Nanotechnology has enabled significant breakthroughs in the early detection and treatment of disease, but many of these advances rely on expensive and less-accessible imaging modalities. Ultrasound, on the other hand, is a noninvasive imaging modality that stands out for its universal availability, cost-effectiveness, and safety. However, harnessing the benefits of nanomaterials for ultrasound has been challenging due to the size and stability constraints of typical ultrasound contrast agents. Recently, an innovative solution has emerged in the form of gas vesicles (GVs), a class of air-filled protein nanostructures found in certain aquatic microbes. These promising next-generation ultrasound contrast agents offer a crucial bridge between nanotechnology and ultrasonography.
In this thesis, we investigate the in vivo behavior of GVs, explore their potential applications as nanodiagnostic agents, and consider key factors for their future clinical deployment. In Chapter 2, we examine the interactions of GVs with blood components, focusing on imaging performance and immunogenicity. In Chapter 3, we show that intravenously injected GVs are cleared by liver-resident macrophages and subsequently undergo lysosomal degradation. We leverage this finding to develop an ultrasound-based method for visualizing cellular degradative processes and demonstrate its potential as a liver disease diagnostic. In Chapter 4, we introduce bicone GVs, the smallest known ultrasound contrast agent. We show that these sub-80 nm particles can penetrate tumors, deliver potent ultrasound-induced mechanical effects, and are readily engineered for molecular targeting, extended circulation time, and payload conjugation.
Together, these findings highlight the tremendous potential of GVs as injectable nanomaterials for ultrasound imaging, laying the foundation for future studies to further refine the design and application of these agents.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/87jm-7v06, author = {Dutka, Przemysław}, title = {Cryo-ET Reveals Molecular Details of Multi-Megadalton Bacterial Protein Complexes}, school = {California Institute of Technology}, year = {2023}, doi = {10.7907/87jm-7v06}, url = {https://resolver.caltech.edu/CaltechTHESIS:05092023-163511031}, abstract = {Cryo-electron tomography (cryo-ET) is a powerful method for investigating the 3D structure of intact cells, organelles, and complex protein macromolecules that cannot be crystallized or are too heterogenous for single-particle cryo-electron microscopy (cryo-EM). However, obtaining high- resolution cryo-ET structures for many biologically important targets is still a challenge. To address this challenge, cryo-ET can be combined with other methods, including X-ray crystallography, single-particle cryo-EM, structure predictions, cross-linking mass spectrometry, biochemistry, and evolutionary analysis to produce integrative models. Recently, with the development of AI-based tools such as AlphaFold2, structure prediction has played an increasingly important role in integrative modeling. The combination of cryo-ET and structure prediction in particular has provided unprecedented insights into the ultrastructure of cellular components. This thesis focuses on two bacterial multi-megadalton protein complexes which are difficult to study by classical structural biology approaches: gas vesicles (GVs) and the Legionella pneumophila Dot/Icm type IV secretion system (T4SS). GVs are gas-filled protein nanostructures that regulate the position of certain microorganisms in water and consequently their access to sunlight and nutrients. Here, we investigate the mechanical properties of GVs and reveal the molecular structure of GVs and its implication for the assembly mechanism. The Dot/Icm T4SS is a macromolecular complex formed by approximately 27 proteins, utilized by L. pneumophila to hijack the host cell’s biology for its replication purposes. A nearly-complete integrative model of this complex provides crucial insights into its structural organization and its evolution from conjugation to secretion, as well as the transportation of substrates into the host cell.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/qs6v-5d67, author = {Hurt, Robert Cooper}, title = {Engineering of Second-Generation Acoustic Reporter Genes}, school = {California Institute of Technology}, year = {2023}, doi = {10.7907/qs6v-5d67}, url = {https://resolver.caltech.edu/CaltechTHESIS:05192023-001330664}, abstract = {A major outstanding challenge in the fields of biological research, synthetic biology, and cell-based medicine is visualizing the functions of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 µm. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles (GVs) to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo.
The goal of my thesis work has been to engineer second-generation ARGs with improved acoustic and expression phenotypes compared to the existing first-generation constructs. I took two complementary engineering approaches to developing these constructs: homolog screening and directed evolution, sometimes referred to as the “nature and nurture” of protein engineering. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue in vivo, stable long-term expression, and compatibility with in vitro multiplexed imaging. In collaboration with others in the lab, we demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.
