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Shortening and dispersion of single-walled carbon nanotubes upon interaction with mixed supramolecular compounds

Shortening and dispersion of single-walled carbon nanotubes upon interaction with mixed... Congo red (CR) dye molecules self-associate in water solutions creating ribbon-like supramolecular structures that can bind various aromatic compounds by intercalation, forming mixed supramolecular systems. Mixed supramolecular systems, such as CR-doxorubicin and CREvans blue, interact with the surface of carbon nanotubes, leading to their stiffening and ultimately to their breaking and shortening. This work presents a simple method of obtaining short and straight carbon nanotubes with significantly better dispersion in aqueous solutions and consequently improved usability in biological systems. Keywords: atomic force microscopy (AFM); Congo red (CR); doxorubicin (DOX); dynamic light scattering (DLS); Evans blue (EB); scanning electron microscopy (SEM); single-walled carbon nanotubes (SWNT); transmission electron microscopy (TEM). Introduction The chemistry of supramolecular compounds describes the organization of individual molecules in macromolecular associates. The formation of complexes is based on noncovalent interactions, such as hydrophobic interactions, electrostatic interactions, hydrogen bonds and van der Waals forces. An example of a compound that forms supramolecular structures is Congo red (CR), which can interact with proteins [1, 2] but also binds to the surface of carbon *Corresponding author: Anna Jagusiak, Medical Biochemistry, Jagiellonian University Medical College, Kraków, Poland, E-mail: anna.jagusiak@uj.edu.pl Barbara Piekarska and Katarzyna Chlopa: Medical Biochemistry, Jagiellonian University Medical College, Kraków, Poland Elzbieta Bielaska and Tomasz Paczyk: Institute of Catalysis and Surface Chemistry, Polish Academy of Science, Kraków, Poland nanotubes [3­5]. CR has been widely known for almost a century as a histochemical stain for detecting amyloid. CR molecule is symmetrical. The central part of the molecule is nonpolar, whereas the charges are distributed diametrically (Figure 1A). The centrally located aromatic rings of CR may be involved in the binding to the nonpolar surface of the nanotubes consisting of the aromatic rings. In aqueous solutions, CR molecules associate creating supramolecular systems, in which individual molecules interact with each other, forming a ribbon-like structure [6, 7] (Figure 1B). The presence of sulfonic groups gives these systems a character of polyanion at physiological pH. The hydrophobic core of this supramolecular structure is formed by stacked aromatic rings. CR molecules interact with each other in a face-to-face manner, engaging p electrons that can lead to the relocation of charges and creation of a dipole within the supramolecular structure of CR [1, 6­8]. The self-association tendency depends strongly on the position of the sulfonic groups in the molecule. Two of the analogues of CR, Evans blue (EB; Figure 1C) and trypan blue (TB; Figure 1D), are isomers differing in the location of sulfonic groups. The centrally located sulfonic groups in TB limit the hydrophobic area of the molecule and prevent the formation of supramolecular structures, whereas EB, just as CR, shows self-assembling properties [2, 9]. CR administered intravenously is bound by serum albumin, taken up by Kupffer cells, and excreted in the bile. CR can also be internalized by the macrophages of other tissues or excreted in the urine [10]. Its ability to penetrate the blood-brain barrier is highly limited [11]. The structural analog of CR ­ EB ­ used for diagnostic purposes, including dissolving assay, serves to determine the blood volume [12]. CR binding assay is used in vivo for the diagnosis of amyloidosis [13]. No toxicity of CR given in vivo is shown, and the excretion of CR by the renal system is described. In vivo experiments with rabbit ear reveal that CR may accumulate in areas characterized by the presence of antibody-antigen complexes (local inflammation, Arthus effect), whereupon 124Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes Figure 1:(A) CR; (B) association of CR molecules in aqueous solutions ­ supramolecular, ribbon-like structure; (C) EB; and (D) TB. it is progressively removed from the host organism [14]. These results pave the way for the further analysis of compounds capable of forming supramolecular structures as potential mechanisms of drug delivery into areas where antibody-antigen complexes are created (e.g. inflamed tissues, tumor cells, amyloid plaques, or bacterial infection sites). These results also show that the use of CR-type supramolecular compounds is safe during intravenous use. The property of CR, which is both interesting and important for practical reasons, is the ability to incorporate into its supramolecular ribbon-like structure a number of compounds containing planar, aromatic and nonpolar areas. This process creates mixed supramolecular systems in which the CR ribbon-like assembly may serve as a carrier of the intercalated compound. This applies for both other supramolecular dyes (like EB) and fluorescent dyes (like rhodamine B [15] as well as some chemotherapeutic drugs, such as doxorubicin (DOX) (Figure 2A and B) [16, 17]. This paper focuses on the analysis of the interaction of mixed supramolecular systems with carbon nanotubes. Carbon nanotubes are intrinsically hydrophobic, and in aqueous solutions, they aggregate into bundles and ropes and create a network composed of tightly packed individual nanotubes interacting via van der Waals forces and also via mechanical connections (Figure 3A). Their separation and their maintenance in the dispersed state lead to many problems. There is a strong demand for developing methods for the effective and efficient dispersion of nanotubes in water solutions. Through a process called functionalization, water-insoluble carbon nanotubes become more hydrophilic and better dispersible in Figure 2:(A) DOX structure and (B) scheme of intercalation of DOX molecules or EB molecules (black dashed lines) between molecules of CR (red solid lines). an aqueous environment; thus, their biocompatibility is improved. Functionalization modifies the outer surface of carbon nanotubes and can be based on numerous covalent and noncovalent modifications [18]. In search for a method allowing to effectively disperse carbon nanotubes, a