Aerosol-made Nanoparticles for Theranostics: Bioimaging, Nanothermometry, and Photothermal Therapy

Particles at the nanoscale have emerged as promising tools for a broad range of applications in medicine. Tailored nanostructures enable novel possibilities in disease therapy and diagnostics (summarized as theranostics), complementing and improving the state of the art techniques. Their unique performance can be attributed to their extremely small size, that not only enables crossing certain biological barriers or higher targeting efficiencies compared to molecular drugs, but also evokes novel effects that only occur at small scales. A highly attractive subgroup among these nanostructures is light-activated theranostics due to their versatility and absence of harmful radiation such as X-rays. As such, they can, for example, be used to localize and image cancerous tissue, selectively destroy cancer cells and simultaneously act as control units during therapy. Owing to the possibility of combining multiple functionalities into one nanostructure, such systems have the potential to address an imminent need in today’s society for advanced, specific and controlled theranostics. In Chapter 1, the interaction of light with biological tissue is discussed in detail. The focus lies on the wavelength-dependent phenomena governing imaging and therapy, specifically absorption, scattering, and autofluorescence. When visible light hits biological tissue, most of the photons are absorbed and scattered, limiting applications to very superficial layers such as during in vitro cell-monolayer experiments. However, based on the spectral properties of selected representative tissues, two most promising spectral regions in the near-infrared (NIR) are identified for light-activated theranostics, namely the NIR-I (650 – 900 nm) and NIR-II (1000 – 1350 nm). Within these regions, light absorption, scattering, and autofluorescence are minimized, enabling light to penetrate much deeper into biological tissue. By tuning the operational range of a theranostic modality into these regions, its working depth in tissue is extended to the centimeter range. In the following, three examples of light-activated theranostics - photothermal therapy, fluorescence imaging, and nanothermometry - are introduced and their underlying principles are elucidated. To enhance the performance or selectivity in light-activated theranostics, exogenous agents such as nanoparticles are often introduced. Therefore, a short overview of currently available classes of nanoparticles is given. Special attention is paid to identify potential drawbacks and shortcomings of different material groups, as well as remaining challenges in terms of material design. Photothermal therapy relies on the conversion of incoming light to heat, thereby locally heating up the surrounding tissue. When targeted to a tumor, this therapy allows to selectively kill diseased cells. To increase specificity, photothermal agents with strong light-absorption such as plasmonic nanoparticles are employed. However, the most commonly used gold particles require sophisticated geometries (i.e. rods, stars, cages) to shift their absorption into the near-infrared. Besides increasing the already high costs of gold-particles, such complex geometries also impair their stability. To overcome these drawbacks, chapters 2 and 3 describe the synthesis of titanium nitride (TiN) nanoparticles as alternative photothermal agents. Titanium nitride represents a low-cost material that exhibits strong NIR-I absorption, even for spherical particles. The synthesis involves first the preparation of TiO2 nanoparticles by flame-spray pyrolysis (FSP), and second the nitridation under ammonia atmosphere into TiN. The influence of the nitridation parameters (holding time, temperature) on the product characteristics are investigated with commercially available, bare TiO2 nanoparticles (P25) as a model system. Longer nitridation at higher temperatures gradually decreases the remaining oxygen in the particles, thereby improving their absorption. However, too high temperatures (> 850 °C) result in strong aggregation and decreased performance. To overcome this problem, an amorphous SiO2-coating is introduced as a spacer layer around the particles. Specifically, TiO2 particles are produced by FSP and in-situ coated with a SiO2 layer. Through close control over process parameters, both the thickness of the SiO2-coating and the TiO2 core particles can be independently controlled. Despite the SiO2-coating, these particles can still be nitrided to form SiO2-coated TiN, whereby the core-shell structure is maintained. These SiO2-coated TiN particles exhibit improved optical absorption and photothermal efficiency, as well as better dispersibility and thermal stability against reoxidation. This makes them superior to well-established materials such as gold nanoshells or other alternative photothermal agents. The interaction of these particles with HeLa cells is investigated in detail in chapter 3. They are taken up by the cells and localized in membrane-enclosed vesicles within the cytoplasm. The particles themselves are well-tolerated by cells. In contrast, when combined with laser irradiation, they can effectively kill HeLa cancer cells through the increase in temperature, making SiO2-coated TiN most appealing as a photothermal agent. The treatment of tumors is typically preceded by a detailed diagnosis based on an imaging modality. Fluorescence imaging is known for its inexpensive real-time imaging possibilities with high spatial and temporal resolution. However, the currently available fluorescent contrast agents operating in the NIR-regions exhibit major limitations, such as poor photostability and quick degradation. To tackle this problem, novel inorganic nanoparticles with NIR-II emission are developed based on Mn5+ (chapter 4). These are doped into a Ba3(VO4)2 host matrix, leading to strong emission at around 1180 nm following 750 nm excitation. However, these particles exhibited substantial dissolution. This, in turn, led to relatively high cytotoxicity, which was traced back to released vanadium ions. To overcome this problem, a bismuth-containing mixed oxide with the composition Ba3(VO4)2:Mn5+ - Bi2O3 (called BaVOMn – BiO) was prepared. The addition of bismuth resulted in higher particle stability, and therefore negligible cytotoxicity. Furthermore, also the brightness of these particles was increased by almost an order of magnitude through the addition of Bi2O3. The particles also exhibited excellent colloidal, chemical and photostability, in contrast to commercial ICG or PbS-CdS quantum dots. Finally, BaVOMn-BiO particles were incubated together with HeLa cells and imaged by fluorescence microscopy, revealing their uptake by cells and accumulation around the nucleus. Finally, their performance for ex vivo deep-tissue imaging was assessed, placing their performance on par with PbS-CdS quantum dots. Interestingly, the emitted fluorescence signal carries more information: The luminescence is typically also affected by the surrounding temperature. Therefore, some luminescent contrast agents can also act as local temperature sensors to allow for non-contact nanothermometry. This enables the local determination of temperature within tissues, which is crucial to control the heating during photothermal therapy (chapters 2 and 3). The most important performance parameter for such thermometers is its sensitivity. In a first approach (chapter 5), the sensitivity is optimized based on the selection of emission lines for a ratiometric read-out of a previously developed NIR-emitting material (BiVO4:Nd3+). Founded on a detailed understanding of the rich variety of involved Nd3+-energy states, the sensitivity can be increased by an order of magnitude, enabling the accurate temperature sensing even within deeper tissues up to 6 mm. In chapter 5, the focus lies on the optimization of thermometric performance. However, other parameters crucial to their use in biomedical imaging are just as important and need to be carefully considered. These include suitable particle sizes, brightness, (chemical, colloidal and photo-)stability, and biocompatibility. As it was suggested to retain from using toxic metals in the formulations at all, regardless of the product stability, we investigate in chapter 6 a promising material composition based on phosphates. For the first time, Ba3(PO4)2 nanoparticles doped with Mn5+ are produced with sizes below 100 nm, suitable for intravascular applications. Besides the optimization of particle emission intensity (λ = 1190 nm), close attention is paid to their stability and biocompatibility. The response of three cell lines (cancer cells (HeLa), fibroblasts (NHDF), monocytes (THP-1)) to particle exposure is analyzed in detail, showing no signs of adverse reaction. Finally, the suitability of these particles for temperature sensing is evaluated, considering different read-out strategies. The result is a bright NIR-II emitting, stable, and biocompatible nanoprobe capable of temperature sensing in deep-tissue