Modulation of gold nanoparticle surface chemistry to target glioblastoma cells for SERS based imaging

Surgery is the mainstay treatment of brain tumors, however complete resection is rarely achieved, especially when dealing with grade IV glioblastoma (GBM). GBM is the most lethal brain tumor worldwide with an average survival not longer than 15 months. A reason of this dismal outcome is the lack of intraoperative visualization techniques for the objective identification of true tumor borders and infiltrating tumor cells. Hence, the improvement of GBM visualization during surgical operation is the motivation of this project.
Recent developments of handheld fiber optic probes and lasers for low cost systems, together with sensitivity enhancement techniques such as surface enhanced Raman scattering (SERS), have ruled Raman spectroscopy as one of the most promising technologies for surgical guidance. This technique could overcome the limitations of current intraoperative modalities such as neuronavigation, magnetic resonance imaging and fluorescence guided surgery. For increased sensitivity, metallic nanostructures are preferred because they strongly interact with light, due to surface plasmon resonance (SPR), and produce a much higher level of amplification compared to flat surfaces. Among the nanostructures, gold nanoparticles (GNPs) have found wide application in SERS based imaging studies for their higher biocompatibility and versatile functionalization. When properly engineered, visualization can be targeted on tumor-specific biomolecules providing an accurate mapping of tumor spread. For optimized intraoperative visualization of GBM, detection can be tuned to the near-infrared (NIR) window by acting both on the size and shape of GNPs. This enables to overcome the interfering autofluorescence and to reach a deeper tissue penetration. However, surface chemistry becomes essential to create SERS tags for a fast, sensitive and specific detection of tumor cells.
Due to the absence of comprehensive studies on the impact of GNPs surface functionalization on SERS based imaging, this thesis elucidates the effect of Raman reporter, inert protective polyethylene glycol (PEG) and anti-epidermal growth factor receptor (EGFR) antibody on colloidal stability, cellular binding specificity, detection sensitivity and speed. EGFR was the target of choice because its gene amplification is the most common molecular hallmark in about 60% of GBM and protein overexpression on cell cytoplasmic membrane makes it easily accessible.
Based on the findings, GNPs with dense surface coverage of Raman reporter or PEG produced the maximum imaging sensitivity and binding specificity, respectively. However, GNPs with dense Raman reporter surface coverage were liable to non-specific binding and colloidal aggregation. Conversely, GNPs with dense PEG surface coverage owned the highest stability but underwent more than 90% reduction of SERS sensitivity. Higher integration time (from 0.05 to 0.5 sec) or shorter working distance (from 16.5 to 0.3 mm) to counteract the decreased SERS sensitivity were not considered in view of the final application in vivo. GNPs with surface coverage made of 50% Raman reporter and 50% PEG owned the optimal mixture for an immediate Raman detection of in vitro human GBM cells (LN229wtEGFR, BS153 and U87MG) while minimizing non-specific binding on EGFR-negative cells (IMA2.1). Further, SERS signal was comparable independently on the different EGFR expression level or the presence of EGFRvIII in BS153. The latter is the constitutively active receptor whose presence is associated to the lack of response during fluorescence based visualization of GBM. It was also shown that excess of Raman reporter did not add any significant contribution to SERS sensitivity. Similarly, the conjugation efficiency decreased by 35% through the addition of 10 times the concentration of antibody, compared to the lower concentration. This excess quantity of antibody showed no improvement of binding affinity of GNPs to tumor cells.
Because the blood brain barrier (BBB) limits the therapeutic access to brain tumor, the ability of GNPs to cross an in vitro BBB made of a monoculture of human endothelial cells (hCMEC/D3) is crucial for successful intraoperative visualization. About 0.1% of GNPs, with specific ratio of immobilized functionalities, was able to cross the cell monolayer preserving its integrity and eliciting no cytotoxic effects. Similar results were obtained in vivo.
By providing an in-depth investigation, this work stresses the significance of identifying the appropriate surface chemistry to improve the biomedical potential of GNPs in SERS based imaging applications. At the same time, it provides an in vitro demonstration that SERS based imaging can be implemented intraoperatively for immediate visualization of GBM offering an adequate alternative for the detection of those GBM or low-grade brain tumors that show variable or no response to fluorescence guided surgery, the current state-of-art for GBM intraoperative visualization.