mixed supramolecular system that non-covalently interacts with the surface of a carbon nanotube is used. These interactions involve hydrophobic interactions, van der Waals force and pi-pi interactions [19]. Adhesion based on a pi-pi type interaction with the outer wall of the carbon nanotube concerns, for example, the compounds with a pyrene ring structure. Surfactants (anionic, cationic and nonionic) and large molecules such as polymers or biopolymers (e.g. nucleic acids) may become wrapped around the nanotubes [20]. The dispersion of carbon nanotubes in aqueous solutions is required for their use in biological systems. The functionalization and modifications of nanotube surface enables their entry into cells [21, 22], and because they can penetrate into the cell nucleus, nanotubes can be used as Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes125 Figure 3:SEM images. (A) undispersed SWNT ­ tangled bundles of carbon nanotubes and (B) SWNT-CR-DOX ­ uneven loading of the mixed supramolecular CR-DOX complex and noticeable tendency to straightening the nanotubes. carriers in gene therapy [23, 24]. The mechanism of penetration of nanotubes into the cell is not yet well understood. Various ways to overcome the cell membrane barrier are discussed, with the two following hypotheses being the most commonly considered. The first way of internalization is through micropinocytosis or endocytosis of membraneadsorbed carbon nanotube [22]. The efficiency of internalization depends on the size, type of functional groups and hydrophobicity of the nanotube. The shorter nanotubes are internalized more efficiently [25]. The second entry method does not depend on endocytosis and the individual nanotubes pass through the membrane as "nanoneedles" without causing damage to the cell. This process is still poorly understood, but atomic force microscopy (AFM) images show nanotubes in a perpendicular position to the cell membrane, suggesting the penetration of nanotubes based on spontaneous diffusion through the lipid bilayer [24, 26­28]. Probably nanotubes may penetrate into the cell by a combination of internalization and free diffusion depending on a number of parameters, including the type of functionalization and the length of the carbon nanotubes [26]. The method of shortening the nanotubes presented here appears to be attractive as it could allow the delivery of some chemicals (including drugs) into the cell. Because the shortening of nanotubes definitely increases their dispersion, our results may be useful for different areas of carbon nanotube research. Microscopy analysis Scanning electron microscopy (SEM; Hitachi S-4700), transmission electron microscopy (TEM; JEOL JSM-7500F) and AFM (Bruker Nano Dimension Fast Scan and Dimension ICON) were used to evaluate the changes in the structure and, in particular, the length of the carbon nanotubes caused by the binding of mixed supramolecular systems: CR-EB and CR-DOX. Samples for SEM, TEM and AFM were prepared by heating 3 mM aqueous solution of CR or CR-EB (molar ratio 1:1) at 100°C for 5 min. After gradual cooling, which allows to achieve an ordered supramolecular structure, the solution was added to 0.2 mg SWNTs. The sample was then sonicated in ice cooling water bath for 1 h and left for 24 h at room temperature. The resulting complexes of SWNT-CR or SWNT-CR-EB were purified from the excess of unbound CR and EB using a pressure filtration on a PTFE membrane (0.2 mm pores; Merck Millipore). This type of membrane is impermeable to carbon nanotubes and does not adsorb CR or EB. The procedure allows to obtain welldispersed carbon nanotubes loaded with supramolecular CR or mixed supramolecular CR-EB complexes. As a control sample, we used nanotubes dispersed with sodium cholate. For this purpose, 0.2 mg SWNT was sonicated in sodium cholate solution and then was filtered to remove excess, unbound cholate. To bind DOX to the SWNT-CR complex, 1 mM DOX was added to the previously created SWNT-CR complex. The sample was sonicated for 30 min in a cooled water bath, incubated for 24 h at room temperature, and then filtered several times to remove excess, unbound DOX until a colorless filtrate was received. Filtration was performed using microfiltration tubes Amicon Ultra (50 kDa MWCO; Merck Millipore), which are impermeable to nanotubes and CR and permeable to DOX. Before analysis, samples were dialyzed for 24 h to water to remove excess salts. For TEM analysis, 1 mL of Materials and methods Reagents CR (purity >96%), EB, DOX hydrochloride and singlewalled carbon nanotubes (SWNTs; purity >90%), were purchased from Sigma-Aldrich Co. (Milwaukee, WI, USA). Other reagents used were of analytical grade. 126Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes nanotube suspension was applied to the surface of the copper grid (300 mesh) and dried in vacuum. For SEM analysis, samples were applied to the respective blocks and dried under vacuum and then sputtered with gold. For AFM measurement, samples were prepared by applying 1 mL of nanotube suspension on ultrasmooth mica surface and dried under vacuum. AFM measurements were performed using a probe SNL-10 (microcantilever, type A) with a tip made of silicon nitride mounted on silicon microcantilever. The constant resistance of cantilever was 0.32 N/m. The nominal radius of the tip before the measurement was 2 nm. Using the PeakForce QNM visualization mode, two maps were generated simultaneously: surface topography and mechanical properties. This allowed for the assessment of modulus (elasticity) of the material (DMT Modulus, LogDMT Modulus), adhesion (as measured by the susceptibility of the interaction of the tip with the analyzed material) and deformation (deformation of the surface under the influence of the tip). nanotube surface produces stably dispersed nanotubes (Figure 4A). The mixed supramolecular systems of CR-EB (Figure 4B) and CR-DOX (Figure 4C) show similar properties. However, buffer (Figure 4D), EB, or DOX alone did not lead to the dispersion effect, suggesting that its interaction with this kind of nanotube surface (with low diameter) is too weak, if any. Microscopy analysis Interestingly, the binding of CR-EB and CR-DOX mixed supramolecular systems to the SWNT surface led to the shortening of carbon nanotubes. The changes in nanotube properties caused by their interaction with CR-EB supramolecular complex are shown in TEM images (Figures 5 and 6). The marked straightening and shortening of nanotubes