3 rd International Summer School Supramolecular Systems in

3 rd International Summer School “Supramolecular Systems in Chemistry and Biology” Lviv (Ukraine), 6 - 10 September 2010 _____________________ SUPRAMOLECULARITY CARBON NANOTUBES Mykola T. Kartel Chuiko Institute of Surface Chemistry, NAS of Ukraine 17 General Naumov Street, Kiev, Ukraine OF

INTRODUCTION Supramolecular chemistry – chemistry of molecular ensembles and intermolecular bonds (J. -M. Lehn, 1978) Other definitions: - Chemistry outside of a molecule; - Chemistry of non-covalent bonds Supramolecule is a complex molecule, usually big (host), bonded other molecule (guest) Supramolecular interactions (on energy of bond): - Interaction ion-ionic (100 -350 k. J/mol), are close to covalent bond - Ion-dipole interaction (50 -200 k. J/mol) - Dipole-dipole interaction (5 -50 k. J/mol) - Hydrogen bond (4 -120 k. J/mol) - Cation-π-interaction (50 -80 k. J/mol) - π-π - Steking-interaction (0 -50 k. J/mol) - Van-der-Vaals forces (< 5 k. J/mol)

Supramolecular phenomena are the interdisciplinary area: supra systems are considered in chemistry, biology, biochemistry, life sciences and materials science. Objects of consideration of supramolecular chemistry can be high disperse and high porous materials possessing a considerable specific surface area. High disperse (nanodisperse) materials with the size of particles ~ 1 nm have an external surface area >100 m 2/g. High porous (nanoporous) materials with the size of holes ~ 1 nm have an internal surface area >1000 m 2/g. Already the morphology (texture, structure, supramolecular structure) of such materials has character of supramolecularity (interaction of elements of a skeleton – chains, tetrahedrons, planes, globules, etc. ) Atoms or the molecules adjoining a surface energetically are not compensated. It is the reason of many superficial phenomena: adsorption, heterogeneous catalysis, double electric layer, adhesion and cohesion, wetting, corrosion, capillary phenomena, flotation, surfactant action, number of biological processes. For example, system “adsorbent / adsorbate” can be considered and discussed in terms "host / guest".

“Classical” objects of supramolecular chemistry are carbon ones: graphite and fullerenes. For “macroobiect” graphite it is known the intercalation phenomenon by acids, salts and metals with reception of numerous inclusion compounds, thermoexfoliated graphite and also extreme display – crystal rupture on separate layers (graphene-like structures). For nanoobjects fullerenes it is known an ability to act as the guest (inclusion compounds with macrocycles of calixarenes, cyclodextrin, hydroquinon), or the host (metalorganic complexes, metallic and bimetallic complexes with properties of a superconductor).

SOME sp 2 -CARBON FORMS Graphene Graphite Nanotubes (CNT) S. Iijima. Helical microtubules of graphitic carbon. -Nature, 1991, - V. 354. - P. 56 -58 (SWCNT) М. Endo et al. Carbon, 1975 (MWCNT) L. V. Radushkevich, V. M. Lukjanovich. About structure of the carbon formed at thermal decomposition of carbon oxide on iron contact. –Russian J. Phys. Chem. , 1952, -V. 26, N 1. - P. 88 -95 Fullerene H. W. Kroto et al. C 60 Buckminster-fullerene. Nature, 1985, -V. 318. P. 162 -163

VERSIONS OF CNT SWCNT MWCNT

CNT are cylindrical structures with a diameter from one to several tens nm and length from several nm to several μm. They consist of one or several graphite layers with hexagonal organization of carbon atoms. Tubes come to an end with the hemispherical head formed from half fullerene. Unlike fullerenes, which represent the molecular form of carbon, CNT combine properties nanoclusters and a massive firm body that causes occurrence special, at times unexpected properties. CNT are characterized absolutely new mechanical, adsorption, optical, electric, magnetic, etc. properties.

