**5.1 Quantum dots**

 

Quantum Dots (QDs) are a unique class of light emitting semiconductor nanoparticles ranging from 2-10 nanometers in diameter and are becoming highly popular for biological imaging due to their high intensity and stable fluorescence profile (most QDs are approximately 10–20× brighter than organic dyes). QDs usually consist of a CdSe core surrounded by a inorganic shell composed of ZnS (Pinaud et al. 2010). For biological imaging applications, they are given hydrophilic coatings of PEG or multiple carboxylate groups.

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 535

internalization by tumor cells, and that siRNA cargo could be co-attached without affecting the function of the peptide (Derfus et al. 2007). Using an EGFP model system, the role of conjugation chemistry was also investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer. These nanoprobes could be used for both active and passive targeting. Therefore, in future research, QDs can be seen as multi-functional platforms focusing on targeted delivery, high transfection efficiency, and multi-modal

Fig. 5. (A): Quantum dots as a multi-functional nanoplatform to deliver siRNA and to elucidate of EGFR knockdown effect of PI3K signaling pathway in brain tumor cells. (B): Detailed structural information of multifunctional siRNA-QDs: CdSe as core with ZnS capping along with thiol reactive linker for siRNA conjugation as well as RGD peptides as shown in red and TAT peptides as shown in green. In order to make QD constructs watersoluble and suitable for conjugating with siRNA, hydrophobic ligands are displaced with a dihydrolipoic acid (DHLA) derivatized with an amine terminated polyethylene glycol (PEG) spacer. (C): Two different strategies for the siRNA-QD conjugate. (C1) Linker for attaching siRNA to QDs through a disulfide linkage which are easily reduced within the cells to release the siRNA. (C2) Linker for covalently conjugating siRNA to

QDs which enable tracking of siRNA-QDs within the cells. (Taken from:

imaging/tracking and treatment of cancer as shown in Figure 5.

Jung et al. 2010).

Compared to the commercially available organic dyes and fluorescent proteins used in medical imaging, QDs provide many advantages (Park et al. 2009;Pic et al. 2010;Rogach and Ogris 2010;Yong et al. 2009). The first and foremost important feature is the long-term photostability of QD imaging probes, which opens the possibility of investigating the dynamics of cellular processes over time, such as continuously tracking cell migration, differentiation, and metastasis. In addition, QD emission wavelengths are size-tunable and extend from visible to near infrared (NIR) (650 nm to 950 nm), to take advantage of the improved tissue penetration depth and reduced background fluorescence at these wavelengths. For example, CdSe/ZnS QDs of approximately 2 nm in diameter produce a blue emission, while QDs approximately 7 nm in diameter emit red light. While fluorescence imaging is often limited by the poor transmission of visible light through biological tissue, there is a NIR optical window in most biological tissue that is suitable for deep tissue optical imaging, where only a few organic dyes emit brightly in this region and generally suffer from photobleaching. In contrast, the novel optical properties of QDs allow the synthesis of bright and stable fluorescent labels that emit in the near infra red spectrum by adjusting their size and composition.

Surface functionalization using peptides, proteins and antibodies, confers the ability of QDs to provide high biological compatibility and capacity to target and image tumors in living subjects through the rapid readout of fluorescence imaging. Moreover, QDs allow imaging of deeper tissues and is also used to image lymph nodes and blood vessels in tissues. A key property for *in vivo* imaging is the unusual QD Stokes shift (measured by the distance between excitation and emission peaks), which can be as large as 300-400nm depending on the wavelength of the excitation light, which can be used to further improve the detection sensitivity. Organic dye signals with a small stoke shift are often buried by strong tissue autofluorescene, whereas QD signal with large Stokes shift are clearly detectable above the background. Unlike traditional dyes which usually show a broad emission band, QDs exhibit narrow sharp emission peaks and broadband absorption, which are ideal for multiplexed multicolor imaging. QDs are thus able to increase the number of labels that can be used simultaneously in a single system. The effective brightness per probe particle is also superior with quantum dots as evidenced by their large molar absorption crosssections which are a consequence of their nanometer size and composition. Different from "soft" organic nanoparticles (e.g., polymers, micelles, liposomes), inorganic nanomaterials with rigid cores usually show inefficient extravasation inside tumors. A number of reports suggest that QDs tend to stay within the tumor vasculatures without getting into the interstitial space or tumor cells, reducing the nonspecific tumor cell labeling in angiogenesis imaging (Liu and Peng 2010). The long term photostability and superior brightness of QDs make them appealing for live animal targeting and imaging. These properties have made QDs a topic of intensive interest in cancer biology, molecular imaging, and molecular profiling.