upon CR-EB binding is observed. The average length of nanotubes loaded with CR-EB complex ranged from 100 to 300 nm. The sodium cholate dispersion of nanotubes was used as a control; in this case, we can see much longer and bent carbon nanotubes (Figure 5A). When supramolecular CR alone is used, the dispersion of nanotubes is observed, but the shortening effect is absent. The properties of nanotube-CR-DOX complexes were analyzed by SEM and AFM. The stiffening and shortening effect similar to that caused by CR-EB loading was observed. The control sample contained SWNT dispersed by cholate, wherein a length of nanotubes ranged from 1 to 2 mm and width from about 0.7 to 1 nm (Figures 7 and 8A1­C1). The loading of nanotubes with the CR-DOX complex resulted in the formation of shortened nanotubes with a length of about 400 nm and width from 1.5 to 2 nm (for places where the loading with dyes was low; Figures 8A2­C2 and 9). Figure 8A2­C2 shows the characteristic bulbs observed at the ends of shortened carbon nanotubes; these are sites where CR-DOX binds, as confirmed by analyzing the mechanical properties (DMT Modulus; Figure 8A). These bulbs, which show much lower stiffness Dynamic light scattering (DLS) analysis The measurement and comparison of hydrodynamic radii of CR, EB and DOX and the analysis of formation of CR-DOX or CR-EB complexes were provided using a DynaPro detector (Wyatt Technology) and the DLS method. The measurement temperature of 25°C was applied at 3 min incubation time within the instrument. Each sample was measured four times with 20 acquisitions lasting 10 s each. Results Dispersion of carbon nanotubes The interaction of supramolecular dye (CR) or mixed supramolecular systems (CR-EB or CR-DOX) with carbon nanotubes leads to their dispersion. CR interaction with Figure 4:Dispersion of carbon nanotubes with CR and its analogues. (A) SWNT-CR, (B) SWNT-CR-EB, (C) SWNT-CR-DOX and (D) SWNT buffer. Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes127 Figure 5:TEM images. (A) SWNT-cholate ­ long, bent and twisted nanotubes and (B) SWNT-CR-EB ­ shortened and straight (stiffened) nanotubes. Figure 6:TEM images. SWNT-CR-EB ­ shortening of carbon nanotubes caused by their interaction with CR-EB complex: (A) single nanotube with CR-EB mixed supramolecular compound on the ends and (B) shortened and straight nanotubes. 6.7 nm pm 0.0 1: Height sensor 2.0 um 50 100 150 200 250 300 nm Figure 7:AFM image and cross-section analysis of carbon nanotubes dispersed by sodium cholate. (A) 3D topography and (B) width of SWNTs dispersed by cholate ranges from 0.7 to 1 nm. 128Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes A1 B1 1.4 Arb 6.2 mV C1 0.9 nm 1.2 Arb ­14.1 mV ­0.5 nm DMT Modulus Adhesion Deformation A2 B2 455.9 mArb 5.7 mV C2 1.1 nm DMT Modulus 876.5 nm Adhesion 876.5 nm Deformation 876.5 nm Figure 8:AFM images of SWNT (A1­C1) and SWNT-CR-DOX (A2­C2) complexes. Maps of mechanical parameters: (A) elasticity (Young's modulus), red indicates the smallest stiffness and purple indicates the highest stiffness; (B) adhesion (susceptibility to react with the tip of the tested material); and (C) deformation (deviation of the surface under the tip), red circles indicate the place of other mechanical properties than the rest of the nanotubes. 2.5 nm 3 2.5 2 1.5 1 0.5 0 nm 100 200 300 400 500 600 nm 467.658 (nm) 2.5 nm 2 1.5 1.685 (nm) 1 0.5 0 nm 876.5 nm ­0.9 nm 50 100 150 200 nm 1: Height sensor 876.5 nm ­0.9 nm 1: Height sensor Figure 9:AFM images and cross-section analysis of nanotubes loaded with CR-DOX complex. SWNT-CR-DOX parameters: (A) length of approximately 470 nm and (B) diameter of about 1.7 nm (measured as the height of the nanotube lying on the surface). than the nanotube itself, are places where large amounts of CR and DOX bind. This suggests that nanotubes break at sites where high loading with CR-DOX is observed. The breaking of nanotubes at sites where the binding of mixed supramolecular complexes such as CR-DOX and CR-EB is observed may be caused by locally increased stiffness of the nanotube. CR-DOX-loaded nanotubes were also analyzed using SEM (Figure 3B). The uneven distribution of CR-DOX as well as the straightening of the nanotubes caused by dye loading was observed. Analysis of CR-DOX complexes: DLS To explain the surprising phenomenon of shortening of the nanotubes caused by the interaction with mixed supramolecular complexes (CR-EB and CR-DOX) and the absence of the similar effect in the case of CR-mediated dispersion of nanotubes, we analyzed how the supramolecular properties of CR change upon the incorporation of molecules like EB or DOX. We assumed that the higher stability of the supramolecular assembly will be reflected by the increased size of the supramolecular complex, which Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes129 in turn can be measured using the DLS method that allows to determine the hydrodynamic radii of molecules or complexes in solution. The presence of the planar, anthracycline system in DOX molecules makes it capable of intercalating the CR ribbon-like structure. The structure of EB also makes it the same capability. DLS results comparing CR, DOX and CR-DOX complex are presented in Figure 10. Results comparing CR, EB and CR-EB complex are presented in Figure 11. The results are shown as both percentage of the 0.10 1.00 10.00 0.01 0.10 1.00 10.00 0.01 0.10 1.00 10.00 0.01 0.10 1.00 10.00 0.01 0.10 1.00 10.00 Figure 10:DLS analysis of hydrodynamic radii of CR, DOX and CR-DOX complexes. (A) CR (DMSO) (% of intensity); (B) CR (DMSO) (% of mass); (C) CR (buffer Tris-HCl, pH 7.4) (% of intensity); (D) CR (buffer Tris-HCl, pH 7.4) (% of mass); (E) DOX (buffer Tris-HCl, pH 7.4) (% of intensity); (F) DOX (buffer Tris-HCl, pH 7.4) (% of mass); (G) CR-DOX (buffer Tris-HCl, pH 7.4) (% of intensity); and (H) CR-DOX (buffer Tris-HCl, pH 7.4) (% of mass). The disappearance of the peak corresponding to single DOX molecules in the sample containing CR-DOX mixture (G) confirms the formation of the mixed supramolecular CR-DOX complex. 130Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes 6 4 2 0 0.01 0.10 1.00 10.00 10.00 0.10 1.00 10.00 0 0.01 60 50 40 30 20 10 10.00 70 60 50 40 30 20 10 0 0.01 0.10 1.00 10.00 10.00 Figure 11:DLS analysis of hydrodynamic radii of CR, EB and CR-EB complexes. (A) CR (buffer Tris-HCl, pH 7.4) (% of intensity); (B) CR (buffer Tris-HCl, pH 7.4) (% of mass); (C) EB (buffer Tris-HCl, pH 7.4) (% of intensity); (D) EB (buffer Tris-HCl, pH 7.4) (% of mass); (E) CR-EB (buffer Tris-HCl, pH 7.4) (% of intensity); and (F) CR-EB (buffer Tris-HCl, pH 7.4) (% of mass). The disappearance of the peak corresponding to single EB and single CR molecules in the sample containing CR-EB mixture (E and F) confirms the formation of the large mixed supramolecular CR-EB complex. intensity and percentage of weight, because the particles of lower hydrodynamic radius give a weaker signal. CR solution in DMSO was a control sample, showing parameters of single, non-self-assembled CR molecules with a hydrodynamic radius (R) ranging from 0.1 to 0.2 nm, which is in accordance with previous results [2] (Figure 10A and B). In contrast, 1.43 mM CR solution prepared in 0.05 M Tris-HCl buffer (pH 7.4, 0.145 M NaCl) was characterized by the presence of fraction with hydrodynamic radii RCR=1.4 nm (predominant in the analysis of the percentage of weight), which is interpreted as large supramolecular structures (Figure 10C and D). DOX solution (1 mM in 0.05 M Tris-HCl buffer, pH 7.4, 0.145 M NaCl; Figure 10E and F) showed no self-associating tendency. The hydrodynamic radius (RDOX) was in the range of 0.5­1 nm. This value corresponds well with that of non-self-associating molecules (e.g. TB; Figure 1D), whose hydrodynamic radius equals 0.8 nm [2]. The sample containing CR-DOX mixture showed no peak corresponding to the fraction of single DOX molecules, which were apparently incorporated (intercalated) into the supramolecular CR (Figure 10G and H). EB solution (1 mM in 0.05 M Tris-HCl buffer, pH 7.4, 0.145 M NaCl; Figure 11C and D) showed self-associating tendency. The hydrodynamic radius (REB) was 1.1 nm. The sample containing CR-EB mixture (1:1 molar ratio) showed no peak corresponding to the fraction of single EB or single CR molecules (Figure 11A­D). Instead, there was a peak of Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes131 large hydrodynamic radius range from 80 to 120 nm. Most probably, EB was apparently incorporated (intercalated) into the supramolecular CR (Figure 11E and F). The CR-DOX and CR-EB samples show hydrodynamic radii (both percent intensity and percent mass peaks) shifted towards higher values compared to CR solutions, suggesting that intercalation of DOX or EB increases the stability of CR ribbon-like structure. Discussion Dispersion and the possibility of introduction of carbon nanotubes to water environment by CR confirm the attachment of this compound, introducing sufficient polarity, to strongly apolar carbon nanotubes. This demonstrates the special properties of CR and the importance of its ribbonlike supramolecular structure. Interestingly, CR disperses carbon nanotubes but does not break them. DOX does not appear effective in the dispersion of carbon nanotubes used in this studies. However, both EB and DOX form with CR supramolecular structures even larger than does CR itself. The analysis of hydrodynamic radii by DLS confirms this phenomenon. Increasing the size of the hydrodynamic radius in the case of CR-DOX and CR-EB means that DOX or EB is integrated into the ribbon-like structure of CR. Binding most probably takes place by the intercalation of the planar molecules of DOX or EB between planar molecules of CR forming supramolecular structures. CR can thus simultaneously bound compounds differing in properties, because such complexes with CR form other compounds of the planar structure of the molecule, such as a fluorescent rhodamine B or Titan yellow [15, 29]. This structure is formed by interacting single molecules of CR with other molecules of CR. The supramolecular nature of this dye compound, also in mixed supramolecular forms, causes the attachment to carbon nanotubes in large packages, making an uneven dye distribution and different intensities of local loading (SEM results). Carbon nanotubes become broken into smaller pieces making dispersion more effective. Based on microscopic images (TEM and AFM), it can be concluded that there is a breaking of nanotubes at points of its greatest loading. The analysis of the mechanical properties by AFM showed significant loading of endings of nanotubes, which confirmed the images obtained by SEM. The effect of breaking the nanotubes can be explained by a mechanical weakening of the nanotubes in the place of binding such a large mixed supramolecular assembly. There is, however, also another reason of this phenomenon. It may be connected with the uneven electron distribution in carbon nanotubes caused by the binding and different local loading of supramolecular compounds, which represent molecular units of changeable electron capacity. Joining mixed CR-EB system or CR-DOX system can cause variations of electron density in the neighborhood of supramolecular assembly associated with nanotubes and lead to the inhibition of binding of other molecules. This would explain the described effect of the uneven packet binding of mixed supramolecular assembly to the surface of the nanotubes and the formation of very heavily loaded places next to not at all binding places (Figures 3B and 6) [8]. The dispersion of nanotubes by increasing their polarity and, in particular, their breaking increases the possibility of introducing them into solutions and also the utilization in biological systems [26, 27, 30, 31]. The described observation allows the use of a simple method of the preparation of broken nanotubes. The presented results also allow to receive short nanotubes connected with the supramolecular system with a high binding capacity of other compounds like medicaments. The presented complexes can potentially easily penetrate into cells and can be easily removed from them without destroying their structure. Acknowledgments: The authors thank LabSoft company for providing AFM analysis and Dr. Magdalena Jarosz (Laboratory of Scanning Electron Microscopy of Biological and Geological Sciences, Faculty of Biology and Earth Sciences, Jagiellonian University) for providing SEM analysis. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: The authors acknowledge the financial support from the project Interdisciplinary PhD Studies "Molecular Sciences for Medicine" (cofinanced by the European Social Fund within the Human Capital Operational Programme) and the Ministry of Science and Higher Education (grant no. K/DSC/001370). Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bio-Algorithms and Med-Systems de Gruyter

Shortening and dispersion of single-walled carbon nanotubes upon interaction with mixed supramolecular compounds

