Investigation of Cellular Uptake Mechanism of Functionalised Gold Nanoparticles into Breast Cancer Using SERS

Gold nanoparticles (AuNPs) are widely used in various applications such as cancer imaging and drug delivery. The functionalisation of AuNPs has been shown to affect their cellular internalisation, accumulation and targeting efficiency. The mechanism of cellular uptake of functionalised AuNPs by different cancer cells is not well understood. Therefore, a detailed understanding of the molecular processes is necessary to improve AuNPs for their selective uptake and fate in specific cellular systems. This knowledge can greatly help in designing nanotags with higher cellular uptake for more selective and specific targeting capabilities with less off-target effects. Here, we demonstrate for the first time a straightforward and non-destructive 3D surface enhanced Raman spectroscopy (SERS) imaging approach to track the cellular uptake and localisation of AuNPs functionalised with an anti-ERα (estrogen receptor alpha) antibody in MCF-7 ERα-positive human breast cancer cells under different conditions including temperature and dynamin inhibition. 3D SERS enabled information rich monitoring of the intracellular internalisation of the SERS nanotags. It was found that ERα-AuNPs were internalised by MCF-7 cells in a temperature-dependent manner suggesting an active endocytosis-dependent mechanism. 3D SERS cell mapping also indicated that the nanotags entered MCF-7 cells using dynamin dependent endocytosis, since dynamin inhibition resulted in the SERS signal being obtained from, or close to, the cell surface rather than inside the cells. Finally, ERα-AuNPs were found to enter MCF-7 cells using an ERα receptor-mediated endocytosis process. This study addresses the role of functionalisation of SERS nanotags in biological environments and highlights the benefits of using 3D SERS for the investigation of cellular uptake processes

Characterisation of Estrogen Receptor Alpha (ERα) Expression in Breast Cancer Cells and Effect of Drug Treatment Using Targeted Nanoparticles and SERS

The detection and identification of estrogen receptor alpha (ERα), one of the main biomarkers in breast cancer, is crucial for the clinical diagnosis and therapy of the disease. Here, we use a non-destructive approach for detecting and localising ERα expression at the single cell level using surface enhanced Raman spectroscopy (SERS) combined with functionalised gold nanoparticles (AuNPs). Antibody functionalised nanotags (ERα-AuNPs) showed excellent biocompatibility and enabled the spatial and temporal understanding of ERα location in breast cancer cell lines with different ERα expression status. Additionally, we developed an approach based on the percentage area of SERS response to qualitatively measure expression level in ERα positive (ERα+) breast cancer cells. Specifically, the calculation of relative SERS response demonstrated that MCF-7 cells (ERα+) exhibited higher nanotag accumulation resulting in a 4.2-times increase in SERS signal area in comparison to SKBR-3 cells (ERα-). These results confirmed the strong targeting effect of ERα-AuNPs towards the ERα receptor. The functionalised ERα-AuNP nanotags were also used to investigate the activity of fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). SERS mapping confirmed that ERα degradation occurred after fulvestrant treatment since a weaker SERS signal, and hence accumulation of nanotags, was observed in MCF-7 cells treated with fulvestrant. Most importantly, a correlation coefficient of 0.9 between the SERS response and the ERα expression level, obtained by western blot, was calculated. These results confirmed the strong relationship between the two approaches and open up the possibilities of using SERS as a tool for the estimation of ERα expression levels, without the requirement of destructive and time-consuming techniques. Therefore, the potential of using SERS as a rapid and sensitive method to understand the activity of SERDs in breast cancer is demonstrated

Detection of Estrogen Receptor Alpha and Assessment of Fulvestrant Activity in MCF-7 Tumor Spheroids Using Microfluidics and SERS

