/ cos
2.3 Applications in the Biomedical Field
Figure 2.10 shows a schematic drawing and an image of a spherical MNP consisted of a magnetic core with biocompatible coating for biomedical applications. MNP, a nanometer-scaled ferromagnetic material, when surface is coated by such coatings of particular drugs or diagnostic
29 reagents, is known as magnetic marker or magnetic tracer. It is useful for a wide range of applications from data storages to motors. In addition, it also offers large number of attractive possibilities to be used extensively in the biomedical fields [4]-[5].
For biomedical applications, MNPs can be used for therapy, e.g. drug delivery and hyperthermia/thermal ablation. On the other hand, they can be used for diagnostics, e.g. in vivo diagnosis such as MRI & MPI, and in vitro diagnosis such as immunoassays, biosensor, cell sorting.
Some features of the special physical properties of MNPs made these potentials feasible for biomedical applications.
First, their controllable sizes ranging from a few nanometers up to tens of nanometers makes them fix dimensions that are smaller or comparable to many biological particles such as cells (10-100 μm), viruses (20-450 μm), proteins (5-50 μm), or genes (2 nm wide and 10-(10-100 μm long). They can get attached closely to any biological entity of interest. Then, they can also be coated with biological molecules to make them interact with or bind to a biological entity, thereby providing a controllable means of tagging/labeling/addressing it.
Second, because of the magnetic behavior of the MNPs, they obey Coulomb’s law and can be easily manipulated by an external magnetic field gradient from far separated distance. When combining this action together with the intrinsic penetrability of magnetic fields into human tissues, many applications involving the transport and/or immobilization of MNPs, or of magnetically tagged biological entities can be emerged. This leads to their function as drug delivery, such as an anticancer drug, or a cohort of radionuclide atoms, to a targeted region of the body, such as a tumor.
Third, the MNPs can be made to resonantly respond to a time-varying magnetic field, with advantageous results related to the nanoparticles. For example, the particle can be made to heat up, which makes them available as hyperthermia agents, to deliver toxic amounts of thermal energy to targeted bodies such as tumors, or as chemotherapy and radiotherapy enhancement agents, where a moderate degree of tissue warming results in more effective malignant cell destruction.
There are many types of MNPs products that have been developed for biomedical applications:
Resovist® (Fujifilm RI Pharma), EndoremTM (Guerbet S. A.), Feraheme® (AMAG Pharmaceuticals), Nanotherm® (MagForce Nanotechnologies) etc. Instead of some are already commercially available on the market for clinical use, however, majorities of them are still remaining at the development level. A few limitations for the clinical translation have to be exceeded such as the Food and Drug Administration (FDA) approval, the toxicity issues, resource availability, etc.
In the next section, the use of MNPs in biomedical field is classified into four concepts: drug delivery, hyperthermia, immunoassay, and imaging (MPI), and is discussed in details.
30 (a) (b) Fig. 2.10: (a) Schematic drawing and (b) an image of a spherical MNP consists of a magnetic core
with biocompatible coating for biomedical applications.
2.3.1 Drug Delivery
The major disadvantage of most chemo-therapies used for tumor treatment is that they are relatively non-specific. Drugs for the therapy purpose will go through the process of absorption, distribution, metabolism, and eventually be cleared from the body through a process of excretion, once they are administered intravenously into the systemic. Harmful side-effect can be yielded from the normal chemo-therapy method as drug attacks normal and healthy cells simultaneously with the targeted tumor cell. For instance, the side effect of anti-inflammatory drugs on patients who have chronic arthritis can lead to the discontinuation of their use. However, if such treatment could be localized, the continued use of these very potent and effective agents could be made possible.
Recognition of this led researches in the late 1970s to propose the use of MNPs as magnetic carriers to target specific sites of cancerous tissues within the body [6]. The objectives are to reduce the amount of systemic distribution of the cytotoxic drug, thus reducing the associated side-effects, and to reduce the dosage required by more efficient, localized targeting of the drug.
