3 Carbon Nanotubes: Nanotoxicity Testing and Bioapplications · Carbon Nanotubes: Nanotoxicity Testing and Bioapplications R ... characteristics of multiwalled CNTs and production

97

Ashutosh Tiwari and Atul Tiwari (eds.) Nanomaterials in Drug Delivery, Imaging, and Tissue Engineering, (97–148) © 2012 Scrivener Publishing LLC

3

Carbon Nanotubes: Nanotoxicity Testing and Bioapplications

R. Sharma1 and S. Kwon2

1Center of Nanomagnetics and Biotechnology, Florida State University, Tallahassee, Florida, USA

2Department of Biological Engineering, Utah State University, Logan, Utah, USA

Abstract With the growing use of nanomaterials in bioapplications, the nanotoxicity of new nanomaterials has become a safety concern when used in various applications. In this chapter, technical developments on carbon nanotubes are described including a historical account, experimental models and potential bioapplications.

Carbon nanotube (CNT) materials display superior properties in elec-tric current carrying capacity, thermal conductivity, and thermal stabil-ity. Due to the unique CNT structure with high-aspect ratio, CNT may show unusual toxicity and complicate its safe use in a target tissue. To test nanotoxicity of CNT, we describe a set of protocols of prior knowledge-based physical and chemical characteristics to develop 3-dimensional in vitro models of the intact skin, and a 3D in vitro model of the human air-way using a co-culture of normal human bronchial epithelial cells and normal human fi broblasts. The human airway 3D model served as a tool of health risk assessment of CNTs on the human respiratory systems. To test functionality at different CNT concentrations in a 3D model, physical characteristics of multiwalled CNTs and production of nitric oxide (NO) served as cell viability and infl ammatory marker; mitochondrial activity (MTT assay) served as the cytotoxic response of the epithelial cell layers; transepithelial electrical resistance (TER) measured nanotoxicity in the changes in airway physiological function. Cytoxicity and infl ammatory responses of CNTs were dependent on different size, mass, shape, and functionality of CNTs as viable in vivo tests were conducted to evaluate the

98 Nanomaterials in Drug Delivery

toxicity of engineered CNTs. We monitored the transport across skin, and the physiological perturbation of transepithelial electrical resistance (TER) during the exposure of different concentrations of CNTs. The mechanisms of CNTs’ toxicity are closely related to their structure, functional group, and surface charge on the molecule. We established the nanoscale toxicity of fullerenes of CNTs.

Keywords: Nanotoxicity, carbon nanotube, silver nanoparticles, cytotoxic-ity, fullerene, drug carrier

3.1 Introduction

Nanomaterials have emerged as potential tools in almost every fi eld from space to the environment and from health to robotics. With the increasing demand for nanomaterials it is necessary to evaluate toxicity carefully before accepting new nanomaterial in wider bioapplications. In this chapter, technical developments on carbon nanotubes are described with an account of their histori-cal development, experimental models and potential applications. The fi rst section describes the carbon nanotubes-based nanocom-posites. This chapter is divided in subsections on the toxic nature of CNT, model CNT-metal and collagen composites, structure of carbon nanotube materials, physical principles of CNT-biosurface interaction, nanoindentation testing and the mechanism of infl am-mation in epithelial cells induced by CNT with an account of bio-physical experiments on CNT exposed to mesenchymal cells, 3D human lung prototype, skin tissues and prototype scaffold nano-materials. A possibility of CNT as a safer drug delivery system is explored that describes the future of possible bioapplications of carbon nanotubes and nanocomposites. A major focus of this chap-ter is the proper selection of sensitive, reproducible, accurate and user-friendly techniques for use in characterizing and testing toxic-ity of nanocomposites at nanoscale. This chapter will also discuss the need to capitalize on CNT nanocomposites as safe delivery systems.

3.1.1 What is Nanotoxicity of Nanomaterials?

The concept of “nanotoxicity” has been well known since 1895 when William Roentgen showed his hand image on a fl uorescent screen due to CRT leaked rays on his hand. However, X-rays were

Carbon Nanotubes: Nanotoxicity Testing 99

found to be toxic and hazardous. Later on rapid progress and early acceptance of nanobiotechnology further increased the potential use of nanomaterials in spite of their adverse effects. Environmental impact of nanomaterials remained a controversial hot topic of increased multiple usages of nanomaterials. In particular, the behavior of nanoparticles sitting inside the stem cells, or meta-bolic consequences and immunological responses in cells induced by nanoparticles still remains poorly understood. Nanotoxicology testing takes up this challenge to explore the molecular events that regulate bioaccumulation of toxic products and events of cytotoxic-ity as a consequence of nanoparticle-induced toxicity. Toxicity test-ing of nanomaterials serves as a testing criterion of designing and characterizing new biocompatible nanomaterials or nanocompos-ites suitable for bioapplications. The following sections describe the cytotoxicity of carbon nanotubes.

3.2 Historical Review of Carbon Nanotube

Carbon nanotubes were discovered in 1991 and their use expanded to make conductive and high-strength composites, energy storage devices, sensors and actuators, fi eld emission displays, nano-scale semiconductor devices, probes with unique physical, mechanical, electrical, and thermal properties. Initially CNTs in powder form were considered to be cytotoxic and DNA mutagenic with risk of inhalation-induced toxicity to workers due to direct skin con-tact. Several studies recently indicated possible toxicity of CNTs based on the facts that: 1) CNTs and fullerenes have produced toxic effects on biological systems [1–5]; 2) CNTs can translocate to bloodstream [6, 7]; 3) CNTs can cross blood brain barrier [8]; and 4) toxicity of CNTs results in pulmonary infl ammation as the main effect on tissues due to the distinct shape and biodegrada-tion properties of CNTs. Donaldson et al. [9] described in detail three main properties of CNTs associated with pathogenicity of particles. The properties were: 1) CNTs showed more toxicity than larger sized particles, 2) fi ber-shaped CNTs behave like asbestos and other pathogenic fi bers which have toxicity associated with their needle-like shape, and 3) biologically biopersistent particles. Authors also pointed out that CNTs are possibly one of the least biodegradable man-made materials ever devised [9]. Other con-cerns over the increased emissions of CNTs into the environmental


compartments (air, water and soil) were due to improper disposal of CNTs [10]. With all these concerns, now safe use of carbon nano-tube material is emerging as state of the art since it was found bio-compatible with hard bony material and nanoscale source of drug delivery carrier in the body. Present view of CNTs is perceived as nanomaterial with double face because of CNT induced toxicity and possibility of safer biomedical applications of new CNT nano-composites. However, science is still discovering new methods of CNT nanocomposite preparations and toxicity testing of carbon nanotubes and CNT composites in biomedical applications as mentioned below.

3.3 Carbon Nanotubes (CNTs) and Other Carbon Nanomaterials

Carbon nanotubes (CNTs) and other carbon nanomaterials are of interest for biological and medical applications because of their high chemical durability, mechanical strength and electrical prop-erties. Several studies on the application of carbon nanomaterials have been reported such as CNT substrates of cell culture [11–20], CNT-based drug delivery systems [21, 22], and medical CNT implant materials [23, 24]. A recent study reported the synergy on the unique properties of carbon nanotubes (CNT) with details of tissue compatibility and osteogenesis of human mesenchymal stem cells (hMSC) [25]. It is believed that CNTs will provide an excit-ing opportunity for novel therapeutic modalities. However, little is known about the impact of CNTs on cellular processes such as adhe-sion, proliferation, and differentiation of mesenchymal cells (MSC). The following questions emerged on the interaction between CNT-based nanomaterial and MSC cells:

1. How are CNT-based composites are prepared and tested for physical properties on surface of CNT-based nanocomposites?

2. How do CNT-based composites affect cellular pro-cesses (e.g., renewal, metabolic activity, and differen-tiation) of MSCs?

3. Which stages of cell division during differentiation of stem cells are most affected by CNT-based materials?


4. Does addition of CNTs into naturally derived polymers result in creating stiffer environment in which progen-itor MSC cells may prefer to differentiate into matured cells such as osteocytes, myocytes, hepatocytes?

Changes in cellular and physiological properties of MSC including adhesion, proliferation, and differentiation are major tests of toxicity during cellular processes. CNT-based nanosub-strates are black in color with low optical transparency for optical microscopic observation of the cultured MSC cells fi xed on the CNT-based substrates. Collagen-SWCNT composites are better alternatives. Single-walled CNTs (SWCNTs) substrates in cell cul-ture are strongly entrapped by collagen in composite. Collagen-based composites showed high mechanical strength and good cell viability [13]. Type I collagen is one of the most biocompatible materials. Collagen-coated cell culture dish is widely used for cell culture. CNTs show a high affi nity for the collagen-coated dish surface.

Recent studies have revealed that stemcells are responsive to the extracellular matrix (ECM). Variation in CNT tube length scale and tube size in contact with ECM or extracellular environment also plays a crucial role in determining cellular behavior in MSC cells. MSC cells are naturally accustomed to interact with nanometer length scale features of CNT. It is possible that carbon nanotubes (CNTs) may mimic nanoscale features of the native extracellular matrix and CNTs may be good candidates with applications in the design of new biomaterials for tissue engineering. In the follow-ing sections, surface properties of CNT-composite materials are described with a use of reconstituted Type I collagen and several different types of CNTs to design extracellular matrix to house MSCs in the scaffold. MSC cells serve as incorporated experimen-tal cells in scaffold preparation. Scaffold serves as a living-tissue analog consisting of cells embedded in a collagen-CNT matrix. The application of collagen-CNT matrix is now growing in biological applications. An intriguing point was that augmenting the proper-ties of naturally derived collagen polymers through incorporation of CNTs might enhance in vitro osteogenic and osteoblastic differ-entiation of MSCs. In following the sections, we will describe in situ microscopic observation of cultured MSC cells in interaction with CNTs to evaluate the CNT-induced toxicity.