This thesis is organized as follows: in the first two chapters, I introduce the key background needed to understand both the importance and properties of ARGS, and how they have been and could be engineered. In the next two chapters, I detail specific efforts to engineer them—one involving the construction of a high-throughput, semi-automated setup for acoustic phenotyping of cells and its application to ARG directed evolution, and another involving the screening of several GV cluster homologs to identify ones suitable for use as improved ARGs. Finally, I conclude with insights gleaned from these two ARG engineering projects and suggestions for future ones.
The approaches, results, and ideas presented in this thesis represent the current state-of-the-art in ARG engineering and application. While recent technology development in this field has unlocked exciting new use cases for ARGs in noninvasive biological imaging, most of their potential for basic science and disease diagnosis and treatment has yet to be realized.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/5w59-0667, author = {Xiong, Lealia Li}, title = {Expanding the Toolbox for Thermal Control of E. coli: Cold-Activated Transcription with Applications in Temperature Self-Regulation}, school = {California Institute of Technology}, year = {2023}, doi = {10.7907/5w59-0667}, url = {https://resolver.caltech.edu/CaltechTHESIS:05272023-161541041}, abstract = {Temperature can be used to control engineered E. coli — for example, the living component of an engineered living material (ELM) - through the use of thermolabile transcription factors. Sharp induction of gene expression with heat has been established using these bacteria- and phage-derived proteins. Here, we expand the toolbox for thermal control of E. coli through both direct cold-induced gene expression and through the construction of genetic circuits to invert heat-induced gene expression.
We accomplish direct induction at low temperatures through the use of temperature-sensitive mutants of Lambda repressor as transcriptional activators. In addition, we show that a temperature-sensitive mutant of Lambda repressor can serve as an activator and a repressor of different genes simultaneously in one genetic circuit, leading to opposite thermal responses and serving as a temperature switch.
Next, we demonstrate inversion of a temperature-sensitive repressor using a temperature insensitive repressor. We apply this multicomponent switch to engineer a temperature self-regulation circuit for E. coli-based ELMs. Seasonal variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment, because E. coli growth rate is impaired both below and above 37°C. Our construct enables E. coli to produce a light-absorptive pigment in response to environmental temperature below 36°C with the goal of allowing the cells to absorb sunlight and locally warm to their optimal growth temperature. We demonstrate the efficacy of our pigment temperature switch in a model flat ELM growing at 32°C and 42°C in a home-built illuminated growth chamber. Below 36°C, our engineered E. coli increase in pigmentation, causing an increase in sample temperature and growth rate above non-pigmented bacteria. On the other hand, above 36°C, they decrease in pigmentation, protecting their growth compared to bacteria with temperature- independent high pigmentation. Integrating our temperature homeostasis circuit into an ELM has the potential to improve ELM performance by optimizing growth and protein production in the face of seasonal temperature changes.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, } @phdthesis{10.7907/5rr6-q625, author = {Abundo, Maria Paulene Bernal}, title = {Ultrasound Controlled Drug Delivery by Acoustically Switchable Hydrogels}, school = {California Institute of Technology}, year = {2023}, doi = {10.7907/5rr6-q625}, url = {https://resolver.caltech.edu/CaltechTHESIS:05272023-093432414}, abstract = {Not only is ultrasound widely used as a diagnostic imaging modality, it can also be focused into deep tissues to perform non-invasive actuation of cells, implants and delivery vehicles and other biological targets. With the addition of gas vesicles (GV), generic hydrogel materials gain the ability to communicate with ultrasound, equipping them with in vivo tracking, targeting and actuation capabilities to safely transport biomolecular cargo. This is possible as GVs function simultaneously as ultrasound contrast agents and steric blockers that can be “erased” by an increase in ultrasound pressure to trigger a rapid outflow diffusion of the payload from within the material. We evaluate this concept through in vitro measurements of ultrasound-modulated diffusion and drug release and targeted in vivo release in the lower gastrointestinal tract. Then we demonstrate the use of orally administered hydrogel particles to deliver etanercept in the duodenum to treat gastrointestinal inflammation in a rat model of colitis. Finally, we explore new directions and applications of GV-hydrogel systems, showcasing their potential for deployment in a wide range of biomedical applications.