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Biological nanoparticles, such as exosomes, offer an approach to drug delivery due to their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes’ ability to target the α(v)β(3) integrin, overexpressed on triple-negative breast cancer cells’ surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 μM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells’ survival.

Integrin targeting, arginine-glycine-aspartic acid (RGD)-based nanocarriers have been widely used for tumor imaging, the monitoring of tumor development, and the delivery of anti-cancer drugs. However, the thermodynamics of an RGD-integrin formation and dissociation associated with binding dynamics, affinity, and stability remains unclear. Here, we probed the binding strength of the binary complex to live pancreatic cancer cells using single-molecule binding force spectroscopy methods, in which the RGD peptides were functionalized on a force probe tip through the PEG-based bifunctional linker molecules. While the density of integrin α(V) receptors on the cell surface varies more than two-fold from cell line to cell line, the individual RGD-integrin complexes exhibited a cell type-independent, monovalent bond strength. The load-dependent, bond strength of multivalent RGD-integrin interactions scaled sublinearly with increasing bond number, consistent with the noncooperative, parallel bond model. Furthermore, the multivalent bonds ruptured sequentially either by one or in multiples, and the force strength was comparable with the synchronous rupture force. Comparison of energy landscapes of the bond number revealed a substantial decrease of kinetic off rates for multivalent bonds, along with the increased width of the potential well and the increased potential barrier height between bound and unbound state, enhancing the stability of the multivalent bonds between them.

The changes in cellular structure play an important role in cancer cell development, progression, and metastasis. By exploiting single-cell, force spectroscopy methods, we probed biophysical and biomechanical kinetics (stiffness, morphology, roughness, adhesion) of brain, breast, prostate, and pancreatic cancer cells with standard chemotherapeutic drugs in normoxia and hypoxia over 12–24 hours. After exposure to the drugs, we found that brain, breast, and pancreatic cancer cells became approximately 55–75% less stiff, while prostate cancer cells became more stiff, due to either drug-induced disruption or reinforcement of cytoskeletal structure. However, the rate of the stiffness change decreased up to 2-folds in hypoxia, suggesting a correlation between cellular stiffness and drug resistance of cancer cells in hypoxic tumor microenvironment. Also, we observed significant changes in the cell body height, surface roughness, and cytoadhesion of cancer cells after exposure to drugs, which followed the trend of stiffness. Our results show that a degree of chemotherapeutic drug effects on biomechanical and biophysical properties of cancer cells is distinguishable in normoxia and hypoxia, which are correlated with alteration of cytoskeletal structure and integrity during drug-induced apoptotic process.

Single-molecule measurements of protein dynamics help unveil the complex conformational changes and transitions that occur during ligand binding and catalytic processes. Using high-resolution single-molecule nanocircuit techniques, we have investigated differences in the conformational dynamics and transitions of lysozyme interacting with three ligands: peptidoglycan substrate, substrate-based chitin analogue, and indole derivative inhibitors. While processing peptidoglycan, lysozyme followed one of the two mechanistic pathways for the hydrolysis of the glycosidic bonds: a concerted mechanism inducing direct conformational changes from open to fully closed conformations or a nonconcerted mechanism involving transient pauses in intermediate conformations between the open and closed conformations. In the presence of either chitin or an indole inhibitor, lysozyme was unable to access the fully closed conformation where catalysis occurs. Instead, lysozymes' conformational closures terminated at slightly closed, "excited" conformations that were approximately one-quarter of the full hinge-bending range. With the indole inhibitor, lysozyme reached this excited conformation in a single step without any evidence of rate-liming intermediates, but the same conformational motions with chitin involved three hidden, intermediate processes and features similar to the nonconcerted peptidoglycan mechanism. The similarities suggest that these hidden processes involve attempts to accommodate imperfectly aligned polysaccharides in the active site. The results provide a detailed glimpse of the enzyme-ligand interplay at the crux of molecular recognition, enzyme specificity, and catalysis.

Although graphene-based biosensors provid extreme sensitivity for the detection of atoms, gases, and biomolecules, the specificity of graphene biosensors to the target molecules requires surface decoration of graphene with bifunctional linkers such pyrene derivatives. Here, we demonstrate that the pyrene functionalization influences graphene's electrical properties by yielding partial formation of bilayer graphene which was confirmed by Raman 2D spectrum. Based on this observation, we introduce quadratic fit analysis of the nonlinear electrical behavior of pyrene-functionalized graphene near the Dirac point. Compared to the conventional linear fit analysis of the transconductance at a distance from the Dirac point, the quadratic fit analysis of the nonlinear transconductance near the Dirac point increased the overall protein detection sensitivity by a factor of 5. Furthermore, we show that both pyrene linkers and gating voltage near the Dirac point play critical roles in sensitive and reliable detection of proteins' biological activities with the graphene biosensors.

Insulator-based dielectrophoretic (iDEP) trapping, separating, and concentrating nanoscale objects is carried out using a non-metal, unbiased, mobile tip acing as a tweezers. The spatial control and manipulation of fluorescently-labeled polystyrene particles and DNA were performed to demonstrate the feasibility of the iDEP tweezers. Frequency-dependent iDEP tweezers' strength and polarity were quantitatively determined using two theoretical approaches to DNA, which resulted in a factor of 2 ~ 40 differences between them. In either approach, the strength of iDEP was at least 4-order of magnitude stronger than the thermal force, indicating iDEP was a dominant force for trapping, holding, and separating DNA. The trapping strength and volume of the iDEP tweezers were also determined, which further supports direct capture and manipulation of DNA at the tip end.

