Investigations of TiO2 NP as radiation dose enhancement agent: in vitro and phantom based studies

Youkhana, E 2017, Investigations of TiO2 NP as radiation dose enhancement agent: in vitro and phantom based studies, Doctor of Philosophy (PhD), Health and Biomedical Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Investigations of TiO2 NP as radiation dose enhancement agent: in vitro and phantom based studies
Author(s) Youkhana, E
Year 2017
Abstract Radiotherapy is one of the basic methods for cancer treatments. This extremely valuable and effective technique deliver high therapeutic doses of ionising radiations to the malignant cells to shrink tumours and kill cancer cells in a way that is safer and more reliable. The source of this ionising radiation is typically high energy photon or electron beams, which are potentially carcinogenic and/or deadly to all cells at high dosage. Therefore, the major obstacle of planning and delivery of radiotherapy is the preservation of healthy surrounding tissues by limiting the delivered radiation dose to the tolerance levels of normal tissues while still ensuring the effective targeting of tumour volumes to eradicate it.

Developments in the field of nanotechnology have potentially provided effective radiotherapy techniques through the use of high and medium atomic number (Z) nanostructure materials as radiosensitisation agents. The high Z nanoparticles (NPs) such as gold, bismuth compound, iodine and gadolinium have already been successfully utilised as radiosensitiser agents when applied to tumours for in vitro, in vivo and even in some preclinical trials. However, the high and medium Z materials are more toxic than low Z elements, therefore investigation of radiosensitasation induced by low Z elements have become more attractive. Several studies have been conducted to test the low Z nanoparticles as dose enhancing agents. Most of these studies were in the field of UV and X-rays of kilovoltage energy ranges. This thesis’ research extends thier application to include the most common form of radiotherapy i.e. using megavoltage range of X-rays. The aims of this thesis are focused on investigations of employing low Z materials and particularly anatase titanium dioxide nanoparticles (TiO2 NPs) as potential radiosensitisation agent and as imaging agent too.

This research was conducted by two main ways, one by using phantoms (PRESAGE® dosimeters) and the other by in vitro using two types of cell lines, cultured human keratinocyte (HaCaT) and prostate cancer (DU145) cells, and both methods were aimed at determining the effects on NPs on the radiation dose enhancement at both low (kilovoltage) and high (megavoltage) radiotherapy X-ray beams. Furthermore, TiO2 NPs were activated via proton beam to investigate for their suitability as diagnostic agent hence this nano-compound qualifies to be true theranostic agent.

Several characteristics of TiO2 NPs, which make them ideally suited for application in radiotherapy, are investigated throughout this research. Anatase TiO2 NPs were synthesised, characterised and functionalised to allow dispersion in culture medium for in vitro studies and halocarbons (PRESAGE® chemical compositions) for phantom based studies. The fabricated PRESAGE® dosimeters/phantoms were scanned to obtain the physical and radiological properties and further to determine the radiation dose enhancement induced by TiO2 NPs. Clonogenic and cell viability assays were employed to determine cells survival curves from which the dose enhancement levels “radiosensitisation” are deduced. The dose enhancement produced experimentally by 0.5, 1 and 4 mM TiO2 NPs concentrations for phantom and in vitro studies irradiated with 1-8 Gy ranges of radiation doses was quantified for kilovoltage and megavoltage energies of external X-ray radiation sources. Furthermore, the TiO2 NPs were activated via proton beam and the energy spectrums were acquired using Germanium detectors. The radiolabeled anatase TiO2 NPs were imaged using positron emission tomography (PET) scanner.

One aspect of this research was to demonstrate that the TiO2 NPs were typically synthesised to achieve highly pure and uniform anatase nanocrystalline structure. This form of NPs creates more free radicals and effectively generates more reactive oxygen species (ROS) since it has larger surface to volume ratio. These features combined have a high impact in damaging DNA molecule of biological system during irradiation.

In phantom studies, the radiation modifying effects of incorporating of PEG functionalised anatase TiO2 NPs in the formulation of the water equivalent 3D PRESAGE® dosimeter were explored. The dose enhancement factors (DEFs) were quantified and then the results were validated with the biological studies. The results clearly demonstrates that the sensitivity of the dosimeter increases to the radiation doses with the concentration of the TiO2 NPs incorporated in the composition of the PRESAGE® dosimeter. Furthermore, the measured DEF was significant at 80 kV compared to the negligible dose enhancement detected at 6 MV X-ray energy beams.