The ability to functionalize as well as control the surface of quantum dots with specific linkers and multi-functional molecules is critical for nanoparticle-based gene therapy. Currently, QDs are used both as a transfection vector as well as a fluorescence label in RNA interference research. Quantum dot conjugates have been successfully used for targeted silencing of bcr/abl gene by RNA interference in human myelogenous leukemia K562 cells (Zhao et al. 2010). In addition, Derfus et al., (2007) using PEGlyated quantum dot core as a scaffold, and conjugating siRNA and tumor-homing peptides (F3) to functional groups on the particle's surface found that the homing peptide was required for targeting 534 Non-Viral Gene Therapy

Compared to the commercially available organic dyes and fluorescent proteins used in medical imaging, QDs provide many advantages (Park et al. 2009;Pic et al. 2010;Rogach and Ogris 2010;Yong et al. 2009). The first and foremost important feature is the long-term photostability of QD imaging probes, which opens the possibility of investigating the dynamics of cellular processes over time, such as continuously tracking cell migration, differentiation, and metastasis. In addition, QD emission wavelengths are size-tunable and extend from visible to near infrared (NIR) (650 nm to 950 nm), to take advantage of the improved tissue penetration depth and reduced background fluorescence at these wavelengths. For example, CdSe/ZnS QDs of approximately 2 nm in diameter produce a blue emission, while QDs approximately 7 nm in diameter emit red light. While fluorescence imaging is often limited by the poor transmission of visible light through biological tissue, there is a NIR optical window in most biological tissue that is suitable for deep tissue optical imaging, where only a few organic dyes emit brightly in this region and generally suffer from photobleaching. In contrast, the novel optical properties of QDs allow the synthesis of bright and stable fluorescent labels that emit in the near infra red spectrum

Surface functionalization using peptides, proteins and antibodies, confers the ability of QDs to provide high biological compatibility and capacity to target and image tumors in living subjects through the rapid readout of fluorescence imaging. Moreover, QDs allow imaging of deeper tissues and is also used to image lymph nodes and blood vessels in tissues. A key property for *in vivo* imaging is the unusual QD Stokes shift (measured by the distance between excitation and emission peaks), which can be as large as 300-400nm depending on the wavelength of the excitation light, which can be used to further improve the detection sensitivity. Organic dye signals with a small stoke shift are often buried by strong tissue autofluorescene, whereas QD signal with large Stokes shift are clearly detectable above the background. Unlike traditional dyes which usually show a broad emission band, QDs exhibit narrow sharp emission peaks and broadband absorption, which are ideal for multiplexed multicolor imaging. QDs are thus able to increase the number of labels that can be used simultaneously in a single system. The effective brightness per probe particle is also superior with quantum dots as evidenced by their large molar absorption crosssections which are a consequence of their nanometer size and composition. Different from "soft" organic nanoparticles (e.g., polymers, micelles, liposomes), inorganic nanomaterials with rigid cores usually show inefficient extravasation inside tumors. A number of reports suggest that QDs tend to stay within the tumor vasculatures without getting into the interstitial space or tumor cells, reducing the nonspecific tumor cell labeling in angiogenesis imaging (Liu and Peng 2010). The long term photostability and superior brightness of QDs make them appealing for live animal targeting and imaging. These properties have made QDs a topic of intensive interest in cancer biology, molecular

The ability to functionalize as well as control the surface of quantum dots with specific linkers and multi-functional molecules is critical for nanoparticle-based gene therapy. Currently, QDs are used both as a transfection vector as well as a fluorescence label in RNA interference research. Quantum dot conjugates have been successfully used for targeted silencing of bcr/abl gene by RNA interference in human myelogenous leukemia K562 cells (Zhao et al. 2010). In addition, Derfus et al., (2007) using PEGlyated quantum dot core as a scaffold, and conjugating siRNA and tumor-homing peptides (F3) to functional groups on the particle's surface found that the homing peptide was required for targeting

by adjusting their size and composition.

imaging, and molecular profiling.

internalization by tumor cells, and that siRNA cargo could be co-attached without affecting the function of the peptide (Derfus et al. 2007). Using an EGFP model system, the role of conjugation chemistry was also investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer. These nanoprobes could be used for both active and passive targeting. Therefore, in future research, QDs can be seen as multi-functional platforms focusing on targeted delivery, high transfection efficiency, and multi-modal imaging/tracking and treatment of cancer as shown in Figure 5.