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de Gruyter
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10.1515/bams-2016-0015
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Abstract

Congo red (CR) dye molecules self-associate in water solutions creating ribbon-like supramolecular structures that can bind various aromatic compounds by intercalation, forming mixed supramolecular systems. Mixed supramolecular systems, such as CR-doxorubicin and CREvans blue, interact with the surface of carbon nanotubes, leading to their stiffening and ultimately to their breaking and shortening. This work presents a simple method of obtaining short and straight carbon nanotubes with significantly better dispersion in aqueous solutions and consequently improved usability in biological systems. Keywords: atomic force microscopy (AFM); Congo red (CR); doxorubicin (DOX); dynamic light scattering (DLS); Evans blue (EB); scanning electron microscopy (SEM); single-walled carbon nanotubes (SWNT); transmission electron microscopy (TEM). Introduction The chemistry of supramolecular compounds describes the organization of individual molecules in macromolecular associates. The formation of complexes is based on noncovalent interactions, such as hydrophobic interactions, electrostatic interactions, hydrogen bonds and van der Waals forces. An example of a compound that forms supramolecular structures is Congo red (CR), which can interact with proteins [1, 2] but also binds to the surface of carbon *Corresponding author: Anna Jagusiak, Medical Biochemistry, Jagiellonian University Medical College, Kraków, Poland, E-mail: anna.jagusiak@uj.edu.pl Barbara Piekarska and Katarzyna Chlopa: Medical Biochemistry, Jagiellonian University Medical College, Kraków, Poland Elzbieta Bielaska and Tomasz Paczyk: Institute of Catalysis and Surface Chemistry, Polish Academy of Science, Kraków, Poland nanotubes [3­5]. CR has been widely known for almost a century as a histochemical stain for detecting amyloid. CR molecule is symmetrical. The central part of the molecule is nonpolar, whereas the charges are distributed diametrically (Figure 1A). The centrally located aromatic rings of CR may be involved in the binding to the nonpolar surface of the nanotubes consisting of the aromatic rings. In aqueous solutions, CR molecules associate creating supramolecular systems, in which individual molecules interact with each other, forming a ribbon-like structure [6, 7] (Figure 1B). The presence of sulfonic groups gives these systems a character of polyanion at physiological pH. The hydrophobic core of this supramolecular structure is formed by stacked aromatic rings. CR molecules interact with each other in a face-to-face manner, engaging p electrons that can lead to the relocation of charges and creation of a dipole within the supramolecular structure of CR [1, 6­8]. The self-association tendency depends strongly on the position of the sulfonic groups in the molecule. Two of the analogues of CR, Evans blue (EB; Figure 1C) and trypan blue (TB; Figure 1D), are isomers differing in the location of sulfonic groups. The centrally located sulfonic groups in TB limit the hydrophobic area of the molecule and prevent the formation of supramolecular structures, whereas EB, just as CR, shows self-assembling properties [2, 9]. CR administered intravenously is bound by serum albumin, taken up by Kupffer cells, and excreted in the bile. CR can also be internalized by the macrophages of other tissues or excreted in the urine [10]. Its ability to penetrate the blood-brain barrier is highly limited [11]. The structural analog of CR ­ EB ­ used for diagnostic purposes, including dissolving assay, serves to determine the blood volume [12]. CR binding assay is used in vivo for the diagnosis of amyloidosis [13]. No toxicity of CR given in vivo is shown, and the excretion of CR by the renal system is described. In vivo experiments with rabbit ear reveal that CR may accumulate in areas characterized by the presence of antibody-antigen complexes (local inflammation, Arthus effect), whereupon 124Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes Figure 1:(A) CR; (B) association of CR molecules in aqueous solutions ­ supramolecular, ribbon-like structure; (C) EB; and (D) TB. it is progressively removed from the host organism [14]. These results pave the way for the further analysis of compounds capable of forming supramolecular structures as potential mechanisms of drug delivery into areas where antibody-antigen complexes are created (e.g. inflamed tissues, tumor cells, amyloid plaques, or bacterial infection sites). These results also show that the use of CR-type supramolecular compounds is safe during intravenous use. The property of CR, which is both interesting and important for practical reasons, is the ability to incorporate into its supramolecular ribbon-like structure a number of compounds containing planar, aromatic and nonpolar areas. This process creates mixed supramolecular systems in which the CR ribbon-like assembly may serve as a carrier of the intercalated compound. This applies for both other supramolecular dyes (like EB) and fluorescent dyes (like rhodamine B [15] as well as some chemotherapeutic drugs, such as doxorubicin (DOX) (Figure 2A and B) [16, 17]. This paper focuses on the analysis of the interaction of mixed supramolecular systems with carbon nanotubes. Carbon nanotubes are intrinsically hydrophobic, and in aqueous solutions, they aggregate into bundles and ropes and create a network composed of tightly packed individual nanotubes interacting via van der Waals forces and also via mechanical connections (Figure 3A). Their separation and their maintenance in the dispersed state lead to many problems. There is a strong demand for developing methods for the effective and efficient dispersion of nanotubes in water solutions. Through a process called functionalization, water-insoluble carbon nanotubes become more hydrophilic and better dispersible in Figure 2:(A) DOX structure and (B) scheme of intercalation of DOX molecules or EB molecules (black dashed lines) between molecules of CR (red solid lines). an aqueous environment; thus, their biocompatibility is improved. Functionalization modifies the outer surface of carbon nanotubes and can be based on numerous covalent and noncovalent modifications [18]. In search for a method allowing to effectively disperse carbon nanotubes, a