3.3.1 Physical Principles of Carbon Nanotube Surface Science

Physical principles of carbon nanotube surface science were explored in detail over a decade. Carbon nanotube surface prop-erty was considered peculiar in enhancing the surface area exposed to any tissue or biological exposure. Physical properties were con-sidered as markers of CNT toxicity characteristics such as electri-cal conductivity of single-walled or multiwalled CNT material. A Drude model for electrical conductivity property of multiwalled CNT is described in the following section for its possible nanotox-icity application.

Let us consider the Drude model in which the electrical conduc-tivity is given by σ=eΝμ where e is the absolute value of the elec-tron charge, N is the spatial carrier density, and μ=eπ/m* denotes carrier drift mobility which, in turn, reads μ where τ designates relaxation time (or momentum-scattering rate) and m* stands for carrier effective mass. Then, by replacing the second formula with the fi rst one, it follows:

ts =

2

*

e Nm

(3.1)

Equation 3.1 is standard in the physics of semiconductors and constitutes a relevant element of reference within the context of macroscopic condensed-matter systems. In particular, let us con-sider a multiwalled carbon nanotube (MWCNT) conceived as a longitudinal quantum box according to Mooney et al. and Durkop et al. [25, 26]; in such a tube, conductance is quantized according to the fact that the involved quantum number coincides with the numbering of the CNT layers (conductance scales with the number of layers) [25–28]. Therefore, quantizing formula (3.1) for a metallic MWCNT, conductivity due to the nth layer will be:

ts =

2n n

n

e Nm

(3.2)

where n designates quantum number (n = 1, 2, ...) and m* has been replaced by the free-electron mass denoted by m. In addition, now τn is transit time or motion time (if MWCNT transport is ballistic) so En = h2n2/(8ml2) that τn = l/vn where l is the length of the tube and vn stands for the magnitude of the quantized Fermi velocity which, for n >> 1, approaches the electron velocity deduced from equating the


quantized electron energy (corresponding to the electron confi ne-ment in the carbon nanotube as a longitudinal ideal quantum box) to (1/2) 2.nmv Hence, it follows:

≈

2n

hnv

ml (3.3)

where h is the Planck constant. We regard our MWCNT as a quasi-one-dimensional structure

so that <<A l where A is the cross-sectional area of the tube. On the other hand, we assume that the number of electrons partici-pating in the conduction process depends upon n in accordance with the distribution of electrons in the atomic shells so that the above number equals 2n2. Therefore, inserting the relation, namely Nn = 2n2/(lA) in Equation 3.2, taking in account that the quantized conductance is given by Gn = σnA/l, and inserting it into equation relations 3.2 and 3.3 with the fact that τn = l/Vn, fi nal output is: Gn = 2G0n where G0 = 2e2/h is the fundamental conductance quantum. The abovementioned expression for the quantized conductance of MWCNT indicates that conductance approximately depends lin-early on n which is acceptable in the quasi-classical case, that is, for suffi ciently large values of the quantum number. At any rate, a formula valid for every n can be given. To get this, we use the fol-lowing relationship concerning the energy-level spacing induced by quantum confi nement, namely:

+ − =1 2

Fnn n

hvE E

l (3.4)

where VFn is the magnitude of the quantized Fermi velocity. By Equation 3.4 and the expression for the quantized energy, one gets:

+= (2 1)4Fn

h nv

ml (3.5)

Notice that the right-hand side of Equation 3.3 coincides approxi-mately with the right-hand side of Equation 3.5 when n >> 1 (quasi-classical case). Repeating the calculation process in light of the Drude model developed previously by employing new Equation 3.5 for the electron velocity, the quantized conductance can be expressed as:

204

2 1n

kG nG

n=

+ (3.6)


where k is a phenomenological parameter such that 0 < k < 1 which is a measure of the interwall (interlayer) coupling in the CNT tube. We assume that k is a uniform continuous random variable so its average or expected value is ⟨k⟩ = 1/2. In the quasi-classical case, that is, when n >> 1, from relationship it follows that Gn ≈ 2kG0n which gives an expected observed value. The above expla-nation and results of multiwalled nanotube surface agree with the experimental data as demonstrated by Grado-Caffaro et al., 2008 [28].

3.4 Motivation – Combining Nanotechnology and Surface Science with Growing Bioapplications

Carbon nanotubes: In recent past fullerenes, annotubes showed potential applications due to their large surface area and free radi-cal chemistry, their strong attraction to electrons, and antioxidant properties. The combination of nanotechnology and surface science toward heterogeneous catalysis was a promising challenge for both technical applications and fundamental research, which is still in its infancy, even more than nano-electronics. Successful combina-tion of these complementary research areas has resolved some mys-teries such as some Carbon-60 fullerenes bind to nucleotides, and hamper self-repair in double-strand DNA [29]. In addition, these CNT materials have high electrical and thermal conductivity, high strength, and rigidity. Medical/nonmedical applications further suggested the occupational, accidental exposure with enormous economic impact. Fullerenes (CNT cages), single-wall nanotubes, and multiwalled CNT nanotubes may show cytotoxicity. On the other hand, these materials were reported as nontoxic and pro-tective against pathologies of acute or chronic neurodegeneration and liver diseases [31–36]. Major concerns emerged because CNT produced superoxide anion, lipid peroxidation, and cytotoxicity in plants and animals [37–40]. Uncoated fullerenes in largemouth bass showed lipid peroxidation in brain tissue and glutathione depletion in gills. It makes a difference if CNT nanomaterial is metal or oxide as shown in inhalation studies [41]. CNT material has promising applications as a component of chemical catalysts for chemical pro-cesses such as alcohol oxidation in direct liquid fuel cells, hydro-gen-related technology, or Fischer-Tropsch synthesis, and CNT


may provide sustainable energy sources for the future. Using CNTs as strong support material is a promising approach. Multiwalled CNT materials are used as models for porous materials and a rare two-dimensional biosystem for theoretical and applied studies. In this direction, remarkable progress is made. In the following sec-tion, preparation and physical characterization of metal-supported CNT-Ni, CNT-Si, CNT-Co composites and polymer supported CNT composites are described with an aim to achieve high value CNTs. For characterization, electron microscopy, Raman spectroscopy for atomic hydrogen, and nanoindentation test are described in the fol-lowing section.

Nanofabrication of CNT materials: Metal-supported multiwalled CNT catalysts can be prepared by metal vapor deposition or by “wet-chemistry” procedures. The fi rst technique leads to ultra-clean materials pertinent for mechanistic studies. In the second approach, ultra clean material can be up-scaled to synthesize bulk quantities. Bulk quantities of CNT-metal composite provide a larger dispersion of the nanometal clusters as realistic model sys-tems for technical applications. A noble approach to electrochemi-cal properties of CNT composites with Nickel is described in the following section. Recently, electrochemical properties across the membrane have shown a new way of measuring nanotoxic-ity of CNTs and composites [58]. Other important approaches using Atomic Force Microscopy image as shown in Figure 3.1; and Electron microscopic SEM image of the Ni catalyst addressed the electrochemical growth process of CNTs after ion beam sput-tering deposition are shown in Figure 3.2. The Ni catalyst was aggregated after the fi lm growth due to the surface tension and the stress induced from the mismatch of the thermal expansion coeffi cients of the silicon substrate and Ni fi lm. The size of the Ni particle was about 5–10 nm. The Ni played an important role in the promotion of CNTs growth. The growth on Ni catalyst was similar to the gas-solid interaction process to obtain a single CNT from every Ni particle.

Figure 3.2a,b shows the top view and the cross-section view, respectively, for SEM images of MWCNTs deposited with C3H8:H2 = 2:3 gas mixture at the ECR power of 400 W for 5 min. The SEM image shows the carbon nanotubes arranged approximately per-pendicular to the substrate surface and form an aligned carbon nanotube array. The nanotube array was grown on the thin uniform catalyst layer.


Carbon nanotube AFM probe

Carbon nanotube

Tip-induced E-field

Absorbed water

TiN

Si (100)

Oxide

AFM probe

SEI 10.0kV x100,000 100nm

–+ V

(a) (b) (c)

2 μm 3 μm600 nm

Figure. 3.1 A simple scheme of carbon nanotube characterization by atomic force microscopy (AFM). Reproduced from reference [40].

Figure. 3.2 A representative SEM image of the CNT-Ni composite; (b) Top view of MWCNT deposited on Ni catalysts fi lms; (c) cross-section view SEM images of MWCNTs deposited on a Ni catalyst fi lm with C3H8:H2 = 2:3 gas mixture at the ECR power of 400 W for 5 min. Reproduced from reference [40].