Integration of microfluidic devices with pressure-driven, self-powered fluid flow propulsion methods has provided a very effective solution for on-chip, droplet blood testing applications. However, precise understanding of the physical process governing fluid dynamics in polydimethylsiloxane (PDMS)-based microfluidic devices remains unclear. Here, we propose a pressure-driven diffusion model using Fick's law and the ideal gas law, the results of which agree well with the experimental fluid dynamics observed in our vacuum pocket-assisted, self-powered microfluidic devices. Notably, this model enables us to precisely tune the flow rate by adjusting two geometrical parameters of the vacuum pocket. By linking the self-powered fluid flow propulsion method to the sedimentation, we also show that direct plasma separation from a drop of whole blood can be achieved using only a simple construction without the need for external power sources, connectors, or a complex operational procedure. Finally, the potential of the vacuum pocket, along with a removable vacuum battery to be integrated with non-PDMS microfluidic devices to drive and control the fluid flow, is demonstrated.

Endoxifen is the primary active metabolite of tamoxifen, a nonsteroidal-selective estrogen receptor modulator (SERM) and widely used medication to treat estrogen receptor-positive (ER+) breast cancer. In this study, endoxifen was conjugated to the surface of polymeric nanoparticles (polymersomes) for targeted delivery of doxorubicin (DOX) to estrogen receptor-positive breast cancer cells (MCF7). Rapid cell growth and insufficient blood supply result in low oxygen concentration (hypoxia) within the solid breast tumors. The polymersomes developed here are prepared from amphiphilic copolymers of polylactic acid (PLA) and poly(ethylene glycol) (PEG) containing diazobenzene as the hypoxia-responsive linker. We prepared two nanoparticle formulations: DOX-encapsulated hypoxia-responsive polymersomes (DOX-HRPs) and endoxifen-conjugated, DOX-encapsulated hypoxia-responsive polymersomes (END-DOX-HRPs). Cellular internalization studies demonstrated eight times higher cytosolic and nuclear localization after incubating breast cancer cells with END-DOX-HRPs (targeted polymersomes) in contrast to DOX-HRPs (nontargeted polymersomes). Cytotoxicity studies on monolayer cell cultures exhibited that END-DOX-HRPs were three times more toxic to ER+ MCF7 cells than DOX-HRPs and free DOX in hypoxia. The cell viability studies on three-dimensional hypoxic cultures also demonstrated twice as much toxicity when the spheroids were treated with targeted polymersomes instead of nontargeted counterparts. This is the first report of surface-decorated polymeric nanoparticles with endoxifen ligands for targeted drug delivery to ER+ breast cancer microtumors. The newly designed endoxifen-conjugated, hypoxia-responsive polymersomes might have translational potential for ER+ breast cancer treatment.

Hyperbranched polymer-derived drug nanocarriers have been synthesized that can change sizes selectively in response to pH. These constructs were composed of tertiary amine-conjugated polycarbonate blocks "grafted from" a hyperbranched polyester polyol core. At neutral pH, unprotonated polycarbonate arms stabilized the copolymer aggregates in the form of nanoparticles with hydrodynamic diameters ranging from 150 to 190 nm. Upon lowering of pH, these larger aggregates disassembled into smaller nanoparticles with diameters of 3-5 nm as directed by protonation of tertiary amine side-chains. The pH-dependent reduction of particle sizes was evident by titrimetric, spectroscopic, dynamic light scattering, transmission electron, and atomic force microscopy-based experiments. We observed that these copolymeric nanoparticles could be loaded with dye and drug molecules either by noncovalent encapsulation or by covalent conjugation. A pH-induced disassembly of the aggregates initiated rapid release of the encapsulated payload, but not of the conjugated ones, thus establishing a controlled rate of therapeutic delivery from the nanostructures over an extended period. We envision that such systems can be used for drug delivery applications where nanoparticle sizes critically govern therapeutic efficiency in a vasculature-poor disease microenvironment such as desmoplasia in pancreatic cancer. Hence, we tested the cellular uptake, cytotoxicity, and chemotherapeutic potential of the size-modifiable nanoaggregates using gemcitabine as a model drug in pancreatic cancer setting. We observed that assembled nanoparticles were biocompatible to noncancerous cells, showed pH-dependent effects on cellular uptake as well as promoted accumulation within cancer cells cultured as 3D spheroids. We also found that when conjugated with gemcitabine, the resulting drug-loaded nanoparticles suppressed proliferation of cancer cells. Collectively, the studies suggested that these synthesized, pH-disassembling nanoscale platform will find applications as biomaterials for constructing a size-transformable drug nanocarriers where reduction of size takes effect near localized disease targets in response to microenvironmental triggers.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in men and women in the U.S., with a 5-year survival rate of 10%. This poor prognosis is partly due to the aggressive nature of the disease, lack of early detection methods, its complex and dense tumor microenvironment (TME), and lack of effective treatment options. Within the PDAC TME, morphologically aberrant leaky vessels are responsible for hypoxia and impaired immune response, which likely reduces the efficacy of cancer therapies. We and others showed that pericyte coverage significantly correlates with vascular integrity/function and intratumoral hypoxia. Therefore, understanding perivascular heterogeneity and the unique contribution of each phenotype to the TME evolution is a critical step towards personalized treatment options for PDAC. Our study revealed that tumor-associated pericytes across all PDAC tumor tissues exhibited ectopic αSMA expression up to 10X higher compared to the normal pericytes. This aberrant pericyte phenotype was correlated with vascular leakiness and hypoxia, whereas the Des+ mature pericyte phenotype was inversely correlated with vessel leakiness. Our attempt to elucidate the underlying mechanism of pericyte phenotype switching using in vitro culture system shows that pancreatic cancer cell-derived extracellular vesicles (PC-Exo) is a potent inducer of αSMA expression in pericyte. PC-Exo was also sufficient enough to induce αSMA expression in normal pericytes within the pancreas when injected into the WT mice. In addition to the changes in pericyte markers, PC-Exo stimulated pericytes exhibited immunomodulatory phenotype with significantly higher expression of HLA-DRA, CD274, CD80, E-selectin, and P-selectin. Considering αSMA is a cytoskeletal protein that can influence cellular stiffness, migration, and downstream signaling, we also examined the biomechanical properties of pericytes with altered phenotype. The results show a significant increase in pericyte stiffness and cellular height of aSMA+ pericyte. These pericytes also have elongated morphology and cytoskeletal reorganization visualized by αSMA and phalloidin immunostaining. A recent study showed that acquired αSMA expression converts capillary pericytes into contractile vascular smooth muscle-like cells. Blood vessels are constricted and collapsed within the PDAC TME, which may be partly caused by an abundance of αSMA+ pericytes. In conclusion, our study indicates that tumor-associated pericytes undergo phenotype switching under the influence of pancreatic cancer cells, and these aberrant pericytes might contribute to non-optimal vascular integrity/function. Together, our study indicates that tumor conditioned αSMA+ pericytes present mechanical abnormalities and immune-suppressive features. Future work includes exploring the vascular normalization approach by suppressing pericyte phenotype switching to enhance vascular function and chemo- and immunotherapeutic efficacy. Citation Format: Vikneshwari Natarajan, Alexander Delgado, Reed Jacobson, Lina Alhalhooly, Yongki Choi, Sangdeuk Ha, Jiha Kim. PDAC derived exosomes manipulate tumor pericyte phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB053.

