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Diagnostic and Therapeutic Systems Using Nanomaterials

There is no standard therapeutic process, since there are so many different schools of therapy.

David D. Burns

1.1 Introduction

Both this book and chapter deal with therapy for various human disorders. Therapy is a word that is used for medical- or health-related treatments intended to relieve disorders. Hence, treatment and therapy can be considered synonyms Therapeutic remediation is associated with or depends on the diagnosis of health problems. The word "theranostic" is used to describe the combined efforts of diagnosis and therapy. Diagnostic and therapeutic devices or methods are often developed together. Useful devices are developed by working closely with the medical scientists who give input into a medical situation. For example, for oncotherapy, developing a therapy that is image guided so as to give localized treatment, especially for tumors and cancer; or image-guided catheters to inject chemotherapy directly into the blood vessels that feed a tumor, reducing the need for systemic chemotherapy.

In this chapter we will be discussing nanotherapy, which is a branch of nanotechnology that uses biocompatible nanoparticles as follows:

1. (i) To deliver a drug to a given target location in the body so as to treat the disease through a process known as targeted drug delivery [1];
2. (ii) For photodynamic chemotherapy [2];
3. (iii) Antibody-conjugated nanoparticles as a theranostic agent for ultrasound contrast imaging [3];
4. (iv) Targeted near-infrared (NIR) nanoparticles for photothermal therapy [4]; and
5. (v) Anticancer or oncotherapy by facilitating cancer cell apoptosis [5].

Drug delivery using nanoparticles and nanoconjugates as well as developing diagnostic devices using nanodiagnostic agents are the major areas of research related to the nanotherapeutic system.

Nanotechnology is already being used for early disease detection and diagnosis, treatment and prevention of disease, and precise and effective therapy [6]. Nanotechnology and various nanoparticles have exhibited potential for detecting disease indicators and markers of early precancerous cells, viruses, specific proteins and antibodies.

Nanodiagnostics involves molecular diagnosis, which helps in developing personalized cancer therapy. A nanodiagnostic device is based on pharmacogenetics, pharmacogenomics, and pharmacoproteomic information as well as environmental factors, which influence response to therapy. In the future, with the advancement in nanodiagnostic technologies, it will be a faster way of doing complete health checkups. Moreover, it will help in tailoring the required medication specifically to the individual based on their genetic makeup, which will prevent unwanted side effects.

1.2 Nanodiagnostic Agents

Nanodiagnostics involves molecular diagnosis, which is mostly used for personalized cancer therapy. This field is currently under development and being researched the world over. It involves the use of nanoparticles (carbon nanotubes, nanoshells, gold, and other metallic nanoparticles, nanopores, graphene, and cantilevers), quantum dots (semiconductor nanocrystals, exhibiting strong light absorbance property that can be used as fluorescent labels for biomolecules), etc., for rapid diagnostic tests and nanorobots as tools to make repairs at the cellular level. Personalized medicine for cancer therapy needs the desired biomarkers. Hence, another very important input of nanodiagnostic technologies that is being envisaged is to refine the discovery of biomarkers using nanoparticles because they have high surface area to volume ratio (SVR) and multifunctionality. nanodiagnostic approach will be future point-of-care (POC) diagnostics and monitoring technologies with the help of nanobiosensors and microarrays of biosensors-based biochip systems and microfluidic platforms for rapid diagnostic tests and rapid detection of various diseases or pathogen-specific biomolecules/markers, such as DNA, proteins, whole cells (e.g., circulating tumor cells), etc. The nanotools will offer the fabrication of small-scale portable devices.

The nanodiagnostic agents that are being researched are discussed in the following subsections for their applicability.