Fig. 5. (A): Quantum dots as a multi-functional nanoplatform to deliver siRNA and to elucidate of EGFR knockdown effect of PI3K signaling pathway in brain tumor cells. (B): Detailed structural information of multifunctional siRNA-QDs: CdSe as core with ZnS capping along with thiol reactive linker for siRNA conjugation as well as RGD peptides as shown in red and TAT peptides as shown in green. In order to make QD constructs watersoluble and suitable for conjugating with siRNA, hydrophobic ligands are displaced with a dihydrolipoic acid (DHLA) derivatized with an amine terminated polyethylene glycol (PEG) spacer. (C): Two different strategies for the siRNA-QD conjugate. (C1) Linker for attaching siRNA to QDs through a disulfide linkage which are easily reduced within the cells to release the siRNA. (C2) Linker for covalently conjugating siRNA to QDs which enable tracking of siRNA-QDs within the cells. (Taken from: Jung et al. 2010).

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 537

ultralight weight, high mechanical strength, high electrical conductivity, and high thermal conductivity (Ji et al. 2010). Carbon nanotubes are the strongest and stiffest materials yet discovered in terms of tensile strength and elastic modulus respectively. As CNTs are intrinsically not water soluble, modification through chemical functionalization can

Imaging functionalities and therapeutics can be incorporated on the same nanoparticle for multifunctional cancer imaging and treatment. SWNT-paclitaxel (PTX) conjugates also showed higher efficacy in suppressing tumor growth than clinical Taxol alone in a murine 4T1 breast cancer model, owing to prolonged blood circulation time and enhanced permeability and retention (EPR) in the tumor (Feazell et al. 2007). Besides, with very high surface area per unit weight, SWNTs provide higher capacity of drug loading, compared to that reported for conventional liposomes and dendrimer drug carriers. Doxorubicin, a commonly used cancer chemotherapy drug, can be loaded on the surface of PEGylated SWNTs with remarkably high loading, up to 4 g of drug per 1 g of nanotube, owing to the ultrahigh surface area of SWNTs. Further, the intrinsic stability and structural flexibility of CNTs may prolong the circulation time as well as improve the bioavailability of drug molecules conjugated to them. Surface-enhanced Raman spectroscopy of carbon nanotubes opens up a method of protein microarray with detection sensitivity down to 1 fmol/L. *In vitro* and *in vivo* toxicity studies reveal that highly water soluble and serum stable nanotubes are biocompatible, nontoxic, and potentially useful for biomedical applications. However, nonfunctionalized nanotubes are toxic to cells and animals and therefore one has to be cautious about the safety aspects of CNTs. If well functionalized, nanotubes may be excreted

Carbon nanotube-based drug delivery has shown promise in various *in vitro* and *in vivo* experiments including delivery of small interfering RNA (siRNA), paclitaxel and doxorubicin (Liu et al. 2009). Multiwalled PEGylated carbon nanotubes are found to be successful, effective and do not alter particle sizes and zeta potentials of carbon nanotubes after PEGylation (Ilbasmis-Tamer et al. 2010). In addition, the propensity to absorb the body transparent NIR radiation also envisages photothermal and photoacoustic therapy using

Thermal ablation therapy is one the most promising of methods in cancer treatment but is limited by incomplete tumor destruction and damage to adjacent normal tissues. Current radiofrequency ablation techniques require invasive needle placement and are limited by accuracy of targeting. Use of nanoparticles has refined noninvasive thermal ablation of tumors, and several nanomaterials have been used for this purpose. These include gold nanomaterials, iron nanoparticles, magnetic nanoparticles, carbon nanotubes and affisomes (thermosensitive liposomes). Heating of the particles can be induced by magnets, lasers, ultrasound, photodynamic therapy and low-power X-rays. The clinical trials include studies of designed nanoparticles such as the thermosensitive liposomal doxorubicin (Thermodox®) as a novel activated therapy using radiofrequency ablation (Wang and Thanou 2010). As gold nanoparticles have evolved other gold structures have also been suggested. Nanorods, with the appropriate PEG stealth layer, are being developed as an improved means of hyperthermia. By attaching monoclonal antibodies (mAbs), which can recognize a specific cancer cell, to gold nanoparticles or nanorods are also used in cancer detection. Gold nanoparticles conjugated to anti-epidermal growth factor receptor (El-Sayed et al. 2006)

increases the solubility of carbon nanotubes in aqueous solutions.

mainly through the biliary pathway in feces.

