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Bifunctional nanomaterials for the imaging and treatment of cancer

Abstract and Keywords

This article examines the potential of bifunctional nanomaterials for the imaging and treatment of cancer. Several nanomaterials possess properties desirable for a cancer therapy and have been the subject of research as anticancer agents. Those that have received the most attention include encapsulated iron oxides, single- and multiwalled carbon nanotubes, gold nanorods and gold nanoshells. This article first considers thermal ablative therapy incancer, focusing on the mechanisms of thermotoxicity and thermoresistance before discussing a number of nanomaterials with applications for cancer treatment. In particular, it evaluates the use of nanomaterials in thermal therapy. It also looks at gold nanoshells and nanorods, taking into account their physical properties, and concludes with an assessment of iron-oxide nanoparticles and future directions for nanomaterials as multifunctional agents for cancer therapy.

Keywords: bifunctional nanomaterials, cancer, nanomaterials, cancer therapy, thermal ablative therapy, cancer treatment, thermal therapy, gold nanoshells, gold nanorods, iron-oxide nanoparticles

13.1 Introduction

Cancer represents a significant burden with regards to both health and economy the world over. The World Health Organization projects global cancer incidence to increase 50% to 15 million new cases by 2020 (Stewart and Kleihues 2003). In the United States alone, the National Cancer Institute documented 1 444 920 new cases resulting in 559 650 disease-related deaths in 2007 (Ries et al. 2007). While incremental improvements in conventional therapeutic approaches have increased overall survival, for many cancers the benefits of treatment can be modest. For example, NCI data indicates that modern treatments fail nearly 40% of cancer patients each year (Ries et al. 2007). Thus, there is ample rationale for the search for novel and more efficacious cancer therapies.

The application of nanoscale materials for the diagnosis and treatment of disease (termed “nanomedicine”) represents one such novel approach to the treatment of cancer (Ferrari 2005; Jain 2008). Following the discovery of carbon nanotubes in 1991 by Iijima, the field of nanotechnology has expanded rapidly, leading to the synthesis and characterization of a variety materials whose principal dimensions are in the nanometer range (Iijima 1991; Dai 2002). Dissimilar to the macroscopic formulation of a given material, the nanomaterial may exhibit beneficial emergent properties that can significantly enhance its therapeutic utility.

Several nanomaterials possess properties desirable for a cancer therapy, and exploration of these materials as anticancer agents has begun. Those that have received the most attention include encapsulated iron oxides, single- and multiwalled carbon nanotubes, gold nanorods and gold nanoshells (Hirsch et al. 2003; Kam et al. 2005; Huang et al. 2006; Torti et al. 2007). This chapter reviews these nanomaterials as they seek to offer clinically relevant, (p. 475) minimally invasive therapeutic options for the treatment of local, non-resectable cancers. Each approach utilizes a unique photosensitizing nanomaterial capable of generating heat in response to exposure to electromagnetic radiation, resulting in the thermal ablation of surrounding tissue. Tumor specificity is achieved through the use of conjugated antibody targeting or passive accumulation via the enhanced permeability and retention effect. In addition, each material can serve as a contrast agent when used in conjunction with existing optical or magnetic resonance imaging modalities, permitting the in vivo-visualization and location of the target lesion. Such nanoscale materials specifically fabricated for the treatment of cancer have a unique potential to provide integrated imaging and treatment modalities.

13.2 Thermal ablative therapy in cancer

Ablation of cancerous lesions and consequent clinical remission of the disease is the express goal of any cancer treatment undertaken with curative intent. Surgery, chemotherapy, and radiation are the tools most commonly used to attain this goal. For most malignancies, surgery, if appropriate, remains the standard of care, as it is the pre-dominant modality through which cure can be achieved (Lenhard et al. 2001). However, sizable subpopulations of cancer patients are ineligible for surgery, due to factors such as location and extent of the primary lesion, poor performance status or other preclusive comorbidities (Lenhard et al. 2001). In recent decades, several approaches have been developed that exploit the differing sensitivities of normal and malignant tissue to a thermal insult to treat such patients (Overgaard and Suit 1979; Dickson and Calderwood 1980). Perhaps the most developmentally advanced applications of this approach (all are clinically available) are interstitial laser coagulation, high-intensity focused ultrasound, radio-frequency and microwave ablation (Izzo 2003; Nikfarjam and Christophi 2003; Kelekis et al. 2006; McDannold et al. 2006). These techniques are only moderately invasive and have generally tolerable toxicities, making them more broadly applicable to the cancer-patient population. Each therapy utilizes a distinct carrier wave to transduce energy through the body to the target lesion, whereupon the energy is absorbed and converted to heat. The heating of cells to supraphysiologic temperatures induces distinct, temperature-dependent alterations in morphology, metabolism and viability that will be addressed in a subsequent section of this chapter.

Thermal treatment of cancer can be divided into two basic types: hyper-thermic and thermal ablative. The principal distinctions between the two lie in the range of temperatures achieved, the duration of treatment and their clinical indications for use. Hyperthermic cancer therapy has been an effective adjuvent treatment (used in conjunction with chemo- and radiotherapy) for many cancers and ongoing clinical trials continue to validate the technique and generate new indications for its use (Datta et al. 1990; Dahl and Overgaard 1995; Overgaard et al. 1996; Sneed et al. 1998). Hyperthermic therapy seeks to elevate the temperature of either the whole body or isolated regions of the body to a range between 40–46 °C from 1 to several hours. (p. 476) This has the direct cytotoxic effects described elsewhere in this chapter in addition to enhancing the efficacy of subsequent regimens of chemo- and radiotherapy (Dewhirst et al. 1997; Dahl et al. 1999). Alternatively, thermal ablative therapy (several of the techniques of this therapy are described above) utilizes differing energy transducers to rapidly heat a small area of tissue (<8 cm in diameter) to much higher temperatures (55–90 °C) for a short period of time, generally <10 min per treatment (Nikfarjam et al. 2005). To date, uses of nanomaterials in cancer therapy have focused on thermal tumor ablation.

Experimental thermal therapies employing nanomaterials must be designed with consideration for the biological environment with which they must contend. This chapter will begin with an introduction to the processes in the tumor that these therapies are designed to disrupt and a discussion of countermeasures a tumor can employ to attenuate these effects. These factors bear directly on the success or failure of the therapy and must be accounted for in the design and capabilities of all nano-based thermal therapies.

13.2.1 Mechanisms of thermotoxicity

Broadly speaking, a therapy (especially when considering a cancer therapy) operates on two primary principles: (1) that there exists a biological target in the organism where the treatment exerts its intended effect, and (2) the sensitivity of the true target differs (ideally appreciably) from that of non-targets such that it gives rise to a useful therapeutic index. Thermal therapies satisfy these criteria, since transformed cells exhibit enhanced susceptibility to supraphysiologic temperatures (Overgaard and Suit 1979; Dickson and Calderwood 1980; Fajardo et al. 1980; Henle and Dethlefsen 1980; Everts 2007). Analogous to therapeutic radiation exposure, heating a cell induces a multitude of alterations in cellular processes as diverse as cytoskeletal dynamics, enzyme structure and function and overall metabolism (Dewey 1989; Harmon et al. 1990; Heisterkamp et al. 1999; Hildebrandt et al. 2002; Nikfarjam et al. 2005; Roti 2008). Thus, despite decades of investigation, the definitive proximate cause of heat-induced cell death remains elusive. Several lines of evidence do, however, implicate selective events that correlate well with cell death, and these are discussed in the following sections.