SPECIFIC PROPERTIES OF CNT 1. Mechanical properties: strength and flexibility (hardening of metals and polymers, polymeric composites, additives to lubricants and oils, etc. ); 2. Electronic properties: semi- and/or metallic conductivity, magnetoresistance, cold emission of electrons (electronic devices of the molecular size, information recording, diodes, field transistors, cold cathodes, materials for displays, quantum dots and wires, cathodes for X-ray radiation, electric probes, etc. ); 3. Optical properties: resonant absorption of IR-radiation (lightemitting diodes, optoelectronic devices, thermal nanobombs); 4. Physical and chemical properties: developed surface and variable surface chemistry, programmed reactivity and carrier for active chemical and biological objects (sorbents, catalysts, chemical sensors, electrode materials, chemical batteries, fuel elements and supercondensers); 5. Biological properties: biocompatibility and toxicity!!!, penetration ability into biological cell!!! (preparations, medical nanoinstruments, biosensors, prosthetics, means of gene engineering).

SCALE OF NANOSIZED OBJECTS 1 nm 1000 nm

METHODS OF RECEPTION OF CNT (1) Voltaic arc dispersion of graphite; electrolytic synthesis (2) Laser evaporation (ablation) of graphite; carbon epitaxy (3) Catalytic decomposition of hydrocarbons and carbon monoxide (CVD-technology)

MECHANISM OF CNT GROWTH The metal drop acts a catalyst role at formation carbon nanotube and defines its size (drawing from a site http: //students. chem. tue. nl/).

AGGREGATES OF NANOTUBES AND NANOFIBERS The ordered and chaotic growth on substrates Kinds of nanofibers in a light microscope «Hair of water-nymph» (Moscow State University Department of Chemistry)

PURIFYING AND ENRICHING CNT q Demineralization by strong acids and alkalis; q Thermoprogrammed annealing of amorphous carbon; q Thermoprogrammed wall-by-wall annealing of multi-walled tubes (enriching by single-walled tubes); q Electrolytic annealing of conducted tubes (enriching by semiconductor tubes); q Chemical updating (increase of solubility of tubes); q Dispersion by means of ultrasound and surfactants, sedimentation separation (ultracentrifugation) individual tubes from aggregates.

SEPARATION OF NANOTUBES (SURFACTANTS, ULTRASOUND, ULTRACENTRIFUGE)

Variants of "wrapping" of nanotubes by surfactants.

AUTOMATED ANALYSIS OF CNT (analysis of data received by TEM, SEM and АFМ methods) q Analysis of diameters of nanotubes q Analysis of thickness and orientation of nanofibers q Definition of length, thickness, curvature and size distribution of nanotubes q Analysis of structure of multiwalled nanotubes q Analysis of impurities in nanotubes

COST OF CNT PRODUCTION AND PRICES № Product name Price, USD per gr. 60 1 SWNT (single wall CNT), as produced from cellular zone, 40 -50% 2 SWNT, purified 80% 380 3 SWNT, purified 90 % Expected 4 SWNT-COOH, purified (70 -80%) with 2 -5% of –COOH groups 380 5 SWNT-NH 2, purified (70 -80%) with 2 -5% of - NH 2 groups 600 6 SWNT-CONH-C 18 H 37, purified (70 -80%) with 2 -5% of -CONH-C 18 H 37 groups 600 7 SWNT-COO-R-OH (2 -5% groups) 600 8 SWNT shorted, purity 90%, length 200 -500 nm, 5 -10% of –COOH groups 1200 9 SWNT shorted, purity 90%, length 200 -500 nm, 5 -10% of –NH 2 groups 1600 11 SWNT-COO-R-OH shorted (200 -500 nm), (5 -10% groups) 1600 12 Solutions of functionalized SWNT in different solvents +150 13 Substituted pyrrolidinofullerene derivatives 2000 14 Lower bulk density SWNT-COOH, purified (70 -80%) with 2 -5% of –COOH groups 460 15 DWNT (double wall CNT), 20 -30% purity 250 16 DWNT (double wall CNT), 90% purity 17 SWNT with attached building blocks from Med. Chem. Labs Inc. , collection 1500 Discussed

COST OF CNT PRODUCT DISCRIPTION CARBONACEOUS PURITY* METAL CONTENT PRICE** MINIMUM ORDER wt % from TGA in air AP-SWNT As prepared 40 -60% 30 $50/g 2 g P 2 -SWNT Purified, low functionality 70 -90% 7 -10 $400/g 0. 5 g P 3 -SWNT Purified, high functionality 80 -90% 5 -10 $400/g 0. 1 g P 5 -SWNT Organic soluble (functionalized with ODA) 80 -90% (50% SWNT loading) 4 $150/ 100 mg 0. 1 g P 7 -SWNT Water soluble (functionalized with PEG) 80 -90% (70% SWNT loading) 6 $150/ 100 mg 0. 1 g P 8 -SWNT Water soluble (functionalized with PABS) 80 -90% (30% SWNT loading) 3 $150/ 100 mg 0. 1 g P 9 -SWNT Amide functionalized SWNTs 80 -90% 6 -8 $150/ 100 mg 0. 1 g