mixed supramolecular system that non-covalently interacts with the surface of a carbon nanotube is used. These interactions involve hydrophobic interactions, van der Waals force and pi-pi interactions [19]. Adhesion based on a pi-pi type interaction with the outer wall of the carbon nanotube concerns, for example, the compounds with a pyrene ring structure. Surfactants (anionic, cationic and nonionic) and large molecules such as polymers or biopolymers (e.g. nucleic acids) may become wrapped around the nanotubes [20]. The dispersion of carbon nanotubes in aqueous solutions is required for their use in biological systems. The functionalization and modifications of nanotube surface enables their entry into cells [21, 22], and because they can penetrate into the cell nucleus, nanotubes can be used as Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes125 Figure 3:SEM images. (A) undispersed SWNT ­ tangled bundles of carbon nanotubes and (B) SWNT-CR-DOX ­ uneven loading of the mixed supramolecular CR-DOX complex and noticeable tendency to straightening the nanotubes. carriers in gene therapy [23, 24]. The mechanism of penetration of nanotubes into the cell is not yet well understood. Various ways to overcome the cell membrane barrier are discussed, with the two following hypotheses being the most commonly considered. The first way of internalization is through micropinocytosis or endocytosis of membraneadsorbed carbon nanotube [22]. The efficiency of internalization depends on the size, type of functional groups and hydrophobicity of the nanotube. The shorter nanotubes are internalized more efficiently [25]. The second entry method does not depend on endocytosis and the individual nanotubes pass through the membrane as "nanoneedles" without causing damage to the cell. This process is still poorly understood, but atomic force microscopy (AFM) images show nanotubes in a perpendicular position to the cell membrane, suggesting the penetration of nanotubes based on spontaneous diffusion through the lipid bilayer [24, 26­28]. Probably nanotubes may penetrate into the cell by a combination of internalization and free diffusion depending on a number of parameters, including the type of functionalization and the length of the carbon nanotubes [26]. The method of shortening the nanotubes presented here appears to be attractive as it could allow the delivery of some chemicals (including drugs) into the cell. Because the shortening of nanotubes definitely increases their dispersion, our results may be useful for different areas of carbon nanotube research. Microscopy analysis Scanning electron microscopy (SEM; Hitachi S-4700), transmission electron microscopy (TEM; JEOL JSM-7500F) and AFM (Bruker Nano Dimension Fast Scan and Dimension ICON) were used to evaluate the changes in the structure and, in particular, the length of the carbon nanotubes caused by the binding of mixed supramolecular systems: CR-EB and CR-DOX. Samples for SEM, TEM and AFM were prepared by heating 3 mM aqueous solution of CR or CR-EB (molar ratio 1:1) at 100°C for 5 min. After gradual cooling, which allows to achieve an ordered supramolecular structure, the solution was added to 0.2 mg SWNTs. The sample was then sonicated in ice cooling water bath for 1 h and left for 24 h at room temperature. The resulting complexes of SWNT-CR or SWNT-CR-EB were purified from the excess of unbound CR and EB using a pressure filtration on a PTFE membrane (0.2 mm pores; Merck Millipore). This type of membrane is impermeable to carbon nanotubes and does not adsorb CR or EB. The procedure allows to obtain welldispersed carbon nanotubes loaded with supramolecular CR or mixed supramolecular CR-EB complexes. As a control sample, we used nanotubes dispersed with sodium cholate. For this purpose, 0.2 mg SWNT was sonicated in sodium cholate solution and then was filtered to remove excess, unbound cholate. To bind DOX to the SWNT-CR complex, 1 mM DOX was added to the previously created SWNT-CR complex. The sample was sonicated for 30 min in a cooled water bath, incubated for 24 h at room temperature, and then filtered several times to remove excess, unbound DOX until a colorless filtrate was received. Filtration was performed using microfiltration tubes Amicon Ultra (50 kDa MWCO; Merck Millipore), which are impermeable to nanotubes and CR and permeable to DOX. Before analysis, samples were dialyzed for 24 h to water to remove excess salts. For TEM analysis, 1 mL of Materials and methods Reagents CR (purity >96%), EB, DOX hydrochloride and singlewalled carbon nanotubes (SWNTs; purity >90%), were purchased from Sigma-Aldrich Co. (Milwaukee, WI, USA). Other reagents used were of analytical grade. 126Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes nanotube suspension was applied to the surface of the copper grid (300 mesh) and dried in vacuum. For SEM analysis, samples were applied to the respective blocks and dried under vacuum and then sputtered with gold. For AFM measurement, samples were prepared by applying 1 mL of nanotube suspension on ultrasmooth mica surface and dried under vacuum. AFM measurements were performed using a probe SNL-10 (microcantilever, type A) with a tip made of silicon nitride mounted on silicon microcantilever. The constant resistance of cantilever was 0.32 N/m. The nominal radius of the tip before the measurement was 2 nm. Using the PeakForce QNM visualization mode, two maps were generated simultaneously: surface topography and mechanical properties. This allowed for the assessment of modulus (elasticity) of the material (DMT Modulus, LogDMT Modulus), adhesion (as measured by the susceptibility of the interaction of the tip with the analyzed material) and deformation (deformation of the surface under the influence of the tip). nanotube surface produces stably dispersed nanotubes (Figure 4A). The mixed supramolecular systems of CR-EB (Figure 4B) and CR-DOX (Figure 4C) show similar properties. However, buffer (Figure 4D), EB, or DOX alone did not lead to the dispersion effect, suggesting that its interaction with this kind of nanotube surface (with low diameter) is too weak, if any. Microscopy analysis Interestingly, the binding of CR-EB and CR-DOX mixed supramolecular systems to the SWNT surface led to the shortening of carbon nanotubes. The changes in nanotube properties caused by their interaction with CR-EB supramolecular complex are shown in TEM images (Figures 5 and 6). The marked straightening and shortening of nanotubes