13.2.1.1 The targets of heat

Cellular constituents most often associated with hyperthermic cell death include alterations in the structure and function of cellular plasma membranes, cytoskeleton, DNA and proteins (Dewey 1989; Heisterkamp et al. 1999; Hildebrandt et al. 2002; Nikfarjam et al. 2005; Coffey et al. 2006; Roti 2008).

13.2.1.2 Heat-induced alterations of the plasma membranes

The plasma membrane of a cell and its various membrane-bound organelles serve the vital purpose of defining the extent of the cytosol and, through its general impermeability, permitting the establishment of essential electrochemical (p. 477) gradients through which work may be done. Thus, the integrity of the plasma membrane is essential to the survival of any cell.

Elevating the temperature of a cell serves to alter the fluidity of its membranes and destabilizes several of these essential electrochemical gradients (Konings and Ruifrok 1985). Indeed, several reports indicate that exposure to hyperthermic and thermal ablative temperatures decreases the functionality of many membrane-bound ion pumps resulting in marked increases in intracellular sodium and calcium concentrations (Konings and Ruifrok 1985; Vidair and Dewey 1986; Majda et al. 1994; Hildebrandt et al. 2002). Of these changes, perhaps the influx of calcium is the most significant, resulting in a marked decline in mitochondrial membrane potential (Δψm) and stalling of ATP synthase (Klaassen 2008). As ion concentrations equilibrate with the extracellular space, sensitive proteins begin to denature and aggregate. Such an event in the mitochondrial membrane is thought to drive the mitochondrial permeability transition, leading to the release of various pro-death factors (such as cytochrome c and the protein Smac/Diablo) (Kroemer et al. 1998; Lemasters et al. 2002; Kim et al. 2003a,b). Higher cellular temperatures, such as those seen during thermal ablative treatment, can expedite this process through lysis of the plasma membrane and direct exposure of the cytosol to the extracellular milieu. The end result of this cascade is either apoptotic (hyperthermia) or necrotic (thermal ablative) cell death (Harmon et al. 1990) (discussed further below).

13.2.1.3 Heat-induced alterations to DNA

During the thermal treatment of cancer, DNA damage is induced both directly and indirectly as determined by the severity of the heat shock (Roti 2008). In the thermal ablative temperature range (55–90 °C) the effect on DNA is direct; resulting in the denaturation of the antiparallel strands and possible strand breakage due to the unstabilized abnormal conformation (Dewey et al. 1971; Defer et al. 1977; Lewis 1977). In the hyperthermic ranges (40–46 °C) the role of DNA damage is less well defined. It is well known that the hyperthermic heat shock of a cell traversing S-phase results in chromosomal aberrations (Dewey et al. 1990; Wong et al. 1993). Although some reports have documented the formation of γH2AX complexes (a phosphate-modified histone that is a surrogate marker for DNA double-strand breaks) following temperatures in the hyperthermic range (Olive 2004; Takahashi et al. 2004), others have failed to observe DNA fragmentation through pulsed gel electrophoresis in heat-shocked cells, despite the detection of γH2AX complexes in those same cells (Hunt et al. 2007). Thus, the role of DNA damage in hyperthermia is uncertain, although it is of clear importance in the response to heat exposure in the thermoablative range.

13.2.1.4 Heat-induced alterations to proteins

Of all the cellular macromolecules that could be critical targets of heat, proteins are perhaps the most likely, due to their abundance and the variety of vital functions they perform, as well as to their dependence on weak molecular interactions for structural and functional integrity (Nelson and Cox 2005).

(p. 478) The nuclear matrix (Berezney and Coffey 1974) is a protein network that provides the structural support and spatial regulation necessary to manage a cell’s 5 × 109 base pairs of DNA as well as an additional mass of associated proteins that is nearly 6 times that of the DNA alone (Roti 2008). This matrix facilitates most vital DNA-related events such as transcription, replication and repair. Differential scanning calorimetry of isolated cell nuclei has demonstrated that the nuclear matrix is one of the most thermally sensitive cellular structures, exhibiting marked changes in response to temperatures as low as 40 °C (Lepock 2003, 2004, 2005). Additionally, as other nuclear proteins unfold and aggregate in response to heat shock, these aggregates bind the nuclear matrix and prevent normal DNA–matrix interactions. These events are especially critical during the S-phase of the cell cycle when DNA replication occurs. At this time, the nuclear matrix serves to scaffold an open DNA conformation and facilitate the assembly of the replicative machinery. Heat-induced alterations to this process are thought to be one of the causative mechanisms of replication fork stalls and DNA strand breaks observed in heat-shocked cells. This likely underlies the observation that S-phase cells are most sensitive to hyperthermic cell death (Wong et al. 1993). This has special significance in regard to the treatment of cancer, where many tumors have high mitotic indices and thus elevated S-phase cell fractions.

Another critical protein target of heat is the cytoskeleton. This structure is composed of three principal elements: microtubules, intermediate filaments and actin filaments, and is responsible for cell morphology, movement and various signalling processes. All three cytoskeletal constituents are susceptible to damage by heat through a function that varies by thermal dose and cell type. Studies have shown that at moderate hyperthermic heat doses (43 °C) microtubules depolymerize, leading to the disruption of microtubule organization centers and mitotic spindles (Coss et al. 1982; Coss and Linnemans 1996). Such an event would have significant consequences in an M-phase cell, and may represent a contributing factor in the death of mitotic cells. Loss of pro-survival signalling mediated by heat-induced shedding of integrin adhesion molecules and depolymerization of actin fibers may also contribute to hyperthermic cell death (Shih-Horng Huang 1999).

13.2.1.5 Terminal events: Apoptosis or necrosis

Pathways of cell death have been broadly grouped into apoptotic or necrotic (Buja et al. 1993; Lenhard et al. 2001). Unlike necrosis, apoptosis is a highly ordered, energy-dependent process that does not induce a host inflammatory response (Bursch et al. 1992; Klaassen 2008). Necrosis generally results from overwhelming cellular trauma, which is believed to trigger cell death at least in part by disrupting mitochondrial function, triggering bioenergetic catastrophe and ultimately cell lysis (Lemasters et al. 2002; Kim et al. 2003a,b). The resulting loss of integrity and dissemination of various bioactive, formerly cytoplasmic macromolecules throughout the interstitium leads to the recruitment of inflammatory immune cells (neutrophils and macrophages).

(p. 479) Available data suggest that both death processes are induced by heat treatments. At hyperthermic temperatures (below 46 °C) apoptosis pre-dominates (Harmon et al. 1990). This has the benefit of not inducing the recruitment of inflammatory cells that, along with removing necrotic cellular debris, also release various pro-growth, pro-angiogenic cytokines that can enhance the expansion and recovery of malignant cells not destroyed in the initial treatment (Bursch et al. 1992; Cain 2003). However, therapies that work predominantly through apoptotic mechanisms often fail, as tumors invariably contain cell populations insensitive to apoptotic stimuli (Boehrer et al. 2006; Eberle et al. 2007). For thermal therapies operating in the ablative temperature range, cellular resistance is not a concern (see below for supracellular mechanisms of resistance) as research has demonstrated they work almost entirely through coagulative necrosis (Heisterkamp et al. 1999; Nikfarjam et al. 2005a,b). As the necrotic process does not require cellular “cooperation” to induce death, the entire tumor is generally susceptible to the pro-death thermal insult (Lemasters et al. 2002; Kim et al. 2003). However, if the ablated region fails to encompass the entire tumor, the residual disease may be stimulated to recur through the eventual influx of inflammatory cells. These will likely remain important considerations for any heat-based cancer therapy.

