Oligo cyc-DEP: On-chip cyclic immunofluorescence profiling of cell-derived nanoparticles.

We present a follow-on technique for the cyclic-immunofluorescence profiling of suspension particles isolated using dielectrophoresis. The original lab-on-chip technique ("cyc-DEP" [cyclic immunofluorescent imaging on dielectrophoretic chip]) was designed for the multiplex surveillance of circulating biomarkers. Nanoparticles were collected from low-volume liquid biopsies using microfluidic dielectrophoretic chip technology. Subsequent rounds of cyclic immunofluorescent labeling and quenching were imaged and quantified with a custom algorithm to detect multiple proteins. While cyc-DEP improved assay multiplicity, long runtimes threatened its clinical adoption. Here, we modify the original cyc-DEP platform to reduce assay runtimes. Nanoparticles were formulated from human prostate adenocarcinoma cells and collected using dielectrophoresis. Three proteins were labeled on-chip with a mixture of short oligonucleotide-conjugated antibodies. The sample was then incubated with complementary fluorophore-conjugated oligonucleotides, which were dehybridized using an ethylene carbonate buffer after each round of imaging. Oligonucleotide removal exhibited an average quenching efficiency of 98 ± 3% (n = 12 quenching events), matching the original cyc-DEP platform. The presented "oligo cyc-DEP" platform achieved clinically relevant sample-to-answer times, reducing the duration for three rounds of cyclic immunolabeling from approximately 20 to 6.5 h-a 67% decrease attributed to rapid fluorophore removal and the consolidated co-incubation of antibodies.

Hybrid Silica-Coated PLGA Nanoparticles for Enhanced Enzyme-Based Therapeutics.

Some cancer cells rely heavily on non-essential biomolecules for survival, growth, and proliferation. Enzyme based therapeutics can eliminate these biomolecules, thus specifically targeting neoplastic cells; however, enzyme therapeutics are susceptible to immune clearance, exhibit short half-lives, and require frequent administration. Encapsulation of therapeutic cargo within biocompatible and biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) is a strategy for controlled release. Unfortunately, PLGA NPs exhibit burst release of cargo shortly after delivery or upon introduction to aqueous environments where they decompose via hydrolysis. Here, we show the generation of hybrid silica-coated PLGA (SiLGA) NPs as viable drug delivery vehicles exhibiting sub-200 nm diameters, a metastable Zeta potential, and high loading efficiency and content. Compared to uncoated PLGA NPs, SiLGA NPs offer greater retention of enzymatic activity and slow the burst release of cargo. Thus, SiLGA encapsulation of therapeutic enzymes, such as asparaginase, could reduce frequency of administration, increase half-life, and improve efficacy for patients with a range of diseases.

Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

Silica cloaking of adenovirus enhances gene delivery while reducing immunogenicity.

Viral gene therapy is a means of delivering genes to replace malfunctioning ones, to kill cancer cells, or to correct genetic mutations. This technology is emerging as a powerful clinical tool; however, it is still limited by viral tropism, uptake and clearance by the liver, and most importantly an immune response. To overcome these challenges, we sought to merge the robustness of viral gene expression and the versatility of nanoparticle technology. Here, we describe a method for cloaking adenovirus (Ad) in silica (SiAd) as a nanoparticle formulation that significantly enhances transduction. Intratumoral injections in human glioma xenografts revealed SiAd expressing luciferase improved tumor transduction while reducing liver uptake. In immune-competent mice SiAd induced no inflammatory cytokines and reduced production of neutralizing antibodies. Finally, SiAd expressing TNF-related apoptosis-inducing ligand inhibited tumor growth of glioma xenografts. These results reveal that silica cloaking of Ad can enhance viral gene delivery while reducing immunogenicity.

Supramolecular self assembly of nanodrill-like structures for intracellular delivery.