Multiwall CNTs typically make composites with Ni, collagen, polymers, etc. Figure 3.3a shows a bundle of FEG-TEM images of MWCNTs deposited with C3H8:H2 = 1:4 gas mixture at the ECR power of 400 W for 5 min. The resolution of a TEM is generally one order of magnitude higher than that in SEM. As shown in the fi gure, each MWCNT in the bundle has a different length with a bamboo-like structure. It indicates that CNTs in the graphitic lay-ers are not perfectly parallel to the tube axis and do not grow from the bottom to the top of fi lms. Figure 3.3b illustrates the closed-end tip images of MWCNTs and shows that the carbon nanotubes have a hollow structure. The outer diameter and inner diameter of this


nanotube are about 20 and 5 nm, respectively. The outer diameter of CNT could be determined by the diameter of the catalytic Ni particle. Amorphous carbon layer in the image is observed and it indicates that the graphitization was not perfect due to the lower reaction temperature.

Figure 3.4a shows the Raman spectra of the CNTs grown on the polymer C3H8 substrate for different ratios of C3H8 to H2 and has two broadband peaks at about 1350 and 1580 cm−1, refer to the D band and the G band, respectively. The D band has disorder-induced features and indicates the existence of defects whereas the G band denotes original graphite sheet features.

The role of atomic hydrogens in CNT structure is responsible for the formation of nanoparticle nuclei. Hydrogen atoms exist in a bal-anced state during the deposition of nanotube fi lm. As an exam-ple, increasing levels of atomic hydrogens are shown to reduce the decomposition rate of propane on the catalyst surface. The relative intensity ratio of the D band to G band (i.e., ID/IG) varies with the fl ow ratio of to C3H8 as shown in Figure 3.4b. It can be seen that the optimum ratio for the cases in the experiment is 2, and it indicates that this condition is most suitable for the growth of CNTs. The images of SThM with resistance of RC= 21.3Ω for MWCNTs depos-ited at the ECR power of 400 W with the gas mixture of C3H8L:H2 = 1:1 and C3H8:H2 = 1:2 are shown in Figure 3.5a,b, respectively.

(a) (b)

50 nm 5 nm

Figure. 3.3 FEG-TEM images of MWCNTs deposited with C3H8:H2 = 1:4 gas mixture at the ECR power of 400 W for 5 min. (a) The bundle of CNTs, and (b) the closed-end tip. Reproduced from reference [40].


1200 1400 1600Wavenumber (cm–1) Flow ratio (H

2/C

3H

8)

I D/I G

Inte

nsi

ty (

arb

. un

its)

1800

C3H8:H2 = 1:1

(a) (b)

C3H8:H2 = 1:2

C3H8:H2 = 1:3

C3H8:H2 = 1:4

C3H8:H2 = 2:3

0

3

2.8

2.6

2.4

2.2

21 2 3 4 5

Figure. 3.4 Raman spectra of MWCNTs grown on Si substrates as (a) a function of the ratio of C3H8 to H2. (b) Variation of ID/IG ratio with the fl ow ratio of to C3H8. Reproduced with permission [40].

Investigators indicated the signifi cance of average size of surface roughness of the fi lms due to the different vacancies and crystalline state of the fi lms. In the fi gures, surface roughness of MWCNTs was lower when the growth condition of gas mixture of C3H8:H2 = 1:2 were used.

“Nanoidentation test” is a technique to test hardness and sur-face morphology of nanocomposites depending on plasma power used. As an example, Figure 3.5a depicts the load-depth curve of nanoindentation test for MWCNTs at the applied force of 250 μN as a function of the ratio of C3H8 to H2. The hardness and the stiff-ness of the fi lms were 0.4~4 GPa and 10~70 kN/m, respectively. The higher hardness of the MWCNTs was deposited at C3H8:H2 = 1:2. These results indicated that the reduction of the sp2 content in MWCNTs might increase the values of hardness without clear rela-tionship between the indentation depth and C3H8 to ratio due to the fi lm defects. However, the load-depth curve with C3H8:H2 = 1:3 gas mixture showed that the indentation depth increased linearly with increasing load as shown in Figure 3.5b. The surface images of the MF-CNTs fi lm grown at different plasma powers of 200W, 300W, 400W and 500W are shown in Figure 3.6. The surface mor-phology differences were evident among the samples under dif-ferent plasma powers. The SEM images showed that the carbon nanotubes are formed as an amorphous carbon fi lm and CNT mate-rial did not produce a hollow tube shape when the fi lm was grown at a plasma power of 200 W.


mm/div mm/div0.035 0.026

00

4.0 mm/div 4.0 mm/div

4.0 mm/div4.0 mm/div

(a) (b)

250(a) (b)

C3H

8:H

2 = 1:1

C3H

8:H

2 = 1:2

C3H

8:H

2 = 1:3

C3H

8:H

2 = 1:4

C3H

8:H

2 = 2:3

8000

6000

4000

2000

00 400 800

Depth (nm)Depth (nm)

Lo

ad (

mN)

Lo

ad (

mN)

1200

200

150

100

50

00 40 80 120 160

Figure. 3.5 SThM Images of with resistance of RC = 21.3Ω for MWCNTs deposited with the gas mixture of (a) C3H8:H2 = 1:1 and (b) C3H8:H2 = 1:2 at the ECR power of 400 W for 5 min. Reproduced with permission [40].

Figure. 3.6 The load-depth curve of nanoindentation test for MWCNTs grown on Si substrates as (a) a function of the ratio of C3H8 to H2 at F = 250 μN and (b) a function of applied load with C3H8:H2 = 1:3 gas mixture. Reproduced from reference [41].

At high plasma power of 200W, the surface density of the vertical nanotubes decreased and therefore the spacing among the tubes became larger due to randomly oriented surface of MF-CNTs. At plasma power of 300W, the ends of the MF-CNTs became straighter and more uniform, with some CNT tube tips protruding on the fi lm’s surface. At plasma power of 500W, the protruding tube tips increased and aggregated due to the higher plasma power providing higher kinetic energy to the carbon atoms to result with aggregation of tubes and lower adhesion in tubes so that the tubes fl ake off the substrate. With increased


deposition time, the length of the MF-CNTs also increased. The surface islands became smaller and the MF-CNTs fi lm contained more amorphous carbon impurities. As an illustration, a growth model can explain the state of Cobalt-fi lled CNTs as follows: After Co layer deposition, the Co catalyst becomes fragmented into sphere-like nanoparticles by electrostatic force and sintering ther-modynamics [41]. When the CNTs are fi lled by the Cobalt, the Co is trapped in the CNTs from the basal side. Therefore, the growth of the Co-fi lled CNTs takes place during the decomposition of the carbon molecules on the Co-CNT nanoparticles surface, and then the carbon atoms diffuse through the Co nanoparticle to pro-duce the MF-CNTs. The hollowness between the basal sides of the CNTs was reported due to the fact that Co is not suffi cient enough to fi ll the entire CNTs [41].

In general, impact of CNT nanotoxicity studies including surface chemistry studies on CNTs in nanotechnology may be summarized with the following projections:

1. Heterogeneous catalysis is a multi-billion dollar industry ($11.2 billions in 2008) of great importance for the US economy.

2. The strategic plan of the National Science Foundation (NSF) states that “nanotechnology could become a $1 trillion/year industry by 2015.”

3. Studies devoted to catalyst improvements, even in the long run, are part of the effort toward “Technology for a Sustainable Environment,” which is one of the new core funding areas of US funding agencies.

4. Nanoscience is highly interdisciplinary; therefore, the results obtained in surface chemistry studies will be of great signifi cance. For example, the gas-surface interaction of O2 with nanostructured sur-faces has been studied to design the next generation of gas sensors. Therefore, we can safely assume that studies on heterogeneous catalysis also will be sig-nifi cant for materials science studies.

5. We can expect that mass production via nanotech-nology of CNT samples tailored toward a given sur-face reaction will be achievable soon. The economic impact will be enormous.


6. Carbon materials (activated carbon, carbon black) have already been used very successfully in cataly-sis. The high surface-to-volume ratio or aspect ratio of CNTs can be even larger than for activated car-bon, i.e., all the advantages of conventionally used carbon are preserved and enhanced.

7. Supported CNTs have a higher throughput per unit volume than activated carbon, a consequence of the larger dispersion and metal-support interactions.

8. The microporous structure11 of activated carbon leads to transport limitations for surface reactions. CNTs have a mesoporous structure that minimizes this limitation.

9. The large diffusion coeffi cients for gas/liquid transport through nanotubes can prevent catalyst poisoning.

10. Functionalization of CNTs by metal nanoparticles allows for catalyst tailoring.

11. The variety in the crystal structures of CNTs may promote catalyst optimization.

12. The high electrical conductivity of CNTs is desirable for electro-chemical applications such as fuel cells.

13. High purity of CNT avoids the self-poisoning of the catalyst.

14. The highly inert with high surface area properties of CNT material makes it suitable for aerospace applica-tions. However, poor knowledge of CNT interaction with molecules in tissues has restricted CNT materi-als for use in drug delivery and medical diagnostics.

3.5 Cytotoxicity Measurement and Mechanisms of CNT Toxicity

In the last decade, several biophysical properties were investigated such as surface chemistry of membrane damage, protein dena-turation, DNA damage, immune reactivity, macrophage action,

1 The following pore sizes, with d for the diameter, are typically used to classify different systems: micro (d < 2 nm), meso (2 to 50 nm), and macro (d>50 nm) porous materials.


infl ammatory action of cells, and the reactive oxygen species show-ing specifi c changes in cells associated with size, mass, and surface area of carbon nanotubes. Molecular mechanisms of cytotoxicity caused by CNT are still not resolved and remain unconfi rmed. The following section is a guideline to explain mechanisms of cytotoxic-ity of CNT composites.