upon CR-EB binding is observed. The average length of nanotubes loaded with CR-EB complex ranged from 100 to 300 nm. The sodium cholate dispersion of nanotubes was used as a control; in this case, we can see much longer and bent carbon nanotubes (Figure 5A). When supramolecular CR alone is used, the dispersion of nanotubes is observed, but the shortening effect is absent. The properties of nanotube-CR-DOX complexes were analyzed by SEM and AFM. The stiffening and shortening effect similar to that caused by CR-EB loading was observed. The control sample contained SWNT dispersed by cholate, wherein a length of nanotubes ranged from 1 to 2 mm and width from about 0.7 to 1 nm (Figures 7 and 8A1­C1). The loading of nanotubes with the CR-DOX complex resulted in the formation of shortened nanotubes with a length of about 400 nm and width from 1.5 to 2 nm (for places where the loading with dyes was low; Figures 8A2­C2 and 9). Figure 8A2­C2 shows the characteristic bulbs observed at the ends of shortened carbon nanotubes; these are sites where CR-DOX binds, as confirmed by analyzing the mechanical properties (DMT Modulus; Figure 8A). These bulbs, which show much lower stiffness Dynamic light scattering (DLS) analysis The measurement and comparison of hydrodynamic radii of CR, EB and DOX and the analysis of formation of CR-DOX or CR-EB complexes were provided using a DynaPro detector (Wyatt Technology) and the DLS method. The measurement temperature of 25°C was applied at 3 min incubation time within the instrument. Each sample was measured four times with 20 acquisitions lasting 10 s each. Results Dispersion of carbon nanotubes The interaction of supramolecular dye (CR) or mixed supramolecular systems (CR-EB or CR-DOX) with carbon nanotubes leads to their dispersion. CR interaction with Figure 4:Dispersion of carbon nanotubes with CR and its analogues. (A) SWNT-CR, (B) SWNT-CR-EB, (C) SWNT-CR-DOX and (D) SWNT buffer. Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes127 Figure 5:TEM images. (A) SWNT-cholate ­ long, bent and twisted nanotubes and (B) SWNT-CR-EB ­ shortened and straight (stiffened) nanotubes. Figure 6:TEM images. SWNT-CR-EB ­ shortening of carbon nanotubes caused by their interaction with CR-EB complex: (A) single nanotube with CR-EB mixed supramolecular compound on the ends and (B) shortened and straight nanotubes. 6.7 nm pm 0.0 1: Height sensor 2.0 um 50 100 150 200 250 300 nm Figure 7:AFM image and cross-section analysis of carbon nanotubes dispersed by sodium cholate. (A) 3D topography and (B) width of SWNTs dispersed by cholate ranges from 0.7 to 1 nm. 128Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes A1 B1 1.4 Arb 6.2 mV C1 0.9 nm 1.2 Arb ­14.1 mV ­0.5 nm DMT Modulus Adhesion Deformation A2 B2 455.9 mArb 5.7 mV C2 1.1 nm DMT Modulus 876.5 nm Adhesion 876.5 nm Deformation 876.5 nm Figure 8:AFM images of SWNT (A1­C1) and SWNT-CR-DOX (A2­C2) complexes. Maps of mechanical parameters: (A) elasticity (Young's modulus), red indicates the smallest stiffness and purple indicates the highest stiffness; (B) adhesion (susceptibility to react with the tip of the tested material); and (C) deformation (deviation of the surface under the tip), red circles indicate the place of other mechanical properties than the rest of the nanotubes. 2.5 nm 3 2.5 2 1.5 1 0.5 0 nm 100 200 300 400 500 600 nm 467.658 (nm) 2.5 nm 2 1.5 1.685 (nm) 1 0.5 0 nm 876.5 nm ­0.9 nm 50 100 150 200 nm 1: Height sensor 876.5 nm ­0.9 nm 1: Height sensor Figure 9:AFM images and cross-section analysis of nanotubes loaded with CR-DOX complex. SWNT-CR-DOX parameters: (A) length of approximately 470 nm and (B) diameter of about 1.7 nm (measured as the height of the nanotube lying on the surface). than the nanotube itself, are places where large amounts of CR and DOX bind. This suggests that nanotubes break at sites where high loading with CR-DOX is observed. The breaking of nanotubes at sites where the binding of mixed supramolecular complexes such as CR-DOX and CR-EB is observed may be caused by locally increased stiffness of the nanotube. CR-DOX-loaded nanotubes were also analyzed using SEM (Figure 3B). The uneven distribution of CR-DOX as well as the straightening of the nanotubes caused by dye loading was observed. Analysis of CR-DOX complexes: DLS To explain the surprising phenomenon of shortening of the nanotubes caused by the interaction with mixed supramolecular complexes (CR-EB and CR-DOX) and the absence of the similar effect in the case of CR-mediated dispersion of nanotubes, we analyzed how the supramolecular properties of CR change upon the incorporation of molecules like EB or DOX. We assumed that the higher stability of the supramolecular assembly will be reflected by the increased size of the supramolecular complex, which Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes129 in turn can be measured using the DLS method that allows to determine the hydrodynamic radii of molecules or complexes in solution. The presence of the planar, anthracycline system in DOX molecules makes it capable of intercalating the CR ribbon-like structure. The structure of EB also makes it the same capability. DLS results comparing CR, DOX and CR-DOX complex are presented in Figure 10. Results comparing CR, EB and CR-EB complex are presented in Figure 11. The results are shown as both percentage of the 0.10 1.00 10.00 0.01 0.10 1.00 10.00 0.01 0.10 1.00 10.00 0.01 0.10 1.00 10.00 0.01 0.10 1.00 10.00 Figure 10:DLS analysis of hydrodynamic radii of CR, DOX and CR-DOX complexes. (A) CR (DMSO) (% of intensity); (B) CR (DMSO) (% of mass); (C) CR (buffer Tris-HCl, pH 7.4) (% of intensity); (D) CR (buffer Tris-HCl, pH 7.4) (% of mass); (E) DOX (buffer Tris-HCl, pH 7.4) (% of intensity); (F) DOX (buffer Tris-HCl, pH 7.4) (% of mass); (G) CR-DOX (buffer Tris-HCl, pH 7.4) (% of intensity); and (H) CR-DOX (buffer Tris-HCl, pH 7.4) (% of mass). The disappearance of the peak corresponding to single DOX molecules in the sample containing CR-DOX mixture (G) confirms the formation of the mixed supramolecular CR-DOX complex. 130Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes 6 4 2 0 0.01 0.10 1.00 10.00 10.00 0.10 1.00 10.00 0 0.01 60 50 40 30 20 10 10.00 70 60 50 40 30 20 10 0 0.01 0.10 1.00 10.00 10.00 Figure 11:DLS analysis of hydrodynamic radii of CR, EB and CR-EB complexes. (A) CR (buffer Tris-HCl, pH 7.4) (% of intensity); (B) CR (buffer Tris-HCl, pH 7.4) (% of mass); (C) EB (buffer Tris-HCl, pH 7.4) (% of intensity); (D) EB (buffer Tris-HCl, pH 7.4) (% of mass); (E) CR-EB (buffer Tris-HCl, pH 7.4) (% of intensity); and (F) CR-EB (buffer Tris-HCl, pH 7.4) (% of mass). The disappearance of the peak corresponding to single EB and single CR molecules in the sample containing CR-EB mixture (E and F) confirms the formation of the large mixed supramolecular CR-EB complex. intensity and percentage of weight, because the particles of lower hydrodynamic radius give a weaker signal. CR solution in DMSO was a control sample, showing parameters of single, non-self-assembled CR molecules with a hydrodynamic radius (R) ranging from 0.1 to 0.2 nm, which is in accordance with previous results [2] (Figure 10A and B). In contrast, 1.43 mM CR solution prepared in 0.05 M Tris-HCl buffer (pH 7.4, 0.145 M NaCl) was characterized by the presence of fraction with hydrodynamic radii RCR=1.4 nm (predominant in the analysis of the percentage of weight), which is interpreted as large supramolecular structures (Figure 10C and D). DOX solution (1 mM in 0.05 M Tris-HCl buffer, pH 7.4, 0.145 M NaCl; Figure 10E and F) showed no self-associating tendency. The hydrodynamic radius (RDOX) was in the range of 0.5­1 nm. This value corresponds well with that of non-self-associating molecules (e.g. TB; Figure 1D), whose hydrodynamic radius equals 0.8 nm [2]. The sample containing CR-DOX mixture showed no peak corresponding to the fraction of single DOX molecules, which were apparently incorporated (intercalated) into the supramolecular CR (Figure 10G and H). EB solution (1 mM in 0.05 M Tris-HCl buffer, pH 7.4, 0.145 M NaCl; Figure 11C and D) showed self-associating tendency. The hydrodynamic radius (REB) was 1.1 nm. The sample containing CR-EB mixture (1:1 molar ratio) showed no peak corresponding to the fraction of single EB or single CR molecules (Figure 11A­D). Instead, there was a peak of Jagusiak et al.: Shortening and dispersion of single-walled carbon nanotubes131 large hydrodynamic radius range from 80 to 120 nm. Most probably, EB was apparently incorporated (intercalated) into the supramolecular CR (Figure 11E and F). The CR-DOX and CR-EB samples show hydrodynamic radii (both percent intensity and percent mass peaks) shifted towards higher values compared to CR solutions, suggesting that intercalation of DOX or EB increases the stability of CR ribbon-like structure. Discussion Dispersion and the possibility of introduction of carbon nanotubes to water environment by CR confirm the attachment of this compound, introducing sufficient polarity, to strongly apolar carbon nanotubes. This demonstrates the special properties of CR and the importance of its ribbonlike supramolecular structure. Interestingly, CR disperses carbon nanotubes but does not break them. DOX does not appear effective in the dispersion of carbon nanotubes used in this studies. However, both EB and DOX form with CR supramolecular structures even larger than does CR itself. The analysis of hydrodynamic radii by DLS confirms this phenomenon. Increasing the size of the hydrodynamic radius in the case of CR-DOX and CR-EB means that DOX or EB is integrated into the ribbon-like structure of CR. Binding most probably takes place by the intercalation of the planar molecules of DOX or EB between planar molecules of CR forming supramolecular structures. CR can thus simultaneously bound compounds differing in properties, because such complexes with CR form other compounds of the planar structure of the molecule, such as a fluorescent rhodamine B or Titan yellow [15, 29]. This structure is formed by interacting single molecules of CR with other molecules of CR. The supramolecular nature of this dye compound, also in mixed supramolecular forms, causes the attachment to carbon nanotubes in large packages, making an uneven dye distribution and different intensities of local loading (SEM results). Carbon nanotubes become broken into smaller pieces making dispersion more effective. Based on microscopic images (TEM and AFM), it can be concluded that there is a breaking of nanotubes at points of its greatest loading. The analysis of the mechanical properties by AFM showed significant loading of endings of nanotubes, which confirmed the images obtained by SEM. The effect of breaking the nanotubes can be explained by a mechanical weakening of the nanotubes in the place of binding such a large mixed supramolecular assembly. There is, however, also another reason of this phenomenon. It may be connected with the uneven electron distribution in carbon nanotubes caused by the binding and different local loading of supramolecular compounds, which represent molecular units of changeable electron capacity. Joining mixed CR-EB system or CR-DOX system can cause variations of electron density in the neighborhood of supramolecular assembly associated with nanotubes and lead to the inhibition of binding of other molecules. This would explain the described effect of the uneven packet binding of mixed supramolecular assembly to the surface of the nanotubes and the formation of very heavily loaded places next to not at all binding places (Figures 3B and 6) [8]. The dispersion of nanotubes by increasing their polarity and, in particular, their breaking increases the possibility of introducing them into solutions and also the utilization in biological systems [26, 27, 30, 31]. The described observation allows the use of a simple method of the preparation of broken nanotubes. The presented results also allow to receive short nanotubes connected with the supramolecular system with a high binding capacity of other compounds like medicaments. The presented complexes can potentially easily penetrate into cells and can be easily removed from them without destroying their structure. Acknowledgments: The authors thank LabSoft company for providing AFM analysis and Dr. Magdalena Jarosz (Laboratory of Scanning Electron Microscopy of Biological and Geological Sciences, Faculty of Biology and Earth Sciences, Jagiellonian University) for providing SEM analysis. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: The authors acknowledge the financial support from the project Interdisciplinary PhD Studies "Molecular Sciences for Medicine" (cofinanced by the European Social Fund within the Human Capital Operational Programme) and the Ministry of Science and Higher Education (grant no. K/DSC/001370). Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Journal

Bio-Algorithms and Med-Systemsde Gruyter

Published: Sep 1, 2016

References