1.2.1 Bio-Barcode Assay (BCA)

Bio-barcode assay is already being used as a tool for rapid detection of protein-specific antigen (PSA), which is a marker for prostate and breast cancers [7]. It offers increased sensitivity and safety. For BCA assay, two different probes are used: (i) a magnetic microparticle conjugated to a PSA monoclonal antibody, and (ii) a gold nanoparticle to which a PSA polyclonal antibody and "barcode" oligonucleotides are attached. During the assay, PSA gets conjugated to gold nanoparticles (second probe) and becomes sandwiched between the two antibodies. Then, using magnet microparticle the antigen-containing complex is separated from the rest of the mixture. Here, the magnetic field draws the unbound magnetic microparticles to the walls of the container. The remaining particles that are left behind are involved in the detection. The separated complex is washed so as to de-hybridize the barcode oligonucleotides from the nanoparticle. The free oligonucleotides enable the detection of PSA. Using conventional DNA detection methods, barcode is detected. It is also detected by silver amplification method [8]. By this method, as low as 30 attomole/L in a 10 µL sample has been detected [7]. The sensitivity of this assay can be increased by manipulating the equilibrium of the reaction by changing the concentration of the magnetic microparticle probe (Figure 1.1).

1.2.2 Cantilever Beam

Cantilevers are a nanomechanical tool used in diagnostics. A cantilever beam is a beam which is anchored or fixed at only one end and the other end is free (Figure 1.2). The fixed end entirely resists the moments and shear generated by the loads acting on the beam. Beam that is fixed at one end cannot rotate or translate in the direction that load is applied. Whereas, the other end is free to rotate and translate in the direction of the applied load. Micromachined silicon cantilever beams are similar to those used in atomic force microscopy, function by use of nanomechanical deflections. Because the beam is anchored only on the one end, thermal expansion and ground movement are fairly simple to sustain.

Figure 1.1 Barcode assay of PSA using nanoparticles. A schematic representation of the PSA AuNP probes (upper); and the PSA bio-barcode assay (lower).(Upper) Barcode DNA-functionalized AuNPs (30 nm) are conjugated to PSA-specific antibodies through barcode terminal tosyl (Ts) modification to generate the coloaded PSA AuNP probes. In a second step, the PSA AuNP probes are passivated with BSA. (Lower) The bio-barcode assay is a sandwich immunoassay. First, MMPs surface-functionalized with monoclonal antibodies to PSA are mixed with the PSA target protein. The MMP-PSA hybrid structures are washed free of excess serum components and resuspended in buffer. Next, PSA AuNP probes are added to sandwich the MMP-bound PSA. Again, after magnetic separation and wash steps, the PSA-specific DNA barcodes are released into solution and detected using the scanometric assay, which takes advantage of AuNP catalyzed silver enhancement. Approximately ½ of the barcode DNA sequence (green) is complementary to the "universal" scanometric AuNP probe DNA, and the other ½ (purple) is complementary to a chip-surface-immobilized DNA sequence that is responsible for sorting and binding barcodes complementary to the PSA barcode sequence. (Source: Scheme 1 from C. Shad Thaxton et al., Nanoparticle-based bio-barcode assay redefines "undetectable" PSA and biochemical recurrence after radical prostatectomy. PNAS; 106(44): 18437-19442, 2009. Open access article ©2009 National Academy of Science).

Figure 1.2 A cantilever beam.

At present, cantilevers are one of the most promising technologies for clinical diagnosis. The micromachined silicon cantilevers are now being used for protein and DNA detection and quantification. The advantage of using a cantilever beam is that there is no toxicity concern. A standard cantilever biosensor can detect multiple target molecules from small biological samples especially for the detection of cancer at an early stage. Once a tumor becomes malignant, it is too late to treat the cancer with a maximum success rate. Since cancers spread through blood and particularly the lymphatic system in the body, it becomes important to measure multiple parameters of biological molecules. In a cantilever, biosensor biomolecules are adsorbed onto one side of the surface of the cantilever, causing a decrease in the surface free energy and generating a differential surface stress between either side of the cantilever beam as a result of adsorption of biomolecules occurring at one side of the cantilever. For DNA detection, the cantilever surface holds a particular DNA sequence capable of binding to a specific target. For DNA detection, at one side of the cantilever layer hybridization occurs between the target probes, changing the intermolecular interactions within a monolayer, which induces surface stress that bends the cantilever beam and initiates a motion (Figure 1.2). The deflection of the cantilever caused by surface stress change, which is in the range of several nanometers, is measured using a piezoelectric readout.

Based on the same principle, specific DNA...