Paper Biology 280 S Minireview Advances In Cancer Detection And Therapeutics

Minireview: Advances of Nanomedicine in Cancer Detection and Therapeutics

Joshua E. Mendoza-Elias§

Biology 280S: Biotechnology and Genetic Engineering Fall 2007

Duke University Dr. Tai-ping Sun, Ph.D.

Trinity School of Arts and Sciences, Biology Department Submitted: Thursday, December 6, 2007

Abstract Nanotechnology is a field that has made significant advances in the engineering of technologies that allow earlier detection and treatment of disease – specifically cancer. In the modern era, these technologies define the emerging field of nanomedicine and seek to redefine detection and treatment with the promise of more effective, sensitive and cost effective high throughput systems. Compared to conventional treatment options, these next generation cancer therapeutics also seek to overcome the invasiveness of surgery, chemotherapy, radiotherapy, immunotherapy, and hormonal therapy. These technologies include nanoshells, quantum-dots, ultraoxide particles, DNA microarrays, liposomes, dendrimers, and fullerines. Highlighted in this paper, microwires and microcantilevers (“biofinger”) show promise in becoming multiplex platforms (“Labs on a chip”) that detect a variety of biological markers at low concentration in real time. In addition, DNA based diagnostic computer constructs (DNAdc) that release therapeutic nucleic acid sequences in a gene expression specific manner will be highlighted as well. The potential for a wide range of clinical applications to disease, including cancer, makes a basic understanding of the field of nanomedicine important to the biomedical sciences. In addition, nanomedicine will have a huge impact in developing a repertoire of nano-based therapeutics for other diseases.

Keywords: “Biofinger”-lab on a chip, DNA diagnostic computer construct (DNAdc), DNA microarrays, nanoshells, quantum-dots, ultraoxide particles,

liposomes, dendrimers, fullerines, single wall carbon nanotubes (SWNT)

§ Joshua Mendoza-Elias, Trinity College of Arts and Sciences, Department of Biology. E-mail: [email protected]. Website: http://www.duke.edu/~jme17

Minireview: Advances of Nanomedicine in Cancer Detection & Therapeutics Mendoza-Elias, Joshua E.

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Table of Contents:

1 Introduction ................................ ................................ ........... 3 Figure 1: Repertoire of Nanoparticles..........................................................2

2 First Generation Nanomedicine ................................ ............... 4

2.1 Detection ................................ ................................ .......... 4 Figure 2: State of the Art – Cancer Nanotechnology. .................................3

2.2 Treatment ................................ ................................ ......... 5 Figure 3: Heat Responsive Liposome Design and Function. ......................5

3 State of the Art ................................ ................................ ....... 6

3.1 Detection – Lab on a Chip: ................................ ................ 6 Figure 4: "Biofinger” Lab on a chip-Nanowires and Nanocantilevers. ......6

3.2 DNA as a nanotechnology ................................ .................. 7 Figure 5: DNA as a nanotechnology. ...........................................................7

4 Conclusion ................................ ................................ .............. 9 5 References ................................ ................................ ............ 10

Mendoza-Elias, Joshua E. Min-review: Advances of Nanomedicine in Cancer Detection & Therapeutics

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1Introduction Nanomedicine is concerned with development of technology that allows the monitoring, repair, construction and control of biological systems at the molecular level, using engineered devices and structures built at the nano level [1] (1-100 nanometers [2] ). In turn, these technologies are intended for introduction and use at the clinical level of medical sciences. The goals of nanomedicine are to develop new and better strategies for diagnosis, prevention, treatment of disease and an increased understanding of the underlying complexity of disease mechanisms. For cancer therapeutics, this technology holds great promise for enhanced detection and treatment. Admittedly, nanotechnology is a newly established discipline whose contribution to nanomedicine is still at a nascent stage of development. Many of the technologies that have been developed (Q-dots, dendrimers, gold nano particles, solubalized single walled carbon nanotubes [SWCNT], DNA microarrays, etc.) (see: Figure 1) are in production and have shown success in human disease models. However, these technologies have not yet been incorporated into a wide range of devices and treatments in the clinical setting [3] due to the demanding requirements of the FDA. Nevertheless, nanomedicine continues to gain new ground in preventing and treating disease through the detection of disease-related and pathogenic particles. This has been primarily accomplished through the development of nanofabricated biomaterials with the unique physical properties of strength, hardness, reduced friction, controlled energy emission and improved biocompatibility, that allow for the development of novel emulsions, drug delivery mechanism and platforms for vaccines (see: Figure 2).

