Role of Nanobiotechnology in Developing Personalized Medicine for Cancer

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1 Technology in Cancer Research & Treatment ISSN Volume 4, Number 6, December (2005) Adenine Press (2005) Role of Nanobiotechnology in Developing Personalized Medicine for Cancer Personalized medicine simply means the prescription of specific therapeutics best suited for an individual. Personalization of cancer therapies is based on a better understanding of the disease at the molecular level. Nanotechnology will play an important role in this area. Nanobiotechnology is being used to refine discovery of biomarkers, molecular diagnostics, drug discovery and drug delivery, which are important basic components of personalized medicine and are applicable to management of cancer as well. Examples are given of the application of quantum dots, gold nanoparticles, and molecular imaging in diagnostics and combination with therapeutics another important feature of personalized medicine. Personalized medicine is beginning to be recognized and is expected to become a part of medical practice within the next decade. Personalized management of cancer, facilitated by nanobiotechnology, is expected to enable early detection of cancer, more effective and less toxic treatment increasing the chances of cure. K. K. Jain M.D. Jain PharmaBiotech Blaesiring 7 CH-4057 Basel, Switzerland Key words: Cancer; Personalized medicine; Nanoparticles; Nanotechnology; Nanobiotechnology; Cancer therapy; Cancer diagnosis; Drug delivery in cancer; Nanodiagnostics; and Drug discovery. Introduction Personalized medicine simply means the prescription of specific therapeutics best suited for an individual. It is usually based on pharmacogenetic, pharmacogenomic, and pharmacoproteomic information but other individual variations in patients are also taken into consideration (1, 2). In case of cancer the variation in behavior of cancer of the same histological type from one patient to another is also taken into consideration. Personalization of cancer therapies is based on a better understanding of the disease at the molecular level and nanotechnology will play an important role in this area. Nanotechnology is the creation and utilization of materials, devices, and systems through the control of matter on the nanometer-length scale, i.e., at the level of atoms, molecules, and supramolecular structures. Nanobiotechnology is the application of nanotechnology in life sciences including molecular diagnostics, drug discovery, drug delivery, and development of nanomedicine. Various components of personalized therapy of cancer that are relevant to nanobiotechnology are shown in Figure 1. This article will first introduce the concept of personalized therapy of cancer and then describe the role of nanobiotechnology in for developing personalized therapy of cancer. Personalized Medicine for Cancer Although remarkable developments have occurred in development of new therapies for cancer, management of cancer is less than satisfactory. There is an increas- * Corresponding Author: K. K. Jain, M.D. jain@pharmabiotech.ch 645

2 646 Jain Figure 1: Role of Nanobiotechnology in the development of Personalized Management of Cancer. ing recognition that no therapeutic agent has the same effect on a large number of patients with the same diagnosis. This has led to efforts to personalize cancer therapy as described in more detail elsewhere (3). Personalized approach to cancer management is facilitated by an improvement in understanding of the molecular mechanisms of cancer, advances in molecular diagnostics and proteomic technologies, which are being further refined by the availability of nanobiotechnologies. The greatest impact of nanobiotechnology is on molecular diagnostics for cancer, also referred to as nanodiagnostics. Nanodiagnostics and Personalized Cancer Therapy Nanobiotechnology has the potential to improve early detection of cancer and facilitate the personalization of cancer therapy. The most important component of personalized medicine is molecular diagnostics and nanobiotechnology will play an import role in refining it and has led to use of the term nanodiagnostics (4, 5). Nanobiotechnologies will also improve detection of cancer biomarkers as basis for devising diagnostics as well as therapeutics. Role of Nanotechnology in Detection of Cancer Biomarkers Detection of cancer biomarker is important for diagnostics as well as development of new therapies for cancer. Nanotechnology can provide new platforms for highthroughput, highly sensitive, functional assays to complement genomic technologies for the identification of new biomarkers of cancer risk. Microfluidic biochips are now being further miniaturized to nanoarrays and handle nanofluids. Optical nanosensors can be used for in vivo analysis of proteins and biomarkers in individual living cells (6). Quantum Dots for Cancer Diagnostics Quantum dots (QD), attached to a molecule that