In magnetically targeted therapy, a cytotoxic drug is attached to a biocompatible MNP carrier.
These drug/carrier complexes-usually in the form of a biocompatible ferrofluid, are injected into the patient via the circulatory system. When the MNPs have entered the blood vessel, high-gradient magnetic fields are externally applied to concentrate the drug/carrier complex at a specific target site within the body, as shown in Fig. 2.11. Once the drug/carrier is concentrated at the target, the drug can be released either via enzymatic activity or changes in physiological conditions such as pH, osmolality, or temperature, and be taken up by the tumor cells [7].
Magnetic targeting therapy is developed based on the physical principles that are derived from the magnetic force exerted on a superparamagnetic nanoparticle by a gradient magnetic field, as given by
31
0 2
μ χ 2
Δ B
V
F
m m (2.22)Or,
V BH
Fm m .
2 χ 1
Δ (2.23)
This effectiveness of the therapy is dependent on several physical parameters: field strength, gradient and volumetric, and magnetic properties of the MNPs.
In general, large particles are more effective at withstanding flow dynamics within the circulatory system-particularly in larger veins and arteries. In most cases the magnetic field gradient is generated by a strong permanent magnet, such as Nd-Fe-B, fixed outside the body over the target site. Preliminary investigations of the hydrodynamics of drug targeting suggest that for most magnetite-based carriers, flux densities at the target site must be of the order of 0.2 T with field gradients of approximately 8 Tm-1 for femoral arteries and greater than 100 Tm-1 for carotid arteries.
This suggests that targeting is likely to be most effective in regions of slower blood flow, particularly if the target site is closer to the magnet source.
Since the first magnetic carrier introduced in the 1970s, a variety of MNPs and microparticles carriers have been developed to deliver drugs to specific target sites in vivo, and the optimization of these carriers continues today. Generally, the magnetic component of the particle is coated by a biocompatible polymer such as PVA or dextran, although recently inorganic coatings such as silica have been developed. The coating acts to shield the MNP from the surrounding environment and can also be functionalized by attaching carboxyl groups, biotins, avidin, carbodi-imide, etc. [8]. These molecules then act as attachment points for the coupling of cytotoxic drugs or target antibodies to the carrier complex.
There are two types of structural configurations of magnetic carrier: (1) a magnetic core (usually magnetite or maghemite) coated with a biocompatible polymer, or (2) a porous biocompatible polymer in which MNPs are precipitated inside the pores. Recent works related on carrier development have largely focused on new polymeric or inorganic coatings on magnetite/maghemite nanoparticles, although metal coatings such as gold are also being considered. Research also continues into alternative magnetic particles such as iron, cobalt, or nickel.
Magnetic carriers were first used to target cytotoxic drugs to sarcoma tumors implanted in rat tails. Since that study, success in cytotoxic drug delivery and tumor remission has been reported by several groups using animals models including swine, rabbits, and rats, even though the studies of magnetic targeting in humans is still rare up to now.
Several problems limit the usage of MNPs in the magnetically targeted drug delivery technique:
the possibility of blood clot in the target region due to accumulation of the magnetic carriers;
32 difficulties in scaling up from animal models due to the larger distances between the target site and the magnet; once the drug is released, it is no longer attracted to the MNPs; toxic responses to the magnetic carriers. Further pre-clinical research and experiment should be done to overcome these limitations and use magnetic targeting to improve drug retention and also address safety issues.
Fig. 2.11: A hypothetical magnetic drug delivery system shown in cross-section: a magnet is placed outside the body so that its magnetic field gradient might capture magnetic carriers flowing in the
circulatory system.
