and a C60 (see MS fragmentation tables, Tables S1 and S2 in the SI), while the peptidic signals remain similar. Larger aggregates of C60 might not “fly” in the ESI-MS under these conditions, but AFM images clearly excluded the possibility of greater aggregates. Molecular Dynamic Simulation of C60@(FGGGF)4 Hybrids. Molecular dynamics simulations in explicit solvent, followed by MM-GBSA analysis, were carried out to determine the geometry and the thermodynamic of binding between the peptides and C60. The peptides assemble around the C60 cage, like in a wrapped candy, creating a supramolecular cavity able to host the C60 molecule. The supramolecular structure is formed also thanks to intermolecular interaction between different peptides, forming a β-sheet like structure (see, for example, in Figure 5a yellow and green peptides), that seals C60 and shields the hydrophobic fullerene cage from the water molecules. The average radius of C60@(FGGGF)4 calculated from molecular dynamics simulation (1.2 ± 0.1 nm) is in agreement with the calculated average height of C60@(FGGGF)4 measured by AFM that is 1.3 ± 0.4 nm. The interaction energy (ΔGbinding) between C60 and the
Figure 4. Superimposition of the 1H NMR spectra for FGGGF (red line) and C60@FGGGFn complex (cyan line).
Figure 5. (a) Geometry of the most stable conformation calculated by MM-GBSA analysis for the C60@FGGGF4 complex. (b) Energy components of ΔGbinding.
Figure 2. Left: Absorbance measured at 341 nm of C60@FGGGF sample, upon pH variation, after 1 day (black) and 1 week (gray). Right: UV−visible spectra of C60@FGGGF in water (black line) and in PBS (red line).
Figure 3. AFM image of C60@FGGGF complex. Left: top view, scale bar 100 nm. Right: height distribution analysis of the AFM image.
C
DOI: 10.1021/acs.bioconjchem.8b00916 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry supramolecular host is 27.1 kcal mol−1, a very high value if compared to the 18.5 kcal mol−1 calculated using the same methodology for the interaction between lysozyme and C60.55 In fact, the more flexible peptides, with respect to a structured protein binding pocket, are able to wrap better around the C60 cage, maximizing the van der Waals interactions. Analysis of the binding components of the energy (Figure 5b) shows that van der Waals interactions are the driving force to the binding (−48.9 kcal mol−1). It is important to underline that in the MM-GBSA calculations scheme, π−π interactions are included in the van der Waals term. In addition, the hydrophobic effect, i.e., nonpolar solvation, assists the binding (−4.9 kcal mol−1), while polar solvation (13.0 kcal mol−1) and entropy (13.8 kcal mol−1) are detrimental to the binding and their contribution is positive. ROS Generation Ability of C60@(FGGGF)4 in PBS Buffer. C60 shows a significant visible light-induced generation of reactive oxygen species (ROS), that can be exploited in photodynamic therapy (PDT).66,71−75 However, the use of C60 molecules as photosensitizers still presents important restrictions in its application due to the (i) dependency of ROS production efficiency to the C60 aggregation state;64,65 (ii) “dark toxicity” of C60 aggregates.66,76−78 The possibility of using monodispersed C60@(FGGGF)4 may overcome the current limitations. To evaluate quantitatively the photosensitizing ability of C60@(FGGGF)4, the Amplex Red assay was used to measure the generation of peroxides upon irradiation with visible light. The Amplex Red assay relies on the 1:1 stoichiometric reaction of the colorless, nonfluorescent Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine) with peroxides, to form colored, fluorescent resorufin, catalyzed by horseradish peroxidase (HRP). The ability of C60@(FGGGF)4 to generate peroxides in PBS at different concentrations was investigated (Figure 6). The concentration
production efficiency depends on C60 aggregation state. Aggregation deactivates the electronically excited states of photosensitizers, drastically decreasing the lifetime of the longlived triplet excited state and consequently reducing the ROS production efficiency. When C60 aggregates are present, the aggregation state of C60 is usually concentration dependent.64,65 At higher concentrations, there should be formation of larger aggregates, decreasing ROS production.64,65 On the other hand, when C60 is present as a monomer, there should be, as observed experimentally, a monotonic increase in the peroxide concentration. The definitive evidence for the existence of C60 as a monomer can be provided by measuring the yield of singlet oxygen generation by the C60@(FGGGF)4.The quantum yield (QY) of singlet oxygen generation by C60@(FGGGF)4 was determined by measuring the nearinfrared phosphorescence spectra originated by the spinforbidden radiative deactivation of the lowest singlet electronic excited state to the triplet electronic ground state of oxygen and by comparing it with the standard: Rose Bengal (RB, QY = 0.75 in D2O solution).79 The phosphorescence spectra of the C60@(FGGGF)4 and of the reference RB, both with the same absorbance at the excitation wavelength (λexc = 514 nm) and buffered at pH 7.4, are shown in Figure 7. In perfect
Figure 7. Emission spectra from singlet oxygen generated upon excitation at 514 nm for solutions with the same absorbance (514 nm) of Rose Bengal (red line) and C60@(FGGGF)4 (black line).