will target a specific type of cancer, can accumulate in the tumor, which will light up. One limitation is that light penetration into the body is limited; one can screen for superficial cancers but it is difficult to reach cancers located deep in the body. This method can be improved by using quantum dots that emit near-infrared light. The work cited here is mostly in experimental animals. QDs were rendered soluble by using a polydentate phosphene coating, engineered to emit near-infrared light, injected into live pigs and followed visually to the lymph system just beneath the skin of the animals (7). The new imaging technique allowed clearly visualization of the target lymph nodes without cutting the animals skin. Sentinel Lymph Node (SLN) mapping, the surgical technique employed in the study, is a common procedure used to identify the presence of cancer in a single, sentinel lymph node, thus avoiding the removal of a patient s entire lymph system. SLN mapping relies on a combination of radioactivity and organic dyes but the technique is inexact during surgery, often leading to removal of more of the lymph system than is necessary, causing unnecessary trauma. The QD approach is a significant improvement over the dye/radioactivity method currently used to perform SLN mapping and would enable tailoring surgery to the needs of a patient. Metallic QDs used in the study have potential toxicity and safer materials need to be developed before they can be used in vivo in humans. QDs can be combined with fluorescence microscopy to follow cells at high resolution in living animals. These offer considerable advantages over organic fluorophores for this purpose. QDs and emission spectrum scanning multiphoton microscopy have been used to develop a means to study extravasation in vivo (8). Tumor cells labeled with QDs and injected intravenously into mice can be followed as they extravasate into lung tissue. Use of spectral imaging enables the simultaneous identification of as many as five different populations of cells by multiphoton laser excitation. Use of Quantum Dots for Labeling HER2 Molecular defects in cancer can potentially be linked to specific drug sensitivities. Such correlations might guide the selection of drugs for therapy based on the molecular characteristics of individual tumors. An example is the treatment of breast cancer with trastuzumab (Herceptin), a humanized monoclonal antibody against the HER2 receptor. HER-2 overexpression, a predictive marker of tumor aggressiveness and responsiveness to therapy, occurs in 20-30% of patients with breast cancer. Molecular diagnostics tests are available for detection of this but greater refinement is desirable for improving the accuracy of testing. QDs, coated with a polyacrylate cap and covalently linked to antibodies or to streptavidin, have been used for immunofluorescent labeling of HER2 (9). This labeling is highly specific

3 Nanobiotechnology in Personalized Medicine for Cancer 647 and is brighter and more stable than that of other fluorescent markers. Recent advances have led to QD bioconjugates that are highly luminescent and stable. These bioconjugates open up new possibilities for studying genes, proteins and drug targets in single cells, tissue specimens and even in living animals and enable visualization of cancer cells in living animals. Use of trastuzumab in prostate cancer has also been explored but monotherapy has not been significantly effective. There is lack of a sensitive marker for evaluating the response to therapy. HER2 and a method for detecting protein analytes has been developed that relies on magnetic nanoparticle probes with antibodies that specifically bind a target of interest such as PSA (prostate specific antigen) in case of prostate cancer. There is substantial amplification and PSA can be detected at 30 attomolar concentration. Alternatively, a polymerase chain reaction on the oligonucleotide bar codes can boost the sensitivity of detection of PSA to 3 attomolar (10). Gold Nanoparticles for Cancer Diagnostics Gold nanoparticles can be conjugated to monoclonal antiepidermal growth factor receptor (anti-egfr) antibodies after incubation in cell cultures with malignant as well as nonmalignant epithelial cell lines. However, the anti-egfr antibody-conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type 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 (SPR) absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells (11). The optimal size of the particles for this application is approximately 35 nm. SPR scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro. Advantages of this technique are: Conjugated gold nanoparticles are not toxic to human cells whereas QDs using semiconductor crystals to mark cancer cells are potentially toxic. The technique requires only a simple, inexpensive microscope and white light to view the results. If a cancerous tissue is sprayed with gold nanoparticles containing the antibody, the results can be seen immediately. In a novel probe, QDs are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to suppress luminescence and signal amplification occurs upon interaction with a targeted proteolytic enzyme (12). A 71% reduction in luminescence is achieved with conjugation of AuNPs to QDs and release of AuNPs by peptide cleavage restores QD photoluminescence. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker and may be useful for imaging in cancer diagnosis. Gold nanoparticles have been investigated mostly in animal cancer models. Although they have a low toxicity, further safety studies are needed before they can be used in humans. Molecular Imaging of Cancer In investigational and clinical oncology there is a need for imaging technologies that will indicate response to therapy prior to clinical evidence of response. The conventional imaging methods such as CT and MRI enable anatomic measurements of the tumor. This may be useful for assessing response to traditional cytotoxic agents where tumor shrinkage occurs early. In contrast to this, molecularly targeted agents tend to induce arrest of cancer cell growth and development, but not necessarily significant tumor shrinkage in the short term. Thus there is a need for functional or molecular imaging methods that would give information about what is happening in the tumor at the molecular level. Nanobiotechnology-based Diagnostics Combined with Therapeutics It is well established that nanoparticles can be used both for the diagnosis and delivery of therapeutic agents to tumors. A polymerized nanoparticle platform technology can enable the placement of different targeting moieties on the surface of particles in addition to loading the particles with different contrast and therapeutic agents. It was demonstrated that these nanoparticles can be targeted to endothelial receptors and different payloads of contrast and therapeutic agents were delivered to target cells with high target to background ratios (13). Use of this combined imaging and therapy approach, would facilitate the development of personalized therapy for cancer. Alphanubeta3-targeted paramagnetic nanoparticles have been employed to noninvasively detect very small regions of angiogenesis associated with nascent melanoma tumors (14). Each particle is filled with thousands of molecules of the metal that is used to enhance contrast in conventional MRI scans. The surface of each particle is decorated with a substance that attaches to newly forming blood vessels that are present at tumor sites. This enables the detection of sparse biomarkers with molecular MRI in vivo when the growths are still invisible to conventional MRI. Earlier detection can potentially increase the effectiveness of treatment, particularly in case of melanoma. Another advantage of this approach is that the same nanoparticles used to detect the tumors can be used to deliver stronger doses of anticancer drugs directly to the tumor site without systemic toxicity.

4 648 Jain The nanoparticle MRI would enable physicians to more readily evaluate the effectiveness of the treatment by comparing MRI scans before and after treatment. This fulfills some of the important components of personalized cancer therapy: early detection, combination of diagnostics with therapeutics and monitoring of efficacy of therapy. Role of Nanoproteomics in Personalized Cancer Therapy Clinical proteomics involves the application of proteomic technologies at the bedside, and cancer is a model disease for studying such applications. Oncoproteomics is the term used for application of proteomic technologies in oncology. Role of proteomics, an important factor in personalized management of cancer, has been described elsewhere (15). Proteomic technologies are being developed to detect cancer earlier, to discover the next generation of targets and imaging biomarkers, and to tailor the therapy to the patient. Proteomic technologies will be used to design rational drugs according to the molecular profile of the cancer cell and thus facilitate the development of personalized cancer therapy. Nanotechnology has refined proteomics and this can justify the use of the term nanoproteomics as it makes it possible to analyze as little as picrogram amounts of proteome samples by minimizing sample handling and maximizing peptide recovery (16). Examples of application of nanotechnology that will facilitate the development of personalized medicine are given in the following paragraphs. Nanoscale Protein Analysis Most current protocols including protein purification/display and automated identification schemes yield unacceptably low recoveries, thus limiting the overall process in terms of sensitivity and speed and require more starting material. Low abundant proteins and proteins that can only be isolated from limited source material (e.g., biopsies) can be subjected to nanoscale protein analysis enabling analysis of low nanogram level proteomic samples with individual