Despite recent advances in the supramolecular assembly of cell-penetrating peptide (CPP) nanostructures, the tuning of size, shape, morphology and packaging of drugs in these materials still remain unexplored. Herein, through sequential ligation of peptide building blocks, we create cell-penetrating self-assembling peptide nanomaterials (CSPNs) with the capability to translocate inside cells. We devised a triblock array of Tat48-59 [HIV-1 derived transactivator of transcription48-59] based CPPs, conjugated to up to four Phenylalanine (Phe) residues through an amphiphilic linker, (RADA)2. We observed that the sequential addition of Phe leads to the transition of CSPN secondary structures from a random coil, to a distorted α-helix, a ß-sheet, or a pure α-helix. This transition occurs due to formation of a heptad by virtue of even number of Phe. Atomic force microscopy revealed that CSPNs form distinct shapes reminiscent of a "drill-bit". CSPNs containing two, three or four Phe, self-assemble into "nanodrill-like structures" with a coarse-twisted, non-twisted or fine-twisted morphology, respectively. These nanodrills had a high capacity to encapsulate hydrophobic guest molecules. In particular, the coarse-twisted nanodrills demonstrate higher internalization and are able to deliver rapamycin, a hydrophobic small molecule that induced autophagy and are capable of in vivo delivery. Molecular dynamics studies provide microscopic insights into the structure of the nanodrills that can contribute to its morphology and ability to interact with cellular membrane. CSPNs represent a new modular drug delivery platform that can be programmed into exquisite structures through sequence-specific fine tuning of amino acids.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma.

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 µL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Selection of DNA nanoparticles with preferential binding to aggregated protein target.

High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life's own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions.

Acoustic Microcannons: Toward Advanced Microballistics.

Acoustically triggered microcannons, capable of loading and firing nanobullets (Nbs), are presented as powerful microballistic tools. Hollow conically shaped microcannon structures have been synthesized electrochemically and fully loaded with nanobullets made of silica or fluorescent microspheres, and perfluorocarbon emulsions, embedded in a gel matrix stabilizer. Application of a focused ultrasound pulse leads to the spontaneous vaporization of the perfluorocarbon emulsions within the microcannon and results in the rapid ejection of the nanobullets. Such Nbs "firing" at remarkably high speeds (on the magnitude of meters per second) has been modeled theoretically and demonstrated experimentally. Arrays of microcannons anchored in a template membrane were used to demonstrate the efficient Nbs loading and the high penetration capabilities of the ejected Nbs in a tissue phantom gel. This acoustic-microcannon approach could be translated into advanced microscale ballistic tools, capable of efficient loading and firing of multiple cargoes, and offer improved accessibility to target locations and enhanced tissue penetration properties.

The influence of distance between microbubbles on the fluid flow produced during ultrasound exposure.

The collapse dynamics of lipid monolayer-coated microbubbles in the clinically-relevant size range under 6 µm in diameter have not been studied directly due to their small size obscuring the collapse visualization. This study investigates the influence of inter-microbubble distance on the shape of lipid debris clouds created by the collapse of the microbubble destroying the microbubble lipid monolayer. The shape was highly influenced by the fluid motion that occurred as the microbubbles collapsed. It was observed that at inter-microbubble distances smaller than 37 µm the microbubbles began to interact with one another resulting in distorted and ellipsoid-shaped debris clouds. At inter-microbubble distances less than 10 µm, significantly elongated debris clouds were observed that extended out from the original microbubble location in a single direction. These distortions show a significant distance-dependent interaction between microbubbles. It was observed that microbubbles in physical contact with one another behaved in the same manner as separate microbubbles less than 10 µm apart creating significantly elongated debris clouds. It can be hypothesized that small inter-microbubble distances influence the microbubble to collapse asymmetrically resulting in the creation of fluid jets that contribute to the formation of debris fields that are elongated in a single direction.

Manipulating nanoscale features on the surface of dye-loaded microbubbles to increase their ultrasound-modulated fluorescence output.

The nanoscale surface features of lipid-coated microbubbles can dramatically affect how the lipids interact with one another as the microbubble diameter expands and contracts under the influence of ultrasound. During microbubble manufacturing, the different lipid shell species naturally partition forming concentrated lipid islands. In this study the dynamics of how these nanoscale islands accommodate the expansion of the microbubbles are monitored by measuring the fluorescence intensity changes that occur as self-quenching lipophilic dye molecules embedded in the lipid layer change their distance from one another. It was found that when the dye molecules were concentrated in islands, less than 5% of the microbubbles displayed measurable fluorescence intensity modulation indicating the islands were not able to expand sufficiently for the dye molecules to separate from one another. When the microbubbles were heated and cooled rapidly through the lipid transition temperature the islands were melted creating an even distribution of dye about the surface. This resulted in over 50% of the microbubbles displaying the fluorescence-modulated signal indicating that the dye molecules could now separate sufficiently to change their self-quenching efficiency. The separation of the surface lipids in these different formations has significant implications for microbubble development as ultrasound and optical contrast agents.

In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents.

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents.

Isolation of rare tumor cells from blood cells with buoyant immuno-microbubbles.