13.2.2 Mechanisms of thermoresistance

When faced with thermal-therapy-induced cytotoxic insults, cancerous cells do not passively transition to apoptosis or necrosis. On the contrary, they trigger evolutionarily conserved cytoprotective mechanisms designed to attenuate cytotoxic effects and maintain viability (Williams et al. 2007). Two of these figure prominently in the failure of heat-based therapies: the heat-sink effect and heat-shock proteins (Nikfarjam et al. 2005).

13.2.2.1 Heat-sink effect

The efficacy of thermal-ablation therapies is dependent on their ability to uniformly reach and maintain a given temperature in a target tissue volume for a defined length of time (Heisterkamp et al. 1999). This can be hindered when the target treatment area abuts a large, well-perfused vessel where the transfer of heat into the blood facilitates its dissipation throughout the body (Patterson et al. 1998; Lu et al. 2002, 2003). This is termed the heat-sink effect. This event is widely encountered in highly perfused tissues such as liver and kidney. Current strategies to mitigate this effect include the surgical isolation and temporary occlusion of the offending vessel, either by clamping or catheter-mediated embolization. The resultant decrease in local blood flow enhances tissue heating and helps to decrease the likelihood of undertreated regions and local recurrence.

13.2.2.2 Heat-shock proteins

A family of stress-inducible proteins known collectively as heat-shock proteins (HSPs) plays a central role in a cell’s response to thermal insult. The protein constituents of this diverse family range in size from 10 to >100 kDa and (p. 480) exist at basal levels throughout the various cellular compartments where they serve a variety of housekeeping functions (Rylander et al. 2005). Induction of HSPs can occur in response to a diverse array of stimuli from environmental stressors like heat shock, a pathologic febrile state or to normal developmental conditions such as cytokine exposure (Schlesinger 1990). Induction of these proteins is believed to occur through a mechanism involving release from cytoplasmic binding proteins into the nucleus, where they induce transcription of cytoprotective proteins, including the heat-shock proteins themselves (Kiang and Tsokos 1998; Morimoto and Santoro 1998; Wang et al. 2003; Rylander et al. 2005). For the purposes of thermotolerance, two HSPs in particular have been implicated: HSP 27 and HSP 70.

The thermoprotective effect of HSP expression is believed to result from their ability to serve as chaperones to misfolded proteins (Alberts et al. 1994; Rylander et al. 2005). This function prevents the lethal accumulation of denatured protein aggregates in the cell. To that same end, HSP may also be involved in the trafficking of misfolded/denatured proteins to other organelles where they are fated for either refolding or degradation (Rylander et al. 2005).

HSPs represent a means by which cancerous cells escape thermally induced death. This protective state is termed thermotolerance and describes the condition in which a cell previously exposed to elevated but sublethal temperatures can resist the effects of further heating for an extended period of time (Subjeck et al. 1982a,b; Li et al. 1995). The duration of the resistive period varies as a direct function of the intensity of the initial thermal insult, with higher initial temperature exposures leading to more protracted periods of thermotolerance that may last as long as 5 days (Wang et al. 2003). During this time, cells are not only desensitized to additional temperature increases, but also to other pro-apoptotic stimuli (Fortin et al. 2000; Latchman 2001; Rylander et al. 2005). Indeed, the elevated expression level of HSPs during the thermotolerant period has been implicated in the resistance of several cancers to both chemo- and radiotherapy, likely through their ability to attenuate the pro-apoptotic effects of p53 (Levine 1989; Levine et al. 1991; Tomei and Cope 1991; Ciocca et al. 1993; Samali and Cotter 1996; Rylander et al. 2005).

It is important to note that the varied activities of the HSPs cannot protect a cell against an acute, high-temperature (55–90 °C) exposure, as would be produced during an optimal course of thermoablative therapy (Nikfarjam et al. 2005). However, current thermoablative techniques and temperature-monitoring strategies conspire to make it difficult to ensure that such temperatures are uniformly reached throughout the entire tumor volume, especially at the marginal extremes of the target neoplasm, and it is in this cell population that the protective effects of HSPs are most worrisome. These undertreated cells on the tumor periphery that are now both thermotolerant and chemo/radioresistant are primed to become sites of treatment failure and, ultimately, disease recurrence.

Interventions preventing or greatly attenuating the ability of a transformed cell to mount an effective heat-shock response could be of great utility in boosting the efficacy of heat-based cancer therapies (Morimoto and Santoro 1998; (p. 481) Calderwood and Asea 2002), and are currently being explored (Rossi et al. 2006; Sanguino et al. 2008).

13.3 Nanomaterial applications

13.3.1 Nanomaterials in thermal therapy

As summarized above, thermal therapy is a commonly accepted technique in the treatment of cancer. Heat is used to both enhance the efficacy of other therapies (hyperthermia) as well as in direct tumor ablation. Limitations of current approaches include inadvertent treatment of non-malignant adjacent tissue, incomplete treatment of tumor margins, and inadequate heat delivery, with consequent activation of resistance mechanisms.

Due to their capacity to deliver substantial heat in a non-invasive manner, their potential to deliver multiple rounds of heat therapy, and the ability to utilize their imaging potential for more precise localization of heat delivery, nanomaterials are being actively investigated for their potential to improve the thermal treatment of cancer. Although this is a relatively new area of investigation, several different types of nanomaterials have been explored in this regard. Each of these is described in greater detail below (Table 13.1).

13.3.1.1 Physical properties of single- and multiwalled carbon nanotubes

Carbon nanotubes (CNTs) are cylindrical, carbon molecules that range from 1.4 nm in diameter for a single tube (SWNT) (Fig. 13.1), to 30 nm to 50 nm for concentrically arranged tubes (MWNT) (Fig. 13.2), with lengths in both cases of micrometers or more (Iijima 1991). These structures exhibit extraordinary strength; CNTs are the strongest known fibers derived from their carbon–carbon graphitic bonds. They also exhibit unique electrical properties including ballistic transport, and are highly efficient conductors of heat.

Table 13.1 Physical properties of nanoparticles designed for thermal ablative cancer therapies.

Particle

Size

Composition

References

Nanoshells

50–500 nm

Silica core surrounded by metal (usually gold) shell

Hirsch et al. (2006)

Gold Nanorods

50–100 nm

Reduced gold

Huang et al. (2007)

Super-paramagnetic Iron Oxide

5–10 nm

Magnetite (Fe3O4) and Maghemite (γ-Fe2O3)

Gneveckow et al. (2004)

Single-Wall Carbon Nanotubes

1.4 nm diameter; lengths to micrometers

Carbon

Iijima (1991) Kam et al. (2005)

Multiwalled Carbon Nanotubes

30–50 nm diameters; Lengths to micrometers

Primarily carbon; may be doped with nitrogen, boron, etc.

Torti et al. (2007) Liu et al. (2005)

(p. 482)

Bifunctional nanomaterials for the imaging and treatment of cancerClick to view larger

Fig. 13.1 As-grown SWNTs imaged by TEM. D. Carroll, unpublished.

Bifunctional nanomaterials for the imaging and treatment of cancerClick to view larger

Fig. 13.2 Multiwalled nanotubes. D. Carroll, unpublished.

(p. 483) The specific heat and thermal conductivity numbers of CNTs are among the highest known in materials (Brigger et al. 2002).