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/dt22-nv14, author = {Wu, Di}, title = {Biomolecular Tools for Noninvasive Imaging and Manipulation of Engineered Cells}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/dt22-nv14}, url = {https://resolver.caltech.edu/CaltechTHESIS:05262021-021059637}, abstract = {Today’s most advanced tools for imaging and controlling cellular function are based on fluorescent or light-controlled proteins, which have limited utility in large organisms or engineered living materials due to the scattering of photons. Deeply penetrant forms of energy such as magnetic fields and sound waves, while routinely used to monitor and treat diseases on the tissue and organism level, do not process the equivalent set of biomolecular tools for interfacing with biology on the molecular and cellular level. Emerging technologies discussed in this thesis aim to bridge this gap by harnessing biomolecules that have the appropriate physical properties to interact with sound waves or magnetic fields in such a way that enables the visualization and control of specific cells (Chapter 1). We describe two additions to the expanding toolkit for noninvasive imaging and control. In the first case, we show that gas vesicles, a class of hollow protein nanostructures naturally found in aquatic single-cell organisms, can be used as acoustic actuators to enable the control of cellular forces, movement, and patterning using ultrasound (Chapter 2). In the second case, we show that aquaporins, a class of membrane water channels, can be used to alter cellular permeability and serve as genetic reporters for magnetic resonance imaging (Chapter 3). These tools provide critical capabilities for interfacing with cellular function noninvasively and could open the door to applications in various research, biomedical, and industrial settings.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/p52e-qv56, author = {Sawyer, Daniel Patrick}, title = {Enhanced Noninvasive Imaging of Acoustic Biomolecules}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/p52e-qv56}, url = {https://resolver.caltech.edu/CaltechTHESIS:06022021-043318684}, abstract = {The extensive scientific interest in cellular and biomolecular processes is due in large part to the importance of such processes deep inside living organisms, in the context of both health and disease. However, most methods for imaging cellular processes such as gene expression have relied on fluorescent proteins and other optical reporters that, while providing a direct optical readout of the biomolecular environment in cells readily exposed to light, have greatly limited performance in large animals due to the poor penetration of visible light beyond 1 mm of biological tissue. In contrast, ultrasound is widely used to noninvasively image tissue deep inside living organisms but has rarely been used to investigate cellular function due a lack of acoustic reporters whose production and properties are coupled to biomolecular events. Recently, the first acoustic reporter genes (ARGs) were developed for ultrasound imaging of a unique class of air-filled protein nanostructures known as gas vesicles, or GVs, which scatter sound waves when expressed in bacterial and mammalian cells. ARGs allow gene expression to be visualized with ultrasound similar to how green fluorescent protein (GFP) allowed gene expression to be visualized with light. However, ARGs will have limited utility in practical applications involving living organisms without ultrasound imaging methods providing the specificity to reliably distinguish GVs from surrounding tissue and the sensitivity to detect GVs at low concentrations.
In this thesis, we present two novel ultrasound imaging methods that exploit the unique nonlinear physical properties of gas vesicles to enhance image quality in situations that pose challenges for conventional imaging methods. In Chapter 1, we address the problem of distinguishing GVs from tissue with cross-Amplitude Modulation (xAM), an ultrasound pulse sequence that uses X-waves to isolate the signal generated by reversible buckling of the GV shell while cancelling scattering and artifacts from tissue. In Chapter 2, we present an application of xAM to imaging of dynamic biomolecular processes. We show that, when GVs are engineered such that buckling is induced by enzyme activity, xAM can visualize enzymatic processes deep inside living animals. In Chapter 3, we address the problem of detecting very low concentrations of ARG-expressing cells with Burst Ultrasound Reconstructed with Signal Templates (BURST), an imaging method that exploits the strong, transient signals generated during sudden GV collapse under acoustic pressure by unmixing the temporal dynamics of such signals from background scattering. BURST imaging improves cellular sensitivity by more than 1000-fold and, in dilute cell suspensions, enables the detection of gene expression in individual bacteria and mammalian cells. In Chapter 4, we present an application of an early formulation of BURST to imaging gene expression in mammalian cells. We use this imaging method to visualize vascularization patterns in tumors containing mammalian cells expressing acoustic reporter genes.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/z7ac-2g66, author = {Abedi, Mohamad}, title = {Thermal Bioswitches for Non-Invasive Control of Cellular Therapies}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/z7ac-2g66}, url = {https://resolver.caltech.edu/CaltechTHESIS:03032021-224823348}, abstract = {Temperature is a unique input signal that could be used by engineered therapeutic cells to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, I present here a new class of thermal bioswitches that enables thermal control over bacterial and mammalian cells. For bacterial applications, we developed two new families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32–46 °C. We integrated these molecular bioswitches into thermal logic circuits and demonstrated their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications.
