Biology subjects

Malounda, D.

Publications and source records attributed to Malounda, D..

At least 19 recordsLinked to original sources

bioRxiv2025

A modular method for rapidly prototyping targeted gas vesicle protein nanoparticles

Gas vesicles (GVs) are air-filled protein nanoparticles which are proving to be useful in a number of biomedical applications. We hypothesized that it could be possible to develop a modular method for creating rapidly prototyped GVs by modifying their surface chemistry to include targeting peptides in an orientation-specific manner. Here, we describe a modular method to create targeted GVs using His-tagged antibody fragments, ensuring that the antibody fragments are connected to the GV in an orientation-specific manner. This is achieved via the functionalization of the GVs with nickel-nitrilotriacetic acid (Ni-NTA) group. First, we validated that these functionalized GVs can bind His-tagged green fluorescent protein and characterized the particle size and surface charge of functionalized GVs. Then, GVs targeted to prostate-specific membrane antigen (PSMA) using a minibody were validated using a knockout validation in vitro.

bioengineering↗

bioRxiv2025

Harmonic Amplitude-Modulated Singular Value Decomposition for Ultrafast Ultrasound Imaging of Gas Vesicles

Ultrafast nonlinear ultrasound imaging of gas vesicles (GV) contrast agents promises high-sensitivity biomolecular visualization with applications such as targeted molecular imaging of tumor markers or real-time tracking of gene expression. However, separating GV-specific signal from tissue remains challenging and requires the implementation of complex transmit schemes. In this work we introduce harmonic amplitude-modulated singular value decomposition (HAM-SVD), which synergizes pulse inversion (PI) with amplitude-modulated singular value decomposition (AM-SVD) to isolate GV-specific second-harmonic signals. In HAM-SVD, single-cycle plane waves at 9.6 MHz and five tilted angles (at a pulse repetition frequency of 2500 Hz) are transmitted under four duty cycles with alternating polarity. Beamformed IQ data are reshaped along a "space x pressure" matrix and decomposed via SVD; tissue background is cancelled by discarding the first and lowest singular modes, yielding an image comprised solely of pressure-dependent second harmonic signals. HAM-SVD sequence enables wide-field, ultrafast imaging without complex transmit sequences. Validation via simulations, in vitro phantoms, and in vivo rat lower limb experiments demonstrates HAM-SVDs outperformance compared to PI and AM-SVD. HAM-SVD is shown to achieve a 19.16 {+/-} 1.63 dB signal-to-background ratio (SBR) in vivo, surpassing PI (14.19 {+/-} 1.41 dB) and AM-SVD (15.79 {+/-} 1.38 dB). HAM-SVD overcomes limitations of conventional nonlinear techniques (e.g., depth restrictions, tissue clutter) by combining PIs harmonic sensitivity with AM-SVDs adaptive clutter filtering of tissue signals. This approach enhances molecular imaging specificity for GVs and holds potential for ultrasound localization microscopy of slow-flowing agents.

bioengineering↗

bioRxiv2024

Acoustic tumor paint for real-time imaging, surgical guidance and recurrence monitoring of brain tumors with ultrasound

The rapid growth, invasiveness, and resistance to treatment of glioblastoma multiforme (GBM) underscore the urgent need for improved diagnostics and therapies. Current surgical practice is limited by challenges with intraoperative imaging, while recurrence monitoring requires expensive magnetic resonance or nuclear imaging scans. Here we introduce "acoustic tumor paint", an approach to labeling brain tumors for ultrasound imaging - a widely accessible imaging modality. We show that gas vesicles (GVs), natural air-filled protein nanostructures, preferentially accumulate in brain tumors following systemic administration in syngeneic and xenograft mouse models of GBM. This enables real-time tumor visualization during surgery and postoperative monitoring of recurrence. We characterize GV uptake and breakdown by tumors and their resident cells and support clinical translatability by documenting non-toxic repeated administration. We also demonstrate the potential for post-operative monitoring in humans by imaging GVs through a human skull and an FDA-approved skull prosthesis. Acoustic tumor paint has the potential to enhance diagnostic accuracy, improve surgical outcomes, make monitoring more accessible, and extend survival in GBM patients.