Figure 1: Repertoire of Nanoparticles. Relative size of an assortment of nanoparticles compared to objects representative of scale. The structures in the lower row are between 1 and 100 ηm in size.

Reprinted from: McNeil et al. (2005) Journal of Leukocyte Biology. 78: 1-10. [19]

Figure 2: State of the Art – Cancer Nanotechnology. Nanomedicine therapeutics focus on earlier detection of cancer and higher specificity in the delivery of therapeutic agents. New treatment platforms have arisen that rely on physical properties of nanomaterials or higher-specificity targeting achieved by conjugation of nanomaterials with antibodies. However, earlier detection methods remain scarce. Cancer microarray technologies remain the gold standard. The “Biofinger” is a promising new technology that can detect cancer related molecules at much lower concentrations, with little or no purification of the sample (e.g., blood, lymph).


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2FirstGenerationNanomedicine 2.1DetectionIn terms of detection, first generation nanotechnology emphasized the development of imaging probes that allowed for greater tissue specificity, greater resolution in tissues of interest and greater control over half-life. These include: the nanoshell, quantum-dots (Q-dots), ultraoxide iron oxide (USPIO) particles, single walled carbon nanotubes (SWNT) and DNA microarrays). The development of these technologies emerged primarily from advances in materials sciences; and therefore, the characteristics of custom designed materials became new tools for biological imaging. More importantly, these technologies came to demonstrate the potential for the replacement of chemical based imaging (contrast agents) as they demonstrated properties of being inert, non-toxic and biocompatible. In general, these technologies are unified by a single principle: a particle is engineered so that it can emit a characteristic wavelength of light which is then conjugated to targeting molecules (e.g., antibodies) to probe tissues or cell types of interest. The earliest of these technologies, the nanoshell, typically made from gold, is the simplest of these technologies. When electromagnetic energy such as UV-light is applied, the particle emits a specific wavelength as a function of spherical radius, shell thickness and dielectric core. Therefore these properties allow the nanoshell excitation and emission energetics to be tailored for different applications. A similar technology, the Quantum-dot (Q-dot) differs in the techniques employed in synthesis. The Q-dot uses semiconductor colloidal crystals that, when exposed to ultraviolet light, will emit a specific wavelength. This characteristic wavelength is a function of size and number of atoms in its core. The tailoring of Q-dots is determined by quantum mechanics of the core atoms. When targeting molecules (antibodies or recombinant proteins) are attached to Q-dots, they become ideal probes for DNA, cell-surface receptors and intracellular enzymes; thus, allowing the tracking of viruses, and antibodies. The key advantage of Q-dots lies in their small size, which allows enhanced visualization of biological tissues that is more efficient than biological stains, contrast agents or fluorescent probes, as they are better able transverse cell membranes and interstitial spaces. USPIO particles are a technology developed specifically for the treatment of metastatic cancer tissues. These nanoparticles differ from the rest because they cannot be modified with targeting molecules. Instead, the function of these nanoparticles lies in their ability to enhance magnetic resonance imaging and staining of target cells and molecular processes.