To apply this technology in a relevant clinical scenario, we sought to engineer microbial immunotherapies that can be thermally controlled with focused ultrasound. This technology was enabled by rapid advances in synthetic biology that are driving the development of genetically modified microbes as therapeutic agents for a multitude of human diseases, including cancer. In particular, the reduced immune surveillance within the core of some solid tumors creates an ideal environment for microbes to engraft and release therapeutic payloads. However, these therapeutic payloads could be harmful if released in healthy tissues where microbes tend to also engraft in smaller numbers. As described in Chapter 2, my colleagues and I introduced a temperature-actuated state switch that enables tight spatiotemporal control over the activity of therapeutic microbes when combined with focused ultrasound hyperthermia. Through a combination of rational design and high throughput screening, we optimized the behavior of this switch to minimize leakage and maximize inducibility. When tested in a clinically relevant in vivo model, engineered microbes, successfully switched states, and induced a marked suppression of tumor growth upon focal activation. This bioswitch provides a critical tool to attain selective and sustained activity of therapeutic microbes in vivo.
Encouraged by the successful development of thermally actuated circuits in microbes, we aimed to establish equivalent technologies for thermal control of human T cells. Genetically engineered T cells are actively being developed to perform a variety of therapeutic functions with great clinical promise. However, no robust mechanisms exist to externally control the activity of T cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. In Chapter 4, my colleagues and I tested the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T cells in the well-tolerated temperature range of 37−42 °C, and we introduced genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrated the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. Overall, the technologies developed here provide critical tools to direct control therapeutic cells after they have been deployed deep inside the body.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/zght-4j47, author = {Farhadi, Arash}, title = {Acoustic Reporter Genes for Noninvasive Imaging of Cellular Function}, school = {California Institute of Technology}, year = {2020}, doi = {10.7907/zght-4j47}, url = {https://resolver.caltech.edu/CaltechTHESIS:04222020-163929825}, abstract = {The study of cellular function within the context of intact living organisms is a grand challenge in biological research. Addressing this challenge requires imaging tools that can visualize cells inside the body. If successful, this would greatly increase our ability to study a battery of processes from brain development to tumorigenesis, to monitoring cell-based therapeutics. To date, most common methods for imaging cellular processes such as gene expression have relied on optical reporters, such as fluorescent or luminescent proteins, which provide high molecular precision for studies in petri dishes and transparent organisms, but have limited performance in large animals due to the poor penetration of light in biological tissue. Conversely, magnetic resonance imaging (MRI) and ultrasound can image tissues at depth with high spatial and temporal resolution, but they lack molecular reporters analogous to the green fluorescent protein (GFP). As a result, they have made limited impact on biological research. To address this, we focus on developing biomolecular reporters for MRI and ultrasound — based on a unique class of air-filled protein nanostructures called gas vesicles — using them to image the location and function of cells deep inside the body.
This thesis begins with a brief review of genetically encoded materials for noninvasive imaging, highlighting key advances over the past two decades and providing context for the work below. We discuss the development of increasingly sophisticated tools starting from early efforts to engineer single molecule reporters to recent work on multi-component genetic machinery (including gas vesicles) with multi-modality capabilities. In Chapter 2, we present a platform for engineering the surface of gas vesicles to modulate their acoustic, surface charge, and molecular- targeting properties as injectable acoustic biomolecules. In Chapter 3, we present the recombinant expression of gas vesicles as injectable contrast agents in common lab strain bacteria to facilitate the genetic engineering of the entire gas vesicle gene cluster and to assist this technology’s adoption by other (non-specialist) research groups. This work characterized the ultrasound and hyperpolarized 129Xenon-MRI contrast of gas vesicles as nanoscale contrast agents.
In a parallel effort, we developed a hybrid gene cluster that when introduced to microbes enables the imaging of their gene expression using ultrasound. These bacterial acoustic reporter genes were used to image the location of probiotic cells inside the gastrointestinal tract of mice. However, the ability for these genes to be expressed in mammalian cells had not been demonstrated and presented a major challenge in synthetic biology. In Chapter 4, we addressed this by introducing the first mammalian acoustic reporter genes — a genetic program whose introduction to mammalian cells resulted in the expression of gas vesicles that can be visualized by ultrasound. These mammalian acoustic reporter genes will enable previously impossible approaches to monitoring the location, viability and function of mammalian cells in vivo.
