Adrienne Celeste Greene

Advanced Optical Imaging Reveals the Dependence of Particle Geometry on Interactions Between CdSe Quantum Dots and Immune Cells

The biocompatibility and possible toxicological consequences of engineered nanomaterials, including quantum dots (QDs) due to their unique suitability for biomedical applications, remain intense areas of interest. We utilized advanced imaging approaches to characterize the interactions of CdSe QDs of various sizes and shapes with live immune cells. Particle diffusion and partitioning within the plasma membrane, cellular uptake kinetics, and sorting of particles into lysosomes were all independantly characterized. Using high-speed total internal reflectance fluorescence (TIRF) microscopy, we show that QDs with an average aspect ratio of 2.0 (i.e., rod-shaped) diffuse nearly an order of magnitude slower in the plasma membrane than more spherical particles with aspect ratios of 1.2 and 1.6, respectively. Moreover, more rod-shaped QDs were shown to be internalized into the cell 2-3 fold more slowly. Hyperspectral confocal fluorescence microscopy demonstrates that QDs tend to partition within the cell membrane into regions containing a single particle type. Furthermore, data examining QD sorting mechanisms indicate that endocytosis and lysosomal sorting increases with particle size. Together, these observations suggest that both size and aspect ratio of a nanoparticle are important characteristics that significantly impact interactions with the plasma membrane, uptake into the cell, and localization within intracellular vesicles. Thus, rather than simply characterizing nanoparticle uptake into cells, we show that utilization of advanced imaging approaches permits a more nuanced and complete examination of the multiple aspects of cell-nanoparticle interactions that can ultimately aid understanding possible mechanisms of toxicity, resulting in safer nanomaterial designs.

In vitro Capture, Transport, and Detection of Protein Analytes Using Kinesin‐Based Nanoharvesters

Miniaturization of lab-on-a-chip devices to nanoscale dimensions necessitates a level of systems integration currently found primarily in biological systems. Such devices will require new modes of transportingmacromolecularmaterials at nanometer length scales. In cells, efficient cytoplasmic transport is achieved by energy-consuming, active transport systems in which motor proteins transport cargo along cytoskeletal filaments. For example, the motor protein kinesin-1 carries cell organelles and macromolecules over considerable distances along microtubule filaments. Microtubules are hollow protein polymeric filaments with a diameter of 25 nm and tens of micrometers in length that form a 3D transportation network within the cell. Small groups of kinesin transport cargo at rates up to 12mms , with a catalytic efficiency (i.e., conversion of chemical energy into work) of 50%. Together, this transport system provides a highefficiency means of transporting macromolecular cargo through the highly viscous medium of cytoplasm. The intriguing and powerful properties of kinesin-based transport have spurred its application in hybrid nanoscale systems. Early work focused on applying microfabrication technologies and surface functionalization to guide the kinesinbased transport of molecular shuttles (i.e., stabilized microtubule filaments) and achieve directed transport ofmaterials at the nanoscale. In this mode of application, commonly referred to as the inverted or glidingmotility geometry, kinesin motor proteins are bound on a solid surface such that their catalytic and microtubule-binding domains extend into the solution. In the presence ofATP,microtubule filaments bind to

Controlling kinesin motor proteins in nanoengineered systems through a metal‐binding on/off switch

A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck‐linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck‐linker of kinesin by genetically engineering three histidine residues to create a Zn2+‐binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by ≥10 µM Zn2+, as well as other divalent metals. Motility was successfully restored by removal of Zn2+ using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn2+/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device. Biotechnol. Bioeng. 2008;101: 478–486.

The role of membrane fluidization in the gel-assisted formation of giant polymersomes

Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.

CRISPR-Based Antibacterials: Transforming Bacterial Defense into Offense

The development of antimicrobial-resistant (AMR) bacteria poses a serious worldwide health concern. CRISPR-based antibacterials are a novel and adaptable method for building an arsenal of antibacterials potentially capable of targeting any pathogenic bacteria.

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (> 4 µm) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (>4 µm) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (>100 µm).

EphA2-Ephrina1 Signaling and PI(4,5)P2 Spatial Organization on Breast Cancer Cells

EphA2 belongs to the largest subfamily - Eph receptors - of the Receptor Tyrosine Kinase (RTK) superfamily and is over-expressed in many cancer cell lines. The major role of Eph receptors is to regulate the dynamics of cellular protrusions and cell migration. Previous research reports that activation of EphA2 by its ligand ephrinA1 increases the activity of Phosphoinositide 3-kinase (PI3K). PI3K is one of the key molecules in regulating cell migration by phosphorylating Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) at the cell edge facing the highest chemoattractant concentration.Here, we recapitulate EphA2-EphrinA1 signaling between cells by presenting breast cancer cells expressing EphA2 with an ephrinA1-displaying supported lipid bilayer. Through live cell labeling of PI(4,5)P2 with the fluorescent PLCδ1-PH domain biosensor, we are able to directly monitor PI(4,5)P2 spatial organization and its role in EphA2 signaling pathway. In addition, PI(4,5)P2 signaling and membrane localization are also examined with a spatial mutation strategy, which presents diffusion barriers, disrupting EphA2-ephrinA1 spatial organization. Our study will further clarify the role of PI(4,5)P2 and PI3K in the EphA2 signaling pathway, and help to understand cancer cell progression and metastasis in the long term.

Differential Uptake and Trafficking of Nanoparticles by Living Cells

Engineered nanoparticles are becoming increasingly commonplace in commercial products as well as biomedical research settings. Surprisingly, however, systematic studies of the health hazards of engineered nanomaterials have severely lagged behind their development and application. It has been shown in some cases that nanomaterials can elicit unique and potentially deleterious physiological responses that are not observed with bulk materials of the same type [1].