Pilot manufacture of MW CNT on OAS “ARTEMOV TAMBOV’ ENTERPRISE “KOMSOMOLETS” Tambov, Russia Productivity 2 -2. 5 t/year Dynamics of world production of CNT (t/year)

Pilot manufacture of MW CNT on experimental section of CHUIKO INSTITUTE OF SURFACE CHEMISTRY and ENTERPRISE "ТМ-SPETSMASH“-LTD Kiev, Ukraine Installation of synthesis multiwalled CNT and CNF by CVD-method (catalytic pyrolysis of hydrocarbons). Productivity of 1 -1, 5 kg/day (> 0. 5 t/year) TEM of CNT CARBON NANOTUBES. Specifications TU U 24. 1 -03291669 -009: 2008

TOXICOLOGY OF CNT RESPONSE OF ORGANISM, BODY OR TISSUE TO ACTION OF NANOPARTICLE AS DAMAGING AGENT THE FACTORS ARE CAPABLE TO LEAD TO DAMAGES IN TISSUES, BODY OR ORGANISM AS A WHOLE: q High indicator of a specific surface (i. e. the relation of the area of a particle to its weight) provides the big area of contact to cellular membranes and causes effective adsorption of substances and influences on their transport q High indicator of keeping time (low mobility in tissues, long time of deducing from an organism): more contact time - more damages q High index of reactivity: reactionary ability is interconnected with an indicator of a specific surface, heterogeneity (deficiency) of a material, and also its chemical cleanliness (for example, presence of nanoparticles of toxic metals)

WAYS OF PENETRATION OF CNT TO THE HUMAN BODY AND ANIMALS q At inhalation (contact to a mucous and pulmonary tissue) q Contact with skin covering q Food intake and water drinking q Intended introduction under skin, in GIТ and in blood Following action on cellular level !

WAYS OF CNT PENETRATION INTO CELL Ø PUNCTURE Ø PERMEATION Ø ENDOCYTOSIS

CYTOTOXICITY OF CNT Authors Material Type of cell Result Shvedova et al, 2003 SW CNT, Fe-catalyst Keratinocytes of human (На. Са. Т) – put in solution with 0. 06 -0. 24 mg/ml CNT, contact 8 h Accelerated oxidative stress (growth of quantity of free radicals and peroxides, an exhaustion of the general antioxidant reserves; losses in viability of cells and morphological changes Monteiro. Riviere et al, 2005 MW CNT, (CVDmethod), purified Keratinocytes of human (НЕК) – put in solution with 0. 1 -0. 4 mg/ml CNT, contact till 48 h Production preinflammatory cytokinin (IL); reduction of viability of cells depending on time and a dose Tamura et al, 2004 CNT, purified Human blood neutrophils – contact with solution CNT during 1 h Production increase superoxide anion-radicals and inflammatory cytokine (TNF-α); decrease in viability of cells Cherukuri et al, 2004 SW CNT, purified Phagocytic cells of mice (J 774. 1) Catching ~50% of nanotubes, no cytotoxic effect Shvedova et al, 2005 SW CNT, Fe-catalysts Macrophagic murine cells (RAW 264. 7) Pro-fibrotic mediator TGF- 1 was increased; no oxidative burst, nitric oxide production or apoptosis was observed Muller et al, 2005 MW CNT, purified Peritoneal and alveolar macrophages – incubation in solution with 20, 50 и 100 mg/ml CNT, contact 24 h Emission of lactate dehydrogenase and inflammatory cytokinin (м. RNA squirrel TNF-α) Jia et al, 2005 SW and MW CNT (arc, CVD), purified Alveolar macrophages – solution of CNT, modified dose regime of contact, conc. 1. 4 -22. 6 mg/cm 2, 6 h Reduction of viability of cells and easing of their functional ability Cui et al, 2005 SW CNT Human embryonic kidney cells (НЕК 293) – put in solution with 0. 78 -200 mg/ml Induction of apoptosis and reduction of ability to adhesion, reduction cellular proliferation (on expression of corresponding genes)