protein identification sensitivity at the low zeptomole level (17). Nanoproteomics can be used to identify how genetic determinants of cancer alter cellular physiology and response to agonists. Role of Nanotechnology in Discovery of Personalized Medicines for Cancer Technical achievements in nanotechnology are being applied to improve drug discovery and some of these are relevant to personalized medicine (18). Miniature devices are being constructed to study synthetic cell membranes in an effort to speed up the discovery of new drugs for a variety of diseases, including cancer. Cell membranes contain a variety of proteins some of which act as tiny pumps that quickly remove chemotherapy drugs from tumor cells, making the treatment less effective. Research aims to find drugs that deactivate the pumps and make chemotherapy drugs more effective. A chip constructed for research in this area measures about 1 cm 2 and holds thousands of cylindrical cavities that are open at the top but sealed at the bottom with alumina (19). The bottom is a nanoporous material, i.e., it contains numerous pores measured in nanometers. The goal is to produce laboratories-on-a-chip that might contain up to a million test chambers, or reactors, each capable of screening an individual drug. The chips could dramatically increase the number of experiments that are possible with a small amount of protein. Some nanomaterials are drug candidates in themselves, e.g., dendrimers and nanobodies. Dendrimers as Anticancer Drugs Dendrimers are a novel class of three-dimensional nanoscale, core-shell structures that can be precisely synthesized for a wide range of applications. Specialized chemistry techniques enable precise control over the physical and chemical properties of the dendrimers. They are most useful in drug delivery but can also be used for the development of new pharmaceuticals with novel activities. Polyvalent dendrimers interact simultaneously with multiple drug targets. They can be developed into novel targeted cancer therapeutics. Dendrimers can be conjugated to different bio-functional moieties such as folic acid using complementary DNA oligonucleotides to produce clustered molecules, which target cancer cells that overexpress the high affinity folate receptor (20). Nanobodies as Anticancer Agents Nanobodies (Ablynx) are the smallest available intact antigen-binding fragments harboring the full antigen-binding capacity of the naturally occurring heavy-chain antibodies. Nanobodies have the potential of a new generation of antibody-based therapeutics as well as diagnostics for diseases such as cancer (21). Nanobodies have high target specificity and low inherent toxicity. They can address therapeutic targets not easily recognized by conventional antibodies such as active sites of enzymes. Nanodies have the potential to be developed as personalized medicines for cancer. Role of Nanotechnology-based Cancer Drug Delivery in Developing Personalized Therapy Various nanotechnology-based methods of drug delivery in cancer have been reviewed recently (22). Targeted drug delivery and specific receptor binding are important aspects of personalized management of cancer. Two examples are given here. The synthetic peptide bearing Arg-Gly-Asp (RGD) sequence is considered to specifically bind to avb3 integrin expressed on endothelial cells in the angiogenic blood vessels, which

5 Nanobiotechnology in Personalized Medicine for Cancer 649 provides a potential to inhibit the tumor growth. Hydrophobically modified glycol chitosan (HGC), capable of forming nano-sized self-aggregates, is used as a carrier for the RGD peptide, which is labeled with fluoresein isothiocyanate (FITC-GRGDS) and loaded into self-aggregates by solvent evaporation methods (23). The self-aggregates loaded with FITC-GRGDS might be useful for monitoring or destroying the angiogenic vessels surrounding the tumor tissue. Smart superparamagnetic iron oxide particle conjugates can be used to locate brain tumors earlier and more accurately than current methods and to target the tumors (24). Use of folic acid combined with PEG can further enhance the specific targeting capability of the nanoparticles and enhance their intracellular uptake. A variety of small molecules targeting tumor receptors and even chemotherapy agents can be attached to the nanoparticles. Role of Nanotechnology in Personalizing Gene Therapy of Cancer Gene therapy can be broadly defined as the introduction of genetic material (genetically engineered cells, genes, DNA) into the body and/or other substances for regulation of gene function. The cells may be genetically modified to secrete therapeutic substances. Ex vivo gene therapy involves the genetic modification of the patient s cells in vitro, mostly by use of viral vectors, prior to reimplanting these cells into the tissues of the patient s body. Some cancer vaccines are made from the cells of the patient s tumor. This is a form of individualized