Single walled carbon nanotubes (SWNTs) have been demonstrated as diagnostic tools and are used primarily in biological assays. SWNTs are composed from a sequence of C60 atoms arranged in long, thin cylindrical structures that are determined by thermodynamic conditions of chemical synthesis. Although toxic in the biological environment, SWNTs can be chemically modified to add antibodies and DNA sequences for the detection of disease-related proteins and genes. SWNT have already been reported as high-specificity sensors of antibody signatures in autoimmune disease [4] and single-nucleotide polymorphisms (SNPs) [5]. However, these detection mechanisms require steps for separation, purification and enrichment of molecules, and thereby do not demonstrate an improved method of performing an assay compared to (steps) existing methods. Biologically, the most important and powerful of these detection technologies is the DNA microarray or gene chip. The microarray contains a repertoire of genes that detect the presence and expression levels of mRNA from various tissue types or cell cultures. The data gathered from this is then use to generate gene expression profiles that correspond to specific tissues, cell types or disease states. For cancer diagnosis, this technology has been used to generate gene expression profiles for different types of cancer. In turn, gene expression profiles have been correlated with established treatments to generate candidate treatment options that have the highest probability of success.


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2.2TreatmentIn terms of treatment, first generation nanotechnology has emphasized the development of targeted drug delivery systems. These include: liposomes, dendrimers, fullerenes, and nanoshells. These therapeutic technologies have a unifying element in that they are inert, non-toxic, biocompatible and can be chemically modified to include molecules for targeting. The liposome is the most basic technology and is biologically inspired in design. (see: Figure 3) Liposomes are spherical vessels composed of a lipid bilayer that can be modified to incorporate different molecules that change properties of the liposome, such as half-life, permeability, diffusion, drug release and targeting. Currently, a new generation of heat-responsive liposome, currently in phase II trials, has been developed that allows for localized release of therapeutic agents where heat is applied [6]. Loaded with the therapeutic agent doxorubicin (DOX), this heat-responsive liposome has been demonstrated to produce complete regression of cancer in 11 of 11 xenografted mouse tumors lasting up to 60 days post treatment. Dendrimers are synthetic tree-shaped macromolecules formed from monomers using a stepwise fabrication process. It is the size of a typical protein, but it does not unfold or come apart easily due to the nature of its stronger chemical bonds. It can be easily attached to a variety of molecules to facilitate the transport and release of medication, contrast agents or DNA for gene therapy [2,7]. Fullerenes have been investigated for use as a drug delivery platform as well. Fullerenes are a class of carbon molecules that form closed, convex cage molecules containing hexagonal and pentagonal faces. However, there have been problems with this technology as lipophllic uncoated fullerenes were found to selectively translocate into the brains of large-mouth bass and cause oxidative damage [8]. Nanoshells can also be modified for targeted drug delivery. Because of their size, nanoshells can easily penetrate several centimetres of tissue and can be functionalized with chemical addition of antibodies [2]. Furthermore, their energy emission can be calibrated so that the nanoshells emit a dose of heat to a defined tissue volume [2] that serves to promote mild hyperthermia, which in turn kills cancer cells and reduces tumors.

Figure 3: Heat Responsive Liposome Design and Function. A Liposome Design- Liposomes can achieve targeting by incorporating targeting molecules (e.g., antibody). Further specificity can be achieved by making the liposome responsive to heat (or irradiated) by incorporating polymers. When heat is applied, in the liposome, these polymers are akin to stitches that functions as a “leaky soccer ball” allowing chemotherapeutics (Drug A/Drug B).

B: Liposomes in action for the next generation. Current heat responsive liposomes are most effective if the tumor is surrounded by a well developed vascular system. Future liposome platforms being researched seek to overcome this and gain higher targeting and efficacy at lower concentrations with through chemotactic extravasations in tissues before activation by heat or irradiation.