The control of barrier function of membranes and activity of mitochondria by EPR of spin probes Intensity EPR Nitroxyl radicals in research of oxidation-reduction status of bioobjects t Time, min h+ h 0 h_ h 0/h_ – Parameter of mobility of a probe, which characterizes microdensity min

EPR-spectra of a spin probe in erythrocyte cytosol of donor blood after incubation 2 days at temperature 6 o. C with CNT of various concentrations: 1 – control; 2 – 10 μg/ml; 3 – 200 μg/ml. Influence of donor blood erythrocytes incubation with CNT of various concentrations on intensity of the central component of EPR-spectrum of radicals: 1) – control; 2) – 10 μg/ml; 3) – 50 μg/ml; 4) – 100 μg/ml; 5) – 200 μg/ml.

Reduction of a spin probe in liver homogenate after 4 hours incubation: ♦ - control; ∆ - about 200 μg/ml CNT. EPR spectra of lipophilic spin probe: in water solution; probe and expander mixes; mixes of probe, expander and CNT; probe in suspension of CNT.

INTERRACTION BETWEEN CNT AND NITROXYL RADICAL (preliminary quantum chemical data)

BIOCOMPATIBILITY OF CNT (to cells) Authors Material Type of cell Result Elias et al, 2002 CNT-containing orthopedic materials Osteoblasts – inoculation on material Increase of osteoblast proliferation, growth of alkaline phosphatase, absence of cytotoxicity Supronowicz et al, 2002 PLA/CNT – nanocomposites Osteoblasts – contact with nanocomposites, action of current Growth of proliferation of osteoblasts, absence of cytotoxicity Price et al, 2003 PU/CNT- nanocomposites Osteoblasts, chondrocytes, fibroblasts, plain muscular cells – contact with PU/CNT Adhesion growth of osteoblasts, easing of adhesion of other cells, absence of cytotoxicity Correa-Duarte et al, 2004 MW CNT, oxidized Fibroblasts of mice (L 929) – inoculation on CNT, observation - 7 days Formation of the isolated cells, fusion after 7 days, absence of cytotoxicity Mc. Kenzie et al, 2004 MW CNT, purified, different diameter Astrocytes ( cells which are responsible for reduction of damages of nervous tissue) – contact to CNT surface Normal proliferation, adhesion and functional activity on tubes, especially with a diameter less than 100 nm. Hu et al, 2004 PU/CNT- nanocomposites Astrocytes, aksons of rats – contact with PU/CNT nanocomposites Adhesion reduction of astrocytes, a growth of inhibition of aksons. Cytotoxicity is absent Gabay et al, 2005 MW CNT, purified and chemically modified Neurons – inoculation on CNT, observation - 4 days Localization on CNT, proliferation of aksons. Cytotoxicity is absent Mc. Knight et al, 2004 Vertically oriented CNT with immobilized DNA Cells of Chinese hamster ovary (CHO) – centrifugation and pressing Part of cells was lost, however cytotoxic effect was noted

Growth of colonies of yeast cells (control) Growth of colonies of yeast cells at presence at a nutrient medium of suspension CNT

Kinetics of mobility (activity) of human spermatozoids in the presence of CNT in various concentrations

POSSIBILITIES OF MEDICAL USE OF CNT Nanocomposites with polymers and alloys (prosthetics) Internal functionalization External functionalization On the ends and defects of tubes On the walls of tubes

GENERAL STRATEGY OF FUNCTIONALIZATION OF CNT

GENERAL STRATEGY OF COVALENT FUNCTIONALIZATION OF CNT

IMMOBILIATION OF STREPTAVIDIN ON CNT

CNT AS KILLERS OF CANCER CELLS Ø NANOBOMBS (near IR-light) Ø HYPERTHERMIA (near IR-light) Ø DELIVERY of radioisotopes, cytochrome C, cytostatics etc. (Folic acid – provides selectivity of meeting CNT with cancer cells) Destruction of cancer cells in blood vascular with use of CNT at illumination by near IR-light (on Balaji Panchapakesan)

TRANSPORT OF OBJECTS INTO CELL BY MEANS OF CNT (In cytoplasm and in nucleus) Ø Transport of medicines (antibiotic – amphotericin B) Ø Transport of vaccines (peptide of virus foot-and-mouth disease virus) Ø Transport of proteins (streptavidin, fibrinogen, protein A, erythropoetin, apolipoprotein) Ø Transport of nucleic acids

ANTICARCINOGENIC PREPARATIONS ON THE BASIS OF PLATINUM cis - [Pt (NH 3) 2 Cl 2)] Cisplatin Complex Pt (IV) and Complex Pt (IV) covalent connected with SWNT. Viability of tumor cells (%) at fourdays influence of free complex Pt (IV) and the similar complex attached to SWNT.