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.

Nanofiber near-field light-matter interactions for enhanced detection of molecular level displacements and dynamics.

We experimentally demonstrate that plasmonic nanoparticles embedded in the evanescent field of subwavelength optical waveguides (WGs) are highly sensitive to distances normal to the propagation of light, showing an ~10× increase in spatial resolution compared to the optical field decay of the WG. The scattering cross-section of the Au nanoparticle is increased by the plasmon-dielectric coupling interaction when the nanoparticle is placed near the dielectric surface of the WG, and the decay of the scattering signal is enhanced, showing angstrom level distance sensitivity within 10 nm from the WG. Numerical studies with the finite-difference time-domain (FDTD) method correlate well with the experimental results. To demonstrate real-time monitoring of a single molecule stretching in the evanescent field, we linked individual single-stranded DNA molecules between the WG and plasmonic nanoparticles and pushed on the nanoparticles with fluidic forces. The simple design and ease of obtaining optical feedback on molecular displacements makes our approach ideal for new in situ force sensing devices, imaging technologies, and high-throughput molecular analysis.

Phospholipid/Carbocyanine Dye-Shelled Microbubbles as Ultrasound-Modulated Fluorescent Contrast Agents.

Fluorescent microbubbles have been fabricated with the capacity to have their emission modulated by ultrasound. These contrast agent particles could potentially be used in the future to extract fluorescence modulation from a strong light background to increase imaging depth and resolution in scattering media. Fluorescence intensity modulation was demonstrated at the ultrasound driving frequency.

Profiling the evanescent field of nanofiber waveguides using self-assembled polymer coatings.

Here we demonstrate a facile method of quantifying the decaying optical field surrounding free-standing tin dioxide (SnO(2)) nanofiber waveguides. Through the use of thin self-assembled polyelectrolyte coatings and fluorescent optical transmitters we map out the optical intensity as a function of distance with nanometer resolution.

Aptamer-crosslinked microbubbles: smart contrast agents for thrombin-activated ultrasound imaging.

Thrombosis, or malignant blood clotting, is associated with numerous cardiovascular diseases and cancers. A microbubble contrast agent is presented that produces ultrasound harmonic signal only when exposed to elevated thrombin levels. Initially silent microbubbles are activated in the presence of both thrombin-spiked and freshly clotting blood in three minutes with detection limits of 20 nM thrombin and 2 aM microbubbles.

Stimulus-responsive light coupling and modulation with nanofiber waveguide junctions.

We report a systematic study of light coupling at junctions of overlapping SnO(2) nanofiber waveguides (WGs) as a function of gap separation and guided wavelength. The junctions were assembled on silica substrates using micromanipulation techniques and the gap separation was controlled by depositing thin self-assembled polyelectrolyte coatings at the fiber junctions. We demonstrate that the coupling efficiency is strongly dependent on the gap separation, showing strong fluctuations (0.1 dB/nm) in the power transfer when the separation between nanofibers changes by as little as 2 nm. Experimental results correlate well with numerical simulations using three-dimensional finite-difference time-domain techniques. To demonstrate the feasibility of using coupled nanofiber WGs to modulate light, we encased the junctions in an environment-responsive matrix and exposed the junctions to gaseous vapor. The nanofiber junctions show an ~95% (or ~80%) modulation of the guided 450 nm (or 510 nm) light upon interaction with the gaseous molecules. The results reveal a unique nanofiber-based sensing scheme that does not require a change in the refractive index to detect stimuli, suggesting these structures could play important roles in localized sensing devices including force-based measurements or novel chemically induced light modulators.

Different effect of hydrogelation on antifouling and circulation properties of dextran-iron oxide nanoparticles.

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this "stealth" effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by cross-linking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages in vivo. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles' long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles.

Characterization of individual ultrasound microbubble dynamics with a light-scattering system.

Ultrasound microbubbles are contrast agents used for diagnostic ultrasound imaging and as carriers for noninvasive payload delivery. Understanding the acoustic properties of individual microbubble formulations is important for optimizing the ultrasound imaging parameters for improved image contrast and efficient payload delivery. We report here a practical and simple optical tool for direct real-time characterization of ultrasound contrast microbubble dynamics based on light scattering. Fourier transforms of raw linear and nonlinear acoustic oscillations, and microbubble cavitations are directly recorded. Further, the power of this tool is demonstrated by comparing clinically relevant microbubble cycle-to-cycle dynamics and their corresponding Fourier transforms.