accordance with previous works, the singlet oxygen emission sensitized by Rose Bengal shows a maximum at ∼1270 nm,80 while the emission generated by C60@(FGGGF)4 is shifted ∼10 nm toward longer wavelengths. This shift is caused by the different local properties of the environment where singlet excited state is formed.81 According to the spectra of Figure 7, the measured quantum yield for the C60@(FGGGF)4 is 0.71, very close to that of RB, a molecule which is considered one of the best photosensitizers for singlet oxygen generation. The yield of singlet oxygen is very high, confirming the high performances of C60@(FGGGF)4 in the visible light-induced generation of ROS and indirectly its existence as a monomer in solution. Determination of Cytotoxicity and Phototoxicity upon Visible Light Irradiation of C60@(FGGGF)4 Hybrids in HeLa Cells. To assess in vitro the cytotoxicity of C60@(FGGGF)4 and phototoxicity upon irradiation, the ability of the hybrid to inhibit growth and induce cell death in dark condition and upon photoexcitation with visible light was tested. HeLa cells, a highly proliferative in vitro human cancer model, were used. Figure 8 shows that no significative
Figure 6. Peroxides generated upon visible light irradiation using different concentrations of C60@(FGGGF)4.
of C60@(FGGGF)4 was estimated using the absorbance band at 341 nm. The peroxide concentration generated during irradiation (Figure 6) is plotted as the difference between the signals of the irradiated C60@(FGGGF)4 samples and that of the reference, i.e., C60@(FGGGF)4 at the same concentration, taken in the dark. It appears that the generated peroxides are proportional to the concentration of C60@(FGGGF)4. The linear trend of peroxide production, as a function of C60@(FGGGF)4 concentration, supports the assignment of a monodispersed state of C60 molecules in solution. ROS D
DOI: 10.1021/acs.bioconjchem.8b00916 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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FGGGF and C60@FGGGF; Experimental Section and Computational details (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Matteo Di Giosia: 0000-0003-3494-298X Tainah Dorina Marforio: 0000-0003-0690-306X Andrea Bottoni: 0000-0003-2966-4065 Francesco Zerbetto: 0000-0002-2419-057X Stefania Rapino: 0000-0001-6913-0119 Matteo Calvaresi: 0000-0002-9583-2146
Figure 8. Effect on cell viability upon photoirradiation. Samples in dark conditions (black bars) or upon visible light irradiation (gray bars) at different C60@(FGGGF)4 concentrations. Each value represents the ± mean value standard deviation (SD) of three replicates.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Matteo Di Giosia was supported by a FIRC-AIRC fellowship for Italy (id. 22318). This study was supported by the Italian Ministry of Education, University and Research (MIUR), SIR Programme no. RBSI149ZN9−BIOTAXI funded to MC.
reduction in cell viability was observed for HeLa cells in dark conditions, even at high C60 concentration (50 μM). The C60@(FGGGF)4 hybrids are perfectly biocompatible and do not show “dark toxicity”. In contrast, the presence of C60@(FGGGF)4 caused an increase of the mortality of HeLa cells in a dose dependent manner, when irradiated (Figure 8). The results show that the cell mortality rate is related to the concentration of the photosensitizer present in the cell medium during incubation. As the concentration of C60@(FGGGF)4 increases, the cell mortality increases, confirming the ability of C60@(FGGGF)4 to generate ROS in a concentration-dependent manner.
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CONCLUSION For the first time, a C60 monodispersion, stable in physiological environments, was obtained by wrapping the C60 cage with peptides. The C60@(FGGGF)4 host−guest complex satisfies all the crucial requirements for the dispersion of fullerene in water: (i) high solubility and stability of the C60 adduct, (ii) monodispersion of C60, (iii) high ΔGbinding, (iv) dispersion by a biocompatible host. The absence of dark toxicity of the C60@(FGGGF)4 and the preservation of the photophysical properties of C60, including the ability to generate ROS upon irradiation, indicate the potential of this hybrid as an innovative agent for photodynamic therapy. The peptidic nature of the host will allow its easy functionalization without modifying the pristine fullerene, opening new and exciting opportunities for technological applications of the peptide-C60 hybrid such as the controlled assembly of C60 on surfaces,82,83 the targeting of C60 molecules to selected cells84,85 or the use of multicomponent selfassembled peptide materials for PDT.86−88
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00916. 13 C spectra of FGGGF and C60@FGGGF; AFM image of C60@FGGGF complex in hydrated state, ESI-MS of E
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