Reprinted from: Ferrari et al. (2005) Nature Reviews 5: 161-171. [20]


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3StateoftheArt 3.1Detection–LabonaChip:As new nanotechnologies develop, the emphasis on enhanced detection and treatment continues. One of the emerging nanotechnology fields is the development of new diagnostic assays. Current diagnostic assays such as Western blots, enzyme-linked immunosorbent assays, cell culture, latex agglutination and polymerase chain reaction (PCR) often require purification, separation, and incubation steps. These steps require time, effort and cost for processing. Development of miniaturized devices using nanotechnology aims to reduce or eliminate these steps by developing new platforms that may provide faster, more accurate and less expensive high throughput technologies for the diagnosis of disease and pathogens [2]. Furthermore, platforms in development have the goal to multiplex (detect a broad spectrum of molecular signals and biomarkers in real time). In short, these new assays aim to be “labs on chips.” In order to fulfill these goals, nanowires [9, 10] and nanocantilever arrays [11, 12] have been developed (see: Figure 4). Exploiting the proteomic data, a nanowire can be coated with antibodies against a known disease-associated protein product. When exposed to disease-associated biomarkers, antibodies will bind the biomarker thereby changing the conductance properties of the wire. In this way, a unique electronic signature can be recorded and used to establish disease profiles or measures against controls in the clinical setting. Equipped with an array of wires and a repertoire of antibodies, the nanowire becomes a multiplex real-time detector of disease-associated biomarkers and reporter of their levels. The Nanocantilever, or “biofinger” also employs a similar strategy. Using proteomic data, a cantilever can be coated with antibodies. When antibodies bind disease-associated biomarkers, the cantilever deflects. The changes in deflection can be observed by detectable

Figure 4: “Biofinger” Lab on a chip – Nanowires and Nanocantilevers. A: Nanowires arranged in an array in a microfluidic system. Different molecules (coloured circles) differentially bind to nanowires coated with antibodies. Binding causes changes in the conductance of wires that correspond to unique electronic signatures. B: Nanocantilever array. Biomarker proteins (coloured circles) are affinity-bound to antibodies and cause the cantilevers to deflect. Deflections can be observed directly: (i.) with lasers; (ii.) observing shifts in resonance frequencies in the nanocantilever array. Nanocantilever technology has gained the advantage in the ability to sense a large number of different proteins at the same time, in real time. Reprinted from: Ferrari et al. (2005) Nature Reviews 5: 161-171. [20]


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shifts in the physical properties of the cantilever by direct inspection with laser or changes in resonant-vibrational frequency. Adding an array of cantilevers and antibodies, a “biohand” can also report on the detection of a variety of disease-associated biomarkers and on their levels. 3.2DNAasananotechnology DNA is a versatile molecule that stores information on many levels. The primary structure of DNA encodes information in base-pair sequences. Scientists, in the development of molecular probes for gene expression, exploited this as testament to in situ hybridizations and gene chips. In addition, the secondary and tertiary structure of DNA also encodes information in its molecular geometry as observed in major and minor grooves, helix-turn-helix motifs and hairpins. Together, these information storage properties have been the characters behind many biologically driven mechanisms such as DNA mismatch repair, non-homologous end joining (NHEJ), recombination repair, maintenance of chromatids by telomerase and the RNA interference pathway. Most important, of the information encoding properties of DNA, is the DNA-folding pathway – the sequence of intermediate structures between stable DNA structures in a thermodynamic environment. Innovative uses of the DNA-folding pathway have led to the design of a DNA device that performs several diagnostics tests for high or low concentrations of certain DNA and RNA structures. This device, hereto referred as DNA diagnostic computer construct (DNAdc), is a simple rudimentary computer that uses (is programmed with) “if-then” statements (Boolean logic gates) (Figure 5). Each statement tests for the presence of a gene (e.g., IF gene X is present, THEN proceed to next test). If all the statements test positive, then the device releases a nucleic acid sequence that serves as a therapeutic drug. In this

Figure 5: DNA as a nanotechnology. A: Logical design and operation of “if-then” statements in diagnosis drug release program of the DNAdc construct. Gene expression profiles of disease-associated mRNA are listed on the right. Upward pointing arrows indicate increasing expression. Downward pointing arrows indicate decreasing gene expression. B: Mode of operation of the DNAcd construct. All inputs are identical and are composed of a hairpin structure with a ”Yes,” if positive and “No,” if negative. Each positive diagnosis causes shortening of the construct until the drug is released.