2.3.2 Hyperthermia
Hyperthermia is a type of medical treatment of localized heating the body tissue to 42-46°C using MNPs to kill or damage tumor cells. One of the principal motivations of this technique is supported by the fact that tumor cells are more heat sensitive, compared to normal tissue, suggesting localized hyperthermia as mode of tumor treatment. Blood vessel in healthy tissue expands when is heated and cools the surrounding cells. This does not occur in cancerous tissue, resulting in difficulty in dissipating heat. Therefore, it is possible to kill selectively cancer cells by selectively heating the cancer tissue surrounding. Hyperthermia may differ from thermoablation, which employs higher temperatures up to 56°C.
Whole body hyperthermia method has been used as the conventional method typically to treat metastatic cancer that has already spread towards many parts of the body. This method heats the entire body to temperatures of about 39-41°C. Techniques involved in whole body hyperthermia include the infrared hyperthermia domes which include the whole body apart from the head, putting the patient in a very hot room, or wrapping the patient in hot, wet blankets. While effective to skin cancer therapy, it cannot be used in deep-lying cancer cells due to the self-heating process of the deep part of tissue itself starts from the surface of the body. Therefore noninvasive magnetic hyperthermia using magnetic nanoparticles has been studied in order to be applied to deep-lying cancerous tissue.
The principle of magnetic hyperthermia is based on the heat generated as hysteresis loss by MNPs interacted with an electromagnetic field. The heat is employed to achieve local heat
33 generation in tissue to kill cancer cells (Fig. 2.12). When a high frequency modulating magnetic field is applied, the magnetization of a superparamagnetic nanoparticle lags behind the applied field.
As a result of this phase lag, the magnetic susceptibility, χ can be given by
''
'
χ
χ
χ i
, (2.24)consisted of a real part, χ', representing the in-phase component, and the imaginary part, χ'', representing the loss-component. χ'and χ'' are given by
0 2'
ωτ ω 1
x
x
(2.25)
0 2''
ωτ 1 ω ωτ
x
x (2.26)
where χ0is the DC magnetic susceptibility. The value of χ'decreases with increasing frequency while χ''exhibits a peak at an angular frequency ofω2πf 1/τ. Here, τ is the effective relaxation time of the MNPs as described in eq. (2.16). The specific loss power for a monodisperse sample of superparamagnetic nanoparticles can be expressed as
f H
P πμ
0 02χ
'' . (2.27)Experimental investigations of magnetic hyperthermia date back to 1957 when Gilchrist et al.
heated various tissue samples with 20-100 nm size maghemite particles exposed to a 1.2 MHz magnetic field [9]. Since then, numerous studies have been explored describing MNPs suitable for hyperthermia and its effectiveness for the treatment of cancer. The major advantages of this application is that MNPs produce zero toxicity to living organisms, the possibility of chemical modifications to the MNPs, MNPs can be administered by injection, MNPs can be integrated in high concentrations to the affected area , and it effectively absorbs the energy of high-frequency magnetic field to generate heat. Therefore, magnetic hyperthermia as a non-invasive method can be seen as a promising cancer treatment in the future.
Fig. 2.12: Magnetic hyperthermia.
Cancer cells MNPs
Heat AC field
34 2.3.3 Immunoassay
An immunoassay is a biochemical test used in a wide field of biotechnology measurement, e.g.
for the detection of pathogens and cancer cells, DNA gene analysis, detection of environmental toxins, and immune response. The immune response is obtained by measuring the binding reaction between a particular bio-substance to be measured, referred as antigen, and the diagnostic reagent that selectively bind to that antigen, referred as antibody. Immunoassay relies on the ability of the antibody to recognize and bind a specific antigen in order to measure the type and amount of the antigen in vitro.
Another key feature of all immunoassays in addition to the binding of an antibody to its antigen is that to develop a means to produce a measurable signal in response to the binding. Generally, immunoassays involve chemically linking antibodies or antigens with some kind of detectable label.