bioengineering↗

bioRxiv2024

Real-time volumetric imaging of cells and molecules in deep tissues with Takoyaki ultrasound

Acoustic contrast agents and reporter genes play a critical role in allowing ultrasound to visualize blood flow, map molecules and track cellular function in opaque living organisms. However, existing ultrasound methods to image acoustic contrast agents predominantly focus on 2D planar imaging, while the biological phenomena of interest unfurl in three dimensions. Here, we introduce a method for efficient, dynamic imaging of contrast agents and reporter genes in 3D using multiplexed matrix array transducers. Our "Takoyaki" pulse sequence uses the simultaneous scanning of multiple focal points to excite contrast agents with sufficient acoustic pressure for nonlinear imaging while efficiently covering 3D space. Through in vitro experiments, we first show that the Takoyaki sequence produces highly sensitive volume images of gas vesicle contrast agents and compare its performance with alternative imaging schemes. We then establish its utility in cellular imaging in vivo by visualizing acoustic reporter gene-expressing tumors in a mouse model of glioblastoma. Finally, we demonstrate real-time volumetric imaging by tracking the dynamics of fluid motion in brain ventricles after intraventricular contrast injection. Takoyaki imaging enables a more comprehensive understanding of biological processes by providing spatiotemporal information in 3D within the constraints of accessible multiplexed matrix array systems.

bioengineering↗

bioRxiv2024

Characterisation of Gas Vesicles as Cavitation Nuclei for Ultrasound Therapy using Passive Acoustic Mapping

Genetically encodable gas filled particles known as gas vesicles (GVs) have shown promise as a biomolecular contrast agent for ultrasound imaging and have the potential to be used as cavitation nuclei for ultrasound therapy. In this study, we used passive acoustic mapping techniques to characterize GV-seeded cavitation, utilizing 0.5 and 1.6 MHz ultrasound over peak rarefactional pressures ranging from 100 to 2200 kPa. We found that GVs produce cavitation for the duration of the first applied pulse, up to at least 5000 cycles, but that bubble activity diminishes rapidly over subsequent pulses. At 0.5 MHz the frequency content of cavitation emissions was predominantly broadband in nature, whilst at 1.6 MHz narrowband content at harmonics of the main excitation frequency dominated. Simulations and high-speed camera imaging suggest that the received cavitation emissions come not from individual GVs but instead from the coalescence of GV-released gas into larger bubbles during the applied ultrasound pulse. These results will aid the future development of GVs as cavitation nuclei in ultrasound therapy.

bioengineering↗

bioRxiv2024

Enhanced Ultrasound Image Formation with Computationally Efficient Cross-Angular Delay Multiply and Sum Beamforming

Ultrasound imaging is a valuable clinical tool. It is commonly achieved using the delay and sum beamformer algorithm, which takes the signals received by an array of sensors and generates an image estimating the spatial distribution of the signal sources. This algorithm, while computationally efficient, has limited resolution and suffers from high side lobes. Nonlinear processing has proven to be an effective way to enhance the image quality produced by beamforming in a computationally efficient manner. In this work, we describe a new beamforming algorithm called Cross-Angular Delay Multiply and Sum, which takes advantage of nonlinear compounding to enhance contrast and resolution. This is then implemented with a mathematical reformulation to produce images with tighter point spread functions and enhanced contrast at a low computational cost. We tested this new algorithm over a range of in vitro and in vivo scenarios for both conventional B-Mode and amplitude modulation imaging, and for two types of ultrasound contrast agents, demonstrating its potential for clinical settings.

