• SAMRAT MAZUMDAR

Abstract

Cancer is by far one of the most challenging diseases for centuries. In the US, it accounts for over a million deaths annually and is expected to rise in the coming future. Therefore, there is vital need to develop novel strategies, which can help in combating the disease at any level. Metallic nanoparticles present an interesting view, which can function as both therapeutic and diagnostic agents due to their unique properties. The main motive of the proposed work is development of gadolinium based magnetic nanoparticles, followed by their surface functionalization which may improve imaging and targeting outcomes. Doped Gadolinium nanoparticles will be prepared by co-precipitation method for optimum magnetic properties. The synthesized particles will be subjected to functionalization with suitable group for specific target in nature for cancer cells. Eventually, in-vitro studies will be carried out to validate the hyperthermia effect on cancer cells.

1. Introduction

Overview

Although, it is difficult to define cancer, but in simple terms, it is a group of related diseases which is characterized by uncontrolled cell proliferation and spread, mostly due to loss of control in the cell cycle (Pérez-Herrero and Fernández-Medarde, 2015). The most commonly detected cancers are lung cancer, breast cancer and skin cancer, etc. A variety of factors contributes to the disease progression, such as genetic changes, infections and exposure to carcinogens. In general, cancer is detected/diagnosed by various techniques like, blood tests, X-ray imaging, Computed Tomography (CT) scanning and Endoscopy etc. Conventional treatment strategies include surgery, chemotherapy and radiation therapy. However, they possess numerous limitations especially dose-related side effects and toxicity (Brigger et al., 2002). Currently, researchers are looking towards newer approaches which are selective, non-invasive, non-toxic and effective. These efforts are led to the development of experimental cancer therapies. These not only improves the curing rate but also, act as a supplement to the conventional therapies. However, it is still early to state that these alternatives can completely replace the existing treatment strategies and its effectiveness in clinical settings, are yet to be determined.

Alternative approaches include Gene therapy (Vile et al., 2000), Photodynamic Therapy (PDT) (Dougherty et al., 1998), Hyperthermia (Urano, 1999) ,Targeted Nano-medicines (Xu et al., 2015). Recently, a tremendous amount of research is being carried out in the field of hyperthermia due to encouraging results and its potential for significantly lowered toxicity.

Hyperthermia

“Hyperthermia” is a very ancient technique which is now regaining popularity in the field of oncology (Seegenschmiedt and Vernon, 1995). It involves the use of heat energy to elevate the temperature inside a tumour tissue and subsequently kill the cancer cells. The desired temperature range for hyperthermia is 42°-44°C which is, greater than the physiological temperature (Wust et al., 2002).There is a variety of factors governing the effectiveness of hyperthermia which includes thermal variables, device characteristics, frequency, current and tumour morphology (Valdagni et al., 1988). At temperatures below 41°C, blood flow increases while tissue oxygenation increases above 41°C providing a dual effect against tumour. Once temperatures are increased above 42.5°C-43°C, the exposure time can be halved for every 1°C rise to provide a similar heating efficiency however, excessive heating should be avoided. The heating device used for hyperthermia should be versatile, comfortable as well capable of exhibiting uniform heating patterns. The applied frequencies may range from 5-500 KHz (Lacroix et al., 2008) while a current of about 100-800A might be sufficient for heating. Studies suggest that enlarged tumour with poor vasculature might be more susceptible to heat treatment (Kim et al., 1982).

Hyperthermia has a radiosensitizing effect which can be advantageous in combination with radiotherapy since most radioresistant cells are heat sensitive.

Classification of Hyperthermia

1. Direct heating/Extracellular method – Heat is applied by means of external sources such as thermostatic water bath, infrared sauna and ultrasound. This approach is limited by the presence of biological barriers which is responsible for insulation. Therefore, excess heat is required to achieve the same which can trigger side effects (burns, bleeding).
2. Indirect heating/Intracellular method – Provides a safer and effective means through the injection of nanoparticles followed by their internalization (Ningthoujam et al., 2012).Ex. Magnetic hyperthermia.

Mechanism of Hyperthermia

Primarily, hyperthermia induce apoptosis, necrosis or autophagy through multiple pathways to cells (Hurwitz and Stauffer, 2014). Reports suggest that it can deliver a higher amount of oxygen into the hypoxic tumour region through changes in blood perfusion. Generally, tumour cells express lower concentration of Heat Shock Proteins (HSP) in comparison to normal cells. Therefore, HSP-peptide complex levels can be increased significantly by the application of hyperthermia, further leading to anti-tumour immunity response (Kobayashi et al., 2014).