**5.3 Gold and other nanoparticles for cancer diagnosis**

nanotubes.

However, there have been some QD concentration dependent toxicity and distribution concerns. QDs have been shown to remain in liver, lymph nodes and bone marrow of mice for 1 month after tail injection, despite of its low affinity to cells and tissues (Ballou et al. 2004;Ballou et al. 2009). Recently, the hydrophilic QDs (diameter <10 nm) have attracted more attention for *in vivo* applications, due to the rapid renal clearance of QDs, minimizing the potential toxicity to the system (Park et al. 2009). Recently, Peng and co-workers developed InAs/InP/ZnSe core/shell/shell QDs with high quantum yield (76%) of NIR fluorescence, ultrasmall hydrodynamic sizes (< 10 nm), and the biocompatibility desired for *in vivo* applications (Liu and Peng 2010). These novel QDs have obviously lower intrinsic toxicity compared to commercial Cd-containing NIR emitting QDs and showed significantly improved circulation half-life with reduced RES uptake. NIR-emitting QDs demonstrate exceptional brightness and fluorescent quantum yields. Also, once capped with a chemically stable shell, QDs can exhibit remarkable photostability, providing continuous fluorescent singles for long-term imaging applications. Other types of novel QDs with entirely different compositions and photoluminescence mechanism such as silicon QDs and carbon dots have also emerged as potential probes in bioimaging applications. Further, typical fluorescence images of a single QD shows changes in emission intensity. The intensity time trace illustrates the random alternation between "on" and "off" states, which is known as blinking and it is a signature feature of an individual QD. Also, reducing size of QDs has proven difficult because of decreased colloidal stability and increased nonspecific interactions. Other major limitations include reproducibility in production, proper control of surface functionality, bulky surface coatings (PEG, multiple antibodies, ampiphilic molecules) which leads to restrictions on studying spatially confined, crowded regions of the cell and may also perturb the behavior of the labeled molecules. Quantum dots are not yet approved for use in humans and much more research is needed in future for this growing field.

#### **5.2 Carbon nanotubes**

Molecular imaging exploits the specific recognition of labeled probes to their biological targets in conventional imaging techniques to monitor biological processes at the molecular level with improved specificity and sensitivity. Conventional clinical cancer imaging techniques, such as X-ray, CT and MRI, do not possess sufficient spatial resolution for early detection of the disease. Positron emission tomography (PET) is a highly sensitive and accurate imaging technology that relies on changes in tissue biochemistry and metabolism. It is the most valuable means we have so far to identify early-stage alterations in molecular biology, often before there is any morphologic change. Nevertheless, fluoro-desoxy-glucose (FDG), the most commonly used PET tracer in clinical oncology (more than 95% of the molecular imaging procedures make use of FDG at present), is not a specific tracer for malignant diseases but for increased metabolism. Therefore, it is imperative to develop new tools for early cancer diagnosis.

CNTs have been explored in almost every single cancer treatment modality, including drug delivery, lymphatic targeted chemotherapy, thermal therapy, photodynamic therapy, and gene therapy. Based on their structure, CNTs can be classified into two general categories: single-walled (SWNTs), which consist of one layer of cylinder graphene (diameter 0.4-2 nm) and multi-walled (MWNTs), which contain several concentric graphene sheets (diameter 2- 100 nm). CNTs have unique physical and chemical properties such as high aspect ratio,