Breast cancer is one of the leading causes of cancer death in women. Novel in vitro tools that integrate three-dimensional (3D) tumor models with highly sensitive chemical reporters can provide useful information to aid biological characterization of cancer phenotype and understanding of drug activity. The combination of surface-enhanced Raman scattering (SERS) techniques with microfluidic technologies offers new opportunities for highly selective, specific, and multiplexed nanoparticle-based assays. Here, we explored the use of functionalized nanoparticles for the detection of estrogen receptor alpha (ERα) expression in a 3D tumor model, using the ERα-positive human breast cancer cell line MCF-7. This approach was used to compare targeted versus nontargeted nanoparticle interactions with the tumor model to better understand whether targeted nanotags are required to efficiently target ERα. Mixtures of targeted anti-ERα antibody-functionalized nanotags (ERα-AuNPs) and nontargeted (against ERα) anti-human epidermal growth factor receptor 2 (HER2) antibody-functionalized nanotags (HER2-AuNPs), with different Raman reporters with a similar SERS signal intensity, were incubated with MCF-7 spheroids in microfluidic devices and spectroscopically analyzed using SERS. MCF-7 cells express high levels of ERα and no detectable levels of HER2. 2D and 3D SERS measurements confirmed the strong targeting effect of ERα-AuNP nanotags to the MCF-7 spheroids in contrast to HER2-AuNPs (63% signal reduction). Moreover, 3D SERS measurements confirmed the differentiation between the targeted and the nontargeted nanotags. Finally, we demonstrated how nanotag uptake by MCF-7 spheroids was affected by the drug fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). These results illustrate the potential of using SERS and microfluidics as a powerful in vitro platform for the characterization of 3D tumor models and the investigation of SERD activity