The unique electronic properties of carbon nanotubes come from the quantum confinement of electrons around the circumference of the nanotube. Radially, electrons are confined to reside on the monolayer thickness of the graphene sheets making up the tube. Because there are various ways in which the lattice of the single-walled nanotube can be twisted down its axis, the overall unit cell of the carbon lattice can be composed of very large or very small numbers of graphene hexagons. This means that nanotubes can range from being very good metals to being semiconductors with rather large bandgaps; extraordinary for a single-element molecule (Brigger et al. 2002). In the case of multiwalled nanotubes, the concentric walls of the tubes usually have one or more that are metals, so all multiwalled nanotubes are metals or small-bandgap semiconductors.

The interaction of light with the nanotubes structure is of particular importance in a number of applications, including biomedical. The photophysical effects related to low dimensionality and quantum confinement of electrons in single-walled nanotubes have been studied through Raman scattering, fluorescence, non-linear optical properties, etc., yet this is still quite an unexplored area of research (Hui 2005). What we do know is that SWNTs exhibit a wide variety of different vibrational modes, which result from the large number of carbon atoms in the unit cell of the nanotube. In general, more phonon modes appear as the nanotube diameter and the size of the unit cell increases, meaning that the ability of heat transport and heat capacity is unique to the nanotube structure.

Likewise in MWNTs a large number of phonon modes mean that these structures are exceptional heat conductors. However, in contrast to the SWNT, the larger numbers of electrons available in the MWNT for transport, together with the small bandgap, or metallic behavior suggests that these materials act much more like a classical antenna; coupling highly efficiently to radiation when the length of the nanotubes are comparable to half that of the wavelength of the incident radiation (Hanson 2005). This means the electrons in the nanotube can more easily become excited and begin releasing excess energy in the form of heat. Further, radiation captured in this way can be scattered (or transferred) efficiently to the surrounding media creating enhanced dielectric loss in the material, serving to enhance the heating of any host material in which the nanotube is embedded.

The substitutional doping of carbon nanotubes is a desirable method to control the electronic properties of the tubes by the chemistry rather than the specific geometry of the tubes. This allows for the engineering electronic band structure to increase the overall conductivity (hence the antenna behavior) of the nanotube. Carbon nanotubes can be doped in different ways, including intercalation of electron donors like alkali metals or acceptors like halogens, substitutional doping, encapsulating in the interior space, coating on the surface, molecular absorption, covalent sidewall functionalization, etc. Substitution of the carbon atoms with electron donors like nitrogen (N) or acceptors like boron (B) is the method of doping we employed. It has been found that the impurity level induced by N is located 0.27 eV below the bottom (p. 484) of the conduction bands and the B-induced level is 0.16 eV above the top of the valence bands (Schonenberger and Forro 2000).

For pure carbon nanotubes, the valence and conduction bands appear to be symmetric about the Fermi level. The metallic behavior of B-doped nanotubes characterized by the local density of states (LDOS) can be investigated by scanning tunnelling microscopy and spectroscopy. It has been found that undoped carbon nanotubes show a small bandgap (semiconducting or semi-metallic behavior), whereas for the B-doped MWNTs the bandgap is filled from the valence-band side with a prominent acceptor-like peak near the Fermi level—this indicates that the outermost tube cylinders are structurally perfect with few defects. For the undoped tubes, the DOS is symmetric around the bandgap for the 1st and 2nd van Hove singularities—asymmetries arise due to the mixing of p and s orbitals (Dai 2002). Doping of the tubes leads to a lowering of the Fermi level into the valence band of the undoped tube. The stronger the doping, the stronger is also the shift of the Fermi level. In relation to transport properties it has also been found that this lowering of the Fermi level by substituted B dopants also increases the number of conduction channels without introducing strong carrier scattering. B-doped nanotubes show metallic behavior with weak electron–phonon coupling and the resistance increases at lower temperatures. Substitutional doping also significantly enhances the value of third-order optical non-linear coefficients. For doped nanotubes this enhancement can be by an order of magnitude with respect to C60. This is particularly useful for photonic applications that require large second hyperpolarizabilities.

Finally, carbon nanotubes are also good candidates for optical-limiting applications as they exhibit non-linear scattering. Enhanced optical-limiting behaviors of B- and N-doped nanotubes are observed by varying the incident energy and measuring the transmitted energy. In comparison with the nonlinear transmittance versus incident fluence of pure and B- or N-doped carbon nanotubes at 532 nm and 1064 nm, doped nanotubes are found to have better optical-limiting properties (lower threshold values) than pure nanotubes (Schonenberger and Forro 2000).

13.3.1.2 Applications of single- and multiwalled carbon nanotubes

Given the multitude of exploitable properties detailed above, it is perhaps not surprising that carbon nanotubes of every variety have been studied as potential means to enhance the treatment of human malignancies (Kim 2007) (Table 13.2). A review of the literature suggests that two forms, the single- and multiwalled variants, have progressed the furthest in development, and this section will focus on these exclusively.

13.3.1.3 Anticancer activity of carbon nanotubes in vitro

The efficacy of both single- and multiwalled carbon nanotubes in the thermal treatment of cancer was initially demonstrated in cell culture. In 2005, the laboratory of Dr. Hongjie Dai published an initial study on the use of single-walled carbon nanotubes (SWNT) for the thermal treatment of cancer. This publication demonstrated that single-walled nanotubes efficiently absorb radiation in the near-infra-red range (λ = 700–1100 nm) and convert it to heat, which is dissipated in the surrounding medium. Absorbance was robust enough (p. 485)

Table 13.2 Use of nanoparticles in anticancer thermal therapy.

Particles

Applications

Imaging modality

Targeting strategies

Advantages

Limitations

Developmental stage

Nanoshells1

  • -Imaging contrast Agent

  • -Photothermal ablation

Optical Coherence Tomography

  • -EPR effect

  • -Antibody conjugation

  • -Direct injection

  • -Tuneable EM absorption and reflection spectra

  • -Ease of gold conjugation chemistry

  • -Uniform particle sizes

Not proven visible by standard clinical imaging modalities

Phase I Clinical Trial: Head and neck cancer

Nanorods2

  • -Imaging contrast agent

  • -Photothermal ablation

Optical Coherence Tomography

  • -Antibody conjugation

  • -Tuneable EM absorption and reflection spectrums

  • -Ease of gold conjugation chemistry

  • -Uniform particle sizes

Not proven visible by standard clinical imaging modalities

Pre-Clinical: Cell-culture systems

Iron-Oxide Nanocrystals3

  • -Imaging contrast agent

  • -Thermal ablation

MRI, CT

  • -Direct injection

  • -No depth of penetration issues with alternating magnetic fields

Requires direct injection

Phase I/II Clinical Trials: Brain and prostate cancers

Single-Wall Carbon Nanotubes4

  • -Imaging agent

  • -Photothermal ablation

  • -Drug carrier

Raman Spectroscopy

  • -EPR effect

  • -Antibody conjugation

  • -Targeting Peptides

  • -Potential for tuneable absorption spectrum.