Nanotoxicity is caused after nanoparticles enter GIT by eating and drinking via mucociliary escalator in the respiratory tract. Mostly CNTs are excreted out by the bowel. CNT tube size and charge surfac-tant effect on CNT determine the CNT uptake and transport through liver, spleen, blood and brush border membrane. Upon ingestion of radiolabeled CNT by an animal, approximately 90% radiolabeled fullerenes remain in the body, 70% in the liver and the other 20% is excreted from the body. CNT functionalized with DTPA and radio-tracer was quickly excreted from blood in mice (Half life = 3 and half hours). In a previous study, the aggregation property of CNT was reported important in toxicity and nanopowder of Zn and caused high mortality in mice, but micropowder of Zn did not show mortal-ity [30]. Based on previous reports, surface chemistry of CNTs plays a signifi cant role in bioapplications as summarized in following section.

• Reactive oxygen makes reactive oxygen species of CNT, which causes toxicity to cells.

• Physical parameters such as size, mass, and surface area of carbon nanotubes play a determinant role and show specifi c effect on macrophage action, and infl am-matory action of cells.

• Membrane damage, protein denaturation, DNA dam-age, immune reactivity are some leading examples of toxicity caused by surface coating of Zn, Cd, and Si around CNTs.

• Surface charge of polycations on membrane plays a signifi cant role in toxicity. Charge density on the sur-face of CNTs nanostructures is associated with magni-tude of toxicity.

Toxicity and structural details of fullerenes further suggest the problems and feasibility of accepting CNT and composites for bio-applications. Multiwall CNT such as C60 showed toxicity due to surface modifi ed by PVP to make highly stable charge transfer com-plexes [31]. A similar concern was expressed that some fullerenes if


suspended in solution by dissolving C60 in THF solvent may not pass through the blood brain barrier, while only THF may pass. Metal catalyst used in nanotube fabrication may be toxic, and sample preparation may exacerbate the toxicity of these metals. However, more derivatized fullerene structures are less toxic due to their low effi ciency in ROS generation. Most of the single-walled and multi-walled CNTs are not water-soluble. CNT toxicity effects may range over seven orders of magnitude for different functionalizing CNT molecules. Aggregation in CNT derivatization on outer surface is important as interior surfaces of CNTs may be less derivatized or not at all derivatized. Sidewall CNT functionalization and low concentrations of CNT are always better options to reduce toxicity than using surfactant coating over CNT. Dose dependent epitheloid granuloma was reported for CNT material with formation of aggre-gates of nanotubes inside macrophages by Varga et al., 2010 [34].

3.1.6 In Vivo Studies on CNT Toxicity

With the advent of continuous monitoring of biological signals by real-time robust automated recording devices, it has become pos-sible to measure CNT toxicity at nanoscale. Few reports are avail-able about airborne CNT nanoparticles (NP) and dermal exposure of CNT and other nanometals. For interested readers, confi rmed reports of other representative nanoparticles are illustrated in airway exposure and nanotoxicity to understand the toxicity mechanisms.

It is a known fact that inhalation of TiO2, carbon black, diesel toxicity causes infl ammation, oxidative stress, and blood clotting ability. Approximately, 2.5 micron nanomaterial particles can reach in alveoli and may cause macrophage phagocytosis, and infl amma-tion and nanoparticles may interfere with its clearance effi ciency from alveoli. Translocation of nanoparticles is also greatly effected in the liver under infl uence of nonparenchymal cells. Conceptually, NP can go to the heart and can cause arrhythmia and coagulation. NP may also translocate through the olfactory nerve ending to the brain. Nanoparticles that usually cause infl ammation and affect the autoimmune system are most likely to damage or alter tissues in the body such as skin, brain, liver, heart, blood, lymph node. Recent studies showed TiO2, ZnO nanoparticle ROS generated and micro-beads can get to the dermis and lymph nodes where proteins affect the autoimmune system [42]. Submicron-sized NP can get through via hair follicles. Small NP may interact with the immune system.


Conceptually, nanoparticles in semiconductors create an electron-hole pair and are detected by photon absorption in UVA or UVB regions. Such behavior indicates that the electron-hole pair acts with water and makes ROS, singlet oxygen, and superoxide [42].

In following the section we will describe an account of the infl am-mation testing protocol of alveolar epithelial cells and mechanism of CNT toxicity in lung fi broblasts initially reported by Stoker et al. [45].

3.1.7 Infl ammatory Mechanism of CNT Cytoxicity

Infl ammatory mechanism of CNT cytoxicity was explored in detail by several investigators in pulmonary infl ammation [46–52]). Monolayer cultures of epithelial, fi broblasts and smooth muscle cells were ideally used as experimental control of the system to test toxicity of CNTs without any cell-cell interactions. Dexamethasone and VEGF controlled delivery was visualized across the fi broblast-embedded collagen gel as multimodal dynamic model to simulate human airways [53–55,]. The infl ammatory lung reactions (alveoli-tis) were used as the source of genetic lesions, which could eventu-ally lead to the development of lung cancer [47]. Another in vitro model of the airway was reported with culturing epithelial cells as monolayer on a membrane and fi broblasts as a monolayer at a fi xed distance away separated by culture media [56, 57]. In vivo stud-ies were performed using guinea pigs and rats. In a recent study, investigators were shown the appearance of multifocal granulo-mas, resulting in infl ammatory reactions of the terminal and respi-ratory bronchials with mild fi brosis in the alveolar septa (Helland et al., 2007) [3]. Another study reported an improved model of fi broblasts following unique characteristics: 1) It maintains the normal anatomical arrangement (orientation and dimensions) of epithelial and fi broblast cells; 2) The fi broblast was embedded in collagen I, yet remained anchored; 3) A thin (10 μm) porous poly-ester membrane separated the epithelial and fi broblast cell layers to allow communication between the epithelium and fi broblast to investigate cell-specifi c protein expression following exposure to external perturbation. Recently, Stoker et al. reported a simple eval-uation method to test the toxicity of engineered CNTs. Cytotoxic/infl ammatory responses and barrier function of the human lung layers following exposure of CNTs were observed using in vitro a co-culture system of airways [45]. Kwon et al. (2009) further estab-lished the role of Nitric oxide (NO) produced by many cells in the

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body. Under normal (basal) conditions, NO was continually pro-duced by cNOS (constitutive nitric oxide synthase). However, dur-ing infl ammation, the amount of NO produced by iNOS can be a 1000-fold greater than that produced by cNOS in basal condition. Investigators predicted that NO production measurement could identify the level of infl ammation in co-culture system [58].

On the other hand, in the last fi ve years CNT was reinvestigated and its inert properties were capitalized on as tiny nanomissiles hit-ting the target and releasing drugs at tissue site very precisely. The electron microscopic structure of CNTs suggested that drug-binding property with CNT does not block or inhibit any drug active group from acting and it remains still unidentifi ed against macrophage and immune defense system in the body. Recently, a nanobiosci-ence group at Rice University and other institutions established that CNT might be used as safe drug carriers in the body [53, 5]. However, the pitfalls of fullerene structures are that they have high binding and high activation energies with a high possibility of binding CNT with circulating free molecules such as hormones, enzymes, peptides and ions. These issues still make the CNT toxic suspects. We established the CNT molecules transporting across the skin layers in time dependent manner using microimaging MRI techniques. Microimaging showed clearly that the epidermis layer of the skin was a prime target of CNT, and CNT can affect the viable skin cells while they are used as drug carriers. Our other direction of CNT effect on live alveolar cells was to observe the infl amma-tory changes in cultured fi broblast cells. The synergy of cytokines, nitric oxide production and cytotoxicity of alveolar cells were the main alterations caused during CNT exposure to alveolar cells. The transepithelial electrical resistance of alveolar cells was a unique index identifi ed as CNT induced cytotoxicity biomarker without changing the drug delivery properties. The fullerenes are main CNT constituents. They are bound with drugs at their hydrophilic –C-COO- or –NH- or –SH sites. The pH, temperature, concentration and charge of drug molecules in blood are the main factors of con-trolled rate of drug delivery.

An ideal approach of toxicity evaluation is the use of cultured mesenchymal cells exposed to CNT material. In the following sec-tion, we describe the method developed at the lab over the years for mesenchymal stemcells to test toxicity of MWCNTs.

Mesenchymal stem cells (MSC) serve as model culture to study cytotoxicity. In a previous study, MSC were isolated from rats


femurs under aseptic conditions and the excised connective tissue or epiphyses were removed from the femurs and the cavity was washed twice with phosphate buffered saline (PBS) solution with 1% penicillin/streptomycin to remove bone marrow as described in detail elsewhere [58]. In the following section we describe in situ microscopic observation of cultured MSC cells in interaction with CNTs to evaluate the CNT toxicity during osteogenesis for inter-ested readers. In brief, solution was centrifuged at 1000 rpm for 10 minutes, and the PBS was extracted. The cells were resuspended in Dulbecco’s Modifi ed Eagle’s Medium (DMEM) with other sup-plements: 10% fetal bovine serum (FBS), 5 ml of penicillin/strepto-mycin, and 1 ml of amphotericin. Investigators maintained cells at or between osteoprogenitor and pre-osteoblast stage and primary cell culture was grown in a 35 mm2 Petri dish incubated at 37°C and 5% CO2. The differentiating media was added to MSCs at approxi-mately 90% confl uence because there is a signifi cant reduction in proliferation as these cells become differentiated. Cells were incu-bated at 37°C and 5% CO2 for 7–16 days. Another study reported MSCs differentiated to the osteoblastic phenotype by addition of 10 mM of β-glycerophosphate, 50 μg/ml of ascorbic acid, and 10 nM of dexamethasone in the media [54, 59].