bioengineering↗

bioRxiv2024

Harmonic imaging for nonlinear detection of acoustic biomolecules

Gas vesicles (GVs) based on acoustic reporter genes have emerged as potent contrast agents for cellular and molecular ultrasound imaging. These air-filled, genetically encoded protein nanostructures can be expressed in a variety of cell types in vivo to visualize cell location and activity or injected systemically to label and monitor tissue function. Distinguishing GVs from tissue signal deep inside intact organisms requires imaging approaches such as amplitude modulation (AM) or collapse-based pulse sequences, however they have limitations in sensitivity or require irreversible collapse of the GVs that restricts its scope for imaging dynamic cellular processes. To address these limitations, this study explores the utility of harmonic imaging to enhance the sensitivity of non-destructive imaging of GVs and cellular processes. Traditional fundamental-frequency imaging utilizing cross-wave AM (xAM) sequences has been deemed optimal for GV imaging. Contrary to this, we hypothesize that harmonic imaging, integrated with xAM could significantly elevate GV detection sensitivity. To verify our hypothesis, we conducted imaging on tissue-mimicking phantoms embedded with purified GVs, mammalian cells genetically modified to express GVs, and live mice after systemic GV infusion. Our findings reveal that harmonic xAM (HxAM) imaging markedly surpasses traditional xAM in isolating GVs nonlinear acoustic signature, showcasing significant enhancements in signal-to-background and contrast-to-background ratios across all tested samples. Further investigation into the backscattered spectra elucidates the efficacy of harmonic imaging in conjunction with xAM. HxAM imaging enables the detection of lower concentrations of GVs and cells with ultrasound and extends the imaging depth in vivo by up to 20% and imaging performance metrics by up to 10dB. These advancements bolster the capabilities of ultrasound for molecular and cellular imaging, underscoring the potential of using harmonic signals to amplify GV detection.

bioengineering↗

bioRxiv2024

Ultrasound-actuated drug delivery with acoustic percolation switches

Devices that can be remote-controlled under image guidance to precisely deliver biomedicines to sites of disease are a major goal of biomedical research. However, most existing externally triggered delivery systems are based on complex micromachines that are controlled with electromagnetic waves and require custom external instrumentation. Here we present a drug delivery platform comprising a simple protein-containing hydrogel that can be both imaged and triggered to release drugs at specific locations using widely available ultrasound imaging devices. This technology is based on the addition of air-filled protein nanostructures called gas vesicles (GVs) to hydrogel delivery vehicles. While intact, GVs sterically block the release of drug payloads and allow the vehicle to be imaged with ultrasound. An increase in ultrasound pressure causes the collapse of GVs within hydrogels present at the desired anatomical location, instantly creating percolation channels and triggering rapid drug release. Both the imaging and release are performed using a common diagnostic ultrasound probe. We implement this concept by establishing ultrasound-controlled drug diffusion and release from hydrogels in vitro and demonstrating targeted image-guided protein delivery in vivo following oral administration. We use this approach to deliver anti-inflammatory antibodies to treat gastrointestinal inflammation in a rat model of colitis. Targeted acoustic percolation switches (TAPS) open a conduit for local, image-guided drug delivery with a simple formulation and commonplace ultrasound equipment.

bioengineering↗

bioRxiv2023

Ultrasonic reporters of calcium for deep tissue imaging of cellular signals

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

bioengineering↗

bioRxiv2023

Gas vesicle-blood interactions enhance ultrasound imaging contrast

Gas vesicles (GVs) are genetically encoded, air-filled protein nanostructures of broad interest for biomedical research and clinical applications, acting as imaging and therapeutic agents for ultrasound, magnetic resonance, and optical techniques. However, the biomedical applications of GVs as a systemically injectable nanomaterial have been hindered by a lack of understanding of GVs interactions with blood components, which can significantly impact in vivo performance. Here, we investigate the dynamics of GVs in the bloodstream using a combination of ultrasound and optical imaging, surface functionalization, flow cytometry, and mass spectrometry. We find that erythrocytes and serum proteins bind to GVs and shape their acoustic response, circulation time, and immunogenicity. We show that by modifying the GV surface, we can alter these interactions and thereby modify GVs in vivo performance. These results provide critical insights for the development of GVs as agents for nanomedicine.