However, there have been some QD concentration dependent toxicity and distribution concerns. QDs have been shown to remain in liver, lymph nodes and bone marrow of mice for 1 month after tail injection, despite of its low affinity to cells and tissues (Ballou et al. 2004;Ballou et al. 2009). Recently, the hydrophilic QDs (diameter <10 nm) have attracted more attention for *in vivo* applications, due to the rapid renal clearance of QDs, minimizing the potential toxicity to the system (Park et al. 2009). Recently, Peng and co-workers developed InAs/InP/ZnSe core/shell/shell QDs with high quantum yield (76%) of NIR fluorescence, ultrasmall hydrodynamic sizes (< 10 nm), and the biocompatibility desired for *in vivo* applications (Liu and Peng 2010). These novel QDs have obviously lower intrinsic toxicity compared to commercial Cd-containing NIR emitting QDs and showed significantly improved circulation half-life with reduced RES uptake. NIR-emitting QDs demonstrate exceptional brightness and fluorescent quantum yields. Also, once capped with a chemically stable shell, QDs can exhibit remarkable photostability, providing continuous fluorescent singles for long-term imaging applications. Other types of novel QDs with entirely different compositions and photoluminescence mechanism such as silicon QDs and carbon dots have also emerged as potential probes in bioimaging applications. Further, typical fluorescence images of a single QD shows changes in emission intensity. The intensity time trace illustrates the random alternation between "on" and "off" states, which is known as blinking and it is a signature feature of an individual QD. Also, reducing size of QDs has proven difficult because of decreased colloidal stability and increased nonspecific interactions. Other major limitations include reproducibility in production, proper control of surface functionality, bulky surface coatings (PEG, multiple antibodies, ampiphilic molecules) which leads to restrictions on studying spatially confined, crowded regions of the cell and may also perturb the behavior of the labeled molecules. Quantum dots are not yet approved for use in humans and much more research is needed in future for this

Molecular imaging exploits the specific recognition of labeled probes to their biological targets in conventional imaging techniques to monitor biological processes at the molecular level with improved specificity and sensitivity. Conventional clinical cancer imaging techniques, such as X-ray, CT and MRI, do not possess sufficient spatial resolution for early detection of the disease. Positron emission tomography (PET) is a highly sensitive and accurate imaging technology that relies on changes in tissue biochemistry and metabolism. It is the most valuable means we have so far to identify early-stage alterations in molecular biology, often before there is any morphologic change. Nevertheless, fluoro-desoxy-glucose (FDG), the most commonly used PET tracer in clinical oncology (more than 95% of the molecular imaging procedures make use of FDG at present), is not a specific tracer for malignant diseases but for increased metabolism. Therefore, it is imperative to develop new

CNTs have been explored in almost every single cancer treatment modality, including drug delivery, lymphatic targeted chemotherapy, thermal therapy, photodynamic therapy, and gene therapy. Based on their structure, CNTs can be classified into two general categories: single-walled (SWNTs), which consist of one layer of cylinder graphene (diameter 0.4-2 nm) and multi-walled (MWNTs), which contain several concentric graphene sheets (diameter 2- 100 nm). CNTs have unique physical and chemical properties such as high aspect ratio,

growing field.

**5.2 Carbon nanotubes** 

tools for early cancer diagnosis.

ultralight weight, high mechanical strength, high electrical conductivity, and high thermal conductivity (Ji et al. 2010). Carbon nanotubes are the strongest and stiffest materials yet discovered in terms of tensile strength and elastic modulus respectively. As CNTs are intrinsically not water soluble, modification through chemical functionalization can increases the solubility of carbon nanotubes in aqueous solutions.

Imaging functionalities and therapeutics can be incorporated on the same nanoparticle for multifunctional cancer imaging and treatment. SWNT-paclitaxel (PTX) conjugates also showed higher efficacy in suppressing tumor growth than clinical Taxol alone in a murine 4T1 breast cancer model, owing to prolonged blood circulation time and enhanced permeability and retention (EPR) in the tumor (Feazell et al. 2007). Besides, with very high surface area per unit weight, SWNTs provide higher capacity of drug loading, compared to that reported for conventional liposomes and dendrimer drug carriers. Doxorubicin, a commonly used cancer chemotherapy drug, can be loaded on the surface of PEGylated SWNTs with remarkably high loading, up to 4 g of drug per 1 g of nanotube, owing to the ultrahigh surface area of SWNTs. Further, the intrinsic stability and structural flexibility of CNTs may prolong the circulation time as well as improve the bioavailability of drug molecules conjugated to them. Surface-enhanced Raman spectroscopy of carbon nanotubes opens up a method of protein microarray with detection sensitivity down to 1 fmol/L. *In vitro* and *in vivo* toxicity studies reveal that highly water soluble and serum stable nanotubes are biocompatible, nontoxic, and potentially useful for biomedical applications. However, nonfunctionalized nanotubes are toxic to cells and animals and therefore one has to be cautious about the safety aspects of CNTs. If well functionalized, nanotubes may be excreted mainly through the biliary pathway in feces.