Reprinted from: A: Beneson et al. (2004) Nature. 429: 423-429. [21] B: Condon et al. (2006) Nature Reviews 7: 565-575. [22]


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way, medical knowledge is condensed in the sequences (program) of the DNAdc. Functioning as a molecular automaton [13, 14, 15, 16] the DNAdc scans for the presence of mRNA of disease-related genes (identified by in silico searches of DNA expression profiles of prostate cancer biopsies). The DNAdc begins in the positive condition (sticky end free) and, then scans or probes for the presence of the relevant mRNA. If the mRNA is detected, given the hybridization properties of DNA, a DNA:RNA duplex will form. The formation of the duplex then allows the restriction enzyme FokI to recognize and cleave the FokI restriction sequences which are accessible only in the duplex state. Once the sequence is cleaved, the process is repeated with another mRNA until a single stranded therapeutic sequence (drug) is released. As per proof of concept, genes associated with prostrate cancer were used to program and design the first DNAdc and the single stranded therapeutic sequence was antisense DNA sequence that down regulated the expression of a target gene MDM2, a negative regulator of the p53 tumor suppressor. An important aspect of the DNAdc program is that they can only proceed with the disease-related mRNA of interest, as logic gates do not permit No YES (No to Yes) transition and if negative state is entered, the computer remains locked in that state. In this way, the “if-then” statements are able to “guard” the remaining sequences from opening and releasing the therapeutic sequence. The DNAdc only releases the therapeutic sequence when all the “if-then” statements are satisfied with the diagnosis.


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4Conclusion Nanomedicine holds great promise for the development of novel, more effective and cheaper detection and treatment of disease. In the hopes of pushing the field farther, the National Institutes of Health (NIH) Roadmap for the Twenty-First Century (http://www.nih.gov) has sought to address the roadblocks and gaps that constrain biomedical research by offering funding towards the development of comprehensive interdisciplinary research programs that combine structural biology, molecular libraries, imaging, bioinformatics and computational biology. In addition to combining NIH centers, the NIH proposes changes in the manner and complexity in which clinical research is carried out, so that these technologies emerge more rapidly in the coming decades and shorten the time of nanomedicine development from concept to commercially viable product.

Given this, it is suggested that the future of nanomedicine would benefit from the development of a robust integrated system that uses the new repertoire of nanomedicine against a disease class, such as cancer. Such a system would cover all of the steps involved in diagnosis, treatment and remission. Furthermore, as one of the goals of nanomedicine is the use of nanotechnology to better understand the fundamental mechanisms of disease, the use of such a system would serve as a model in the developing nano-based therapeutics for other diseases. In terms of cancer therapeutics, nanomedicine has already demonstrated the ability to enhance existing techniques of detection with enhanced imaging. More importantly, the successful demonstration of site-specific hyperthermic treatment and encouraging phase II clinical trials highlight the importance and potential success of nanomedicine in treating cancer. Diagnostic assays, such as nanowires and nanocantilevers, also show progress towards the development of more sensitive multiplexing assays that will not only allow for earlier diagnosis of cancer, but enhance prevention capabilities, allow for impediment of cancer stage advancement and maintenance of remission.

With the advent of more powerful tools of prevention, more specialized therapeutics must arise to complement them the treatment of disease. The DNAdc shows great promise in achieving a high degree of treatment efficacy. The DNAdc demonstrates that diagnostic/analytical or “smart” delivery of drugs is possible. Although considerable work still remains in developing this technology for in vivo applications, Dittmer et al. [17, 18] suggests that the instructions for the synthesis of a DNA machine, like the DNAdc, could be achieved by encoding it in an artificial gene that could then control the working of the device. Although the platforms for targeted drug delivery already exist and are being improved upon, the coupling of the DNAdc with new vectors that establish synthetic gene networks may allow for the creation of a new, more powerful and versatile tool for cancer therapeutics.