synthetic biology↗

bioRxiv2022

Geometric effects in gas vesicle buckling under ultrasound

Acoustic reporter genes based on gas vesicles (GVs) have enabled the use of ultrasound to noninvasively visualize cellular function in vivo. The specific detection of GV signals relative to background acoustic scattering in tissues is facilitated by nonlinear ultrasound imaging techniques taking advantage of the sonomechanical buckling of GVs. However, the effect of geometry on the buckling behavior of GVs under exposure to ultrasound has not been studied. To understand such geometric effects, we developed computational models of GVs of various lengths and diameters and used finite element simulations to predict their threshold buckling pressures and post-buckling deformations. We demonstrated that the GV diameter has an inverse cubic relation to the threshold buckling pressure, whereas length has no substantial effect. To complement these simulations, we experimentally probed the effect of geometry on the mechanical properties of GVs and the corresponding nonlinear ultrasound signals. The results of these experiments corroborate our computational predictions. This study provides fundamental insights into how geometry affects the sonomechanical properties of GVs, which, in turn, can inform further engineering of these nanostructures for high-contrast, nonlinear ultrasound imaging. STATEMENT OF SIGNIFICANCEGas vesicles (GVs) are an emerging class of genetically encodable and engineerable imaging agents for ultrasound whose sonomechanical buckling generates nonlinear contrast to enable sensitive and specific imaging in highly scattering biological systems. Though the effect of protein composition on GV buckling has been studied, the effect of geometry has not previously been addressed. This study reveals that geometry, especially GV diameter, significantly alters the threshold acoustic pressures required to induce GV buckling. Our computational predictions and experimental results provide fundamental understanding of the relationship between GV geometry and buckling properties and underscore the utility of GVs for nonlinear ultrasound imaging. Additionally, our results provide suggestions to further engineer GVs to enable in vivo ultrasound imaging with greater sensitivity and higher contrast.

biophysics↗

bioRxiv2022

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically-encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryo-electron tomography to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder--a site that may act as an elongation center. High-resolution subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a {beta}-sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes.

molecular biology↗

bioRxiv2022

Magneto-acoustic protein nanostructures for non-invasive imaging of tissue mechanics in vivo

Measuring cellular and tissue mechanics inside intact living organisms is essential for interrogating the roles of force in physiological and disease processes, and is a major goal in the field of mechanobiology. However, existing biosensors for 3D tissue mechanics, primarily based on fluorescent emissions and deformable materials, are limited for in vivo measurement due to the limited light penetration and poor material stability inside intact, living organisms. While magneto-motive ultrasound (MMUS), which uses superparamagnetic nanoparticles as imaging contrast agents, has emerged as a promising modality for real-time in vivo imaging of tissue mechanics, it has poor sensitivity and spatiotemporal resolution. To overcome these limitations, we introduce magneto-gas vesicles (MGVs), a unique class of protein nanostructures based on gas vesicles and magnetic nanoparticles that produces differential ultrasound signals in response to varying mechanical properties of surrounding tissues. These hybrid protein nanostructures significantly improve signal strength and detection sensitivity. Furthermore, MGVs enable non-invasive, long-term, and quantitative measurement of mechanical properties within 3D tissues and organs in vivo. We demonstrated the performance of MGV-based mechano-sensors in vitro, in fibrosis models of organoids, and in vivo in mouse liver fibrosis models.

biochemistry↗

bioRxiv2021

Ultrafast amplitude modulation for molecular and hemodynamic ultrasound imaging

Ultrasound is playing an emerging role in molecular and cellular imaging thanks to new micro- and nanoscale contrast agents and reporter genes. Acoustic methods for the selective in vivo detection of these imaging agents are needed to maximize their impact in biology and medicine. Existing ultrasound pulse sequences use the nonlinearity in contrast agents response to acoustic pressure to distinguish them from mostly linear tissue scattering. However, such pulse sequences typically scan the sample using focused transmissions, resulting in a limited frame rate and restricted field of view. Meanwhile, existing wide-field scanning techniques based on plane wave transmissions suffer from limited sensitivity or nonlinear artifacts. To overcome these limitations, we introduce an ultrafast nonlinear imaging modality combining amplitude-modulated pulses, multiplane wave transmissions and selective coherent compounding. This technique achieves contrast imaging sensitivity comparable to much slower gold-standard amplitude modulation sequences and enables the acquisition of larger and deeper fields of view, while providing a much faster imaging framerate of 3.2kHz. Additionally, it enables simultaneous nonlinear and linear image formation, and allows concurrent monitoring of phenomena accessible only at ultrafast framerates, such as blood volume variations. We demonstrate the performance of this ultrafast amplitude modulation (uAM) technique by imaging gas vesicles, an emerging class of genetically encodable biomolecular contrast agents, in several in vitro and in vivo contexts. These demonstrations include the rapid discrimination of moving contrast agents and the real-time monitoring of phagolysosomal function in the mouse liver.