High recurrence and metastasis to vital organs are the major characteristics of triple-negative breast cancer (TNBC). Low vascular oxygen tension promotes resistance to chemo- and radiation therapy. Neuropilin-1 (NRP-1) receptor is highly expressed on TNBC cells. The tumor-penetrating iRGD peptide interacts with the NRP-1 receptor, triggers endocytosis and transcytosis, and facilitates penetration. Herein, we synthesized a hypoxia-responsive diblock PLA-diazobenzene-PEG copolymer and prepared self-assembled hypoxia-responsive polymersomes (Ps) in an aqueous buffer. The iRGD peptide was incorporated into the polymersome structure to make hypoxia-responsive iRGD-conjugated polymersomes (iPs). Doxorubicin (DOX) was encapsulated in the polymersomes to prepare both targeted and non-targeted hypoxia-responsive polymersomes (DOX-iPs and DOX-Ps, respectively). The polymeric nanoparticles released less than 30% of their encapsulated DOX within 12 hours under normoxic conditions (21% oxygen), whereas under hypoxia (2% Oxygen), doxorubicin release remarkably increased to over 95%. The targeted polymersomes significantly decreased TNBC cells' viability in monolayer and spheroid cultures under hypoxia compared to normoxia. Animal studies displayed that targeted polymersomes significantly diminished tumor growth in xenograft nude mice. Overall, the targeted polymersomes exhibited potent anti-tumor activity in monolayer, spheroid, and animal models of TNBC. With further developments, the targeted nanocarriers discussed here might have the translational potential as drug carriers for the treatment of TNBC.

Uncontrolled cell growth, division, and lack of enough blood supply causes low oxygen content or hypoxia in cancerous tumor microenvironments. 17β-Estradiol (E2), an estrogen receptor (ER) ligand, can be incorporated on the surface of nanocarriers for targeted drug delivery to breast cancer cells overexpressing ER. In the present study, we synthesized estradiol-conjugated hypoxia-responsive polymeric nanoparticles (polymersomes) encapsulating the anticancer drug doxorubicin (E2-Dox-HRPs) for targeted delivery into the hypoxic niches of estrogen-receptor-positive breast cancer microtumors. Estradiol-conjugated polymersomes released over 90% of their encapsulated Dox in a sustained manner within hypoxia (2% oxygen) after 12 h. However, they released about 30% of Dox in normal oxygen partial pressure (21% oxygen, normoxia) during this time. Fluorescence microscopic studies demonstrated higher cytosolic and nuclear internalization of E2-Dox-HRPs (targeted polymersomes) compared to those of Dox-HRPs (nontargeted polymersomes). Monolayer cell viability studies on ER-positive MCF7 cells showed higher cytotoxicity of targeted polymersomes in hypoxia compared to in normoxia. Cytotoxicity studies with hypoxic three-dimensional spheroid cultures of MCF7 cells treated with targeted polymersomes indicated significant differences compared to those of normoxic spheroids. The novel estradiol-conjugated hypoxia-responsive polymersomes described here have the potential for targeted drug delivery in estrogen-receptor-positive breast cancer therapy.

Immobilizing enzymes on nanoparticles (NPs) enhances the cost-efficiency of biocatalysis; however, when the substrates are large, it becomes difficult to separate the enzyme@NP from the products while avoiding leaching or damage of enzymes in the reaction medium. Metal-Organic Framework (MOF) coated magnetic nanoparticles (MNPs) offers efficient magnetic separation and enhanced enzyme protection; however, the involved enzymes/substrates have to be smaller than MOF apertures. A potential solution to these challenges is co-precipitating metal/ligand with enzymes on MNP surface, which can partially bury (protect) the enzyme below the composite surface while exposing the rest of the enzyme to the reaction medium for catalysis of larger substrates. Here, to prove this concept, we show that using Ca(2+) and terephthalic acid (BDC), large-substrate enzymes can be encapsulated in the CaBDC-MOF layers coated on MNPs via enzyme-friendly, aqueous phase one-pot synthesis. Interestingly, we found that using MNP as the nuclei of crystallization, the composite size can be tuned so that nanoscale composites were formed under low Ca(2+)/BDC concentrations while microscale composites under high Ca(2+)/BDC concentrations. While the microscale composites showed significantly enhanced reusability against a non-structured large substrate, the nanoscale composites displayed enhanced catalytic efficiency against a rigid, crystalline-like large substrate, starch, likely due to the improved diffusivity of the nanoscale composites. To our best knowledge, this is the first report on aqueous phase one-pot synthesis of size-tunable enzyme@MOF/MNP composites for large substrate biocatalysis. Our platform can be applied to immobilize other large substrate enzymes with enhanced reusability and tunable sizes.