In Chapter 5, we explore a new paradigm in MRI by taking advantage of the acousto-magnetic property of gas vesicles. Here, we present background-free MRI to address a longstanding challenge in untangling the signal of exogenous contrast agents from the endogenous MRI contrast produced by biological tissues. Chapter 6 explores the optical properties of gas vesicles as genetically encodable phase contrast agents in digital holographic imaging. Chapter 7 is a brief discussion of the potential future directions for this work.
The data presented in this thesis lays the ground for exciting new research on developing noninvasive biomolecular tools that will enable the discovery of novel biological processes.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/9QEJ-6H55, author = {Davis, Hunter Cole Davis}, title = {Mechanistic Insights for Magnetic Imaging and Control of Cellular Function}, school = {California Institute of Technology}, year = {2020}, doi = {10.7907/9QEJ-6H55}, url = {https://resolver.caltech.edu/CaltechTHESIS:10072019-141728052}, abstract = {The vast biomolecular toolkit for optical imaging and control of cellular function has revolutionized the study of in vitro samples and superficial tissues in living organisms but leaves deep tissue unexplored. To look deeper in tissue and observe system-level biological function in large organisms requires a modality that exploits a more penetrant form of energy than visible light. Magnetic imaging with MRI reveals the previously unseen, with endogenous tissue contrast and practically infinite penetration depth. While these clear advantages have made MRI a cornerstone of modern medical imaging, the sparse library of molecular agents for MRI have severely limited its utility for studies of cellular function in vivo. The development of new molecular agents for MRI has suffered from a lack of tools to study the connection between changes in the microscale cellular environment and the corresponding millimeter-scale MRI contrast. Bridging this gap requires revisiting the mechanistic underpinnings of MRI contrast, casting aside some of the simplifications that smooth over sub-voxel heterogeneity that is rich with information pertinent to the underlying cell state.
Here, we will demonstrate theoretical, computational, and experimental connections between subtle changes in microscale cellular environment and resultant MRI contrast. After reviewing some foundational principles of MRI physics in the first chapter, the second chapter of the thesis will explore computational models that have significantly enhanced the development of genetically encoded agents for MRI, including the first genetically encoded contrast agent for diffusion weighted imaging. By improving the efficacy of these genetically encoded agents, we unlock MRI reporter genes for in vivo studies of cellular dynamics much in the same way that the engineering of Green Fluorescent Protein has dramatically improved in vitro studies of cellular function.
In the third chapter, we introduce our study that maps microscale magnetic fields in cells and tissues and connects those magnetic fields to MRI contrast. Such a connection has previously been experimentally intractable due to the lack of methods to resolve small magnetic perturbations with microscale resolution. To overcome this challenge, we leverage nitrogen vacancy diamond magnetometry to optically probe magnetic fields in cells with sub-micron resolution and nanotesla sensitivity, together with iterative localization of field sources and Monte Carlo simulation of nuclear spins to predict the corresponding MRI contrast. We demonstrate the utility of this technology in an in vitro model of macrophage iron uptake and histological samples from a mouse model of hepatic iron overload. In addition, we show that this technique can follow dynamic changes in the magnetic field occurring during contrast agent endocytosis by living cells. This approach bridges a fundamental gap between an MRI voxel and its microscopic constituents and provides a new capability for noninvasive imaging of opaque tissues.
In the fourth chapter, we focus on the use of magnetic fields to perturb, rather than image, biological function. Recent suggestions of nanoscale heat confinement on the surface of synthetic and biogenic magnetic nanoparticles during heating by radiofrequency alternating magnetic fields have generated intense interest due to the potential utility of this phenomenon in non-invasive control of biomolecular and cellular function. However, such confinement would represent a significant departure from classical heat transfer theory. We present an experimental investigation of nanoscale heat confinement on the surface of several types of iron oxide nanoparticles commonly used in biological research, using an all-optical method devoid of potential artifacts present in previous studies. By simultaneously measuring the fluorescence of distinct thermochromic dyes attached to the particle surface or dissolved in the surrounding fluid during radiofrequency magnetic stimulation, we found no measurable difference between the nanoparticle surface temperature and that of the surrounding fluid for three distinct nanoparticle types. Furthermore, the metalloprotein ferritin produced no temperature increase on the protein surface, nor in the surrounding fluid. Experiments mimicking the designs of previous studies revealed potential sources of artifacts. These findings inform the use of magnetic nanoparticle hyperthermia in engineered cellular and molecular systems and can help direct future resources towards tractable avenues of magnetic control of cellular function.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/71ak-w328, author = {Mittelstein, David Reza}, title = {Modifying Ultrasound Waveform Parameters to Control, Influence, or Disrupt Cells}, school = {California Institute of Technology}, year = {2020}, doi = {10.7907/71ak-w328}, url = {https://resolver.caltech.edu/CaltechTHESIS:05242020-045332969}, abstract = {Ultrasound can be focused into deep tissues with millimeter precision to perform non-invasive ablative therapy for diseases such as cancer. In most cases, this ablation uses high intensity ultrasound to deposit non-selective thermal or mechanical energy at the ultrasound focus, damaging both healthy bystander tissue and cancer cells. Here we describe an alternative low intensity pulsed ultrasound approach known as “oncotripsy” that leverages the distinct mechanical properties of neoplastic cells to achieve inherent cancer selectivity. We show that when applied at a specific frequency and pulse duration, focused ultrasound selectively disrupts a panel of breast, colon, and leukemia cancer cell models in suspension without significantly damaging healthy immune or red blood cells. Mechanistic experiments reveal that the formation of acoustic standing waves and the emergence of cell-seeded cavitation lead to cytoskeletal disruption, expression of apoptotic markers, and cell death. The inherent selectivity of this low intensity pulsed ultrasound approach offers a potentially safer and thus more broadly applicable alternative to non-selective high intensity ultrasound ablation.