The In vitro studies, TiO2 NPs proved to be cytocompatible to cells, even at very high concentrations. The DEFs were deduced from the data analysed in the form of cell survival curves. The result indicates that radiosensitisation induced by TiO2 NPs was significant at kilovoltage range of energy which the maximal dose enhancement was observed at 80 kV. Furthermore, significant radiosensitisation was observed for in vitro study at megavoltage energy beam. Higher radiosensitisation were obtained for low energy x-rays compared to the high energy ones. Generally, with the inclusion of TiO2 NPs in the target, same fraction of cells were destroyed with lower radiation doses compared to the case of absence of TiO2 NPs. This means if the TiO2 NPs are added to the biological target, a reduction of external dose of an order of magnitude can be achieved to deliver the same local control as without the inclusion of TiO2 NPs for treatments with kilovoltage and megavoltage X-rays beams. This reduction of delivered radiation dose to the target results in reducing the dose to the surrounding normal tissues during treatment that is the primary concern in all radiotherapy treatment procedures. Hence, TiO2 NPs are considered to be an efficient dose enhancer agent and have a great potential value for future clinical radiotherapy applications.

In addition, the radiobiological effect of amino functionalised anatase TiO2 NPs on HaCaT and DU145 cell lines were investigated. The linear (α) and the quadratic (β) radiobiological-parameters were extracted from the cell survival curves in order to describe the DNA damage by radiation. The results clearly demonstrate that α value significantly increases with the inclusion of TiO2 NPs while β values do not show any predictable trend. This increase in α value indicates that the probability of double strand DNA breakage increases with the presence of NPs in the target. Accordingly, the DEF results for in vitro and phantom based studies showed good agreement with the hypothesis of α value increases as a consequence of inclusion TiO2 NPs in the target.

There are measurable differences in the level of produced DEF between biological and phantom based studies at MV energy. The PRESAGE® dosimeters showed lower enhancements in radiosensitivity than cell in culture studies. This is due to PRESAGE® dosimeters is only able to detect the free electron generated as a result of photoelectric effect, Compton scatter and/or Auger effect and not being suited to detect the generated ROS (Biochemical effects) due to a lack of free water molecules in its structure, whereas cells can be affected by many other biochemical factors, such as the generated ROS which is added to the stress caused by generated electron free radicals, and this result in higher radiosensitivity. Therefore, the ROS generated from amino functionalised anatase TiO2 NPs upon exposed to radiotherapy X-ray energy beam was investigated. Aqueous solutions without and with the presence of TiO2 NPs was exposed to 6 MV beam. The result clearly shows that the level of generated ROS was proportionally dependent on the TiO2 NPs concentration. This explains that biochemical effects need to be considered as a key factor for enhancing the cellular radiosensitivity with the presence of nanoparticles, which would be an important consideration for in vitro and in vivo radiosensitivity measurements.

Finally, the TiO2 NPs are proposed as a reliable potential candidate for producing nuclear medical radioisotopes via proton activation. The results demonstrate that intense peak was observed at 511 keV which correspond to the γ-ray resulted from electron-positron annihilation. This γ-ray peak is the most important radioisotope for potential nuclear medicine imaging applications using PET. Recently, several innovative for new radiopharmaceutical evolution potentially suggest β- and α emitters. Therefore, the produced 47Sc radionuclide is a promising therapeutic agent for preparing radiolabeled antibodies due to its favorable β- emission energy (162 keV) which decays to stable 47Ti (100% β- emission ), and to its moderate half-life (T1/2 = 3.35 d).

To conclude, this research shows that TiO2 NPs improve the efficiency of dose delivery, which has implications for future radiotherapy treatments. The TiO2 NPs can also be used as a potential imaging agents hence with these findings renders these NPs as theranostic agents with dual effects (i.e. imaging and dose enhancer agent) simultaneously if it is in the targets.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Health and Biomedical Sciences
Subjects Radiation Therapy
Medical Physics
Nuclear Medicine
Keyword(s) Titanium Dioxide
Nanoparticles (NPs)
PRESAGE® dosimeters/phantoms
Dose enhancement
Reactive Oxygen Species (ROS)
Proton Activation
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Created: Thu, 12 Oct 2017, 07:26:05 EST by Denise Paciocco
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