Exosomes are naturally secreted extracellular bilayer vesicles (diameter 40–130 nm), which have recently been found to play a critical role in cell-to-cell communication and biomolecule delivery. Their unique characteristics—stability, permeability, biocompatibility and low immunogenicity—have made them a prime candidate for use in delivering cancer therapeutics and other natural products. Here we present the first ever report of echogenic exosomes, which combine the benefits of the acoustic responsiveness of traditional microbubbles with the non-immunogenic and small-size morphology of exosomes. Microbubbles, although effective as ultrasound contrast agents, are restricted to intravascular usage due to their large size. In the current study, we have rendered bovine milk-derived exosomes echogenic by freeze drying them in the presence of mannitol. Ultrasound imaging and direct measurement of linear and nonlinear scattered responses were used to investigate the echogenicity and stability of the prepared exosomes. A commercial scanner registered enhancement (28.9% at 40 MHz) in the brightness of ultrasound images in presence of echogenic exosomes at 5 mg mL−1. The exosomes also showed significant linear and nonlinear scattered responses—11 dB enhancement in fundamental, 8.5 dB in subharmonic and 3.5 dB in second harmonic all at 40 μg mL−1 concentration. Echogenic exosomes injected into the tail vein of mice and the synovial fluid of rats resulted in significantly higher brightness—as much as 300%—of the ultrasound images, showing their promise in a variety of in vivo applications. The echogenic exosomes, with their large-scale extractability from bovine milk, lack of toxicity and minimal immunogenic response, successfully served as ultrasound contrast agents in this study and offer an exciting possibility to act as an effective ultrasound responsive drug delivery system.

In pancreatic ductal adenocarcinoma (PDAC), early onset of hypoxia triggers remodeling of the extracellular matrix, epithelial-to-mesenchymal transition, increased cell survival, the formation of cancer stem cells, and drug resistance. Hypoxia in PDAC is also associated with the development of collagen-rich, fibrous extracellular stroma (desmoplasia), resulting in severely impaired drug penetration. To overcome these daunting challenges, we created polymer nanoparticles (polymersomes) that target and penetrate pancreatic tumors, reach the hypoxic niches, undergo rapid structural destabilization, and release the encapsulated drugs. In vitro studies indicated a high cellular uptake of the polymersomes and increased cytotoxicity of the drugs under hypoxia compared to unencapsulated drugs. The polymersomes decreased tumor growth by nearly 250% and significantly increased necrosis within the tumors by 60% in mice compared to untreated controls. We anticipate that these polymer nanoparticles possess a considerable translational potential for delivering drugs to solid hypoxic tumors.

Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often "harsh" reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal-Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.

Protein-polymer conjugates are attractive biomaterials which combine the functions of both proteins and polymers. The bioactivity of these hybrid materials, however, is often reduced upon the conjugation. It is important to determine and monitor the protein structure and active site availability, in order to optimize the polymer composition, attachment point, and abundance. The challenges in probing these insights are the large size and high complexity in the conjugates. Here, we overcome the challenges by combining Electron Paramagnetic Resonance (EPR) spectroscopy and Atomic Force Microscopy (AFM) and characterize the structural insights of antibacterial hybrids formed by polyethylene glycol (PEG) and an antibacterial protein. We discovered that the primary reasons for activity loss were PEG blocking the substrate access pathway and/or altering protein surface charges. Our data indicated that the polymers tended to stay away from the protein surface and form a coiled conformation. The structural insights are meaningful for and applicable to the rational design of future hybrids.

This work establishes a relationship between the electronic structure, polaron formation, and electrochemical activity in transition-metal tungstates., Transition-metal tungstates MWO4 (M = Co, Ni, Cu, Zn) have applications in many areas, including supercapacitors. A good understanding of the electronic structure is essential to understanding their functional properties. Here, we report a first-principles study of the materials using hybrid density-functional calculations. The electronic structure is analyzed with a focus on the nature of the electronic states near the band edges. We find that hole polarons can form at the Co lattice site in CoWO4 and the O site in NiWO4, CuWO4, and ZnWO4, resulting in the formation of Co3+ in the former and O– in the latter. The electrochemical activity observed in certain tungstate compounds, but not in others, appears to be related to the ability to form hole polarons on the transition-metal ions. The formation energy and migration barrier of the hole polaron in CoWO4 are also calculated and the results are employed to understand the reported p-type conductivity.

The dynamic interactions of an individual matrix metalloproteinase-1 were imaged and monitored in the presence of either triple-helical or non-triple-helical, partially-structured collagen-mimic substrates. The enzyme exhibited ten-fold increased catalytic turnover rates with the structurally modified substrate by skipping the triple-helix unwinding step during the catalytic pathway.

Silica nanoparticles (SiNPs) are important nano-sized, solid-state carriers/hosts to load, store, and deliver biological or pharmaceutical cargoes. They are also good potential solid supports to immobilize proteins for fundamental protein structure and dynamics studies. However, precaution is necessary when using SiNPs in these areas because adsorption might alter the activity of the cargoes, especially when enzymes are loaded. Therefore, it becomes important to understand the structural basis of the cargo enzyme activity changes, if there is any. The high complexity and dynamics of the nano-bio interface present many challenges. Reported here is a comprehensive study of the structure, dynamics, and activity of a model enzyme, T4 lysozyme, upon adsorption to a few surface-modified SiNPs using several experimental techniques. Not surprisingly, a significant activity loss on each studied SiNP was found. The structural basis of the activity loss was identified based on results from a unique technique, the Electron Paramagnetic Resonance (EPR) spectroscopy, which probes structural information regardless of the complexity. Several docking models of the enzyme on SiNPs with different surfaces, at different enzyme-to-SiNP ratios are proposed. Interestingly, we found that the adsorbed enzyme can be desorbed via pH adjustment, which highlighted the potential to use SiNPs for enzyme/protein delivery or storage due to the high capacity. In order to use SiNPs as enzyme hosts, minimizing the enzymatic activity loss upon adsorption is needed. Lastly, the work outlined here demonstrate the use of EPR in probing structural information on the complex (inorganic)nano-bio interface.