Imaging of breast cancer using SERS and SESORS

This thesis was previously held under moratorium from 27/11/19 to 27/11/21Breast cancer is one of the leading causes of oncologic mortality and morbidity among women worldwide. It is estimated that every 10 minutes one person is diagnosed with the disease in the UK, while 1 in 8 women will develop breast cancer at some point in their lives. Although different techniques, for the characterisation of cancer phenotype, exist there are still limitations as these approaches are destructive, require processed/fixed samples and are not suitable for 3D tumour samples and in vivo models. Surface enhanced Raman spectroscopy (SERS) overcomes these limitations as a non-destructive bioanalytical method that offers high specificity, selectivity and multiplex capacities, in comparison to conventional imaging techniques. The main aim of this research is to create a platform for targeting, detecting and tracking the intracellular distribution of estrogen receptor alpha (ERα) biomarker in breast cancer, using SERS combined with antibody functionalised gold nanoparticles (AuNPs). Specifically, the anti-ERα antibody functionalised AuNPs (ERα-AuNPs) were conjugated with 1,2-bis(4-pyridyl)ethylene (BPE) Raman reporter that enabled the spatial and temporal understanding of where ERα was located at a single cell level. The nanotags showed excellent biocompatibility with no cellular toxicity. 3D SERS cell mapping, under different endocytosis inhibition conditions, confirmed that ERα-AuNPs were using a temperature-dependent way for their uptake. Additionally, dynamin and membrane ERα were shown to be responsible, at least in a part, for the nanotags’ uptake in MCF-7 cells. Therefore, SERS provided an excellent biological insight of ERα-AuNPs uptake by generating 3D images of the entire cell volume, without the need for destructive, time consuming and expensive imaging methods such as transition electron microscopy (TEM). 2D and 3D SERS also confirmed the strong targeting effect of ERα-AuNPs against ERα since a higher SERS signal and nanotag accumulation were observed in MCF-7 cells (ERα+) compared to SKBR-3 (ERα-) breast cancer cells. SERS was also used for investigating the efficacy of fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). The results confirmed that ERα-AuNPs can be used as a tool for identifying and characterising different breast cancer cells, based on ERα expression, and informing about SERDs activity in breast cancer. SERS also provided an excellent bioanalytical tool for the characterisation of breast cancer phenotype and the assessment of fulvestrant activity in a 3D environment using live MCF-7 spheroids formed in a microfluidic device. The results confirmed the great penetration capabilities and strong targeting effect of ERα-AuNPs towards ERα, compared to nonspecific anti-HER2 antibody functionalised AuNPs (HER2-AuNPs). Additionally, fulvestrant activity was found to have a lower therapeutic effect the 3D MCF-7 spheroids in comparison to the 2D cell cultures demonstrating that 2D and 3D tumour models had different biological and architectural behaviours that affected their sensitivity to fulvestrant. Therefore, SERS and microfluidics were used as a powerful analytical tool, that effectively bridged the gap between the 2D monolayer cultures and animal models, for breast cancer cells characterisation and investigation of fulvestrant efficacy. Finally, this thesis investigated the potentials for detection of ERα ex vivo and in vivo using a handheld SORS instrument with back scattering optics. SESORS allowed the detection of ERα-AuNP nanotags through tissue barriers of up to 15 mm thickness. Most importantly, it was possible to detect and track ex vivo the ERα-AuNPs incubated in live breast tumour spheroids buried at 10 mm porcine tissue. The in vivo work indicated that SESORS was detecting scattered photon from areas deeper than the breast cancer tumour, mainly due to the fixed optical arrangements of the spectrometer. Nevertheless, a higher signal was detected ex vivo in breast tumours in comparison to the liver after their removal from sacrificed animals, suggesting the strong targeting effect of ERα-AuNP nanotags to the tumour site. This thesis highlights the performance and capabilities of SERS, microfluidics and SESORS on detecting, targeting and tracking ERα and opens up exciting opportunities for using these techniques as non-destructive and sensitive tools for improved biomedical imaging in a clinical environment.Breast cancer is one of the leading causes of oncologic mortality and morbidity among women worldwide. It is estimated that every 10 minutes one person is diagnosed with the disease in the UK, while 1 in 8 women will develop breast cancer at some point in their lives. Although different techniques, for the characterisation of cancer phenotype, exist there are still limitations as these approaches are destructive, require processed/fixed samples and are not suitable for 3D tumour samples and in vivo models. Surface enhanced Raman spectroscopy (SERS) overcomes these limitations as a non-destructive bioanalytical method that offers high specificity, selectivity and multiplex capacities, in comparison to conventional imaging techniques. The main aim of this research is to create a platform for targeting, detecting and tracking the intracellular distribution of estrogen receptor alpha (ERα) biomarker in breast cancer, using SERS combined with antibody functionalised gold nanoparticles (AuNPs). Specifically, the anti-ERα antibody functionalised AuNPs (ERα-AuNPs) were conjugated with 1,2-bis(4-pyridyl)ethylene (BPE) Raman reporter that enabled the spatial and temporal understanding of where ERα was located at a single cell level. The nanotags showed excellent biocompatibility with no cellular toxicity. 3D SERS cell mapping, under different endocytosis inhibition conditions, confirmed that ERα-AuNPs were using a temperature-dependent way for their uptake. Additionally, dynamin and membrane ERα were shown to be responsible, at least in a part, for the nanotags’ uptake in MCF-7 cells. Therefore, SERS provided an excellent biological insight of ERα-AuNPs uptake by generating 3D images of the entire cell volume, without the need for destructive, time consuming and expensive imaging methods such as transition electron microscopy (TEM). 2D and 3D SERS also confirmed the strong targeting effect of ERα-AuNPs against ERα since a higher SERS signal and nanotag accumulation were observed in MCF-7 cells (ERα+) compared to SKBR-3 (ERα-) breast cancer cells. SERS was also used for investigating the efficacy of fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). The results confirmed that ERα-AuNPs can be used as a tool for identifying and characterising different breast cancer cells, based on ERα expression, and informing about SERDs activity in breast cancer. SERS also provided an excellent bioanalytical tool for the characterisation of breast cancer phenotype and the assessment of fulvestrant activity in a 3D environment using live MCF-7 spheroids formed in a microfluidic device. The results confirmed the great penetration capabilities and strong targeting effect of ERα-AuNPs towards ERα, compared to nonspecific anti-HER2 antibody functionalised AuNPs (HER2-AuNPs). Additionally, fulvestrant activity was found to have a lower therapeutic effect the 3D MCF-7 spheroids in comparison to the 2D cell cultures demonstrating that 2D and 3D tumour models had different biological and architectural behaviours that affected their sensitivity to fulvestrant. Therefore, SERS and microfluidics were used as a powerful analytical tool, that effectively bridged the gap between the 2D monolayer cultures and animal models, for breast cancer cells characterisation and investigation of fulvestrant efficacy. Finally, this thesis investigated the potentials for detection of ERα ex vivo and in vivo using a handheld SORS instrument with back scattering optics. SESORS allowed the detection of ERα-AuNP nanotags through tissue barriers of up to 15 mm thickness. Most importantly, it was possible to detect and track ex vivo the ERα-AuNPs incubated in live breast tumour spheroids buried at 10 mm porcine tissue. The in vivo work indicated that SESORS was detecting scattered photon from areas deeper than the breast cancer tumour, mainly due to the fixed optical arrangements of the spectrometer. Nevertheless, a higher signal was detected ex vivo in breast tumours in comparison to the liver after their removal from sacrificed animals, suggesting the strong targeting effect of ERα-AuNP nanotags to the tumour site. This thesis highlights the performance and capabilities of SERS, microfluidics and SESORS on detecting, targeting and tracking ERα and opens up exciting opportunities for using these techniques as non-destructive and sensitive tools for improved biomedical imaging in a clinical environment