Magnetic Hyperthermia

In order to prevent damage to surrounding healthy tissues from the hyperthermia effect, nanoparticles should be confined to a defined area (tumour region). These are achieved through targeting of nanoparticles by functionalization and application of magnetic fields to specified regions (Bañobre-López et al., 2013). Metallic magnetic nanoparticles under the influence of oscillating magnetic field undergo a change in magnetic moment attributed to Neel and Brownian fluctuations. These fluctuations are responsible for heat generation through friction, which might be effective in damaging the cancer cells.

Limitations of Magnetic Hyperthermia

There are technical problems which may act as a barrier towards effective treatment. The two main aspects include uniform heat distribution and desired target temperature (Brusentsova et al., 2005). Treatment might be a failure in case of insufficient thermal dose .There are no well-defined methods used to evaluate the temperature distribution in the target area but, Magnetic Resonance Imaging (MRI) can be used to generate a temperature profile corresponding to hyperthermia. MRI can also be helpful in tracking the release of drug from a formulation (Tashjian et al., 2008).

MRI Contrast Agents

In the Magnetic Resonance Imaging (MRI) system, most of the magnetic materials (iron based materials) act as T₂ contrast agents which give rise to darkened image/negative contrast. Subsequently, this is mode is useful for tracking purpose. However, there are a few disadvantages which limit their usability in clinical settings. Firstly, the dark images accompanied by low signal intensity may often lead to misdiagnosis and secondly, the large magnetic susceptibility can produce MRI artifacts making it increasingly difficult to determine the exact state of the injury or damage. T₁ contrast agents (Gadolinium, Manganese) provide a brighter signal, which can be easily observed in the MRI due to their paramagnetic nature which do not disrupt the magnetic homogeneity (Gallo and Long, 2015). Through nanotechnology, it is also possible to simultaneously carry out imaging and drug delivery further, overcoming the limitations posed by the conventional system.

2. Hypothesis/Rationale

The paramagnetic Gadolinium exhibits excellent MRI imaging capabilities which can be exploited for several purposes and possesses high magnetic moment. Due to its limited inter-atomic interactions, it is unable produce hyperthermia. We hypothesize that by modifying the properties of gadolinium, it may serve a dual purpose i.e. hyperthermia and imaging. Furthermore, these particles can be tagged with various targeting moieties or loaded with anti-cancer drugs to increase the effectiveness of the therapy.

3. Objectives

On the basis of above background, the objectives are as follows.

• Synthesis and Optimization of Gadolinium-based ferrite nanoparticles.
• Surface modification of prepared nanoparticles.
• Folate conjugation to the modified surface coating.
• Optimization of hyperthermia
• Characterization and in-vitro studies

4. Plan of work

4.1 Synthesis and Optimization of Gadolinium-based ferrite nanoparticles

Gadolinium based ferrite nanoparticles will be synthesised using suitable mechanisms such as chemical co-precipitation method and optimized.

4.2 Surface modification of prepared nanoparticles

Surface modification will be carried out by layer by layer (LBL) synthesis.

4.3 Folate conjugation to the modified surface coating

Since most cancer cells overexpress folate receptor, folic acid will be conjugated to nanoparticles through amine functionalization.

4.4 Optimization of hyperthermia

The process will be optimized by monitoring the parameters affecting it.

4.5 Characterization and in-vitro studies

4.5.1 Characterization

The developed nanoparticle will be characterized by the following techniques.

• Particle size analysis -Zetasizer.
• Chemical Composition determination-Fourier Transform Infrared Spectroscopy (FTIR),
• Structural and Crystalline analysis- X-ray Diffraction pattern.
• Surface Morphology-Scanning Electron Microscopy, Transmission Electron Microscopy.
• Magnetic Property Testing- Vibrating Sample Magnetometry.

4.5.2 In vitro studies

• Cytotoxicity studies – MTT Assay will be performed to assess the cytotoxicity and biocompatibility of nanoparticles.
• In-vitro hyperthermia studies with cancer cell lines
• Cellular uptake studies- Performed using Transmission electron microscopy and Electron Dispersive X-ray spectroscopy.
• Magnetic Resonance Imaging studies.

5. Expected Outcomes

The developed nanoparticles might exhibit

• Improved magnetic hyperthermia in comparison to unmodified gadolinium particle.
• Target localization may be observed through Magnetic Resonance Imaging.