Carbon nanotube-based drug delivery has shown promise in various *in vitro* and *in vivo* experiments including delivery of small interfering RNA (siRNA), paclitaxel and doxorubicin (Liu et al. 2009). Multiwalled PEGylated carbon nanotubes are found to be successful, effective and do not alter particle sizes and zeta potentials of carbon nanotubes after PEGylation (Ilbasmis-Tamer et al. 2010). In addition, the propensity to absorb the body transparent NIR radiation also envisages photothermal and photoacoustic therapy using nanotubes.

#### **5.3 Gold and other nanoparticles for cancer diagnosis**

Thermal ablation therapy is one the most promising of methods in cancer treatment but is limited by incomplete tumor destruction and damage to adjacent normal tissues. Current radiofrequency ablation techniques require invasive needle placement and are limited by accuracy of targeting. Use of nanoparticles has refined noninvasive thermal ablation of tumors, and several nanomaterials have been used for this purpose. These include gold nanomaterials, iron nanoparticles, magnetic nanoparticles, carbon nanotubes and affisomes (thermosensitive liposomes). Heating of the particles can be induced by magnets, lasers, ultrasound, photodynamic therapy and low-power X-rays. The clinical trials include studies of designed nanoparticles such as the thermosensitive liposomal doxorubicin (Thermodox®) as a novel activated therapy using radiofrequency ablation (Wang and Thanou 2010). As gold nanoparticles have evolved other gold structures have also been suggested. Nanorods, with the appropriate PEG stealth layer, are being developed as an improved means of hyperthermia. By attaching monoclonal antibodies (mAbs), which can recognize a specific cancer cell, to gold nanoparticles or nanorods are also used in cancer detection. Gold nanoparticles conjugated to anti-epidermal growth factor receptor (El-Sayed et al. 2006)

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 539

nanoparticulates appear to be superior in terms of optical performances, they are marred by their heavy metal composition and high propensity for toxicity. It is therefore reasonable to be concerned about the ineffectual clearance and long-term accumulation in untargeted

With the understanding of the genetic origins of certain cancers, an entirely new approach to the treatment of this disease has evolved, employing nanoparticle-based gene therapy. Numerous nanoparticle based cancer gene therapy strategies are already in clinical trials. The key to the success of any new therapeutic is to maximize safety without compromising efficacy, which has led to growing interest in non-viral gene delivery systems (such as liposomes) over the viral gene delivery systems. Grafting biorecognition molecules (ligands, antibodies) onto the nanoparticles (i.e active targeting) aims to improve targeting by specific cell uptake and using hydrophilic polymer coating, PEG, which aims to further enhance biocompatibility. To overcome other challenges of gene therapy, such as escape from endosome and other nuclear and cytosolic barriers, next generation vectors are being designed with use of gene regulatory elements (promoters and enhancers) to restrict gene expression to specific cells, along with nuclear localization signal peptides for nuclear

There has been substantial interest in dual purpose nanoparticle based gene therapy for both diagnostic (imaging) and therapeutic purposes (drug/gene delivery). Newer technologies for cancer detection/diagnosis using metallic and semiconducting nanoparticles are also under intense investigation. These nanoparticles for *in vivo* application targeting cancer are amenable to different size structures and possess tunable properties. Quantum dots possess unique size- and composition- dependent optical and electrical properties. In addition to quantum dots, carbon nanotubes, paramagnetic nanoparticles, nanoshells and nanosomes represent just a few of these novel technologies,

The imminent research challenge facing investigators moving forward is the expansion of the knowledge and understanding of the chemical and physical properties associated with these nanoparticle systems toward the design of superior cancer therapy modalities that

Aggarwal,S., Yadav,S. and Gupta,S. (2011) EGFR targeted PLGA nanoparticles using gemcitabine for treatment of pancreatic cancer. *J. Biomed. Nanotechnol.* 7, 137-138. Alivisatos,P. (2004) The use of nanocrystals in biological detection. *Nat. Biotechnol.* 22, 47-52. Ashihara,E. (2010) [RNA interference for cancer therapies]. *Gan To Kagaku Ryoho.* 37, 2033-

