The method of preparation of highly stable reproducible C₆₀ fullerene aqueous colloid solution is described. The structural organization of C₆₀ fullerenes in aqueous solution was studied and analyzed in detail using various techniques such as chemical analysis, UV/VIS spectroscopy, atomic force and scanning tunneling microscopy, dynamic light scattering, and zeta potential method

Cherepanov, V. V.

Davidenko, N. A.

Evstigneev, M .P.

Prylutskyy, Y. I.

Ritter U.

DSpace at Belgorod State University

DOI: 10.1080/1536383X.2013.870900Structural Features of Highly Stable Reproducible C60 Fullerene Aqueous Colloid Solution Probed by Various TechniquesU. RITTER 1, YU. I. PRYLUTSKYY2, M. P. EVSTIGNEEV3’4, N. A. DAVIDENKO2, V. V. CHEREPANOV5, A. I. SENENKO5, O. A. M ARCHENKO5, and A. G. NAUM OVETS5lIlmenau University o f  Technology, Weimarer Str. 25, 98693 Ilmenau, Germany 2Taras Shevchenko National University o f  Kyiv, Volodymyrska Str., 64, 01601 Kyiv, Ukraine 3Sevastopol National Technical University, Sevastopol 99053, Crimea, Ukraine 4Belgorod State University, Pobedy Str.85, 308015 Belgorod, Russia 5 Institute o f  Physics o f  N A S  o f  Ukraine, Nauky Aven., 46, 03680 Kyiv, UkraineThe method of preparation of highly stable reproducible C60 fullerene aqueous colloid solution is described. The structural organization of C6o fullerenes in aqueous solution was studied and analyzed in detail using various techniques such as chemical analysis, UV/VIS spectroscopy, atomic force and scanning tunneling microscopy, dynamic light scattering, and zeta potential methods.Keywords: atomic force microscopy, dynamic light scattering, C60 fullerene aqueous colloid solution, chemical analysis, scanning tunneling microscopy, UV/VIS spectroscopy, zeta potentialIntroductionC60 fullerenes have been intensively investigated in the last decades mainly because of the vast range o f their potential applications in biomedicine (1, 2). Due to its nanom eter size, the pristine C60 fullerenes are able to  interact with biom ole­cules (3, 4) and penetrate through the cell m em brane (5, 6). They exhibit antioxidant properties (7) and, being non-toxic (at low concentration at least) (8-13), exert specific health effects (e.g., suppress the growth o f m alignant tum ors (14, 15)). A lthough these molecules have extremely low water solubility (16), they form  stable colloid solutions containing individual C60 fullerenes as well as C60 fullerene aggregates (clusters) in water (17, 18), when subjected to extended m ix­ing, sonication, or solvent exchange (19, 20). To understand behavior o f C6o fullerene in the biological m edium  (at the lev­els o f cell, tissue and organ), it is necessary to know exactly its concentration in water (dose effect), distribution in size and shape (size effect). Because the biomedical effects o f the C60 fullerene nanoparticles directly depend on theseAddress correspondence to M. P. Evstigneev, Department of Physics, Sevastopol National Technical University, Universitet- skaya str., 33, Sevastopol, 99053, Ukraine. Fax: +38(0692)243590 E-mail: max_evstigneev@mail.ruColor versions of one or more of the figures in the article can be found online at www.tandfonline.com/lfnn.properties (14, 21, 22), their knowledge will enable under­standing o f “which form  o f C60 fullerene is bioactive, namely a single molecule or its cluster?”The aim  o f this paper is to describe in detail the technology of relatively cheap production o f highly stable C60 fullerene aqueous colloid solution (C60FAS), comprising the m ethod of preparation and detailed analysis (characterization) o f the m orphological properties o f the prepared solution. It should be noted tha t the m ethod for obtaining aqueous colloid solu­tion o f C60 fullerene was first proposed by Andrievsky et al. (23), however, the procedure outlined in the cited paper was not reproducible. W ater soluble C6o fullerene prepared from toluene is know n in literature as nano-C60 (9, 10).ExperimentalMethod o f  Preparation o f  CM Fullerene Aqueous Colloid SolutionC arbon soot was generated by evaporating spectral carbon rods (Fa. Schunk) in a d.c. arc at 24 V in a He atmosphere (0.2 bar). The soot was extracted for 6 h in boiling toluene. Undissolved soot particles were eliminated by filtration. The filtrate was then gently w arm ed under flowing nitrogen to evaporate the solvent. Preparative separation of C60 and C70 fullerenes was perform ed through flash chrom atography on silica gel/activated carbon with toluene as eluent. The C60 fullerene fraction had  a purity of >98%. For further531separation and analysis the fullerene m ixture was dissolved in