3.1.8 Characterization and Toxicity of SWCNT and MWCNT Carbon Nanotubes

The high-purity single-walled carbon nanotubes (SWCNTs) serve as nanomaterial available from Carbon Nanotechnologies Inc, pre-pared by HiPCO process with residual metal content 3–12% by weight, individual nanotubes 0.8–1.2 nm in diameter and 100-1000 nm in length. In following section, we describe atomic force micros-copy observation of cultured MSC cells in interaction with CNTs to evaluate the CNT toxicity. To prepare nanomaterial, SWCNTs are added to distilled water, and the mixture may be sonicated for 60 minutes using Sonicator 3000 manufactured by Misonix (Farmingdale, NY). Other study reported formation of nanoropes from nonionic octylphenol ethoxylate surfactant, Triton™ X-100 added to the dilute aqueous SWCNT solution to facilitate the sepa-ration of individual nanotubes [56]. The degree of SWCNT disper-sion in the aqueous solution is ideally evaluated using an atomic force microscope (AFM) MultiMode II developed by Veeco Digital Instruments Group (Woodbury, NY). Investigators used several


drops of nanotube suspension applied on to a silicon substrate and suspension was allowed to dry in open air, leaving nanotube agglomerates. The silicon substrate was observed under the AFM, and the nanotube rope sizes were measured at various locations.

Recently, toxicity measurement of CNTs was established using protocol of different cytotoxicity and infl ammatory response mea-surement tests as acceptance testing of CNT material [56]. Physical methods and cellular techniques emerged as potential methods of cytotoxicity testing. The following section describes routine MTT assay, in vivo microimaging technique and transepithelial resistance monitoring across cells developed by our team. MTT assay (Sigma) was used to evaluate the changes in cellular metabolic (mitochon-drial) activity of cells as a cytotoxic response. Cells were exposed to varying concentrations of SWCNTs after 48 hours. In brief, 150 μL of MTT (5 mg/ml) was added to each well and incubated for 4 hours. Afterward, 850 μL of the MTT solubilization solution (10% Triton X-100 in 0.1 N HCl in anhydrous isopropanol) was added to each well. The resulting formazan crystals was solubilized in acidic isopropanol and quantifi ed by measuring absorbance at 570 nm. Data were calibrated to the appropriate calibration curve as stated in Sigma protocols as described by Kwon et al., 2009 [56]. Formation of formazan crystals represents the toxicity of CNT.

Magnetic resonance imaging (MRI) of rat skin tissues exposed to CNTs serves as experimental model to monitor CNT transport across the skin barrier. At our lab, MRI was performed by high res-olution 3D FLASH T1 weighted MRI in a 21.1T MR scanner using an Rf birdcage Rf 15/900 coil (Bruker Biospin) and PARAVISION 3.2 software at NHMFL, Florida State University at Tallahassee. The MRI microimaging was performed before and after placing 10 nm CNT in glass capillaries at different intervals of 2, 4, 6 hours using scan parameters: TR/TE/fl ip angle = 750ms/ 4.18ms/25°, FOV/matrix size/spatial resolution = 2.6×3.4 cm/ 256×256/0.015 mm, and the inversion time (TI approximately 250 ms) set to null normal skin. Epidermis and hair follicle were measured as reported by Sharma et al. (2010) [60].

Measurement of Transepithelial Electrical Resistance (TER) is a new development used in isolated cells [58]. Investigators reported human bronchial epithelial cells grown at the interface of air and liq-uid. In this technique, culture media was provided from the bottom through the porous membrane. TER of human bronchial epithelial cell with fi broblasts-embedded collagen layers cultured in TranswellTM


was monitored using a portable Voltohmmeter (Millipore, Bedford, MA) attached to a dual “chopstick” or transcellular resistance mea-surement chamber (Millipore, Bedford, MA) as shown in Figure 3.13. In brief, different concentrations of CNTs were exposed to the co-cul-ture layers for 6 hours. Each of the two electrode systems contained Ag/AgCl electrode for measuring voltage and a concentric spiral of silver wire for passing current across the epithelium. Electric current passed across the epithelium was used to measure TER (ohms.cm2). TER values higher than the background fl uid resistance indicated a confl uent airway epithelium with enlarged cell-cell tight junctions due to CNT toxicity. TER was monitored to identify the perturbation in the normal physiology and permeability due to CNT toxicity on human bronchial epithelial cells (Kwon et al., 2009) [58].

Metabolizing enzymes or intracellular enzymes such as alka-line phosphatase, leucine aminopeptidases, hepatic lysosomal enzymes, esterases are other targets involved in CNT induced cellular changes. Enhanced enzymes represent the cell differen-tiation, phagocytosis and proliferation in cells. The measurement methods of intracellular enzymes. Our team established Alkaline Phosphatase concentration in cell lysate measured by using chemi-luminesce in epithelial cells after adding 100 μl of CSPD substrate to 20 μl sample of epithelial cells (Kwon et al., 2009) [58].

Hypoxia state of cells is another form of cytotoxicity testing to capitalize the potential of hypoxia in measuring the cytotoxicity effect of CNT on epithelial cell survival. Initially, hypoxia induced by CNT was estimated by clonogenic assay of clonogenic cell sur-vival [61–63]. Oxygen pressure measurement of poor oxygenation in tumor cells is other method of cytotoxicity testing. Hypoxia was measured in tumor cells using OxyLite pO2 system (Oxford Optronix Ltd, UK) attached with four oxygen probes implanted in mice tumors to measure tumor oxygen by using fl uorescence quenching technique under aneuroleptanalgestia of animals placed in thermal blanket at 37oC. For 10 minutes, probes measured the100 pO2 readings in tumor cells [64]. Radiopharmaceutical radiolabeled nanoparticles are now mostly evaluated by measuring oxygen pres-sure of cells at different locations in tumors.

Cell proliferation measurement is another unique choice of determining cytotoxicity. Cell proliferation assays provide infor-mation of fast cell divisions induced by CNTs in contact. The rate of cell proliferation may be measured using the Picogreen dsDNA bioassay kit (Invitrogen, CA). Cellular DNA concentration from cell lysate represents the nuclear integrity and its slow degeneration or


programmed cell death indicates the apoptosis in cell. Cell prolifer-ation and apoptosis assays distinguish the cell damage from necro-sis if it occurs due to severe cell injury by CNTs on epithelial cells.

Multiwalled MWCNTs may make composite MWCNT-collagen materials. Baktur et al. (2010)1 established a technique of MWCNT-collagen composite preparation at fi xed concentration of colla-gen Type I (1 mg/ml) mixed with MWCNTs. Different MWCNTs forms included MWCNTs, MWCNTs-OH, MWCNTs-COOH, with two different sizes (OD:20-30nm. Length: 0.5-2.0μm, 10-30μm). Originally, MSCs were seeded on the different types of MWCNTs-collagen scaffolds and method was described [59]. Investigators believe that MWCNTs-collagen scaffolds signifi cantly enhanced the differentiation of MSCs at 10 ppm. In another study, investiga-tors showed that all types of MWCNT-collagen scaffolds induced the higher level of MSC differentiation compared to controls [60]. However, MWCNTs-collagen scaffolds were less likely to affect MSCs proliferation with no signifi cant difference in MSC mor-phology before and after differentiation. In the following sections, unpublished data by Baktur et al., 20102 showed signifi cant differ-ence in the level of alkaline phosphatase enzyme concentration as shown in Figure 3.7. The following section describes the observation

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Figure. 3.7 The effect of MWCNT-collagen scaffolds on MSCs differentiation. Concentrations of MWCNT-COOH was varied on the fi xed concentration of collagen (1mg/ml). * signifi cantly different than day 0 control (p<0.05). # signifi cantly different than plastic and collagen control at day 16 (p<0.05). Reproduced with permission [54].


of MSCs Differentiation on CNT-collagen scaffolds and toxicity of carbon nanotubes.

3.6 MSCs Differentiation and Proliferation on Different Types of Scaffolds

In previous reports, MSC cells differentiate into osteocytes by slow process of osteogenesis on surface of MWCNT scaffolds in presence of collagen or plastic composite material in scaffolds. The progress of osteogenesis was sensitive to released alkaline phosphatase concen-tration from MSC cells in medium [57]. Alkaline phosphatase enzyme concentration serves as biomarker of MSC differention and osteo-genesis to compare the quality and biocompatibility of MW-CNTs in scaffold support material as shown in Figure 3.8 [43, 57].

Another important cellular process is proliferation of MSCs represented by DNA concentration in MSCs exposed to different CNT Scaffolds. The MSC cell proliferation was related with type of MWCNT-collagen scaffold as shown in Figure 3.9 [45].

