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

Acetone Sensing and Catalytic Conversion by Pd-Loaded SnO2

Noble metal additives are widely used to improve the performance of metal oxide gas sensors, most prominently with palladium on tin oxide. Here, we photodeposit different quantities of Pd (0–3 mol%) onto nanostructured SnO2 and determine their effect on sensing acetone, a critical tracer of lipolysis by breath analysis. We focus on understanding the effect of operating temperature on acetone sensing performance (sensitivity and response/recovery times) and its relationship to catalytic oxidation of acetone through a packed bed of such Pd-loaded SnO2. The addition of Pd can either boost or deteriorate the sensing performance, depending on its loading and operating temperature. The sensor performance is optimal at Pd loadings of less than 0.2 mol% and operating temperatures of 200–262.5 °C, where acetone conversion is around 50%

Acetone Sensing and Catalytic Conversion by Pd-Loaded SnO2

Noble metal additives are widely used to improve the performance of metal oxide gas sensors, most prominently with palladium on tin oxide. Here, we photodeposit different quantities of Pd (0–3 mol%) onto nanostructured SnO2 and determine their effect on sensing acetone, a critical tracer of lipolysis by breath analysis. We focus on understanding the effect of operating temperature on acetone sensing performance (sensitivity and response/recovery times) and its relationship to catalytic oxidation of acetone through a packed bed of such Pd-loaded SnO2. The addition of Pd can either boost or deteriorate the sensing performance, depending on its loading and operating temperature. The sensor performance is optimal at Pd loadings of less than 0.2 mol% and operating temperatures of 200–262.5 °C, where acetone conversion is around 50%.ISSN:1996-194

Light Gold: A Colloidal Approach Using Latex Templates

ISSN:1616-3028ISSN:1616-301

Nd3+-Doped BiVO4 luminescent nanothermometers of high sensitivity

Neodymium-doped BiVO4nanoparticles are explored for luminescentnanothermometry in the first and second biological windows. Thenanothermometer sensitivity can be increased by an order of magnitudethrough careful selection of excitation wavelength and emission peaks,leading to sub-degree resolution and penetration depth up to 6 mm inbiological tissues.ISSN:1359-7345ISSN:1364-548

Silica-Coated TiN Particles for Killing Cancer Cells

ISSN:1944-8244ISSN:1944-825

Single-Nanoparticle Thermometry with a Nanopipette

Thermal measurements at the nanoscale are key for designing technologies in many areas, including drug delivery systems, photothermal therapies, and nanoscale motion devices. Herein, we present a nanothermometry technique that operates in electrolyte solutions and, therefore, is applicable for many in vitro measurements, capable of measuring and mapping temperature with nanoscale spatial resolution and sensitive to detect temperature changes down to 30 mK with 43 μs temporal resolution. The methodology is based on local measurements of ionic conductivity confined at the tip of a pulled glass capillary, a nanopipettete, with opening diameters as small as 6 nm. When scanned above a specimen, the measured ion flux is converted into temperature using an extensive theoretical support given by numerical and analytical modeling. This allows quantitative thermal measurements with a variety of capillary dimensions and is applicable to a range of substrates. We demonstrate the capabilities of this nanothermometry technique by simultaneous mapping of temperature and topography on sub-micrometer-sized aggregates of thermoplasmonic nanoparticles heated by a laser and observe the formation of micro- and nanobubbles upon plasmonic heating. Furthermore, we perform quantitative thermometry on a single-nanoparticle level, demonstrating that the temperature at an individual nanoheater of 25 nm in diameter can reach an increase of about 3 K. © 2020 American Chemical Society.ISSN:1936-0851ISSN:1936-086

Simultaneous Nanothermometry and Deep‐Tissue Imaging

Bright, stable, and biocompatible fluorescent contrast agents operating in the second biological window (1000–1350 nm) are attractive for imaging of deep‐lying structures (e.g., tumors) within tissues. Ideally, these contrast agents also provide functional insights, such as information on local temperature. Here, water‐dispersible barium phosphate nanoparticles doped with Mn5+ are made by scalable, continuous, and sterile flame aerosol technology and explored as fluorescent contrast agents with temperature‐sensitive peak emission in the NIR‐II (1190 nm). Detailed assessment of their stability, toxicity with three representative cell lines (HeLa, THP‐1, NHDF), and deep‐tissue imaging down to about 3 cm are presented. In addition, their high quantum yield (up to 34%) combined with excellent temperature sensitivity paves the way for concurrent deep‐tissue imaging and nanothermometry, with biologically well‐tolerated nanoparticles.ISSN:2198-384

Precision in Thermal Therapy: Clinical Requirements and Solutions from Nanotechnology

The heating of diseased tissue as a therapeutic measure has gained increased clinical attention, mostly due to its target‐specificity that minimizes side effects. However, to ensure a successful therapy, heating has to be homogeneous and highly localized, as well as, within a certain temperature range. Therefore, precise control over thermal treatments is a clinical prerequisite to minimize treatment and safety margins. Although this requirement is mentioned frequently, past research has focused predominantly on improving thermometry resolution and heating efficiency through tedious material optimization. Here, current clinical applications of thermal therapy with their challenges are first highlighted, especially with respect to treatment control and margins. Thereafter, it is quantitatively shown that clinically available thermometry fulfills the requirements and future research should focus on achieving better temperature control instead. With nanotechnology, novel strategies based on self‐limiting nanoparticle systems and particle‐based thermometers with active feed‐back control have also become available and are discussed. All of these approaches are systematically compared and analyzed with respect to their clinical applicability. The extent to which control over thermal therapy is necessary is also discussed alongside a presentation of the existing methods which fulfill the set requirements for clinical success and what issues remain to be tackled by research in the near future.ISSN:2366-398

Bi2O3 boosts brightness, biocompatibility and stability of Mn-doped Ba3(VO4)2 as NIR-II contrast agent

Deep-tissue fluorescence imaging remains a major challenge as there is limited availability of bright biocompatible materials with high photo- and chemical stability. Contrast agents with emission wavelengths above 1000 nm are most favorable for deep tissue imaging, offering deeper penetration and less scattering than those operating at shorter wavelengths. Organic fluorophores suffer from low stability while inorganic nanomaterials (e.g. quantum dots) are based typically on heavy metals raising toxicity concerns. Here, we report scalable flame aerosol synthesis of water-dispersible Ba3(VO4)2 nanoparticles doped with Mn5+ which exhibit a narrow emission band at 1180 nm upon near-infrared excitation. Their co-synthesis with Bi2O3 results in even higher absorption and ten-fold increased emission intensity. The addition of Bi2O3 also improved both chemical stability and cytocompatibility by an order of magnitude enabling imaging deep within tissue. Taken together, these bright particles offer excellent photo-, chemical and colloidal stability in various media with cytocompatibility to HeLa cells superior to existing commercial contrast agents.ISSN:2050-7518ISSN:2050-750