  • -Low toxicity

Particle sizes not uniform

Pre-Clinical: Animal models

Multiwalled Carbon Nanotubes5

  • -Imaging contrast agent

  • -Photothermal Ablation

  • -Drug carrier

MRI

  • -Direct injection

  • -EPR effect

  • -Antibody conjugation

  • -Efficient heat generation

  • -Doping adds new functionalities

  • -Low toxicity

Particle sizes not uniform

Pre-Clinical: Animal models

Key References: (1)) Hirsch et al. (2006),

(p. 486) to raise the temperature of the SWNT-containing solution to over 55 °C (i.e. into the established thermoablative range) during 60 s of exposure to 808-nm laser radiation (Kam et al. 2005). The significance of strong absorbance in this wavelength range is considerable, since biological bodies themselves generally have weak absorbance in the near-infra-red segment of the EM spectrum (Konig 2000; Weissleder 2001). This property is fundamental to the application of this and other nanomaterials (indeed, most studies discussed in this chapter utilize NIR to stimulate the release of heat from their respective nanomaterials) that seek to treat non-superficial cancerous lesions in vivo.

This initial study also demonstrated that the heat generated following laser radiation of SWNT was sufficient to kill HeLa cells (a well-characterized cervical carcinoma cell line) that had internalized SWNT during an incubation period (Kam et al. 2005). Additionally, SWNTs could be selectively targeted to HeLa cells that overexpressed the folate receptor (FR+) by conjugating a folate moiety to polyethylene glycol-functionalized (PEGylated) SWNT. Folate-functionalized SWNTs were preferentially taken up by the FR+ cells, and these cells were killed with considerable selectivity upon illumination with 808-nm laser radiation (Kam et al. 2005). In a later study, it was also demonstrated that functionalized SWNTs can be conjugated with platinum-based chemotherapeutics that can enhance cell kill in when used in conjunction with NIR-stimulated heating (Feazell et al. 2007). Given the structural similarities between SWNT and MWNT, this approach would likely be applicable to multiwalled carbon nanotube (MWNT)-based cancer therapeutics as well.

Recently, it has been demonstrated that SWNTs can be heated through the use of a non-invasive radio-frequency field, and that this capability can be used to successfully kill cancer cells in vitro and VX liver tumors in vivo (Gannon et al. 2007). This demonstration suggests SWNTs may be capable of non-invasively treating tumors in any part of the body, a capability currently not shared by NIR laser-based treatments.

Similar to SWNT, MWNT also have demonstrated utility in the realm of experimental cancer thermotherapy. The coupling of MWNT to NIR and its application to killing of cancer cells was recently demonstrated (Torti et al. 2007). In keeping with the predictions of classical antenna theory, this paper confirmed that MWNT with a length of 330 nm failed to generate appreciable heat in response to illumination with a 1064-nm laser, while MWNT with average lengths of 700 and 1100 nm, respectively, readily raised the temperature of the solution to over 50 °C (Torti et al. 2007). Cell-culture experiments using CRL 1932 cells (an established human clear cell carcinoma line) demonstrated that cells treated with either 700 or 1100-nm MWNT and NIR were readily killed, whereas treatment with 330-nm MWNT and NIR failed to elicit a cell-death response. There was no toxic effect of either MWNT or NIR when used individually (Fig. 13.3). MWNTs exhibit two potential advantages: first, due to the unique structure of MWNTs they absorb NIR more efficiently than SWNTs, thus requiring less incident radiation to generate an equal increase in temperature. In addition, they do not require internalization by the target cell to induce its death (Torti et al. 2007). An additional potential advantage of (p. 487)

Bifunctional nanomaterials for the imaging and treatment of cancerClick to view larger

Fig. 13.3 The combination of MWNT and laser irradiation induces a temperature increase and cell death of human kidney cancer cells. S. Torti, D. Carroll, unpublished.

MWNT is their ability to be modified by dopants such as nitrogen or boron, which may augment their thermal properties, although to date this has not been explicitly investigated (Liu et al. 2005).

13.3.1.4 Pharmacokinetics and biodistribution of carbon nanotubes

Pharmacological properties are critical in the assessment of any therapeutic material, including nanomaterials. Several groups have investigated the disposition of both single- and multiwalled nanotubes in rodent models following intravenous delivery.

Early studies focused on the distribution of nanotubes in non-disease-state model systems. Singh et al. described an approach by which ammonium-functionalized single- and multiwalled carbon nanotubes were covalently modified with diethylenetriaminepentaacetic (DTPA) dianhydride chelated 111In. (p. 488) Dilute solutions were injected intravenously into cohorts of mice and tissues were harvested at 0.5, 3 and 24 h time points. These tissues were then read by a gamma counter (Singh et al. 2006). Surprisingly, both nanomaterials demonstrated little uptake by the reticuloendothelial system (RES), with renal excretion being the dominant form of systemic clearance. Moderate uptake was also seen in blood, muscle, skin and bone 30 min postinjection, with minimal activity observed at later time points. From this data the authors calculated a blood circulation half-life of 3–3.5 h (Singh et al. 2006).

A more recent publication utilizing Raman spectroscopy to measure tissue distribution of PEG-functionalized SWNT came to rather different conclusions (Liu et al. 2007). Contrary to the previous report, Liu et al. noted extended blood circulation times in excess of 20 h, with eventual nanotube clearance performed almost exclusively by organs of the RES (specifically liver and spleen) (Liu et al. 2008). In addition, significant quantities of nanotubes were detected in these organs up to three months postinjection. Encouragingly, the authors noted no resultant pathologies in the liver or spleen as determined by histological examination at the 100-day conclusion of the study. Together, these reports suggest that the method of nanomaterial functionalization strongly impacts its final disposition in the body. Thus, each new approach to functionalization will likely require new pharmacokinetic assessments.

Both of the previous studies were performed in tumor-free mice. Since the eventual goal is the targeting of nanomaterials to tumors, studies have also been initiated to assess the distribution of nanotubes in tumor-bearing models (Liu et al. 2007). Using PEGylated and radiolabelled SWNTs, mice bearing subcutaneous tumor xenographs were injected intravenously and the tissue distribution was tracked by positron emission tomography. Concordant with results described above (Liu 2008), blood-circulation times were extensive, and excretion was primarily hepatobiliary in nature. They also demonstrated that about 5% of the injected dose of nanotubes passively accumulated in the tumors, and that this could be increased to over 15% if the nanotubes were conjugated to RGD, a small peptide that targets integrins (Liu et al. 2007). These reports suggest that systemic delivery of nanotubes is a viable option. Section 13.3.1.6 will discuss ongoing efforts to enhance the uptake of these materials by their target lesions.

13.3.1.5 Toxicology of nanotubes

The inherent toxicity of any agent is a key attribute of its overall profile as a therapeutic. The debate over the intrinsic biocompatibility of carbon nanotubes has intensified following recent reports comparing long nanotubes (15–20μm) to asbestos particles (Poland et al. 2008). At present, the toxicologic profiles of SWNT and MWNT remain poorly understood, in part due to the diversity of nanotube preparations, the difficulty in obtaining pure, homogeneous samples, the uncertain contribution of coating and functionalization to toxicity and biodistribution, and the unclear impact of contaminants such as residual catalyst or solvent to observed toxic effects.

(p. 489) Nanotubes are not new materials: MWNT have been observed in ice samples dating from the Neolithic Stone Age, and are present as combustion products in both indoor and outdoor environments (Lam et al. 2006). Nevertheless, their deliberate manufacture has raised concerns over hazards posed by occupational exposure. Toxicologic studies have focused primarily on the lung, since inhalation is the most likely route of occupational exposure, and epidemiological studies of nanoscale exhaust particles in the air have already demonstrated an association between materials of this scale and cardiovascular and respiratory morbidity and mortality (Samet et al. 2000). Early in-vitro studies using rat macrophage and human lung cell lines demonstrated that exposing cells to doses of SWNT and MWNT ranging from 5–100μg/mL was not acutely toxic and did not result in marked decreases in cell viability, despite appreciable uptake of the material into the cytosol (Pulskamp et al. 2007). The authors did note that unpurified nanotubes increased intracellular levels of reactive oxygen species and attenuated mitochondrial membrane potential; however, these effects were not observed in cells treated with purified nanotubes and the authors attributed those toxicities to residual metal catalyst on the nanotube exterior and not the tubes themselves.