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5References [1] Webster, P. 2005. World Nanobiotechnology Market. Frost and Sullivan. [2] Gordon, A.T., Lutz, G.E., Cooper, R.A., and Boninger, M.L. 2007. Introduction to Nanotechnology: potential application in physical medicine and rehabilitation. American Journal of Physical Medicine and Rehabilitation. 86:225-241. [3] Morrow, K.J., Bawa, R., Wei, C. 2007. Recent advances in basic and clinical nanomedicine. Medical Clinics of North America. 91: 805-843. [4] Chen, R. J. & Hongjie, D. 2003. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proceedings of the National Academy of Sciences. USA. 100: 4984–4989. [5] Woolley, A., Guillemette, C., Cheung, C.L., Housman, D.E., and Lieber, C.M. 2000. Direct haplotyping of kilobase-size DNA using carbon nanotube probes. Nature Biotechnology. 18: 760–764. [6] Needham, D., Anyaramhatla, G., Kong, G., Dewhirst, M.W. 2000. A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model. Cancer Research. 60; 1197-1201. [7] Dufes, C., Uchegbu, I.F., Schatzlein, A.G. Dendrimers in gene delivery. 2005. Advanced Drug Delivery Reviews. 57: 2177-2202. [8] Oberdorster, E. 2004. Manufactured nanomaterials (fullerens, C60) induce oxidative stress in the brain of juvenile large-mouth bass. Environmental Health Perspectives. 2: 1-15. [9] Cui, Y., Qingqiao W., Hongkun, P. & Lieber, C. M. 2001. Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species. Science. 293: 1289–1292. [10] Adar, R. Beneson, Y., Linshiz, G., Rosner, A., Tishby, N., Shapiro, E.. 2004. Stochastic computing with biomolecular automata. Proceedings of the National Academy of Sciences. USA (submitted). [11] Hansen, K. M., Ji, H., We, G., Datar, R., Cote, R., Majumdar, A., and Thundat, T. 2001. Cantilever-based optical deflection assay for discrimination of DNA single-nucleotide mismatches. Analytical Chemistry. 73: 1567–1571. [12] Wu, G., Datar, R.H., Hansen, K., Thundat, T., Cote., R.J., and Majumdar, A. 2001. Bioassay of prostate-specific antigen (PSA) using microcantilevers. Nature Biotech. 19: 856–860. [13] Benenson, Y., Adar, R., Paz-Elizur, T., Livneh, Z. & Shapiro, E . 2001. Programmable and autonomous computing machine made of biomolecules. Nature. 414: 430–434. [14] Benenson, Y., Adar, R., Paz-Elizur, T., Livneh, Z. & Shapiro, E. 2003. DNA molecule provides a computing machine with both data and fuel. Proceedings of the National Academy of Scences. USA 100: 2191–2196.


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[15] Adar, R. Benenson, Y., Adar, R., Paz-Elizur, T., Livneh, Z. & Shapiro, E. Stochastic computing with biomolecular automata. Proceedings of the National Academy of Sciences. USA (submitted). [16] Benenson, Y. & Shapiro, E. 2004. Dekker Encyclopedia of Nanoscience and Nanotechnology (eds Schwarz,J. A., Contescu, C. I. & Putyera, K.) 2043–2056 (Dekker: New York). [17] Dittmer, W. U. & Simmel, F. C. 2004. Transcriptional control of DNA-based nanomachines. Nano Letters. 4: 689–691. [18] Dittmer, W. U., Kempter, S., Radler, J. O. & Simmel, F. C. 2005. Using gene regulation to program DNA-based molecular devices. Small 1: 709–712. [19] McNeil S.E., Nanotechnology for the biologist. Journal of Leukocyte Biology. 2005. 78: 1-10. [20] Ferrari, Mauro. Cancer nanotechnology: opportunities and challenges. 2005. Nature Reviews. 5: 161-171. [21] Beneson, Y., Gil, B., Ben-Dor, U., Rivka, A. and Shapiro, E. An autonomous molecular computer for logical control of gene expression. 2004. Nature. 429: 423-429.

[22] Condon, Anne. Designed DNA molecules: principles and applications of molecular nanotechnology. 2006. Nature Reviews 7: 565-575.

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