bioengineering↗

bioRxiv2021

Measuring gas vesicle dimensions by electron microscopy

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that during air drying on the EM grid, GVs flatten, leading to a ~1.5-fold increase in their apparent diameter. We demonstrate that GVs diameter can be accurately determined by direct measurements from cryo-EM samples or alternatively indirectly derived from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported data and provide accurate methods to measure GV dimensions.

biochemistry↗

bioRxiv2019

Acoustic biomolecules enhance hemodynamic functional ultrasound imaging of neural activity

Hemodynamic functional ultrasound imaging (fUS) of neural activity provides a unique combination of spatial coverage, spatiotemporal resolution and compatibility with freely moving animals. However, deep and transcranial monitoring of brain activity and the imaging of dynamics in slow-flowing blood vessels remains challenging. To enhance fUS capabilities, we introduce biomolecular hemodynamic enhancers based on gas vesicles (GVs), genetically encodable ultrasound contrast agents derived from buoyant photosynthetic microorganisms. We show that intravenously infused GVs enhance ultrafast Doppler ultrasound contrast and visually-evoked hemodynamic contrast in transcranial fUS of the mouse brain. This hemodynamic contrast enhancement is smoother than that provided by conventional microbubbles, allowing GVs to more reliably amplify neuroimaging signals.

neuroscience↗

bioRxiv2019

Genetically encoded nanostructures enable acoustic manipulation of engineered cells

The ability to mechanically manipulate and control the spatial arrangement of biological materials is a critical capability in biomedicine and synthetic biology. Ultrasound has the ability to manipulate objects with high spatial and temporal precision via acoustic radiation force, but has not been used to directly control biomolecules or genetically defined cells. Here, we show that gas vesicles (GVs), a unique class of genetically encoded gas-filled protein nanostructures, can be directly manipulated and patterned by ultrasound and enable acoustic control of genetically engineered GV-expressing cells. Due to their differential density and compressibility relative to water, GVs experience sufficient acoustic radiation force to allow these biomolecules to be moved with acoustic standing waves, as demonstrated within microfluidic devices. Engineered variants of GVs differing in their mechanical properties enable multiplexed actuation and act as sensors of acoustic pressure. Furthermore, when expressed inside genetically engineered bacterial cells, GVs enable these cells to be selectively manipulated with sound waves, allowing patterning, focal trapping and translation with acoustic fields. This work establishes the first genetically encoded nanomaterial compatible with acoustic manipulation, enabling molecular and cellular control in a broad range of contexts.

bioengineering↗

bioRxiv2019

Acoustically Detonated Biomolecules for Genetically Encodable Inertial Cavitation

Recent advances in molecular engineering and synthetic biology have made it possible for biomolecular and cell-based therapies to provide highly specific disease treatment. However, both the ability to spatially target the action of such therapies, and their range of effects on the target tissue remain limited. Here we show that biomolecules and cells can be engineered to deliver potent mechanical effects at specific locations inside the body under the direction of focused ultrasound. This capability is based on gas vesicles, a unique class of air-filled protein nanostructures derived from buoyant photosynthetic microbes. We show that low-frequency ultrasound can convert these nanoscale biomolecules into micron-scale cavitating bubbles, as demonstrated with acoustic measurements and ultrafast optical microscopy. This allows gas vesicles targeted to cell-surface receptors to serve as remotely detonated cell-killing agents. In addition, it allows cells genetically engineered to express gas vesicles to be triggered with ultrasound to lyse and release therapeutic payloads. We demonstrate these capabilities in vitro, in cellulo, and in vivo. This technology equips biomolecular and cellular therapeutics with unique capabilities for spatiotemporal control and mechanical action.

bioengineering↗