Using single-molecule approaches, we directly observed the dynamic interaction between HDAC8 and various ligands as well as conformational interconversions during the catalytic reaction. Statistical analysis identified key kinetic parameters, demonstrating that the enzymatic activity is highly sensitive to both minor variations in the ligand structures and small synthetic molecules.

Prostate cancer cells overexpress the prostate-specific membrane antigen (PSMA) receptors on the surface. Targeting the PSMA receptor creates a unique opportunity for drug delivery. Docetaxel is a Food and Drug Administration-approved drug for treating metastatic and androgen-independent prostate cancer, and mocetinostat is a potent inhibitor of class I histone deacetylases. In this study, we prepared reduction-sensitive polymersomes presenting folic acid on the surface and encapsulating either docetaxel or mocetinostat. The presence of folic acid allowed efficient targeting of the PSMA receptor and subsequent internalization of the polymeric vesicles in cultured LNCaP prostate cancer cell spheroids. The intracellular reducing agents efficiently released docetaxel and mocetinostat from the polymersomes. The combination of the two drug-encapsulated polymersome formulations significantly (p < 0.05) decreased the viability of the LNCaP cells (compared to free drugs or control) in three-dimensional spheroid cultures. The calculated combination index value indicated a synergistic effect for the combination of mocetinostat and docetaxel. Thus, our PSMA-targeted drug-encapsulated polymersomes has the potential to lead to a new direction in prostate cancer therapy that decreases the toxicity and increases the efficacy of the drug delivery systems.

Considering the systemic toxicity of chemotherapeutic agents, there is an urgent need to develop new targeted drug delivery systems. Herein, we have developed a new nuclear targeted, redox sensitive, drug delivery vehicle to simultaneously deliver the anticancer drugs gemcitabine and doxorubicin to the nuclei of pancreatic cancer cells. We prepared polymeric bilayer vesicles (polymersomes), and actively encapsulated the drug combination by the pH gradient method. A redox-sensitive polymer (PEG-S-S-PLA) was incorporated to sensitize the formulation to reducing agent concentration. Acridine orange (AO) was conjugated to the surface of the polymersomes imparting nuclear localizing property. The polymersomes' toxicity and efficacy were compared with those of a free drug combination using monolayer and three-dimensional spheroid cultures of pancreatic cancer cells. We observed that the redox sensitive, nuclear-targeted polymersomes released more than 60% of their encapsulated contents in response to 50 mM glutathione. The nanoparticles are nontoxic; however, the drug encapsulated vesicles have significant toxicity. The prepared formulation can increase the drug's therapeutic index by delivering the drugs directly to the cells' nuclei, one of the key organelles in the cells. This study is likely to initiate research in targeted nuclear delivery using other drug formulations in other types of cancers.

Lithium-rich complex transition-metal oxides Li$_2$MoO$_3$, Li$_2$RuO$_3$, Li$_3$RuO$_4$, Li$_3$NbO$_4$, Li$_5$FeO$_4$, Li$_5$MnO$_4$ and their derivatives are of interest for high-capacity battery electrodes. Here, we report a first-principles density-functional theory study of the atomic and electronic structure of these materials using the Heyd-Scuseria-Ernzerhof (HSE) screened hybrid functional which treats all orbitals in the materials on equal footing. Dimerization of the transition-metal ions is found to occur in layered Li$_2$MoO$_3$, in both fully lithiated and partially delithiated compounds. The Ru--Ru dimerization does not occur in fully lithiated Li$_2$RuO$_3$, in contrast to what is commonly believed; Ru--Ru dimers are, however, found to occur in the presence of lithium vacancies caused by lithium loss during synthesis and/or lithium removal during use. We also analyze the electronic structure of the complex oxides and discuss the delithiation mechanism in these battery electrode materials., Comment: 7 pages, 6 figures, 1 table

Chemotherapeutic agents for treating cancers show considerable side effects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport and subsequently release the encapsulated anticancer drugs within the nuclei of pancreatic cancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3-polyethylene glycol (PEG)-polylactic acid (PLA) copolymer employing the Cu2+ catalyzed "Click" reaction. We prepared polymersomes from the dexamethasone-PEG-PLA conjugate along with a synthesized stimuli-responsive polymer PEG-S-S-PLA. The dexamethasone group dilates the nuclear pore complexes and transports the vesicles to the nuclei. We designed the polymersomes to release the encapsulated drugs in the presence of a high concentration of reducing agents in the nuclei of pancreatic cancer cells. We observed that the nucleus-targeted, stimuli-responsive polymersomes released 70% of encapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulated the cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608 encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. The polymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medical ultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have the potential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Often cancer relapses after an initial response to chemotherapy because of the tumor’s heterogeneity and the presence of progenitor stem cells, which can renew. To overcome drug resistance, metastasis, and relapse in cancer, a promising approach is the inhibition of cancer stemness. In this study, the expression of the neuropilin-1 receptor in both pancreatic and prostate cancer stem cells was identified and targeted with a stimuli-responsive, polymeric nanocarrier to deliver a stemness inhibitor (napabucasin) to cancer stem cells. Reduction-sensitive amphiphilic block copolymers PEG(1900)-S-S-PLA(6000) and the N(3)-PEG(1900)-PLA(6000) were synthesized. The tumor penetrating iRGD peptide-hexynoic acid conjugate was linked to the N(3)-PEG(1900)-PLA(6000) polymer via a Cu(2+) catalyzed “Click” reaction. Subsequently, this peptide-polymer conjugate was incorporated into polymersomes for tumor targeting and tissue penetration. We prepared polymersomes containing 85% PEG(1900)-S-S-PLA(6000), 10% iRGD-polymer conjugate, and 5% DPPE-lissamine rhodamine dye. The iRGD targeted polymersomes encapsulating the cancer stemness inhibitor napabucasin were internalized in both prostate and pancreatic cancer stem cells. The napabucasin encapsulated polymersomes significantly (p < 0.05) reduced the viability of both prostate and pancreatic cancer stem cells and decreased the stemness protein expression notch-1 and nanog compared to the control and vesicles without any drug. The napabucasin encapsulated polymersome formulations have the potential to lead to a new direction in prostate and pancreatic cancer therapy by penetrating deeply into the tumors, releasing the encapsulated stemness inhibitor, and killing cancer stem cells.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles (liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and the hydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have been widely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of the polymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic copolymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigated acoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acoustically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibited strong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental and subharmonic, respectively, at 5-MHz excitation from 20 µg/mL polymers in solution). Unlike echogenic liposomes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrast agents, which was also confirmed by clinical ultrasound imaging.