In this dissertation, I describe the oncotripsy theory in its initial formulation, the experimental validation and investigation of testable predictions from that theory, and the refinement of said theory with new experimental evidence. Throughout, I describe how careful modifications to the ultrasound waveform directly can significantly impact how the ultrasound bio-effects control, influence, or disrupt cells in a selective and controlled manner.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, } @phdthesis{10.7907/ASX5-KB62, author = {Lakshmanan, Anupama}, title = {Engineering Acoustic Protein Nanostructures for Non-Invasive Molecular Imaging using Ultrasound}, school = {California Institute of Technology}, year = {2019}, doi = {10.7907/ASX5-KB62}, url = {https://resolver.caltech.edu/CaltechTHESIS:06062019-165907194}, abstract = {
Visualizing biomolecular and cellular processes in real time within deep tissues is fundamental to our understanding of the normal and pathological activity underlying health and disease. Ultrasound provides the ability to non-invasively image deep inside biological tissues with high spatial and temporal resolution. However, this technology has limited capacity to monitor molecular and cellular processes, due to the lack of appropriate intra-cellular and endogenously producible nanoscale contrast agents, which can directly couple sound waves to the activity or concentration of physiologically relevant molecules. This problem could in principle be solved by developing genetically encodable ultrasound sensors – biomolecules that can get illuminated in ultrasound imaging in response to specific cellular or molecular activity. This thesis describes the engineering and characterization of acoustic protein nanostructures called ‘gas vesicles’, or ‘GVs’, to accomplish this task.
GVs are protein-shelled gas-filled nanostructures produced by buoyant microbes, and were recently shown to be capable of scattering sound waves to produce ultrasound contrast. Owing to this property, they were initially conceptualized as a new class of ultrasound contrast agents. However, little was known about their tunability to enable molecular ultrasound imaging for a wide range of applications. In this thesis, we leveraged the genetic encodability of GVs to modify them at the level of their DNA sequence and constituent proteins, and thereby tune their mechanical, acoustic, surface and targeting properties. We accomplished this by establishing a facile and modular molecular engineering platform, to produce GVs that provide enhanced nonlinear signals for sensitive and specific detection in deep tissues, target specific cell types such as cancer and immune cells, and also provide distinct acoustic collapse spectra for multiplexed imaging. We then extended this platform to build GV-based biosensors that modulate their nonlinear ultrasound signals in response to changes in the activity or concentration of specific molecules in their environment. Specifically, we engineered acoustic sensors for three different types of enzymes and for calcium – whose activity or flux underlie a wide range of important cellular processes. Furthermore, we succeeded in transferring the genetic code of gas vesicles from their species of origin into a variety of other microbes that do not naturally produce them, in order to unlock their potential as ultrasound reporter genes. Our results establish GVs as reliable acoustic biomolecules, and thereby extend the capabilities of ultrasound for molecular and cellular imaging in a manner analogous to green fluorescent protein (GFP) and its derivatives in optical microscopy. When combined with the advantages of ultrasound for non-invasive imaging, this work facilitates novel technology to significantly enhance our understanding of molecular and cellular processes in basic biology, as well as enable improved diagnosis, monitoring and treatment of diseases.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/KY00-7Y74, author = {Ramesh, Pradeep}, title = {Imaging and Control of Engineered Cells using Magnetic Fields}, school = {California Institute of Technology}, year = {2019}, doi = {10.7907/KY00-7Y74}, url = {https://resolver.caltech.edu/CaltechTHESIS:06052019-181520170}, abstract = {Making cells magnetic is a long-standing goal of synthetic biology, aiming to enable the separation of cells from complex biological samples and their non-invasive visualization in vivo using Magnetic Resonance Imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have largely been unsuccessful due to the stringent required chemical conditions. In this thesis, we introduce an alternative approach to making cells magnetic, focusing on biochemically maximizing cellular paramagnetism. Here, we show that a novel genetic construct combining the functions of ferroxidation and iron-chelation enables engineered bacteria to accumulate iron in ‘ultraparamagnetic’ macromolecular complexes, which subsequently allows for these cells to be trapped using strong magnetic field gradients and imaged using MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, an array of spectroscopic techniques, biochemical assays, and computational modeling to elucidate the unique mechanisms and implications of this ‘ultraparamagnetic’ concept.