More recently, studies using a human mesothelioma cell line reached markedly different conclusions (Wick et al. 2007). In this study, doses of SWNT between 7.5–30μg/mL substantially reduced cell viability in a dose-dependent manner, to below 25% of control for the highest dose. An important result was that the degree of nanotube agglomeration affected the outcome, with large aggregates decreasing viability to a greater extent than small bundles of nanotubes. This observation highlights the need for extensive characterization of the carbon nanotubes under investigation, since small changes in material state can significantly impact the study outcome.

Recent in-vivo studies have generally supported the conclusion that non-functionalized carbon nanotubes are detrimental to their host organism when directly instilled in the lung. For example, mice tracheally instilled with a 500 μg/kg bolus of SWNT developed multifocal granulomas with extensive macrophage infiltration and lung inflammation over the 14-day course of the study (Chou et al. 2008). Studies by others with doses ranging up to 10 mg/kg in both mice and rats showed similar trends, with animals developing extensive granulomas and fibrosis (Muller et al. 2005; Shvedova et al. 2005). Carrero-Sanchez et al. were able to replicate these findings with high doses (1–5 mg/kg) of multiwalled carbon nanotubes instilled tracheally. Interestingly, nitrogen-doped MWNTs failed to induce granuloma formation and only provoked moderate inflammation in mice challenged with the highest dose (Carrero-Sanchez et al. 2006).

In contrast to these studies, MWNT administered by inhalation did not lead to significant lung inflammation or tissue damage (Mitchell et al. 2007). Unlike previous studies that relied on single bolus tracheal instillations of carbon nanotubes, Mitchell and colleagues exposed mice by an inhalation chamber (a more directly relevant exposure technique) to doses of MWNTs up to the comparable allowed occupational limit of human exposure to nuisance dust (5 mg/m3: equivalent to a calculated dose of 2.7 mg/kg for the study animals). After 14 days of exposure, mice were sacrificed and extensively examined. (p. 490) Surprisingly, there was no difference in the number of inflammatory cells between the lungs of control and MWNT-exposed mice or any overt lung damage as determined by histological analysis, although there was an overall decline in immune cell function. The authors suggested that the lack of toxic effect in the lung was likely due to the lower doses used. However, as their highest dose was already 100 times greater than that detected in a recent industrial hygiene report (Maynard et al. 2004) they questioned the necessity of using higher doses due to the enhanced risk of false-positive results.

In a recent provocative study, 50 μg of long (15–20μm) MWNTs injected intraperitoneally into mice induced an inflammatory reaction in the mesothelium similar to that seen in asbestos-injected mice, suggesting, by extension, that long MWNTs might be similarly carcinogenic to that tissue (Poland et al. 2008). The relevance of this finding to currently proposed biomedical applications (including those detailed in this chapter) of carbon nanotubes is uncertain, since no studies have suggested using nanotubes longer than 1μm; a length that failed to provoke any detrimental response in this study. Nevertheless, this result underscores the need for more extensive longitudinal studies that may uncover such long-term toxicities.

Overall, current data regarding the toxicity and biocompatibility of carbon nanotubes are sparse and conflicting. There does appear to be a pre-ponderance of evidence that high doses (>0.5mg/kg) directly instilled in a target tissue such as the lung can provoke a toxic response. The data are less clear at more therapeutically relevant doses. Additionally, agents used to coat and functionalize nanotubes are likely to have a large impact on their toxicity, and the contribution of these agents is only beginning to be appreciated (Zhou et al. 2008). There is also a disconnect between nanotube preparations used for toxicity studies and those used for therapeutic intent: for example, the toxicology studies detailed above used either uncoated nanotubes or nanotubes solubilized with atypical surfactants (PS80); in contrast, nanotube solutions described elsewhere in this chapter that are being investigated as cancer therapies rely on PEG or Pluronic surfactants to coat and functionalize the nanotubes. At a minimum, coating agents will affect bundling, which has been shown to affect toxicity (Wick et al. 2007); likely, coatings will also affect other key parameters that influence toxicity, such as clearance and biodistribution. Thus, in addition to the general need for more extensive toxicologic studies, there is a need for studies that directly focus on the precise materials that are envisioned as therapeutic agents. These will be essential for the future translation of investigational carbon-nanotube-based cancer therapies into clinical application.

13.3.1.6 In-vivo targeting strategies employing nanotubes

To date, two methods have pre-dominated in the effort to selectively target intravenously delivered nanomaterials to tumors; passive targeting and active targeting through the use of conjugated targeting peptides.

The first strategy is a passive approach that takes advantage of what is termed the enhanced permeability and retention effect (EPRE) to deposit nanomaterials in the tumor bed. The EPR effect derives from structural (p. 491) abnormalities in tumor vasculature: tumor vessels are characterized by discontinuous endothelial tight junctions, and a lack of uniform mural cell reinforcement, which together lead to vessels of poor integrity that are highly permeable to large molecules. This action is complemented by deficiencies in tumor lymphatic structure, such that molecules that have extravasated in the tumor are not readily removed through lymphatic drainage (Maeda et al. 2000; Maeda 2001). The dissimilar competencies of normal and tumor-associated vessels can be exploited to selectively target materials of a particular size range to a tumor, while sparing normal tissues. Indeed, it has been shown that particles with apparent molecular masses >45 kDa or sizes in the range of hundreds of nanometers (these sizes exceed the renal clearance threshold, preventing them from being rapidly cleared from circulation by glomerular filtration) can achieve concentrations tenfold greater than both plasma and normal tissue within 24 h of intravenous administration (Matsumura and Maeda 1986; Maeda and Matsumura 1989; Maeda et al. 2003). With specific reference to its application in the targeting of systemically delivered carbon nanotubes, Liu et al. demonstrated that 5% of an intravenous dose of PEG-functionalized SWNTs passively accumulated in an experimentally induced rodent tumor within 6 h of induction (Liu et al. 2007).

The second strategy is an active targeting strategy, in which a tumor-targeting ligand is attached to the nanotube in order to direct the nanotube to the tumor. For example, by covalently modifying PEGylated SWNTs with RGD peptides (these bind tightly to the angiogenesis and metastasis-related integrin αvβ3) tumor accumulation of SWNTs amounting to 15% of the total injected dose was achieved (Liu et al. 2007). While this technique has great potential for the active targeting of drugs to a given tissue, it relies on either the tumor-specific expression or tumor overexpression of a known target.

13.3.1.7 Use of carbon nanotubes in tumor imaging

The refinement of existing medical imaging modalities (primarily MRI, CT and PET) has greatly enhanced their utility in detecting indwelling tumors and tracking their response to therapy. In relation to the proposed use of carbon nanotubes (particularly MWNTs) for the thermal treatment of cancer, MRI has capabilities that best complement the envisioned therapeutic strategies. Two in particular (its amenability to contrast enhancement and use in non-invasive thermometry) will be briefly considered.