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Figure. 3.8 The effect of MWCNT-collagen scaffolds on MSCs differentiation. Concentrations of MWCNT-COOH was varied on the fi xed concentration of collagen (1mg/ml). * signifi cantly different than day 0 control (p<0.05). # signifi cantly different than plastic and collagen control at day 16 (p<0.05). Reproduced with permission [54].

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Morphology of MSCs depends on the type of dish material used in culturing MSCs and material of scaffold used. Different scaffold materials of type 10 PPM, collagen and plastic showed different AFM images of SWCNTs dried on silicon after 12 days as shown in Figure 3.10.

Characterization of Carbon Nanotubes supplements signifi cant information such as high fi eld microscopy of CNT. Dark, uniform solution suggested the CNT nanotubes well dispersed in the medium. The surfactant-aided nanotube suspension remained stable for at least two months. The image of SWCNTs dried on a silicon substrate is shown in Figure 3.10. The average length and diameter of nanotube ropes were about 500 nm and less than 10 nm, respectively. A more detailed study on the distributions of nanotube dimensions using image analysis is discussed else-where [65].

Infl ammatory and cytotoxic responses in MSCs were observed by increased nitric oxide (NO) production following exposure of increased concentration of single-walled carbon nanotubes (SWCNTs) to epithelial cells (Figure 3.11a). At higher concentra-tions of SWCNTs, MSC cells showed cytotoxic response and por-tions of cell layers were detached as reported by [45]. Each of the

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Figure. 3.9 The effect of MWCNTs-collagen scaffolds on MSCs proliferation. Different types of MWCNTs at 10 ppm on the fi xed concentration of collagen (1 mg/ml). * represents signifi cantly different than day 0 control (p < 0.05). Reproduced with permission [45].


NO production rate was normalized by total proteins. Cellular metabolic activity was observed following exposure of different concentrations of SWCNTs to both MSC cell layers. MTT activity in MSC cells was decreased with increased concentration of SWCNTs, especially for epithelial cells (Figure 3.11b).

3.6.1 An In Vivo Model CNT-Induced Infl ammatory Response in Alveolar Co-culture System

We present two models (static and dynamic models) developed in the lab to address the following questions: 1) How do two differ-ent types of lung cells interact with each other to respond to CNT exposure? 2) What are the cellular and molecular mechanisms of cytotoxic response and interaction in the human respiratory sys-tem? 3) How will different size and structure of CNTs be translo-cated and accumulated to alter the mechanisms of cellular response and specifi c gene expression pattern? The dynamic cell growth system displayed better cellular responses over static cell growth system and similar to animal studies. The dynamic cell growth sys-tem can be considered as a viable alternative to in vivo test system

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Figure. 3.10 High Field Microscopy images of SWCNTs dried on silicon substrate. SWCNTs materials of 500 nm length and 10 nm diameter in size were fi xed on scaffold made of plastic, collagen and 10 PPM. The changes in MSC cells on different scaffolds were observed on fi rst and 12 days postincubation period. Notice the remarkable change in MSC cells on collagen-CNT scaffold in middle panel. Reproduced with permission [45].


in combination with existing in vitro static cell growth systems to evaluate the cellular responses on the respiratory system following exposure of different types of CNTs.

Monitoring of CNT-induced infl ammatory response under different exposure conditions, multiple infl ammatory markers and various candidate biological markers (cellular component,

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Figure. 3.11 Effect of SWCNTs on NO production and cell viability from epithelial cell layers. NO production following exposure of SWCNTs to epithelial cells was dramatically increased as the concentration of SWCNTs increased (A). At higher concentrations of SWCNTs, cells showed cytotoxic response (B). Each NO production was normalized by total proteins. MTT assay was used to show cytotoxic response. * denotes a signifi cant difference from the control (0% SWCNT) (p < 0.05). Results were presented as mean ± SD. Number of replicates for (A) was four. Number of replicates for (B) was sixteen. Reproduced with permission [45].


macromolecules, other metabolic parameters, and cellular func-tion) explain the mechanistic details. In addition, comparative studies with normal cell lines and novel experimental set-ups for a multiplexed screening of biological markers may explore CNT-induced infl ammatory responses. Such epidemiological studies may also establish associations between exposures to engineered nanoparticles and adverse health effects and to assess other poten-tial exposure-response relationships.

3.6.2 Static Model: 3-Dimensional Tissue Engineered Lung

In laboratory, a prototype of monolayered epithelial cells co-cultured with fi broblast-embedded collagen gel was designed to simulate human airway [137]. Fibroblasts contract the extracel-lular matrix to close a wound and function perfectly upon placed in collagen gel. The model served as ideal example of isolated mediators participating in epithelial-fi broblast communication. It had several features: 1) It places the normal anatomical orienta-tion of epithelial and fi broblast cells; 2) Fibroblasts embedded in anchored collagen I separated by 10 micrometers polyester mem-brane serves the purpose to study cell-specifi c protein expression or communication between epithelium and fi broblasts exposed to CNTs.

We describe a working protocol of co-culture technique used for 3D-tissue engineered lung in the following section [137].

The co-culture technique in this review offers several distinct advantages over earlier models [137]. The airway epithelial cells are cultured as a monolayer over a thin (10 mm) porous polyester membrane. A thin lung fi broblast-embedded collagen layer was attached to the opposing side of membrane. In this fashion, the epithelial cells and fi broblast cells maintain the normal anatomical arrangement, but the polyester membrane allows easy separation of the cell types for cell-specifi c gene expression and proteomics analysis (Figure 3.12). Fibroblast-embedded Collagen I gels were prepared using rat-tail tendon collagen (RTTC; Collaborative, Bedford, MA, USA). Normal human lung fi broblasts (NHLF) were harvested upon reaching 75–80% confl uence, and added (seeding density of 5 x 104 fi broblasts/mL of fi nal volume) to an iced mixture of collagen (fi nal concentration 2.0 mg/ml), 5X


concentrated DMEM, and 10X reconstitution buffer comprised of NaHCO3, HEPES buffer (Gibco, Grand Island, NY, USA) and NaOH. Aliquots of the mixture were pipetted onto the underside of a 1 cm2 Transwell (Costar, Cambridge, MA, USA) polyester membrane (0.4 μm pore). The outer rim of the membrane was fi t-ted with a highly porous polyethylene ring. The liquid gel then swept into the porous ring at the edge and upon “gelling” was able to keep the fi broblast-embedded gel from contracting. The collagen mixture was then allowed to “gel” (non-covalent cross-link) at 37oC in 5% CO2 for 10–15 minutes. Harvested primary human bronchial epithelial (HBE) cells (passage 2–3) were then seeded (1.5 x 105 cells/cm2) directly on top of the polyester mem-brane. The entire tissue was submerged in media for 5 days and the epithelium was allowed to attach and become confl uent. For the fi rst 48 hours, the media was basal epithelial growth medium (BEGM, Clonetics, USA) and a low retinoic acid concentration. For days 3–5 (and days 6–21), the media was a 50:25:25 mixture of BEGM:DMEM:Hams F12 with a high retinoic acid concentration. At day six, an air-liquid interface was established (media main-tains a high retinoic acid concentration) and the epithelium was allowed to differentiate for approximately two weeks at which time it is ready for experimentation.

Human bronchial epithelial cells were grown at the interface of air and liquid. Culture media was provided from the bottom through the porous membrane. Transepithelial electrical resistance (TER) of human bronchial epithelial cell with fi broblasts-embedded collagen layers cultured in TranswellTM could be monitored using a portable Voltohmmeter (Millipore, Bedford, MA, USA) attached to

Figure. 3.12 Preparation of tissue engineered bronchial mucosa follows three major steps and three weeks in culture.


a dual “chopstick” or transcellular resistance measurement cham-ber (Millipore, Bedford, MA, USA). Each of the two electrode sys-tems contained Ag/AgCl electrode for measuring voltage and a concentric spiral of silver wire for passing current across the epithe-lium. Electric current could then be passed across the epithelium to measure TER (ohms.cm2). It was perceived that TER values higher than the background fl uid resistance indicate a confl uent airway epithelium with tight junctions. TER was monitored to identify the perturbation in the normal physiology and permeability of human bronchial epithelial cells. In a recent study, different concentrations of CNTs were exposed to the co-culture layers for 6 hours. The TER of the controls (5% and 20% of Triton X-100 and 0% of CNTs) were stable around 500 ohms.cm2 (resistance of epithelial-free tis-sue was subtracted) for 48 hours. 10–20% of CNTs rapidly compro-mised the barrier function of the epithelium and the TER decreased to 120 ohms.cm2. After removing CNTs, the TER completely recov-ered to the control level.

3.6.3 Dynamic Model: Integration of 3D Engineered Tissues into Cyclic Mechanical Strain Device

Dynamic cell growth condition served as more realistic in vitro viable alternative to in vivo model. We established a dynamic cell growth environment to mimic the dynamic changes in the amount of circumferential and longitudinal expansion and contraction that occur during normal breathing movement in the lung (Figure 3.13). Flexcell Tension Plus system can also be used to implement 5% cyclic equibiaxial elongation, which is equivalent to 45% of total lung capacity, and the amount of stretching experienced during nor-mal breathing condition. Patel and co-workers recently showed the differences in cellular responses (cell proliferation, cellular infl am-mation, reactive oxygen species (ROS), and glutathione (GSH)) to air pollutants including CNTs between dynamic and static cell growth environments, and demonstrated that implementing dynamic cell growth conditions was a closer approximation of in vivo conditions. This study provided one of the alternative ways to evaluate CNT-induced effects on human respiratory systems and a detailed insight for the development of a viable alternative to exist-ing static in vitro or in vivo tests.