In addition to making cells magnetic, remote control of cellular localization in deep tissue is another long-standing goal of synthetic biology. Such an ability to non-invasively direct cells to sites of interest will not only improve therapeutic outcomes by minimizing off-target activity, but more broadly enable new research on complex cellular communities, such as the gut microbiome, in living animals. Given their deep penetrance through tissues, magnetic fields are ideally suited for facilitating non-invasive targeting of cells; however, the rapid decay of magnetic flux density from its source currently limits the depths to which magnetic targeting can be employed to within 1-2 mm from the surface. Here, we demonstrate a new approach wherein the retention of orally-administered and synthetically magnetized cell-like-particles is selectively enhanced within the murine intestinal tract to depths of up to 13 mm from the surface. Our cellular localization assisted by magnetic particles (CLAMP) strategy can potentially be generalized to any cell (bacterial, mammalian) or drug-containing nanoparticle of interest, and can be combined with existing non-invasive imaging modalities thereby facilitating remote environmental sensing at sites of interest.
Finally, while magnetic fields in MRI scanners are widely used today to safely and non-invasively image anatomical structures in living animals, much of the image contrast in MRI is the result of microscale magnetic-field variations in tissues. However, the connection between these microscopic patterns and the appearance of macroscopic MR images has not been the subject of direct experimental studies due to a lack of methods to map microscopic fields in biological samples under ambient conditions. Here, we optically probed magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy (NV) diamond magnetometry and combined these measurements with simulations of nuclear-spin precession to predict the corresponding MRI contrast. Additionally, we demonstrate the broad utility of this technology for imaging an in vitro model of cellular iron uptake, as well as imaging histological samples from a mouse model of hepatic iron overload. Taken together, our approach bridges a fundamental intellectual gap between a macroscopic MRI voxel and its microscopic constituents.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/DH10-6N61, author = {Piraner, Dan Ilya}, title = {Tunable Thermal Bioswitches as a Control Modality for Next Generation Therapeutics}, school = {California Institute of Technology}, year = {2019}, doi = {10.7907/DH10-6N61}, url = {https://resolver.caltech.edu/CaltechTHESIS:05312019-045041264}, abstract = {
Synthetic biology is rapidly contributing to the field of therapeutic development to create increasingly potent agents for the treatment of a variety of diseases. These living “designer therapeutics” are capable of integrating multiple sensory inputs into decision making processes to unleash an array of powerful signaling and effector responses. Included in the great therapeutic potential of these agents, however, is a cognate risk of severe toxicity resulting from runaway on-target or erroneously induced off-target activity. The ability to remotely control engineered therapeutic cells after deployment into patient tissue would drastically reduce the potential dangers of such interventions. However, among existing biological control methods, systemic chemical administration typically lacks the spatial precision needed to modulate activity at specific anatomical locations, while optical approaches suffer from poor light penetration into biological tissue. On the other hand, temperature can be controlled both globally and locally — at depth — using technologies such as focused ultrasound, infrared light and magnetic particle hyperthermia. In addition, body temperature can serve as an indicator of the patient’s condition. Overall, temperature is a versatile signal which can provide a handle to actuate a biological response for the control of therapeutic agents.