toluene and fractionated with a preparative high-perfor- m ance liquid chrom atography (Jasco PU-2086) coupled to  a multi-wavelength U V /V IS  detector (Jasco UV-2077) and an autosam pler. A preparative Cosmosil Buckyprep Packed Colum n with toluene mobile phase was used. The flow rate was set to 20 m L /m in . The resultant C60 fullerene fraction had a purity o f >99.5%. Further purification was done by sublim ation o f the C60 fullerene in a high vacuum  (purity 99.99% by H PLC  analysis).For the preparation of C60FAS we used a saturated solu­tion of pure C6o fullerene (purity > 99.99%>) in toluene with a C60 molecule concentration corresponding to m axim um  solu­bility near 2.9 mg m L -1 , and the same am ount o f distilled water in an open beaker. The two phases formed were treated in ultrasonic bath. The procedure was continued until the to l­uene had completely evaporated and the w ater phase became yellow colored. F iltration o f the aqueous solution allowed to separate the product from  undissolved C60 fullerene. The pore size of the filter during the filtration of the aqueous solu­tion was smaller then 2 fim  (Typ W hatm ann 602 h i /2).D ifferent concentrations o f C60 fullerene in w ater (from 0.1 to  0.01 m g m L -1) are obtained by this m ethod. The concentration  o f C60 fullerene in the prepared C60FAS sample was determ ined as the concentration o f to tal organic carbon in aqueous solution and equals to  0.1 mg m L -1 (1.39-10-4  M ) (A nalytik Jena TO C  Analyser m ulti N /C  3100). The obtained C60FA S is stable for about 12 m onths at T  — 277 K.UV/VIS MeasurementsU V /V IS  absorption spectrum o f C60 fullerene in water was recorded using a double-beam  spectrophotom eter SQ-4802 (U N ICO , USA). The solution was poured into a polymethy- lacrylate cuvette (Spain) having an optical path  length of 1 cm, which enabled to perform  the m easurem ents in the range of 200-700 nm. The tem perature was m aintained con­stant at T  — 298 K.AFM  and STM  MeasurementsThe state o f C60 fullerene was m onitored using atomic force m icroscopy (AFM ; ‘‘Solver Pro M ” system; N T -M D T , R us­sia) and scanning tunneling microscopy (STM; N T -M D T , Russia) technique. U nder A FM  and STM study the samples were deposited by precipitation from  an aqueous solution droplet onto a cleaved m ica substrate (V-l G rade, SPI Sup­plies) or A u (111) surface (SPI Supplies), respectively. M eas­urem ents were performed after complete evaporation of the solvent. The sample visualization in the A FM  experiments was carried out in a semicontact (tapping) m ode using N SG 10 (N T-M D T) probes. Typical values o f the tunneling current and voltage in the STM  experiments were chosen within the ranges of 0.01-0.1 nA and 0.1-0.8 V, respectively.DLS MeasurementsM easurem ent of the size distribution for C60 fullerenes in aqueous solution was performed by dynamic light scattering(DLS) at T  — 298 К  on a Zetasizer Nano-ZS90 (M alvern, W orcestershire, U K ). D LS instrum ent equipped with a He- N e laser (max 5 mW ) operating at the wavelength o f 633 nm, was used.Zeta Potential MeasurementsZeta potential m easurem ent for C60FAS was carried out on a Zetasizer N ano-ZS90 (M alvern, W orcestershire, U K ) at T  — 298 K. The results were evaluated using the Smoluchowski approxim ation, which is known to be rigorously valid only for spherical-like particles.Results and DiscussionChemical Analysis o f  C60 Fullerene Aqueous Colloid SolutionThe purity  o f prepared C60FA S sample (i.e., the presence/ absence o f any residual im purities, for exam ple carbon black, toluene phase) is an  im portan t factor, which influ­ences its toxicity (8, 13). The purity  o f prepared C 60FAS sample was determ ined by H PLC  and G C /M S  using stan­dard  program s. Insoluble im purities were determ ined by ultra-centrifugation.Insoluble impurities in the prepared solution were found to be less than  1 j i g  m L -1 . Toluene from  the synthesis could not be detected in the water by G C /M S  analysis. 