6. Future Prospects

Based on in-vitro results in-vivo studies can be performed in animals. This treatment modality can be combined with Photodynamic Therapy and Chemotherapy for better results.

7. References

Bañobre-López, M., Teijeiro, A. & Rivas, J. 2013. Magnetic Nanoparticle-Based Hyperthermia For Cancer Treatment. Reports Of Practical Oncology & Radiotherapy, 18, 397-400.

Brigger, I., Dubernet, C. & Couvreur, P. 2002. Nanoparticles In Cancer Therapy And Diagnosis. Advanced Drug Delivery Reviews, 54, 631-651.

Brusentsova, T. N., Brusentsov, N. A., Kuznetsov, V. D. & Nikiforov, V. N. 2005. Synthesis And Investigation Of Magnetic Properties Of Gd-Substituted Mn–Zn Ferrite Nanoparticles As A Potential Low-T C Agent For Magnetic Fluid Hyperthermia. Journal Of Magnetism And Magnetic Materials, 293, 298-302.

Dougherty, T. J., Gomer, C. J., Henderson, B. W., Jori, G., Kessel, D., Korbelik, M., Moan, J. & Peng, Q. 1998. Photodynamic Therapy. Journal Of The National Cancer Institute, 90, 889-905.

Gallo, J. & Long, N. J. 2015. Nanoparticulate Mri Contrast Agents. The Chemistry Of Molecular Imaging, 199-224.

Hurwitz, M. & Stauffer, P. Hyperthermia, Radiation And Chemotherapy: The Role Of Heat In Multidisciplinary Cancer Care. Seminars In Oncology, 2014. Elsevier, 714-729.

Kim, J. H., Hahn, E. W. & Ahmed, S. A. 1982. Combination Hyperthermia And Radiation Therapy For Malignant Melanoma. Cancer, 50, 478-482.

Kobayashi, T., Kakimi, K., Nakayama, E. & Jimbow, K. 2014. Antitumor Immunity By Magnetic Nanoparticle-Mediated Hyperthermia. Nanomedicine, 9, 1715-1726.

Lacroix, L. M., Carrey, J. & Respaud, M. 2008. A Frequency-Adjustable Electromagnet For Hyperthermia Measurements On Magnetic Nanoparticles. Rev Sci Instrum, 79, 093909.

Ningthoujam, R., Vatsa, R., Kumar, A., Pandey, B., Banerjee, S. & Tyagi, A. 2012. Functionalized Magnetic Nanoparticles: Concepts, Synthesis And Application In Cancer Hyperthermia. Functionalized Materials, 229-260.

Pérez-Herrero, E. & Fernández-Medarde, A. 2015. Advanced Targeted Therapies In Cancer: Drug Nanocarriers, The Future Of Chemotherapy. European Journal Of Pharmaceutics And Biopharmaceutics, 93, 52-79.

Seegenschmiedt, M. & Vernon, C. 1995. A Historical Perspective On Hyperthermia In Oncology. Thermoradiotherapy And Thermochemotherapy. Springer.

Tashjian, J. A., Dewhirst, M. W., Needham, D. & Viglianti, B. L. 2008. Rationale For And Measurement Of Liposomal Drug Delivery With Hyperthermia Using Non-Invasive Imaging Techniques. International Journal Of Hyperthermia, 24, 79-90.

Urano, M. 1999. Invited Review: For The Clinical Application Of Thermochemotherapy Given At Mild Temperatures. International Journal Of Hyperthermia, 15, 79-107.

Valdagni, R., Liu, F.-F. & Kapp, D. S. 1988. Important Prognostic Factors Influencing Outcome Of Combined Radiation And Hyperthermia. International Journal Of Radiation Oncology* Biology* Physics, 15, 959-972.

Vile, R., Russell, S. & Lemoine, N. 2000. Cancer Gene Therapy: Hard Lessons And New Courses. Gene Therapy, 7, 2-8.

Wust, P., Hildebrandt, B., Sreenivasa, G., Rau, B., Gellermann, J., Riess, H., Felix, R. & Schlag, P. 2002. Hyperthermia In Combined Treatment Of Cancer. The Lancet Oncology, 3, 487-497.

Xu, X., Ho, W., Zhang, X., Bertrand, N. & Farokhzad, O. 2015. Cancer Nanomedicine: From Targeted Delivery To Combination Therapy. Trends In Molecular Medicine, 21, 223-232.

8. Requirements

Chemicals

Instruments