A variety of different labels are employed to allow for detection of antibodies and antigens: enzymes, radioactive isotopes, DNA reporters, fluorogenic reporters, and electrochemiluminescent tags. Each of these labels utilizes different methods for detection. Most of the labels are detectable because they either emit radiation from radioactive isotopes that can be incorporated into in immunoassay reagents to produce a radioimmunoassay (RIA), or produce a color change in a solution for enzyme-linked immunosorbent assays (ELISA) or sometimes known as enzyme immunoassays (EIAs), or fluoresce under light for fluorogenic reporters, or because they can be induced to emit detectable light from electrochemiluminescent tags in response to electrical current.
Magnetic immunoassay is a novel type of diagnostic immunoassay using magnetic marker as label instead of conventional labels as have been mentioned previously. As shown in Fig. 2.10, magnetic marker is made of MNPs encapsulated or glued together with polymers. In magnetic immunoassay, it involves the specific binding of an antibody to its antigen, where a magnetic marker is conjugated to one element of the pair. The presence of magnetic marker is then detected by a magnetometer which measures the magnetic field change induced by the magnetic marker. The signal measured by the magnetometer is proportional to the antigen quantity in the sample to be measured.
Magnetic markers exhibit several features very well suited for immunoassay. First, the magnetic properties are not affected by reagent chemistry or photo-bleaching, therefore, stable over time. Then, the magnetic background in a biological sample is usually insignificant as they are weakly diamagnetic. They are not screened by aqueous reagents or biomaterials, therefore turbidity or staining in sample have no impact on magnetic properties, thus eliminating any interference signals.
Finally, magnetic markers can be manipulated remotely by magnetism.
In recent years, request of detecting small amount of antigen-antibody binding reaction at high speed with high sensitivity becomes in demand. However, the challenge of developing an effective
35 magnetic immunoassay system is to separate ambient magnetic noise from the weak signal of the magnetically labeled target. Hence, various testing devices utilizing different approaches and instruments have been developed to address the problem. For instance, giant magnetoresistive sensors and spin valves, piezoresistive cantilevers, inductive sensors, superconducting quantum interference devices (SQUID), anisotropic magnetoresistive rings, and miniature Hall sensors have been employed to achieve a significant signal-to-noise ratio (SNR) for bio-sensing applications.
Figure 2.13 shows an example of biological immunoassay utilizing SQUID sensor and magnetic marker developed by Enpuku et al. [10]. By using magnetic marker as label, immunoassay method can be approached with comparable high sensitivity detection with conventional method of optical marker method, however with more rapid detection as the time-consuming pre-requisite bound/free process in regular immunoassay is not involved. Besides, binding reaction can be detected in a wider dynamic range.
Fig. 2.13: Principle of magnetic immunoassay detected by a SQUID magnetometer.
2.3.4 Magnetic Particle Imaging (MPI)
In medical imaging, various imaging tools that use a variety of electromagnetic radiations exist.
For instance, Positron emission tomography (PET) using high energy γ-rays radiation, computed tomography (CT) using x-rays radiation, and magnetic resonance imaging (MRI) using radio-waves radiation. Here, contrast agents and tracers are known to play significant role in providing critical information for diagnostics and therapy. Spatial resolution and detection sensitivity are the two inter-related keywords that define the applicability of the medical imaging tools existed.
For instance, while producing a high spatial resolution 3D image without use of ionizing radiation, detection sensitivity in MRI is limited by the background signal from the host tissue [11].
Alternatively, measurements of the magnetic relaxation of MNPs as novel tool for high-resolution in vivo diagnostics, while affective, is associated with difficulties in back transforming the data to retrieve a high spatial resolution image [12]. Therefore, magnetic particle imaging (MPI) is proposed as a new imaging tool to overcome these limitations by employing the magnetization response of SPIO nanoparticles to generate a tomographic image. The image is characterized qualitatively by both high spatial resolution which is comparable to that of CT image, and high sensitivity as good as
36 PET image. Besides, no tissue contrast affects the imaging method. 3D-high resolution-real time imaging can be provided without ionizing radiation.
References
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