APPLICATION OF CNT AS CARRIERS OF BIOPREPARATES Authors Conjugates Result Pantarotto et al, 2003 Functionalized SW CNT + small peptide sequence from the foot-and-mouth disease virus (FMDV) SW CNT-FMDV peptide complex induced a specific antibody response in vivo. It was maintained and recognized by mono- and polyclonal antibodies Pantarotto et al, 2004 Functionalized SW CNT + peptide fragment from the α-subunit of the Gs protein (αs) SWCN-αs complex was able to cross the cell and nucleic membranes (human 3 T 6 and murine 3 T 3 cells) Kam et al, 2004 Purified and shortened SW CNT + streptavidin SW CNT- streptovidin conjugate caused extensive cell death, which was attributed to the delivery of streptavidin to the cells (proleukemia cells of human and T-lymphocytes) Wu et al, 2005 CNT + amphotericin B; Amphotericin B got into various cells and increased its activity Bianco et al, 2005 CNT + different proteins of < 80 k. Da (fibrinogen, protein A, erythropoietin, and apolipoprotein) CNT-TEG-short protein complex quickly got at fibroblasts and other cells, sometimes migrated to their nuclei. Proteins executed own biological functions Lu et al, 2004 SW CNT + RNA polymer Successful transportation of SW CNT-RNA polymer complex into cytoplasm and nucleus of cell Pantarotto et al, 2004 SW CNT and MW CNT + plasmid DNA All conjugates influenced on regulative expression of marker genes in human cells Cai et al, 2005 SW CNT + plasmid DNA, with nickel under the influence of a magnetic field High efficiency of transduction of SW CNT-DNA conjugates in lymphoma cells (Ball 7 B-lymphoma) Kam et al, 2005, 2006 SW CNT + cytochrome C, RNA, DNA CNT transferred cytochrome C to the cancer cells; accumulation of SW CNT-RNA conjugates in cytoplasm and nucleus of He. La cells

NANOSENSORS (vertical oriented CNT on surface of electrode)

NANOSENSORS orientation of CNT on electrode) (vertical

NANOSENSORS (usage of CNT in form of fibers or nets) Joining of antibodies to grids from CNT allows to create nanosensors. At linkage of antibodies with a corresponding antigens (for example, the specific protein of cancer cells) changes conductivity of fibers from CNT, that is fixed by a current between electrodes. (Balaji Panchapakesan, University of Delaware, USA)

CONCLUSIONS: Manufacture and use of carbon nanotubes are: Expensive (small yields, special physical, chemical and biological methods of enriching, fractioning, separations, reception of great volumes with reproduced properties; the powerful and expensive equipment for the control of properties and characterization is also required); Unsafe (influence on alive organisms at cellular level and on environment; necessity to develop nanotoxicology with all expenses needed for it); Perspective (for medicine it is new class of preparations, delivery systems of medicines, vaccines, serums, a vector for gene engineering, nanosensors, nanoinstruments).

CONCLUSIONS q We actually use the first method of spin probes to study the cytotoxicity of nanomaterials (CNT) and the development of nanotechnology (drug delivery systems, medical biotechnology) has shown its great potential. q Advantages of the method associated with an instant assessment of the influence of parameters of the microenvironment of the probe (microviscosity, polarity, micro -relief surface, red-ox potential) on the parameters of their EPR spectra, as well as the small size of the probes (<< 1 nm), which fits well with the size range studied nanostructures and significantly less than the size of biological objects (proteins, cells, subcellular structures, etc. ). q Another important aspect of the effectiveness of spin probes is that the method allows to work with very complex, optically opaque biological objects and to judge the status of their individual structures or fragments by detecting changes in the parameters of the microenvironment of the paramagnetic proper tags.

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