In this thesis, a tunable and modular system is developed to respond to thermal perturbations in cellular environments and affect a biological response. At the core of this system is a pair of single-component thermosensing proteins whose dimerization is strongly and sharply coupled to their thermal environment. These domains are first utilized in their native context as negative regulators of transcription in prokaryotes, wherein they are integrated into genetic circuits to control expression of reporter genes. These gene circuits show strong and sharp thermal activation and can be utilized in multiplex to affect higher order logical operations. Cells imbued with these circuits demonstrate transcriptional activation upon global thermal elevation within the host animal within which they reside (fever) or upon a spatiotemporally localized temperature shift imparted by focused ultrasound hyperthermia. In subsequent work, one of these bioswitches is introduced into mammalian cells where it functions as a modular Protein-Protein Interaction (PPI) domain, conferring temperature-dependent protein localization.
The work conducted in this thesis demonstrates the feasibility of utilizing temperature as a stimulus for biological activity. This technology can be harnessed to regulate therapeutically relevant processes in bacterial and mammalian cells such as transcriptional regulation and protein localization, and potentially broader protein function. The thermal bioswitches described herein could be utilized to engineer an array of research tools and biological therapies with actuation driven by spatiotemporally precise noninvasively applied stimuli or by real-time sensing of host conditions.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Shapiro, Mikhail G.}, } @phdthesis{10.7907/Z9P55KJ7, author = {Monge Osorio, Manuel Alejandro}, title = {Localization and Stimulation Techniques for Implantable Medical Electronics}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z9P55KJ7}, url = {https://resolver.caltech.edu/CaltechTHESIS:05312017-143935777}, abstract = {Implantable medical devices (IMDs) are emerging as one of the keystones of tomorrow’s medical technology. Although they have enabled a revolution in medicine, from research to diagnosis to treatment, most of today’s devices have critical limitations. They are bulky, have low resolution, and, in some cases, are limited to basic functionality. Miniaturization of IMDs will have an enormous impact not only on the technology itself and the medical procedures they enable, but also on the lives of patients, who will be more comfortable, have greater confidence in their medical treatments, and enjoy an overall improvement in their quality of life. The path towards miniaturized bioelectronic devices requires a reevaluation of existing paradigms to reach a seamless integration of electronics and biology. Miniaturization of medical electronics then involves an exploration of advanced integrated circuit processes and novel circuit and system level architectures. In this dissertation, we provide an overview of implantable medical devices and present novel circuit and system level techniques for the miniaturization of medical electronics.
The function of wireless miniaturized medical devices such as capsule endoscopes, biosensors, and drug delivery systems depends critically on their location inside the body. However, existing electromagnetic, acoustic, and imaging-based methods for localizing and communicating with such devices with spatial selectivity are limited by the physical properties of tissue or imaging modality performance. In the first part of this dissertation, we introduce a new approach for microscale device localization by embodying the principles of nuclear magnetic resonance in a silicon integrated circuit. By analogy to the behavior of nuclear spins, we engineer miniaturized RF transmitters that encode their location in space by shifting their output frequency in proportion to the local magnetic field. The application of external field gradients then allows each device’s location to be determined precisely from the frequency of its signal. We demonstrate the core capabilities of these devices, which we call addressable transmitters operated as magnetic spins (ATOMS), in an integrated circuit smaller than 0.7 mm^3, manufactured through a standard 180 nm complementary metal-oxide-semiconductor (CMOS) process. We show that ATOMS are capable of sub-millimeter localization in vitro and in vivo. As a technology that is inherently robust to tissue properties and scalable to multiple devices, ATOMS localization provides an enabling capability for the development of microscale devices to monitor and treat disease.
In neuroprosthetics, retinal prostheses aim to restore vision in patients suffering from advanced stages of retinal degeneration (e.g., retinitis pigmentosa) by bypassing the damaged photoreceptors and directly stimulating the remaining healthy neurons. In the second part of this dissertation, we describe a fully intraocular self-calibrating epiretinal prosthesis that reduces area and power consumption, and increases the functionality and resolution of traditional implementations. We introduce a novel novel digital calibration technique that matches the biphasic stimulation currents of each channel independently while sharing the calibration circuitry among every 4 channels. The system-on-chip presents dual-band telemetry for power and data with on-chip rectifier and clock recovery. These techniques reduce the number of off-chip components and achieve a power conversion efficiency >80% and supporting data rates up to 20 Mb/s. The system occupies an area of 4.5 x 3.1 mm2 and is implemented in 65 nm CMOS . It features 512 independent channels with a pixel size of 0.0169 mm2 and arbitrary waveform generation per channel. The chip is integrated with flexible MEMS origami coils and parylene substrate to provide a fully intraocular implant.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Emami, Azita}, }