Azad,N.S., Posadas,E.M., Kwitkowski,V.E., Steinberg,S.M., Jain,L., Annunziata,C.M.,

Minasian,L., Sarosy,G., Kotz,H.L., Premkumar,A., Cao,L., McNally,D., Chow,C., Chen,H.X., Wright,J.J., Figg,W.D. and Kohn,E.C. (2008) Combination targeted therapy with sorafenib and bevacizumab results in enhanced toxicity and

maximize efficiency of treatment, while maintaining a superior safety profile.

antitumor activity. *J. Clin. Oncol.* 26, 3709-3714.

organs and tissues of these particulates for *in vivo* use.

used for both diagnostic and delivery purposes.

**6. Conclusion**

targeting.

**7. References** 

2041.

mAbs specifically and homogeneously bind to the surface of the cancer cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found to give a relatively sharper surface plasma resonance (Hinz et al. 2006) absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells . Surface plasma resonance scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles may be useful in molecular biosensor techniques for the diagnosis and investigation of cancer cells *in vivo* and *in vitro*.

These inorganic nanoparticles represent a different class of nanoparticles that are usually much smaller, 5–40 nm and they do not have the flexibility observed in liposomes and polymeric nanoparticles. Inorganic nanoparticles have made their appearance in cancer therapy during the last decades in a number of applications. The main type of inorganic nanoparticles—the iron oxide nanoparticles, has been used for imaging tumor (Wang and Thanou 2010). The main advantage of magnetic nanoparticles is their ability to be visualised by Magnetic Resonance (MR) imaging. Additionally, iron oxide nanoparticles can be guided to target sites (i.e. tumor) using external magnetic field and they can be also heated to provide hypethermia for cancer therapy. Yu et al. reported thermally crosslinked superparamagnetic iron oxide nanoparticles that could carry a Cy5.5 near infra-red probe (dual imaging) and doxorubicin for the imaging and treatment of cancer. The nanoparticles substantially diminished tumor size and provided the proof of concept that they can combine several modalities for maximum antitumor effect (Yu et al. 2010). Magnetic nanoparticles have been used in the development of dual purpose probes for the *in vivo* transfection of siRNA. The iron nanoparticles deliver siRNA at the same time as imaging their own accumulation in tumor sites. Hence, multifunctional nanoparticles have emerged that are capable of cancer targeting and simultaneous cancer imaging and therapy.

Metal nanoshells are another class of nanoparticles with tunable optical resonances. Metal nanoshells consist of a spherical dielectric core nanoparticle, in this case silica, which is surrounded by a thin metal shell, such as gold. These particles possess a highly tunable plasmon resonance, a resonant phenomenon whereby light induces collective oscillations of conductive metal electrons at the nanoshell surface. Nanoshells derived from gold provide an attractive system for imaging applications owing to the established ease of preparation, chemical inertness, good biocompatibility, and surface functionalization. Further, nanoparticle based near infrared imaging (NIR) is steadily presenting itself as a powerful diagnostic technique with the real potential to serve as a minimally invasive, nonionizing method for sensitive, deep tissue diagnostic imaging that are not prone to the rapid photobleaching and instability of their organic counterparts. NIR laser treatment of the bulk tissue selectively heats and destroys the nanoshell-laden tumor regions within the tissue, while leaving surrounding tissue intact. Nanoshells are currently evaluated in a number of clinical settings after a 5-year period of intensive preclinical development. Such development of nanoshells included the combination of nanoshells with cancer antibodies. Anti-HER2 antibody conjugated onto nanoshells provides the potential of combining antibody therapy with imaging and hyperthermia. NIR dye-encapsulating nanoparticles also demonstrate improved optical performances compared to unencapsulated organic fluorophores. Specifically, the encapsulation shields the dye molecules from unfavorable environmental influences that normally hinder fluorescence signals, thereby enhancing quantum yields, emission brightness, and fluorescent lifetime. While, at present, these NIR nanoparticulates appear to be superior in terms of optical performances, they are marred by their heavy metal composition and high propensity for toxicity. It is therefore reasonable to be concerned about the ineffectual clearance and long-term accumulation in untargeted organs and tissues of these particulates for *in vivo* use.
