Raman spectroscopy for the investigation of gold nanoparticles in Glioblastoma

Glioblastoma (GBM) is a stage IV, aggressive form of brain cancer that leads to poor patient prognosis and life expectancy. Although it is classified as being a rare form of cancer, there are over 3,000 cases diagnosed every year in the UK. Patients suffering from a GBM have an average survival of approximately 15 months, and less than 10% will live for more than 5 years with the current standard treatment. The treatment for GBM requires significant improvements as there is difficulty in distinguishing between cancerous and non-cancerous tissues using conventional imaging techniques. Surface enhanced Raman scattering (SERS) uses gold nanoparticles (AuNPs) to enhance the Raman scattered light and allow for a greater signal to be produced. Spatially offset Raman scattering (SORS) allows for the detection of scattered photons through depth, such as an obscuring barrier. Combining these two techniques, surface enhanced spatially offset Raman scattering (SESORS) provides enhanced Raman signals allowing for even deeper detection capabilities of SORS. The main aim of this research was to investigate AuNPs as cancer imaging and treatment agents to allow for detection and understanding of localisation through the use of SERS and SESORS. Specifically, SESORS was used to accurately locate a AuNP inclusion, buried within a 3D tissue matrix. An effect we named ‘linear offset induced image drag’ was identified which refers to a spatial shift in SESORS images caused by the magnitude and direction of the linear offset, which falsely depicts the location of the inclusion at depth. This finding resulted in the application of a 360° ring-collection SESORS geometry, which subsequently led to locating the position of two NP imaging templates, labelled with different Raman reporters, buried at different depths. Using SERS, the detection, uptake, distribution, and localisation of AuNPs incubated in already formed U87-MG glioblastoma multicellular tumour spheroids (MTS) was demonstrated. Immunohistochemistry revealed that the compactness of the cells in the MTS, the oxygen and nutrient gradients and the proliferation status of the MTS limited the uptake of the AuNPs into the core and indicated that they would remain within the proliferating, outer layer of the MTS. An antibody targeting tenascin-C was used to aid in cellular uptake, however, this led to no additional improvement in uptake compared to a non-targeting antibody. This work emphasises the importance of bridging the gap between 2D cellular monolayers and in vivo tumour models. As a natural progression, an in vivo chicken egg embryo tumour model was used for the detection and localisation of AuNPs. Using SERS to detect the AuNPs distribution throughout a dissected tumour revealed similar findings to that of the in vitro MTS, where the NPs were located around the periphery of the tumour. Using a handheld SORS spectrometer, AuNPs incubated in an in vivo live tumour in the chicken egg embryo model were detected through 12 mm of tissue for the first time. Finally, the AuNPs were incubated in MTS and ionising radiation therapy was used to investigate radiosensitisation effects. Two sets of nanotags, with and without a silica shell were investigated and compared at different concentrations to determine an optimum concentration of nanotag in combination with varying doses of radiation. The radiation doses used were believed to be too high and they caused a significant level of cell death such that it was impossible to identify any radiosensitisation through the addition of the AuNPs. Lower doses were demonstrated, however showed similar results and further investigation will be required to improve uptake of the nanotags and subsequently, their opportunity for radiosensitisation. Overall, we have demonstrated the exciting potential of AuNPs as cancer imaging agents for inclusion detection through depth in a 3D matrix, in 3D in vitro MTS and finally in in vivo chicken egg embryo tumour models for the tracking and detection of AuNPs.Glioblastoma (GBM) is a stage IV, aggressive form of brain cancer that leads to poor patient prognosis and life expectancy. Although it is classified as being a rare form of cancer, there are over 3,000 cases diagnosed every year in the UK. Patients suffering from a GBM have an average survival of approximately 15 months, and less than 10% will live for more than 5 years with the current standard treatment. The treatment for GBM requires significant improvements as there is difficulty in distinguishing between cancerous and non-cancerous tissues using conventional imaging techniques. Surface enhanced Raman scattering (SERS) uses gold nanoparticles (AuNPs) to enhance the Raman scattered light and allow for a greater signal to be produced. Spatially offset Raman scattering (SORS) allows for the detection of scattered photons through depth, such as an obscuring barrier. Combining these two techniques, surface enhanced spatially offset Raman scattering (SESORS) provides enhanced Raman signals allowing for even deeper detection capabilities of SORS. The main aim of this research was to investigate AuNPs as cancer imaging and treatment agents to allow for detection and understanding of localisation through the use of SERS and SESORS. Specifically, SESORS was used to accurately locate a AuNP inclusion, buried within a 3D tissue matrix. An effect we named ‘linear offset induced image drag’ was identified which refers to a spatial shift in SESORS images caused by the magnitude and direction of the linear offset, which falsely depicts the location of the inclusion at depth. This finding resulted in the application of a 360° ring-collection SESORS geometry, which subsequently led to locating the position of two NP imaging templates, labelled with different Raman reporters, buried at different depths. Using SERS, the detection, uptake, distribution, and localisation of AuNPs incubated in already formed U87-MG glioblastoma multicellular tumour spheroids (MTS) was demonstrated. Immunohistochemistry revealed that the compactness of the cells in the MTS, the oxygen and nutrient gradients and the proliferation status of the MTS limited the uptake of the AuNPs into the core and indicated that they would remain within the proliferating, outer layer of the MTS. An antibody targeting tenascin-C was used to aid in cellular uptake, however, this led to no additional improvement in uptake compared to a non-targeting antibody. This work emphasises the importance of bridging the gap between 2D cellular monolayers and in vivo tumour models. As a natural progression, an in vivo chicken egg embryo tumour model was used for the detection and localisation of AuNPs. Using SERS to detect the AuNPs distribution throughout a dissected tumour revealed similar findings to that of the in vitro MTS, where the NPs were located around the periphery of the tumour. Using a handheld SORS spectrometer, AuNPs incubated in an in vivo live tumour in the chicken egg embryo model were detected through 12 mm of tissue for the first time. Finally, the AuNPs were incubated in MTS and ionising radiation therapy was used to investigate radiosensitisation effects. Two sets of nanotags, with and without a silica shell were investigated and compared at different concentrations to determine an optimum concentration of nanotag in combination with varying doses of radiation. The radiation doses used were believed to be too high and they caused a significant level of cell death such that it was impossible to identify any radiosensitisation through the addition of the AuNPs. Lower doses were demonstrated, however showed similar results and further investigation will be required to improve uptake of the nanotags and subsequently, their opportunity for radiosensitisation. Overall, we have demonstrated the exciting potential of AuNPs as cancer imaging agents for inclusion detection through depth in a 3D matrix, in 3D in vitro MTS and finally in in vivo chicken egg embryo tumour models for the tracking and detection of AuNPs