Improving the therapeutic index of anticancer agents is an enormous challenge. Targeting decreases the side effects of the therapeutic agents by delivering the drugs to the intended destination. Nanocarriers containing the nuclear localizing peptide sequences (NLS) translocate to the cell nuclei. However, the nuclear localization peptides are nonselective and cannot distinguish the malignant cells from the healthy counterparts. In this study, we designed a "masked" NLS peptide which is activated only in the presence of overexpressed matrix metalloproteinase-7 (MMP-7) enzyme in the pancreatic cancer microenvironment. This peptide is conjugated to the surface of redox responsive polymersomes to deliver doxorubicin and curcumin to the pancreatic cancer cell nucleus. We have tested the formulation in both two- and three-dimensional cultures of pancreatic cancer and normal cells. Our studies revealed that the drug-encapsulated polymeric vesicles are significantly more toxic toward the cancer cells (shrinking the spheroids up to 49%) compared to the normal cells (shrinking the spheroids up to 24%). This study can lead to the development of other organelle targeted drug delivery systems for various human malignancies.

Exosomes are naturally secreted bilayer vesicles ranging in size from 40 to 200 nm that play a critical role in cell-to-cell communications and protein and RNA delivery. Researchers have explored exosomes as potential drug delivery vehicles due to their natural morphology and small size. Here, for the first time, bovine milk derived exosomes have been modified to be acoustically responsive as potential ultrasound contrast agents or a drug carrier. The echogenic exosomes were formed through a freeze-drying process in the presence of mannitol. The size and morphology of the particles were assessed with a qNano™ and atomic force microscopy (AFM). The ultrasound response of these particles was characterized through linear and non-linear scattering behaviors. The presence of the echogenic exosomes enhances the scattered signal by 11.4 ± 6.3 dB. The stability of these particles under constant ultrasound exposure were assessed to be similar to that of echogenic polymersomes. The variation of mannitol concentration was assessed. To assess the imaging improvement of ultrasound imaging, the exosomes were injected through a tail vein in mice. The modification of the echogenic exosomes shows to have great promise as potential ultrasound contrast agents or ultrasound responsive drug delivery system.

Solid tumors are often poorly irrigated due to structurally compromised microcirculation. Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack of enough oxygen and nutrients lead to the development of hypoxic regions in the tumor tissues. As the partial pressure of oxygen drops below the necessary level (10 psi), the cancer cells modulate their genetic makeup to survive. Hypoxia triggers tumor progression by enhancing angiogenesis, cancer stem cell production, remodeling of the extracellular matrix, and epigenetic changes in the cancer cells. However, the hypoxic regions are usually located deep in the tumors and are usually inaccessible to the intravenously injected drug carrier or the drug. Considering the designs of the reported nanoparticles, it is likely that the drug is delivered to the peripheral tumor tissues, close to the blood vessels. In this study, we prepared lipid nanoparticles (LNs) comprising the synthesized hypoxia-responsive lipid and a peptide-lipid conjugate. We observed that the resultant LNs penetrated to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia-responsive lipid undergoes reduction, destabilizing the lipid membrane, and releasing encapsulated drugs from the nanoparticles. We demonstrated the results employing spheroidal cultures of the pancreatic cancer cells BxPC-3. We observed that the peptide-decorated, drug encapsulated LNs reduced the viability of pancreatic cancer cells of the spheroids to 35% under hypoxic conditions.

Hypoxia in tumors contributes to overall tumor progression by assisting in epithelial-to-mesenchymal transition, angiogenesis, and metastasis of cancer. In this study, we have synthesized a hypoxia-responsive, diblock copolymer poly(lactic acid)–azobenzene–poly(ethylene glycol), which self-assembles to form polymersomes in an aqueous medium. The polymersomes did not release any encapsulated contents for 50 min under normoxic conditions. However, under hypoxia, 90% of the encapsulated dye was released in 50 min. The polymersomes encapsulated the combination of anticancer drugs gemcitabine and erlotinib with entrapment efficiency of 40% and 28%, respectively. We used three-dimensional spheroid cultures of pancreatic cancer cells BxPC-3 to demonstrate hypoxia-mediated release of the drugs from the polymersomes. The vesicles were nontoxic. However, a significant decrease in cell viability was observed in hypoxic spheroidal cultures of BxPC-3 cells in the presence of drug encapsulated polymersomes. These polymersomes have potential for future applications in imaging and treatment of hypoxic tumors.

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein’s activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF’s base-by-base activity. After measuring as few as 20 copies, the template length can be determined with

DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by τclosed. Under Vmax conditions, the average time of τclosed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by τopen, which includes molecular recognition of the incoming dNTP. All α-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair.

© 2015 American Chemical Society. Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozymes opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzymes state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozymes two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 μs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzymes state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.