1II N M R  spec­trum  (400 M Hz) of CgoFAS recorded in heavy water did not reveal any residual proton signals.Absorption Spectrum o f  C60 Fullerene Aqueous Colloid SolutionTo confirm the presence o f C60 fullerene in water, the U V / VIS absorption spectrum was recorded (0.1 mg m L -1) in the range of the wavelengths A =  200-700 nm  (Figure 1). Three208 nmwavelength, nm Fig. 1. UV/VIS absorption spectrum of C60FAS (0.1 mg mL-1 ).532intense broad UV absorption bands with m axim a at 208, 265, and 347 nm  dom inate which is in good agreement with litera­ture data  (24).A I 'M and STM  Characterization o f  C60 Fullerene Aqueous Colloid SolutionIn  order to  additionally characterize the com position of the prepared C60FAS, the state of C60 fullerene was m onitored using atomic force microscopy and scanning tunneling microscopy technique.The A FM  picture in Figure 2(a) corresponds to  the con­centration of C60 fullerene in water equal to  0.1 mg m L -1 . Figure 2(b), which corresponds to  ten times less concentra­tion of C60 fullerene in water (0.01 mg m L -1), dem onstrates random ly arranged individual C6o molecules with diameter ~0 .7  nm  and their bulk sphere-like aggregates with a height of 2-50 nm. Figure 3(a) shows the STM image o f surface area covered by lone particles and their clusters. Analysis of~ 15 nm л■ 50 nm- 3  nm- 0 , 7  nm - 2  nm(b)0 10 20 30 40  50 nmFig. 3. STM image of C60 fullerenes on gold surface (a) and its profile along the marked line (b). C6o fullerenes were precipitated from C60FAS with 0.01 mg mL-1 concentration.the image in the cross-section (Figure 3(b)) indicates that the observed objects have the height o f ~0 .7  nm, which corre­sponds to the size o f lone C60 fullerene. The obtained results are in good agreement with theoretical calculations (25) and literature data  from  electron microscopy (26, 27).It is im portan t to  note tha t some individual C6o aggre­gates w ith a height o f ~100 nm  were also seen in the probe m icroscopy images. N um erous experim ental investi­gations have dem onstrated the polydisperse nature of C60FA S, including either m onom ers or aggregates having diam eters ranging from  several to  hundreds o f nanom eters (26, 28, 29), which fully agrees w ith our A F M /S T M  results presented above.DLS Study o f  C60 Fullerene Aqueous Colloid SolutionA typical result o f D LS experiment shown in Figure 4 gives the distribution o f light scattering particles according to  their hydrodynam ic diameters at a fixed solute concentration (0.1 mg m L -1). The m ain fraction of light scattering particles had diameters in the range of 100 nm. This result is in a good agreement with literature data (30) and the data  o f laser2018161412£/л§ 10 а0.1 10 100 1000 10000d. шFig. 2. AFM  images of C60 fullerenes on mica surface, which were precipitated from C60FAS with 0.1 (a) and 0.01 (b) mg m L-1 concentration.Fig. 4. Distribution of the scattered light intensity according to the diameters of light scattering C60 fullerene particles (0.1 mg m L-1 ).533-30 -20 -10 0Zeta Potential (mV)Fig. 5. Zeta potential of C6oFAS (0.1 mg m L_1).correlation spectroscopy of pristine C60 fullerene water col­loid solution, which confirm that the average hydrodynam ic radius of nanoparticles is 50 nm  and no further agglom era­tion is observed (31).Zeta Potential Study o f  C60 Fullerene Aqueous Colloid SolutionThe m agnitude of the zeta potential is related to  the stability o f colloid dispersions, because it determines the degree and nature of the interaction between the particles o f the disperse system. The value of zeta potential for C60FAS was equal to —23 mV (Figure 5). This result satisfactory agrees with the previously published data for colloid C60 fullerenes in water: —30 mV (24) and —38 mV (32). A t the same time, Mched- lov-Petrossyan et al. reported m uch smaller zeta potential of —9 mV (33). A high negative charge of colloid clusters (or, m ore strictly, the electrostatic repulsion between the nega­tively charged clusters) seems to play significant role in the stabilization of C60FAS (i.e., it disfavors the aggregation and m akes the solution electrically stable).ConclusionsThe m ethod o f p reparation  o f highly stable, purified, and reproducible C60 fullerene aqueous colloid solution is described. The m orphological properties o f the prepared C6oFAS have been investigated by m eans of chemical analysis, U V /V IS  spectroscopy, atom ic force and scan­ning tunneling m icroscopy, dynam ic light scattering, and zeta potential m ethods. It was found tha t the properties o f the prepared aqueous colloid solution well agree with literature data.The suggested m ethod of preparation is relatively cheap, reproducible and allows getting highly concentrated fullerene solutions ready for further use in various fields of nanobiotechnology.References1. Cataldo, F. and Da Ros, T. 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Structural features of highly stable reproducible C₆₀ fullerene aqueous colloid solution probed by various techniques

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Structural features of highly stable reproducible C₆₀ fullerene aqueous colloid solution probed by various techniques

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