The first problem to tackle for biomedical applications of SWNTs is to solubilize and disperse carbon nanotubes in aqueous solutions and functionalize them with biomolecules such as proteins/antibodies, nucleic acids and carbohydrates. Past studies using SWNT-antibody conjugates for specific photothermal ablation of cancer cells attached antibodies to SWNTs either noncovalently through adsorption  or indirectly via streptavidin-biotin interaction . Direct adsorption of antibodies to SWNTs is simple to execute but the weak interaction between the antibody and the nanotubes raises the possibility of loss of the targeting function of the antibodies. Indirect conjugation via streptavidin-biotin interaction involves an additional step of preparing the antibody-biotin complex. A method for direct covalent attachment of antibodies to SWNTs for specific tumor targeting has been reported  that involves four reactive steps. Here, we used a simpler method for direct covalent conjugation of antibody to SWNTs. HiPco SWNTs were first dispersed in water through microwave-assisted carboxylation, activated by EDC and NHS, and reacted with HER2 IgY antibody to form the covalent complex. Microwave-assisted functionalization has several advantages over conventional chemical techniques, such as rapidness and environmental friendliness . However, the functionalization process causes some changes in the optoelectronic properties of the SWNTs, such as increase in the disorder mode (D-band) at ~1300 cm-1 (Fig. 3A) and loss of interband transitions between van Hove singularities in the absorption spectrum (Fig. 3B). Similar changes have been reported previously for covalently functionalized carbon nanotubes [27, 35, 36]. Nevertheless, the resultant IgY-SWNT complexes retain a significant portion of the optic properties of SWNTs, as evidenced by the fairly strong Raman and NIR absorbance.
The characteristic G band at ~1590 cm-1 was detected in HER2-expressing SK-BR-3 cells treated with the IgY-SWNT complexes (Fig. 4) but not in the similarly treated receptor-negative MCF-7 cells, indicating the ability of Raman spectroscopy to specifically detect cancer cells in vitro. As a nondestructive optical spectroscopic technique that does not require extrinsic contrast-enhancing agents, the use of Raman spectroscopy has seen a remarkable increase during the last decade in its application to the field of medicine . In particular, Raman spectroscopy has shown great promise as a new tool for detection of malignant and premalignant tissues and as a real-time guidance tool during oncosurgical procedures . However, most of these studies are based on spectral differences between normal and neoplastic tissues that result from compositional changes in the affected tissues, and thus, in most cases, the detection is not highly specific and only possible at later stages of tumor progression. In the current study, characteristic Raman signals (at ~1590 cm-1) are collected at the single-cell level from cancer cells targeted by the IgY-SWNT complexes, thus opening the possibility of using Raman spectroscopy for targeted molecular detection of tumors at the incipient stage. An added advantage of Raman spectroscopy lies in its potential for in vivo applications for which limited penetration depth is a fundamental barrier. Until recently, Raman spectroscopy has been generally restricted to probing surface or near-surface areas of biological tissues with penetration depth of only several hundred microns into tissue. This limitation mainly stems from the diffuse scattering nature of tissue which leads to random propagation of photons within its matrix and prevents the formation of sharp images required to discriminate signals emerging from deeper areas. Several methods have been developed recently for the retrieval of Raman signals from deep areas thus enhancing tissue penetration of Raman spectroscopy. These deep Raman techniques discriminate between Raman signals emerging from different depths within the sample using temporal or spatial gating . For instance, combining spatially offset Raman spectroscopy (SORS) with three-dimensional tomographic imaging, it was possible to image a canine hind limb section of a thickness of up to 45 mm using transmission Raman spectroscopy [40, 41]. Therefore, combined with advances in Raman spectroscopic technologies for deep tissue imaging , SWNTs functionalized with antibody specific for tumor cell receptors may be exploited for in vivo specific detection of cancer cells at early stages.
The present study demonstrates very high specificity of the HER2 IgY-SWNT complexes for HER2-expressing cancer cells, indicating the potential usefulness of the IgY antibody for selective targeting of cancer cells. IgY antibodies offer many advantages over their mammalian IgG counterparts in terms of both production and biochemical and immunological properties. IgY antibodies can be isolated in large quantities from egg yolk using simple separation methods; the non-invasive production method also brings the great benefit concerning the welfare of the immunized animals . IgY antibodies can also be used to avoid interference in immunological assays caused by the human complement system, rheumatoid factors, human anti-mouse IgG antibodies (HAMA) or human and bacterial Fc-receptors . Similarly, for clinical use as antibody-based therapeutics, they neither activate mammalian complement nor interact with mammalian Fc receptors that could mediate inflammatory responses . Despite these advantages, the application of IgY antibodies in research and medicine has been very limited . Oral administration of IgY antibodies have shown great promise as immunotherapy for the prevention and treatment of enteric, respiratory, and dental infections in humans and animals [44–47]. As eggs are normal dietary components, there is practically no risk of toxic side effects of oral administration of IgY antibodies [44, 46]. However, the phylogenetic distance between birds and mammals implies potential concerns over the immunogenicity of IgY antibodies in human. So far, there has been no report on intravenous administration of IgY antibodies in human and the associated immune responses. Nevertheless, concerns over IgY immunogenicity in human should be completely cleared out before any clinical application of IgY should be attempted. The results presented here and in a previous study  may bring more attention to this class of antibodies and promote studies on the immunogenicity of IgY preparations in human.
Temperature measurement of the IgY-SWNT complex solution at the nanotube concentration of 4 mg/L showed an increase of ~14°C in the bulk solution, indicating the temperature rise of the surrounding environment would not cause harm to normal cells that do not bind to the SWNT-containing complex in the short time period (2 min). On the other hand, the same result also hinted that the thermal destructive effect to cancer cells must be microscopic rather than macroscopic. We hypothesize that temperature rise in the nanoscale vicinity of individual nanotubes can be dramatic. The sharp local temperature increase may cause damage to subcellular structures such as cell membranes ultimately leading to cell death. The ability to directly measure temperature of an individual nanoparticle will help to validate the hypothesis, and such an endeavor is currently underway .
The method described here for selective cancer cell destruction differs from the previously published ones [7, 20–22] in that our method does not require internalization of SWNTs into tumor cells. HER2 is a transmembrane glycoprotein with the receptor motif extended outside the cell membrane . The reason for the lack of internalization of the SWNT complex by the cancer cells after binding to the cell surface receptor is not known; however, it is likely due to the surface chemistry of the SWNTs used here [27, 32]. It has been reported the surface chemistry has a profound impact on the cellular uptake of nanoparticles such as quantum dots . Although the exact mechanism may differ for various nanoparticles, the surface dependent cellular uptake may be a common phenomenon for all nanoparticles . It is very important to note that the functionalization method used in the current study is different from those published previously where internalization of SWNTs after binding to the cell surface receptors have been reported [7, 20, 22]. In the study by Chakravarty et al. , cellular localization of SWNTs after incubation with cancer cells was not reported.
The method described here for selective photothermal ablation of cancer cells without the need of internalization by the cells has the advantage of being more easily extended to other types of cancer cells over agents that need internalization, as cellular internalization is not always achievable with all cancer types. Many cancer cells overexpress specific tumor markers (receptors) on their surface for which IgY antibody with high specificity and sensitivity can be developed. Thus, the IgY-SWNT complex, as exemplified in this study by the anti-HER2 IgY antibody, has the potential to become a novel, generic modality for detection and therapy of various cancer types. Our next step is to evaluate the pharmacokinetics, biodistribution, cytotoxicity and activity of such IgY-SWNT complexes in vivo using animal models.