3.6.4 In Vivo MR Microimaging Technique of Rat Skin Exposed to CNT

In the following section, a microimaging technique of rat skin tis-sues is described to visualize the cellular damage at CNT-skin tis-sue interface. The technical development was based on the fact that CNT molecule size was important and we established that the skin epidermis layer 150–175 micron thick is a combined layer of viable cells [60]. The CNT size between 35–100 nanometers was suitable material to pass across the skin epidermis barrier. The rate of dif-fusion and transport of CNT was also a size-dependent criterion

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Figure. 3.13 The apparatus to apply tensional stress. (a) Schematic of the constant or cyclic tensional stress driven by a vacuum. (b) Circular Tissue Train™ Culture System for the mechanical loading of cells in a 3D matrix or for culturing cells in a mechanically active environment. (c) A computer-regulated bioreactor that uses vacuum pressure to apply cyclic or static strain to cells cultured on fl exible-bottomed culture plates.


to evaluate the CNT material as biocompatible drug carrier. Skin cell membrane is made of phospholipids, cholesterol and lipopro-teins. Cell membrane plays an active role in controlling the ion transport inside and outside across the channels or ion pores using active energy against concentration gradient. The CNT molecules displayed very small size 35 nanometers in our experiment so we believed that drug molecule (105–200 Angstrom) size attached with insert CNT does not expose its hydrophilic bonds so inert CNT molecules keeps the bound drug molecule safe while CNT passes across the membrane and later releases drug at suitable pH to act at the tissue target site. Our technique was based on ex vivo MRI 3D FLASH images of skin showing axial, sagittal and coronal images. The coronal images of skin are shown in Figure 3.14. The images showed distinct morphological and structural features of three main skin layers. The ex vivo excised skin MRI of the ventral abdomen skin showed different structures of epidermis, dermis, hair follicle, and sebaceous oil gland as shown in Figure 3.14. The CNT-skin interface showed consistent damage to skin tissue on MRI microimages shown by arrows in Figure 14. The hair follicles remained intact while epidermis membrane and dermis vascula-ture was badly damaged. The skin features were distinct and mea-surable. The dimensions of skin layers were measured as epidermis (150–200 micrometers); hair root (300 micrometers); hair follicle (50 micrometers); dermis (600–650 micrometers). The 35 nanometer-sized CNT drug carrier passed through epidermis in 2 hours and whole dermis in 6–8 hours.

Effect of CNTs may be monitored based on physiological function of airway epithelial cells. Effect of CNTs on physiological function

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Figure. 3.14 The skin microimaging at 21 Tesla MRI using fast 3D FLASH technique shows control without carbon nanotubes; (b) SWCNT sample in tube placed for 3 minutes on skin top; (c) after 15 minutes SWCNT sample in tube placed on skin top; (d) after 6 hours SWCNT stayed on top of the skin. Notice the slow damage to epidermis and dermis by SWCNT caused shown by arrow.


of airway epithelial cells showed transepithelial resistance (TER) specifi c to different concentrations of CNTs exposed to the epithe-lial co-culture layers for six hours. In a previous study, TER of the controls (5% and 20% of Triton X-100 and 0% of CNT) were stable at 500 ohms.cm2 resistance of epithelial-free tissue for 48 hours. Signifi cantly, 10–20% of CNTs compromised the barrier function of the epithelium and the TER decreased rapidly to120 ohms.cm2 as shown in Figure 3.15. After removing CNTs, the TER completely recovered to the control level.

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Figure. 3.15 Exposure of SWCNTs to co-culture layers impacts transepithelial electrical resistance (TER). Human bronchial epithelial cells were grown at the interface of air and liquid. Culture media was provided from the bottom through the porous membrane. TER of human bronchial epithelial cell with fi broblasts-embedded collagen layers cultured in TranswellTM was monitored using a portable Voltohmmeter (Millipore, Bedford, MA) attached to a dual “chopstick” or transcellular resistance measurement chamber (Millipore, Bedford, MA). Different concentrations of SWCNTs were exposed to the co-culture layers for 6 hours. The TER of the controls (5% and 20% of Triton X-100 and 0% of SWCNT) were stable around 500 ohms.cm2 (resistance of epithelial-free tissue was subtracted) for 48 hours. 10–20% of SWCNTs rapidly compromised the barrier function of the epithelium and the TER decreased to120 ohms.cm2. After removing SWCNTs, the TER completely recovered to the control level.


3.7 New Lessons on CNT Nanocomposites

Presently it is not clear if CNT materials are really toxic and to brand them as such is not useful for applications. In the last fi ve years, CNTs were tested for toxicity and also used for applications. New lessons are timely for testing their toxicity and to evaluate them for bioapplications. Toxicity of CNT and its timely testing both are a challenge of in vivo testing to evaluate the toxicity of engineered CNTs. Experimental access to the airways pose health risk of CNTs. In general, there are two routes of human exposure to CNT: accidental exposure to aerosol in context of CNT production and handling; and CNT used as lighter bone replacement material. Unfortunately, the status of CNT material toxicity is unclear and its use as drug delivery carrier material is uncertain. Many stud-ies suggested potential cytotoxic levels when inhaled or skin con-tacted. The small size of CNTs makes them readily airborne and a potential risk to workers. In addition, uncertainities arise because of no clear knowledge of contributing factors essential for health risk prediction such as routes of exposure, magnitude and duration of exposure, and translocation and persistence of materials after entry. More experimental and population research data on health risks associated with CNT exposure is needed. The size of CNTs and their quick diffusibility makes CNT more readily to become airborne and presents a risk of inhalation. Other factors are route of exposure, translocation and tissue interaction of materials in the body. The potential health risk also depends on the magnitude of toxicity, duration of exposure and persistence of CNT material in the body. High aspect ratio (fi ber-The infl ammatory shape) and large surface area raise further concerns for human health. Our recent studies showed toxic effects of CNT in pulmonary infl amma-tion (Kwon et al., 2012). In previous studies, investigators described three properties of CNTs associated with pathogenecity in particles: 1) more toxicity of nanoparticles; 2) fi ber shaped particles behave like asbestos, and other particles behave needle-like shaped or less toxic; and 3) persistence in biological systems or least biodegrad-able [124]. In this direction, monolayer culture of individual cell types (primarily epithelial, fi broblast, and smooth muscle) provide fundamental information of possible CNT toxicity without details of cell-cell interactions in vivo. A thin lung fi broblast-embedded col-lagen layer attached to the opposing side of membrane may serve as normal anatomical arrangement (barrier at the interface between


air and lung tissues in response to CNTs and air polluants) suitable for separation of the cell types for cell-specifi c gene expression and proteomics analysis. Based on this idea, our co-culture model indi-cated the expression of three important phenotypic markers such as mucin (mucous production), F-actin (tight junction), and tubu-lin (cilia). Human lung carcinoma epithelial cell line (A549) pro-vides valuable information regarding in vitro cytotoxicity testing of SWCNTs [66]. Transformed cell lines are less resistant to toxic effects of CNTs than other cells derived from normal tissues, and also these cells did not fully exhibit normal phenotypic markers. To test cytotoxicity of SWCNTs on the barrier function of lung epi-thelial cell layers, normal human bronchial epithelial cells can form F-actin or tight junction and present a suitable model to observe the perturbation of TER following exposures of SWCNTs.

Over the years our team focus was to derive mechanism of alveo-lar infl ammation in terms of cytokine synergy, nitric oxide produc-tion and hypoxia using TER and cytotoxicity measurements. Major fi ndings were: 1) TER of co-culture layers showed distinct reduced airway physiological function after exposure of different concen-trations of CNTs; 2) Co-cultures indicated that high concentration of CNT debilitated the barrier function of airway epithelial cell lay-ers; 3) Nitric oxide (NO) was other infl ammatory marker and MTT activity signifi ed the cytotoxic response of the cell layers following exposure of different concentrations of CNTs; 4) The airway epithe-lial cells showed infl ammatory response as they increased NO pro-duction; 5) Airway epithelial cells also showed cytotoxic response, while fi broblasts showed mild infl ammatory and mild cytotoxic response; 6) Non-ionic detergent (Triton X-100) used to avoid the aggregation of CNTs did not show any signifi cant cellular toxicity and infl ammatory response.

In other previous studies, investigators emphasized the need of positive control to distinguish CNT toxicity and testing of nanopar-ticle toxicity [67]; (Davoren et al., 2007) [66]. Further studies will be required about the different effects of shape, size and aggrega-tion of CNTs on airway function and cellular toxicity. In a previous study, aggregation of CNT was not detected with phase contrast microscope over a two month period. However, CNT aggregation was observed after two months of the initial CNT solution prepa-ration with Triton X-100 without any cellular toxicity (Figure 3.7). Another group of investigators suggested that CNT aggregation or


extent of aggregation might generate an adverse effect on cell func-tion and cell viability [68].