Sunday, February 8, 2015 experiments with [U-15N,2H,13C]-BsPFK. The assignments allowed for the mapping of peaks representing isoleucine residues onto the crystal structure. This analysis has allowed specific regions of the enzyme involved in the bind- ing of allosteric ligands and the propagation of the allosteric effect to be iden- tified. Funding: NIH-GM33216, NIH-CBI, Welch-A1543. 148-Plat Microsecond-Resolution Recording of T4 Lysozyme Observes a Brownian Ratchet Maxim V. Akhterov 1 , Yongki Choi 2 , Tivoli J. Olsen 3 , Patrick C. Sims 1 , Mariam Iftikhar 3 , O. Tolga Gul 1 , Brad L. Corso 1 , Gregory A. Weiss 3 , Philip G. Collins 1 . Department of Physics and Astronomy, University of California, Irvine, Irvine, CA, USA, 2 Department of Physics, North Dakota State University, Fargo, ND, USA, 3 Department of Chemistry, University of California, Irvine, Irvine, CA, USA. Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the res- olution to directly observe the underlying transition pathway or any intermedi- ate conformational dynamics. We have recently described a single-molecule electronic technique that breaks this barrier (1-3). Using a 1 MHz-bandwidth carbon nanotube transistor, the transition dynamics of T4 lysozyme have been recorded with microsecond resolution. We directly resolve a smooth, continuous transition with an average duration of 37 microseconds that sug- gests a concerted mechanism for glycoside hydrolysis. Unexpectedly, the me- chanical closing and re-opening of the enzyme have identical distributions of transition durations, and the motions do not depend on whether the enzyme is in its catalytic or non-productive state. These results illustrate the principle of microscopic reversibility applied to a Brownian ratchet, with lysozyme tracing a single pathway for closing and the reverse pathway for enzyme open- ing, regardless of its instantaneous catalytic productivity. 1. Y. Choi, et. al., ‘‘Single-Molecule Lysozyme Dynamics Monitored by an Electronic Circuit,’’ Science 335, 319 (2012). 2. T.J. Olsen, et. al., ‘‘Electronic Measurements of Single-Molecule Processing by DNA polymerase I (Klenow fragment),’’ JACS 135, 7855 (2013). 3. P.C. Sims, et. al., ‘‘Electronic Measurements of Single-Molecule Catalysis by cAMP-Dependent Protein Kinase A,’’ JACS 135, 7861 (2013). 149-Plat Human Neuraminidase Enzymes alter the Lateral Mobility and Function of Integrin Receptors Christopher W. Cairo. Chemistry, University of Alberta, Edmonton, AB, Canada. Glycolipids and glycoproteins are important components of membrane struc- ture. Mechanisms which alter the structure of glycans at the membrane could influence cellular responses. For example, by removing an important binding epitope or else by unmasking a new one, protein-glycan interactions may be disrupted or reinforced. Our group has been working to understand the role of human neuraminidase enzymes (hNEU) in regulating cellular adhesion and migration through integrin receptors. Of the four hNEU isoenzymes, three have activity at the plasma membrane and lysosome; thus, these enzymes could regulate the composition of the plasma membrane by stripping neuraminic acid (Neu5Ac; also known as sialic acid) from membrane glycans. Using recombi- nant enzymes and selective hNEU inhibitors developed within our group, we can selectively probe increased or decreased activity of individual isoenzymes in vitro; allowing us to test the effect of specific enzymes. Previous studies have suggested that integrin-mediated adhesion may be altered through hNEU activ- ity. We measured the lateral mobility of integrins in cells treated with NEU3 and NEU4 using single-dye tracking (SDT) by total-internal-reflection fluores- cence microscopy (TIRF). We find that hNEU can dramatically change the diffusion of integrin receptors, and that the effect is dependent on the cell type and the isoenzyme used. Adhesion and cell migration assays of cells treated with chemical inhibitors of the enzymes reveal that hNEU activity is intimately involved in the regulation of integrin adhesion to their native li- gands. We will present lateral mobility and cell migration assays with enzyme and inhibitor conditions. Our results suggest an important role for hNEU as reg- ulators of membrane composition and the activity of adhesion receptors. Platform: Membrane Dynamics 150-Plat Nothing to Sneeze at: A Full-Scale Computational Model of the Human Influenza Virion Tyler Reddy 1 , David Shorthouse 1 , Daniel Parton 2 , Elizabeth Jefferys 1 , Philip W. Fowler 1 , Matthieu Chavent 1 , Marc Baaden 3 , Mark S.P. Sansom 1 . 31a SBCB Unit, Biochemistry, University of Oxford, Oxford, United Kingdom, Memorial Sloan Kettering Cancer Center, New York, NY, USA, 3 Institut de Biologie Physico-Chimique, Paris, France. Tackling the ongoing challenge of influenza infectivity would benefit greatly from a molecular understanding of why the influenza A virion exhibits seasonal patterns of infectivity and has wide-ranging survival times in different environ- ments. A computational approach to the study of the behaviour of the virion that focuses on the poorly-understood structural and dynamic role of the lipids is presented here. We have combined experimental data from X-ray crystallog- raphy, NMR spectroscopy, cryoelectron microscopy, and lipidomics to build a full-scale computational model of the influenza A virion. This represents the first set of microsecond-scale molecular dynamics simulations of an enveloped virion in explicit solvent that we are aware of. We report results for a set of simulations at different temperatures and with varying lipid compositions. The Forssman glycolipid, which is prevalent in the influenza A lipidome, influ- ences several biophysical characteristics of the virion model, including diffu- sion and clustering of proteins and lipids. The distribution of peplomers on the virion surface is consistent with accessibility to bivalent antibodies. The distances of binding sites for host cell sialic acid-containing receptors have been analyzed in the virion model for a variety of planar host cell membrane attack orientations. The relatively rigid membrane of the influenza A virion model exhibits a number of biophysical properties consistent with experimental measurements, and may serve as a useful platform for in silico assessment of virion behaviour. 151-Plat Role Of Membrane-Bending Proteins as Membrane Tension Sensors in Cell Migration Toshiki Itoh. Biosignal Research Center, Kobe University, Kobe, Japan. The plasma membrane (PM) tension has emerged as a key regulator for funda- mental cellular functions. However, a missing link between PM tension and biochemical reaction precludes our understanding of how PM tension is coupled to cellular events like directed migration. We found that FBP17, an F-BAR domain protein, acts as a membrane tension sensor that organizes cell polarity during cell migration. The mechanism is based on membrane- bending activity of the F-BAR domain that is counteracted by PM tension. Because FBP17 binds and activates WASP/N-WASP to promote actin poly- merization, it corresponds to the local activator of actin polymerization in the feedback loop regulated by PM tension for self-organized formation of the leading edge. These findings provide an important mechanistic insight into cell migration underpinning a wide variety of physiological events such as development, immune response, and cancer metastasis. 152-Plat Experiment and Simulation Reveal the Bending Properties of Nanoscopic Lipid Domains Jonathan D. Nickels 1 , Michael Ohl 2 , Xiaolin Cheng 1 , Christopher Stanley 3 , Frederick Heberle 3 , Robert Standaert 1 , John Katsaras 1 . Oak Ridge National Lab/Univ. of Tennessee, Knoxville, Oak Ridge, TN, USA, 2 Oak Ridge National Lab/ Julich Center for Neutron Science, Oak Ridge, TN, USA, 3 Oak Ridge National Lab, Oak Ridge, TN, USA. There has great interest in studying the mechanisms that support nanoscopic lipid domain size using model mixtures. To date, experimental studies have focused in large part on structural parameters such as thickness mismatch between phases, lacking access to mechanical properties in direct, experimental ways. The Neutron Spin Echo technique can directly measure the bending modulus of such bilayers systems by observing shape fluctuations. Furthermore, this method permits us to utilize hydrogen-deuterium contrast matching to make direct obser- vations of the mechanical properties of nanoscopic domains themselves. I will present results detailing for the first time the bending modulus of an in- dividual lipid domain. We have also measured both the structural and mechan- ical properties of each pure phase compositions of the three component lipid mixture which spontaneously forms these domains. Our results demonstrate that the liquid-ordered phase is more rigid than the liquid disordered phase. Interestingly, the value of the bending modulus in vesicles where these phases coexist as nanoscopic domains approaches that of the liquid ordered-phase, whereas our sample contrast matched so that we only probe the disordered phase in the coexisting system have a bending modulus similar to what is seen in the liquid-disordered only system. We analyze our results with the help of all atom MD simulations which accurately reproduce the structural properties of our system and agree with the trend in mechanical properties that we observe experimentally. These results have implications in deter- mination of important properties such as the line tension which may drive domain size.