The spatial resolution of proton magnetic resonance is the physical basis of the medical imaging modality known as MRI. Following the application of the pulsed magnetic field and the consequent excitation of the proton to a high-energy state two relaxation times, termed T1 and T2, can be determined through specific pulse sequences as the proton falls back to low-energy equilibrium. These relaxation times are a function of the proton’s immediate environment, and thus change from tissue to tissue and between normal and malignant. Contrast agents can be used to further highlight areas of the body that preferentially uptake or exclude the material. This technique has proven especially useful in detecting indwelling tumors, as their dysfunctional vasculatures facilitate the accumulation of contrast agent in the diseased tissue.

(p. 492) Gadolinium and iron are T1 and T2 contrast agents, respectively, that have both been successfully incorporated into single- and multiwalled carbon nanotubes (Sitharaman et al. 2005; Bai et al. 2008) (Fig. 13.3). Incorporation was achieved either during the growth phase or in postgrowth processing, with the result being the creation of MR contrast agents that are several times more efficient than those currently in clinical use (Sitharaman et al. 2005; Son et al. 2006; Bai et al. 2008). If one considers this functionality in conjunction with the tumor-targeting strategies detailed above, it is not difficult to imagine the use of these materials to both enhance the detection, treatment and follow-up imaging of a given tumor.

One additional synergy can be envisioned in the combined use of magnetic resonance imaging with nanotube-mediated thermal-ablative therapy. Recent advancements in the design of new MR pulse sequences have made noninvasive thermometry a reality (Samset 2006; de Senneville et al. 2007). These techniques are readily applicable to 1.5 and 3 Tesla clinical MR equipment. Due to minimal postacquisition processing, use of MR thermometry during therapy with nanomaterials has the potential to allow near-real-time temperature mapping, minimizing the risk of undertreating regions of tumor, such as those abutting well-perfused vessels. MR-based thermometry will help by providing the clinician a means by which the anticipated effectiveness of the intervention can be readily assessed, giving them the option to adjust the therapy midtreatment as the need arises. With this capability it becomes reasonable to envision the use of nanotubes as a minimally invasive, multimodal therapeutic approach to the treatment of non-resectable solid cancers.

13.4 Gold nanoshells and nanorods

13.4.1 Physical properties of gold nanoshells and nanorods

Nanoscale formulations of gold and other noble metals exhibit unique properties that make them useful for various biomedical applications (Fig. 13.4). Two in particular, gold nanoshells and gold nanorods, have emerged in recent years as viable contenders in the application of nanomaterial-mediated photothermal therapy.

Gold nanoshells consist of dielectric silica cores onto which gold colloids of variable thickness are adsorbed (Hirsch et al. 2006). These structures range in size from 50–500 nm with alterations in growth conditions determining the size range of the final product. Nanoshells exhibit a particularly robust surface plasmon resonance (the oscillation of conducting electrons at the gold surface interface) in response to incident electromagnetic radiation, resulting in the generation and transduction of heat into their immediate surroundings. By varying the principal dimensions of the silica core and outer gold shell, this effect can be tuned to respond maximally at a discrete portion of the EM spectrum. This property is of particular use when considering the application of nanoshells as mediators of photothermal therapy in vivo. Untuned gold nanoparticles exhibit strong absorbance in the visible segment of the EM spectrum, limiting their utility in vivo due to the natural abundance of biological (p. 493)

Bifunctional nanomaterials for the imaging and treatment of cancerClick to view larger

Fig. 13.4 Gold nanorods imaged by TEM. Used with permission of Dr. Mostafa, A. El-Sayed group, Laser Dynamics Laboratory, Georgia Institute of Technology.

chromophores that absorb in this range (Huang et al. 2007). By increasing the diameter of the silica core and varying the thickness of the gold shell, the absorption spectrum of these particles can be redshifted into the NIR, where tissue absorption is minimal. Additionally, nanoshells can be further tuned to scatter rather than absorb NIR, making them applicable as contrast agents for optical tomography imaging techniques.

Perhaps not surprisingly, gold nanorods retain many of the same physical properties as nanoshells due to the commonality of their external composition. Like nanoshells, nanorods exhibit strong surface plasmon resonance in response to incident light and the absorption spectrum can be controlled by altering the aspect ratio (length/width) of the material, with a larger ratio redshifting peak absorption into the near-infra-red range (Huang et al. 2007).

Reduced gold is a stable and generally inert material highly suitable for medical applications. It has long been used in dentistry for the manufacture of biocompatible prosthetics that have little demonstrated local toxicities. There is some concern over systemic dosing of gold nanoparticles, as colloidal gold is a well established nephrotoxin and inducer of peripheral neuropathies (Klaassen 2008). These are concerns that will have to be addressed in future toxicology studies as both materials progress to in-vivo applications.

Finally, conjugation chemistries involving gold have been described extensively, making the process of attaching targeting moieties fairly straightforward, assuming the proper functional groups are accessible. Thiol-containing proteins have a demonstrated affinity for gold particles, to which they form very tight, non-covalent bonds (Nuzzo et al. 1987). This property is useful for the purposeful conjugation of desired polypeptides for targeting or to enhance biocompatibility. However, it may also enhance the rate of opsonization and subsequent RES clearance when these particles are used in living systems. PEGylation or other means of “stealthing” may constitute one way to address this concern.

(p. 494) 13.4.1.1 Anticancer activity of gold nanoparticles in vitro

The use of gold nanoparticles for the photothermal treatment of cancer was initially described by Hirsch et al. in 2003. In this study 130-nm PEG-passivated gold nanoshells were incubated with SK-BR-3 breast cancer cells and exposed to 820 nm at 35 W/cm2 for seven minutes. Calcein fluorescence and dextran exclusion assays demonstrated complete cell kill within a region corresponding to the area impacted by the laser beam (Hirsch et al. 2003). Huang et al. generated similar findings through the use of gold nanorods. Normal human keratinocytes and the oral squamous cell carcinoma cell line HSC 3 were incubated gold nanorods conjugated to an anti-EGFR antibody (an antibody directed against the epidermal growth factor receptor). Prior to 800-nm laser irradiation (up to 20 W/cm2 for four minutes), cells were washed with buffered saline to remove unbound nanorods. Following laser exposure, cell viability was assessed by trypan blue exclusion. HSC 3 cells, which overexpress EGFR, were readily killed at lower laser powers, while normal keratinocytes were relatively unharmed, due to the low expression of that receptor (Huang et al. 2006). This group went on to demonstrate that their cell lines required temperatures in the range of 70–80 °C to effect complete cell death, and that these temperatures could be reached in nanorod-loaded cells following four minutes of near-infra-red laser irradiation (Huang et al. 2006).

A novel targeting strategy for gold nanoshells is through the use of monocytes/macrophages as a “Trojan horse” (Choi et al. 2007). Using a tumor spheroid model system of breast cancer, Choi and colleagues demonstrated that macrophages “pre-loaded” (through incubation) with gold nanoshells will migrate into a simulated tumor mass, and that the spheroid can then be ablated by irradiating it with a near-infrared laser (Choi et al. 2007). Additional work will be needed to confirm the utility of this strategy in vivo; however, it represents a potential method through which the limitations of previously discussed targeting schemes may be overcome.

13.4.1.2 Anticancer activity of gold nanoparticles in vivo

Initial work described by Hirsch et al. demonstrated that in a mouse model, solutions of gold nanoshells injected directly into a subcutaneous tumor followed by near-infra-red laser illumination for 4–6 min resulted in a temperature increase within the tumor of over 35 °C. Control tumors that did not receive an injection of nanoshells saw average temperature increases of only 7 °C (Hirsch et al. 2003). Histological analyses revealed that the zone of thermal injury in the nanoshell-treated tumors extended about 4 mm below the dermal surface. The authors noted that this could likely be increased through better distribution of the nanoshells throughout the tumor volume and longer treatment times (Hirsch et al. 2003).

