Rare-earth-doped nanoprobes as optical contrast agents for fluorescence-guided surgery

According to the World Health Organization, cancer is the second leading cause of mortality worldwide with more than 9 million deaths each year1. In the case of many solid tumors, treatment involves surgical resection with the goal of achieving negative margins while also sparing healthy tissue. To this end, surgeons rely on visual inspection, palpation, and intraoperative histopathology of frozen tumor sections. However, due to the lack of a method for real-time intraoperative comprehensive tissue assessment, in some cases positive margins are left, which may require a re-excision surgery. New methods are being developed, such as fluorescence-guided surgery (FGS), to improve the outcome of surgical resections2. FGS is an emerging technique that uses fluorescent contrast agents to label tumors, enhancing their distinction from healthy tissue when viewed in real-time with optical imaging systems. Recent work on FGS has explored the use of visible and near-infrared (NIR) fluorophores such as fluorescein, indocyanine green (ICG), and IRDye800 among others, to enhance the contrast between healthy tissue and tumor. However, these contrast agents have limitations in the depth at which they can be imaged and the definition of the edges of the tumor margins. Contrast agents that emit light at longer wavelengths have been explored to overcome the current limitations of visible and NIR fluorophores. For example, rare-earth-doped nanoparticles (RENPs), when excited at a wavelength of 980 nm, emit at short-wave infrared (SWIR) wavelengths (900 – 1,700 nm) that undergo less scattering in tissue than visible or NIR light. The use of SWIR wavelengths enables deeper tissue penetration, improved image resolution, and reduced background from tissue autofluorescence. These characteristics motivate the investigation of RENPs as a new contrast agent to identify tumor margins in FGS. The overall hypothesis in this thesis is that RENPs can be used to identify surgical margins during resection of tumors with higher accuracy and at greater depths within tissue than the currently used NIR fluorophores, or white light evaluation alone. The use of this technology could decrease the number of re-excision surgeries due to positive margins in tissue-sparing resections, lowering costs and benefiting patients. This study first developed a wide-field imaging system for use with SWIR-emitting contrast agents, as described in Chapter 2. This system provides real-time feedback on RENP location in phantoms and preclinical in vivo (mouse) models. The system consists of a laser that excites RENPs at a wavelength of 980 nm and an InGaAs camera that collects emissions at SWIR wavelengths. Real-time image analysis generates a RENP location map, which is displayed on a monitor for visual guidance to locate tumor margins. This system differs from current commercially available FGS systems, which are configured for imaging visible or NIR fluorophores and cannot image SWIR emitting contrast agents. A study comparing images obtained with SWIR and NIR contrast agents using tissue simulating phantoms was conducted and this is described in Chapter 3. Before proceeding to in vivo studies, RENPs were encapsulated in albumin for biocompatibility forming rare-earth albumin nanocomposites (REANCs). These were benchmarked against the most common clinical NIR contrast agent (ICG) by fabricating phantoms with REANCs, ICG, and control solutions embedded at varying subsurface depths beneath ex vivo tissue. These phantoms were imaged and analyzed to generate a quantitative comparison of signal-to-background ratio (SBR), contrast, and edge sharpness between labeled inclusions and the surrounding tissue. The performance of SWIR and NIR contrast agents in fluorescence-guided surgery was investigated in Chapter 4. An in vivo murine mammary fat pad breast cancer model was used to induce tumors which were labeled with REANCs or ICG. Tumors were imaged with the system described in Chapter 2 at 0, 24, and 48 hours post contrast agent administration. These studies established the optimal surgical window by identifying the time point with the highest SBR ratio on imaging. A quantitative comparison of SBR ratio, contrast, and edge sharpness was performed using both contrast agents to evaluate which agent can better identify tumor margins. Following tumor resection using SWIR or NIR fluorescence guidance, evaluation of fluorescence intensity levels in excised tumors and in the surgical bed demonstrated higher contrast between tissues at these sites with REANC contrast compared to ICG. REANCs also demonstrated outstanding photostability over 2 hours of continuous illumination, as well as the ability to perform FGS under ambient lighting. In summary, this thesis developed and evaluated SWIR-emitting contrast agents and imaging systems for fluorescence-guided surgery applications. Direct comparisons were made between this new SWIR platform and existing NIR systems in tissue phantoms and in vivo studies. The use of SWIR-based contrast agents and imaging systems may offer improved contrast and SBR levels compared to existing systems, with benefits for real-time surgical applications.Ph.D.Includes bibliographical reference