therapy. Nanotechnology has been used to refine gene therapy of cancer, which can also be viewed as a sophisticated form of drug delivery. Nanoparticles have been used for p53 gene therapy of cancer and an intravenous nanoparticle formulation of the tumor suppressor gene FUS1 has been tested in experimental animals. Examples of other techniques are integrin-targeted nanoparticles for site-specific delivery and immunolipoplex. Targeted Site-specific Delivery of Anticancer Genes by Nanoparticles A technology has been described that is based on integrintargeted nanoparticles for site-specific delivery of a therapeutic payload by using an anticancer gene (25). This technology provides a carrier particle that is only nm in size but can still carry a large number of therapeutic/targeting molecules. The particles attach to multiple disease-specific receptors on targeted cell surfaces. This payload carried by a nanoparticle can be encapsulated within the nanoparticle or attached to the surface, and can include radiation, therapeutic drugs, cytotoxic drugs, DNA, et cetera. In those cases where the targeting particle binds to the receptor but has suboptimal efficacy, the ability to deliver a payload can offer an important therapeutic advantage. This can also be an important complementary product for combination therapy. Immunolipoplex for Delivery of p53 Gene A sterically stabilized immunolipoplex, containing an antitransferrin receptor single-chain antibody fragment-peg molecule, has been developed to specifically and efficiently deliver a therapeutic gene to tumor cells (26). Lipoplex nanoparticles resembling virus particles can penetrate deeply into the tumor and move efficiently into cells. The immunolipoplex is spiked on the outside with antibody molecules that will seek out, bind to, and then enter cancer cells including metastases wherever they hide in the body. These molecules bind to the receptor for transferrin that is present in large numbers on cancer cells. Once inside, the immunolipoplex will deliver its payload, the p53 gene, whose protein helps to signal cells to self-destruct when they have the kind of genetic damage characterized by cancer and by cancer therapies. Immunolipoplex has shown promising results in animal tumor models and a phase I clinical trial in patients with advanced solid cancers is due to start later in Immunolipoplex-based gene transfer represents an advance over the viral vectors that have been used to deliver gene therapy, because these liposomes do not produce the immunologic response seen when disabled viruses are used to carry the payload. Immunolipoplex also substantially improves the anticancer effects of both chemotherapy and radiation therapy. These agents work synergistically with traditional therapies because the restoration of p53 protein helps push cancer cells that are now damaged to selfdestruct. This approach will make it difficult for the cancer cells to become resistant to therapy, and will be less likely to recur after therapy is complete. Concluding Remarks and Future Prospects Personalized medicine is beginning to be recognized and is expected to become a part of medical practice within the next decade. However, not all treatments need to be personalized and this applies to cancer as well. For cancer it would be important to match the right therapy to the right type of cancer taking into consideration the patient s individual characteristics as well. The treatments would be targeted to increase efficacy and reduce toxicity. As nanobiotechnologies advance, one of the important areas of application include diagnosis and drug delivery in cancer. As miniaturization continues to be integrated into the practice of medicine, it is within the realm of possibility to use molecular tools to design a miniature device that can be introduced in the body, locate and identify cancer cells and finally destroy them. The device would have a nanobiosensor to identify

6 650 Jain cancer cells and a supply of anticancer substance that could be delivered by nanotechnology-based methods on encountering cancer cells. A small computer could be incorporated to program and integrate the combination of diagnosis and therapy and provide the possibility to monitor the in vivo activities by an external device. Since there is no universal anticancer agent, the computer program could match the type of cancer to the most appropriate agent available. By the time cancer has produced signs and symptoms and is diagnosed, it is already late. The ideal would be to detect cancer before clinical manifestations. A nanodevice as envisaged could be implanted as a prophylactic measure in persons who do not any obvious manifestations of cancer. It would circulate freely and could detect and treat cancer at the earliest stage. Such a device could be reprogrammed through remote control and enable change of strategy if the lesion encountered is other than cancer. 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