Nanoelectronic devices integrated with dielectrophoresis (DEP) have been promoted as promising platforms for trapping, separating, and concentrating target biomarkers and cancer cells from a complex medium. Here, we visualized DEP and DEP gradients in conventional nanoelectronic devices by using multi-pass atomic force microcopy techniques. Our measurements directly demonstrated a short range DEP only at sharp step edges of electrodes, frequency dependent DEP polarity, and separation distance dependent DEP strength. Additionally, non-uniform DEP along the edges of the electrodes due to a large variation in electric field strength was observed. The strength and apparent working distance of DEP were measured to be an order of a few nN and 80 nm within the limited scale of particles and other parameters such as an ionic strength of the medium. This method provides a powerful tool to quantify the strength and polarity of DEP and allows optimizing and calibrating the device's operating parameters including the driving field strength for the effective control and manipulation of target biomolecules.

Here, we present electronic-type biosensors for ultra-sensitive detection of pancreatic cancer cells, based on graphene field effect transistors. The devices was decorated with bi-functional pyrene-iRGD peptides in which the pyrene moiety binds to the graphene though pi-pi interaction and the iRGD peptides binds to neuropilin receptors in the pancreatic cancer cells. Real-time electronic detection enabled detecting few cancer cells in the presence of the human serum, which is a critical for the early detection of cancer diagnosis and treatments. We acknowledge the support of ND EPSCoR New Faculty Award and ND NASA EPSCoR RID Grant.

Two-dimensional crystal is tested in the system of several millimeter size polystyrene spheres. Large numbers of same size spheres are triboelectrically charged through rubbing on the substrate plate. Therefore, one can observe the two-dimensional system without the help of any instrument and can easily micro-control the condition of two-dimensional system. The Fourier transform spectra, the pair distribution and the bond-orientational correlation function data show that the system is in near hexatic phase after fully charged, which is predicted by the KTHNY theory.

Nanoscale electronic devices like field-effect transistors have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes have the requisite sensitivity to detect single molecule events and sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of DNA polymerase I. In each case, recordings resolved detailed trajectories of tens of thousands of individual chemical events and provided excellent statistics for single-molecule events. This electronic technique has a temporal resolution approaching 1 microsecond, which provides a new window for observing brief, intermediate transition states. In addition, the devices are indefinitely stable, so that the same molecule can be observed for minutes and hours. The extended recordings provide new insights into rare events like transitions to chemically-inactive conformations.

Single molecule bioelectronic circuits provide an opportunity to study chemical kinetics and kinetic variability with bond-by-bond resolution. To demonstrate this approach, we examined the catalytic activity of T4 lysozyme processing peptidoglycan substrates. Monitoring a single lysozyme molecule through changes in a circuit's conductance helped elucidate unexplored and previously invisible aspects of lysozyme's catalytic mechanism and demonstrated lysozyme to be a processive enzyme governed by 9 independent time constants. The variation of each time constant with pH or substrate crosslinking provided different insights into catalytic activity and dynamic disorder. Overall, ten lysozyme variants were synthesized and tested in single molecule circuits to dissect the transduction of chemical activity into electronic signals. Measurements show that a single amino acid with the appropriate properties is sufficient for good signal generation, proving that the single molecule circuit technique can be easily extended to other proteins.