The fullerenes constitute major components of CNT material. Fullerens cause generation of cytotoxic anion species. The major mechanisms are believed to support CNT-induced toxicity as follow-ing: 1) Fullerenes of nanotubes show free radical chemistry, attrac-tion to electrons, and antioxidant properties; 2) Some Carbon-60 fullerenes bind to nucleotides, hamper self-repair in double–strand DNA; 3) CNT display high electrical and thermal conductivity, high strength, and rigidity. Medical/nonmedical applications suggest occupational, accidental exposure; 4) Fullerens (cages), single-wall nanotubes, and multi-walled nanotubes show toxicity. CNT mate-rial can produce superoxide anion, lipid peroxidation, and cyto-toxicity in plants and animals; 5) Uncoated fullerenes in largemouth bass fi sh showed lipid peroxidation in brain tissue and glutathione depletion in gills. 6) C60 toxicity increases by Poly Vinyl Propylene due to stable charge transfer complexes. 7) Metal catalysts like THF may pass through blood-brain barrier, commonly used in nanotube fabrication; 8) More derivatized fullerenes are less toxic, due to low effi ciency in ROS generation; 9) CNT showed toxicity effects, dose dependent experimental epitheloid granuloma; 10) At optimized CNT single-walled CNT concentrations, low Taxotere quantities encaged inside may target breast tumor tissue more effi ciently; 11) Cultured alveolar fi broblasts following exposure of CNTs showed possibility of transplanting CNT encaged fi broblasts. In conclusion, medical/nonmedical drug delivery system applications of CNT suggest its use with care due to occupational, accidental exposure and nanotoxicity as health concern.

Another side of fullerene-based drug delivery systems is suc-cessful drug transport, delivery and controlled release without any change or deactivation of hepatic drug metabolizing enzymes. Several drug metabolizing enzymes such as esterases, peptidases, NADPH oxidases and diphorases biotransform the drugs into bioactive metabolites. Drug biotransformation makes the drugs either to compete with natural enzymes and intermediary meta-bolic pathway(s) or act as effectors (stimulators or inhibitors) at certain biochemical metabolic step(s). Fullerene molecules in CNT are found not to participate in such metabolic enzyme reactions and they are excreted out unnoticed leaving behind the drug at site. Such inert behavior of carbon nanotubes is an excitement to capitalize them as most suitable drug delivery systems with new


possibilities of other new options of safe nanopolymer composites as delivery nanoballs.

In this chapter, we presented different viable alternatives to in vivo tests to evaluate the toxicity of engineered CNTs [137]. In vitro co-culture confi guration signifi cantly separated the epithelial and fi broblast cell allowing communication between the epithelium and fi broblast. Such confi guration provided clean access to investi-gate cell-specifi c protein expression, following exposure to external perturbation, in the human respiratory system. Co-culture system will address the following important questions: 1) How do two different types of lung cells interact with each other to respond to CNT exposure? 2) What is the cellular and molecular mechanisms of cytotoxic response and interaction in the human respiratory system?; 3) How will different size and structure of CNT will be translocated and accumulated to alter the mechanisms of cellular response and specifi c gene expression pattern?; and 4) What cyto-toxic/infl ammatory responses and barrier function of the human lung layers can be observed following exposure of CNTs using in vitro co-culture system of airway? Further studies are required in the future on nanoparticle-related cellular toxicity and functional relations between the size or structure of CNTs and the perturba-tion of cellular or physiological functions.

1. Development of a 3D collagen scaffold coated with multiwalled CNT nanomaterials in bone regeneration

2. MWCNT-coated collagen sponge is useful 3D scaf-fold for cell cultivation described in sections 3.6.2 and 3.6.3 [137]. MWCNTs have attractive biochemical properties such as strong cell adhesion and protein absorption. In a recent study, a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) was prepared to improve the surface prop-erties of the collagen sponge, and its cell culturing properties were examined. The suface of the collagen sponge was homogeneously coated with MWCNTs by dispersion. MC3T3-E1 cells were cultured on and inside the MWCNT-coated sponge. The DNA content on the MWCNT-coated sponge after 1 week of culture was signifi cantly higher than on an uncoated collagen sponge (p < 0.05). There was no signifi cant difference between the estimated ALP activity normalized by


DNA quantity on the MWCNT-coated sponge and that on the uncoated collagen sponge used as best scaffold for cell cultivation. In addition, the MWCNT-coated surface showed strong cell adhesion. Therefore, the MWCNT-coated collagen sponge is expected to be a useful 3D scaffold for cell cultivation [138, 139].

Carbon nanotubes (CNTs) are composed of two-dimensional hexagonal graphite sheets rolled up to form a seamless hollow tube or cylinder of diameters ranging from 0.7–100 nm and a length of several micrometres up to several millimetres. CNTs can be synthe-sized in two confi gurations, as single-walled nanotubes (SWCNTs) and multi-walled nanotubes (MWCNTs). Whereas SWCNTs are made of one tubular structure, MWCNTs consist of concentrically arranged carbon tubes with a typical spacing of ≈ 0.34 nm between the different layers. Owing to their remarkable structural character-istics (light weight, high aspect ratio, high specifi c surface area), as well as attractive mechanical (high stiffness and strength), electri-cal (high conductivity) and chemical (versatile surface chemistry, easily to functionalise) properties [2], there is increasing interest in biomedical applications of CNTs [142].

Functionalized carbon nanotubes are becoming potential mate-rials in drug design and discovery, and medicinal applications. Covalent functionalization of carbon nanotubes mostly takes advantage of the reactivity of carboxylic acid moieties created by acid oxidation of carbon nanotubes [141].

Functionalized carbon nanotubes display unique properties that enable a variety of medicinal applications, including the diagno-sis and treatment of cancer, infectious diseases and central nervous system disorders, and applications in tissue engineering. These potential applications are particularly encouraged by their abil-ity to penetrate biological membranes and relatively low toxicity. High aspect ratio, unique optical property and the likeness as small molecule make carbon nanotubes an unusual allotrope of element carbon. After functionalization, carbon nanotubes display potential for a variety of medicinal applications, including the diagnosis and treatment of cancer, infectious diseases and central nervous system disorders, and applications in tissue engineering. These potential applications are particularly encouraged by their ability to pene-trate biological membranes and their relatively low toxicity [142]. In recent years, application of carbon nanotubes in drug delivery and carrier systems has been reviewed as safer material with caution.


Functionalized carbon nanotubes were reported to design molec-ular hydrogen sensors [143]. Electronic biosensing applications were developed for assaying surface-protein and protein-protein binding to design antibodies detector using polyethylene oxide-functionalized nanotubes [145].

Recently, functionalized carbon nanotubes were specifi cally investigated for activity of ligand-receptor protein system bound to single-walled carbon nanotubes detection of viral proteins as bio-sensors. Carbon nanotubes as immobilization surface were func-tionalized for fl uorescent-labeled Knob protein or CAR protein via diimide-activated amidation in antibody linked protein detection [145]. The interaction of carbon nanotube with biological macro-molecules is signifi cant for development of nanovector design for gene and drug delivery. In a recent study, molecular dynamic simulations of POPC/cholesterol lipid bilayer and POPC showed that CNTs increased the poration in presence of cholesterol [147]. Carbon nanotubes have proven as superlight weight hard materials in aero- and space industries [146]. Recently, multiwalled carbon nanotubes coated with collagen sponge have emerged as tool in 3D dynamic fl ow cell culture system in tissue engineering using enzymes as markers [148, 137]. In a recent study, osteocytes were cultured on an MWNT-coated collage sponge in a 3D dynamic fl ow cell culture system to generate honeycomb structure in femur and alkaline phosphatase, osteopontin, calcium as differentiation markers were measured to evaluate the use of MWNT-collagen as implant. Investigators reported implantation of one day osteoblast culture reconstituted femur bone tissue after 28–56 days [138, 139].

3.8 Conclusions

Carbon nanotubes make composites with metals and combine with cellulose polymers. Carbon nanotubes exhibit cytotoxicity at vari-ous levels and CNT composites may serve as safe drug delivery systems. Surface properties and structure of CNT play a signifi cant role in designing biocompatible composites such as bones, hard drug delivery carrier, molecular hydrogen sensors, electronic bio-sensing applications, detection of viral proteins, nanovector design, and superlight weight hard materials. Biophysical epithelial resis-tance measurements and cytotoxicity tests may be used in mesen-chymal cells as toxicity markers of carbon nanotube composites. Still mechanism of CNT-induced toxicity action and safe delivery


options of CNT composites remain unresolved as hype. Silver nanoparticles serve as potential antimicrobial agents

In the future, toxicity of carbon nanotubes will be tested in a time and concentration dependent manner using more sensi-tive biophysical and molecular imaging methods to detect cellu-lar infl ammation, metabolic integrity status and cell proliferation, and developing new potential CNT composite materials for wider applications such as electronics, safe delivery carriers and tissue targeting. ofi lr nanopll be explored in context with cell damage and cell cycle.

Acknowledgements

The authors acknowledge the experimental assistance in their graduate work, technical know how and their publish data in dif-ferent journals included in this chapter from both students Mrs. Stoker and Mr. Forrest at the Department of Biological Engineering at Utah State University. The authors also acknowledge the permis-sions to reproduce fi gures and results from previously published data in literature as cited references in the text. Authors appreci-ate the encouragement of Dean Dr. Ching J. Chen, former direc-tor of Center of Nanomagnetics and Biotechnology at Florida State University while preparing this manuscript.

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