Enabling Tools for Disease Diagnosis, Imaging and Intervention

Abstract

The ability to create and use tools differentiates humans from all the other living beings. As a result, tools have been playing an important role in human history. Millions of years ago, our ancestors created the very first piece of tools with stone. Since then, many other tools have been created to change our life. In the field of biomedicine, the creation and use of molecular tools, such as molecular probes, nanoscale agents, and drugs, have significantly benefited the modern society. Specifically, these tools have been applied for both the scientific discoveries and clinical advancements. This dissertation elaborates on three molecular tools’ applications - disease diagnosis, imaging and intervention - towards the healthcare revolution. First of all, in terms of disease diagnosis, the ability to detect biomarkers with ultrahigh sensitivity has radically transformed biology and disease diagnosis. However, owing to incompatibilities with infrastructure in current biological and medical laboratories, recent innovations in analytical technology have not received a broad adoption. Here, we have developed a simple yet universal ‘add-on’ technology (dubbed EASE) that can be directly plugged into the routine practices of current research and clinical laboratories and convert the ordinary sensitivities of common bioassays to the extraordinary level. The assay relies on the bioconjugation capabilities and ultrafast, localized deposition of polydopamine at the target site, which permits a large number of reporter molecules to be captured and lead to detection-sensitivity enhancements exceeding 3 orders of magnitude. The application of EASE in the enzyme-linked-immunosorbent-assay-based detection of the HIV antigen in blood from patients leads to a sensitivity lower than 3 fg ml-1. It is illustrated through experiments that EASE enables the direct visualization of the Zika virus in tissues and of low-abundance biomarkers related to neurological diseases and cancer immunotherapy. Besides the diagnosis sensitivity, molecular probes allow advanced on-surface assay formats to implement, but impose often underappreciated size-associated constraints, especially on assay kinetics and sensitivity. We evaluated and presented substantially slower diffusion limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction-limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme-linked immunosorbent assays and immunofluorescence, we demonstrate that this conceptually distinct approach effectively “erases” size-dependent diffusion constraints, providing a straightforward route to rapid on-surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single-cell molecular profiling. For proof of-concept, this study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with around eight times lower nanoprobe concentration. The findings are expected to stimulate realization of novel assay formats and development of rapid on-surface bioassays with nanoparticle probes. Secondly, in terms of molecular imaging, directly visualizing cellular functions and molecular processes have the potential to transform disease diagnosis, stratify therapy, and aid in drug discovery and validation. Photoacoustic imaging has emerged as a highly promising tool to visualize molecular events with deep tissue penetration. Like many other modalities, however, image contrast under in vivo conditions is far from optimal due to background signals from the tissue. Using iron oxide-gold core-shell nanoparticles, we have previously demonstrated the concept of magnetomotive photoacoustic (mmPA) imaging, which is capable of dramatically reducing the influence of background signals and producing high-contrast molecular images. We achieve two significant advancements on the clinical translation of this technology. On one hand, we introduce a new class of compact, uniform, magneto-optically coupled coreshell nanoparticles that are prepared through localized copolymerization of polypyrrole (PPy) on an iron oxide nanoparticle surface. The resulting iron oxide-PPy nanoparticles feature high colloidal stability and solve the photo-instability and small-scale synthesis problems previously encountered by the gold coating approach. On the other hand, we have developed a new generation of mmPA featuring cyclic magnetic motion and ultrasound speckle tracking (USST), whose imaging capture frame rate is several hundred times faster than the photoacoustic speckle tracking (PAST) method we demonstrated before. These improvements further the robust artifact elimination caused by physiologic motions and validate the application of the mmPA technology for in vivo sensitive tumor imaging. Along with the new nanoprobe design, we also solved the long-standing problem of conducting polymers, the poor resistance to de-doping that directly affects their signature electrical and optical properties. This problem is particularly noteworthy for biomedical uses because of the fast leaching of dopant ions in physiological environments. We develop a new approach to engineer multimodal core−shell nanoparticles with a stably doped conductive polymer shell in biological environments. The stable doping was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. We also found out its outstanding non-fouling property, surprisingly outperforming polyethylene glycol. This platform technology is also expected to create exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing. Also, In contrast to prior efforts to overcome PANI’s doping instability issue, we convert its drawback in pH sensitivity to a unique strength to address an important clinical problem. The structural, spectral, and chemical properties of the core–shell nanoparticles were systematically characterized, and a gastric acid secretory testing protocol simulating current clinical practice was developed for live mouse imaging. These nanometer-sized particles are sufficiently large to avoid passive diffusion through the gastrointestinal mucosa membranes, yet the PANI nanoshell is thin for fast proton diffusion and penetration. Complete nanoparticle elimination after imaging and no systematic toxicity may potentially enable this technology in humans, particularly for elderly and infants, to help reduce the suffering caused by gastrointestinal intubation. We expect this strategy to be readily extended to other pH sensitive polymers and dye molecules (converting a disadvantage into an advantage) for functional stomach imaging. Last but certainly not least, in terms of disease intervention, over the course of evolution, nature has developed sophisticated biological systems built of networks of cells that form electrical, mechanical, and communication infrastructure with functionality and efficiency often greatly surpassing the most advanced engineered systems. The main objective of this research is to develop a novel physical interface for hijacking the primary actuators of the infrastructure - individual live cells - to enable non-invasive in vivo modulation of cellular signaling at currently unattainable levels of spatial and temporal resolution. Fully-developed and -evaluated nanoacoustic technology promises to yield better therapies for diseases, such as cancers, Parkinson’s, Alzheimer’s and drug resistant hypertension.