Following this initial report, additional studies explored the impact of nanoshell-mediated thermal therapy on tumor regression and overall survival. O’Neal et al. demonstrated that PEGylated nanoshells could be injected intravenously in tumor-bearing mice and that within 6 h they would passively accumulate in the tumors in therapeutically relevant concentrations (a function of (p. 495) the enhanced permeability and retention effect discussed in a previous section). Subsequent exposure of these same tumors (diameters at time of treatment 3–5 mm) to an 810-nm laser at 4 W/cm2 for 4 min induced complete remission of the animals for the entire length of follow-up (3 months) (O’Neal et al. 2004). Similar results were observed by a separate group using a heterotopic murine model of prostate cancer, and by yet another group using a colon cancer xenograft system (Gobin et al. 2007; Stern et al. 2008).

13.4.1.3 Use of gold nanoshells and nanorods in cancer imaging

Dissimilar to the carbon-nanotube-based nanotherapeutics discussed in previous sections, gold nanomaterials are invisible to most clinically relevant imaging modalities (MRI and CT). However, their usefulness in optics-based imaging techniques has been shown and several papers have documented applications of these particles for in-vitro analyses.

The early detection of cancer is often cited as one of the best predictors of successful treatment. To accomplish this, screening assays need to be developed that have the sensitivity to distinguish a small population of transformed cells from a background of normal tissue constituents. Current techniques that involve the use of oncoprotein-directed antibodies conjugated to conventional chromophores lack the signal intensity necessary to generate the desired sensitivity. Two recent publications suggest that antibody conjugated gold nanorods may offer a promising solution. Huang et al. discovered that anti-EGFR conjugated nanorods could be used to successfully identify malignant oral squamous carcinoma cells from their normal counterparts. It was shown that following the incubation of the targeted nanorods with the two cell types and subsequent analysis by Raman spectroscopy, the cancerous cells exhibited a distinct polarized Raman spectra that was absent in the normal samples (El-Sayed et al. 2005; Huang et al. 2007; Oyelere et al. 2007). A similar study involving anti-HER2 (a clinically relevant breast cancer marker) targeted nanoshells demonstrated their ability to selectively identify HER-2+ breast cancer cells from normal cells (Loo et al. 2005). These initial studies suggest such strategies may become useful in the screening of patient tissue biopsies and other clinical samples.

Optical coherence tomography (OCT) is a relatively new clinical imaging technique that analyzes the reflections of low coherence light off of underlying tissue components to provide cross-sectional images of the subsurface down to micrometer resolution (Huang et al. 1991; Fujimoto et al. 1995). The utility of this system can be further enhanced through the use of contrast agents that serve to highlight specific biological structures. Gobin and colleagues demonstrated that gold nanoshells have the capacity to serve as OCT contrast agents due to their ability to reflect and scatter light in a tunable manner (Gobin et al. 2007). While OCT has been used to image superficial cancers such as gastrointestinal and oral mucosal, its use in the detection of deeper-seated tumors is constrained by a maximum imaging depth of 2mm (Li et al. 2000). Beyond this threshold, the reflected light is too greatly attenuated by the intervening tissue to be of use.

(p. 496) 13.5 Iron-oxide nanoparticles

Iron-oxide nanoparticles have received considerable attention as a vehicle for the generation of tumor-specific heat. Because these particles have been extensively reviewed recently (Kumar 2006), they are not discussed in detail here. Briefly, this technique involves the application of alternating magnetic fields to nanoscale crystals of iron oxide (Fe3O4 or γ-Fe2O3) to generate heat through the process of hysteresis and relaxation losses (Jordan et al. 1993). In this approach, ultrafine (5–20 nm) iron-oxide crystals are coated with a functionalizing material (either dextran or aminosilane) to prevent aggregation and suspended in saline to create a magnetic fluid (MF). This fluid is directly injected into a target tissue and is later heated when placed in a magnetic-field applicator (Gneveckow et al. 2004). Additionally, the MF functions as a contrast agent in both CT and MR imaging protocols. The primary advantage of this technique over other nanomaterial-based thermoablative methods described earlier in this chapter is its ability to theoretically treat any region of the body. Unlike NIR-based therapies, this method does not suffer from depth of penetration issues as the body is functionally transparent to magnetic fields. This approach has received considerable support in Europe, where it has rapidly transitioned into the clinic. Phase I trials have already been completed in glioblastoma multiforme, prostate, esophageal, pancreatic and various sites of recurrent cancers and Phase II trials are underway for both glioblastoma and prostate cancers (MagForce 2008). However, no trials are open in the United States.

13.6 Conclusions and future directions

The nanoparticles described in this review have each demonstrated potential as multifunctional agents for the treatment of cancer. The promised package of tumor specificity, therapeutic efficacy and imaging enhancement makes a compelling argument for their continued development. Indeed, in recognizing the significant impact nanomaterials will have in the diagnosis and treatment of cancer, the National Cancer Institute has pledged support of research into nanomedicine in cancer through its Alliance for Nanotechnology in Cancer. However, it is clear that several areas of concern must be addressed before these technologies can make the desired “bench to bedside” transition.

Fundamental to the success of these therapies will be their ability to demonstrate an acceptable toxicity profile. It is clear that both oncologists and their patients favor efficacy over safety, and it is encouraging that the limited data that is currently available from studies in mice has not pointed to dramatic toxicity of any of the nanomaterials highlighted in this review (SWNT, MWNT, nanoshells, nanorods and encapsulated iron oxides). However, data is still sparse, and precludes the drawing of firm conclusions on toxicity. An additional limitation is the current inability to reproducibly manufacture identical batches of nanoparticles (more a concern for carbon-nanotube-based therapies) for assessment. Advancements in manufacturing and purification techniques will likely redress this in the future; however, it remains a concern in the near term.

(p. 497) Future work will also likely include expanded research on the use of these particles, particularly carbon nanotubes, as targetable transporters of chemotherapeutics and for use as chemo/radiosensitizers through their ability to generate localized heat. Published studies have already demonstrated the ability of carbon nanotubes to be filled with drugs such as carboplatin, and indicated that these formulations effectively inhibit the growth of cancers (Hampel et al. 2008). Confirmation of the effectiveness of this strategy in vivo will be necessary; however, this is likely to be a very promising application.

Additionally, work will be required to integrate the imaging and treatment elements inherent in these new strategies. MRI appears to be the most promising imaging modality, as it provides high-resolution anatomical images, offers a thermometric capability that synergizes well with proposed thermal treatments, and does not suffer from a limited depth of penetration that constrains techniques like optical coherence tomography. Of course, the strong magnetic field required for MR imaging will necessitate the use of only non-ferromagnetic materials in laser delivery.

Although the use of nanomaterials in cancer therapy is still in the early stages, it is encouraging that Nanospectra Biosciences has recently received FDA approval to begin a Phase I clinical trial in recurrent or refractory head and neck cancer patients with a nanoshell-based photothermal therapy (Nanospectra 2008). The lessons learned from this and future trials will undoubtedly foster the creation of a new generation of purpose-built nano-materials with enhanced capabilities for the imaging and effective treatment of cancer.

Acknowledgments

This work was supported in part by National Institutes of Health grants R01 CA12842 (SVT) and T32 CA079448 (AB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

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