A microfluidic platform for the synthesis of polymer and polymer-protein-based protocells.

In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.

Label-Free Multitarget Separation of Particles and Cells under Flow Using Acoustic, Electrophoretic, and Hydrodynamic Forces.

Multiplex separation of mixed biological samples is essential in a considerable portion of biomedical research and clinical applications. An automated and operator-independent process for the separation of samples is highly sought after. There is a significant unmet need for methods that can perform fractionation of small volumes of multicomponent mixtures. Herein, we design an integrated chip that combines acoustic and electric fields to enable efficient and label-free separation of multiple different cells and particles under flow. To facilitate the connection of multiple sorting mechanisms in tandem, we investigate the electroosmosis (EO)-induced deterministic lateral displacement (DLD) separation in a combined pressure- and DC field-driven flow and exploit the combination of the bipolar electrode (BPE) focusing and surface acoustic wave (SAW) sorting modules. We successfully integrate four sequential microfluidic modules for multitarget separation within a single platform: (i) sorting particles and cells relying on the size and surface charge by adjusting the flow rate and electric field using a DLD array; (ii) alignment of cells or particles within a microfluidic channel by a bipolar electrode; (iii) separation of particles based on compressibility and density by the acoustic force; and (iv) separation of viable and nonviable cells using dielectric properties via the dielectrophoresis (DEP) force. As a proof of principle, we demonstrate the sorting of multiple cell and particle types (polystyrene (PS) particles, oil droplets, and viable and nonviable yeast cells) with high efficiency. This integrated microfluidic platform combines multiple functional components and, with its ability to noninvasively sort multiple targeted cells in a label-free manner relying on different properties, is compatible with high-definition imaging, showing great potential in diverse diagnostic and analysis applications.

Design and Application of Stimulus-Responsive Droplets and Bubbles Stabilized by Phospholipid Monolayers.

Biomimetic colloidal particles are promising agents for biosensing, but current technologies fall far short of Nature's capabilities for sensing, assessing, and responding to stimuli. Phospholipid-containing cell membranes are capable of binding and responding to an enormous variety of biomolecules by virtue of membrane organization and the presence of receptor proteins. By tuning the composition and functionalization of simulated membranes, soft colloids such as droplets and bubbles can be designed to respond to various stimuli. Moreover, because lipid monolayers can surround almost any hydrophobic phase, the interior of the colloid can be selected to provide a sensitive readout, for example in the form of optical microscopy or acoustic detection. In this work, we review some advances made by our group and others in the formulation of lipid-coated particles with different internal phases such as fluorocarbons, hydrocarbons, or liquid crystals. In some cases, binding or displacement of stabilizing lipids gives rise to conformational changes or disruptions in local membrane geometry, which can be amplified by the interior phase. In other cases, multivalent analytes can promote aggregation or even membrane fusion, which can be utilized for optical or acoustic readout. By highlighting a few recent examples, we hope to show that lipid monolayers represent an extremely versatile biosensing platform that can react to and detect biomolecules by leveraging the unique capabilities of phospholipid membranes.

Engineering the Echogenic Properties of Microfluidic Microbubbles Using Mixtures of Recombinant Protein and Amphiphilic Copolymers.

Microbubbles are used as ultrasound contrast agents in medical diagnosis and also have shown great promise in ultrasound-mediated therapy. However, short lifetime and broad size distribution of microbubbles limit their applications in therapy and imaging. Moreover, it is challenging to tailor the echogenic response of microbubbles to make them suitable for specific applications. To overcome these challenges, we use microfluidic flow-focusing to prepare monodisperse microbubbles with a mixture of a recombinant amphiphilic protein, oleosin, and a synthetic amphiphilic copolymer, Pluronic. We show that these microbubbles have superior uniformity and stability under ultrasonic stimulation compared to commercial agents. We also demonstrate that by using different Pluronics, the echogenic response of the microbubbles can be tailored. Our work shows the versatility of using the combination of microfluidics and protein/copolymer mixtures as a method of engineering microbubbles. This tunability could potentially be important and powerful in producing microbubble agents for theranostic applications.

Brain Targeted Xenon Protects Cerebral Vasculature After Traumatic Brain Injury.

Abstract Cerebrovascular dysfunction following traumatic brain injury (TBI) is a well-characterized phenomenon. Given the therapeutic potential of xenon, we aimed to study its effects after localized delivery to the brain using microbubbles. We designed xenon-containing microbubbles stabilized by dibehenoylphosphatidylcholine (DBPC) and polyethylene glycol (PEG) attached to saturated phospholipid (DPSE-PEG5000). Using a pig model of TBI, these microbubbles were intravenously injected, and ultrasound was used to release xenon at the level of the carotid artery. The control group received perfluorobutane containing microbubbles. Diffusion tensor imaging (DTI) showed areas of higher fractional anisotropy for pigs receiving xenon microbubbles compared to the control group at 1 day after injury. Radial diffusivity analysis showed that this effect was mainly the result of acute edema. Pigs were euthanized at 5 days, and the brain tissues of xenon-treated animals showed reduction of perivascular inflammation and blood-brain barrier disruption. Endothelial cell culture experiments showed that glutamate reduces tight junction protein zona occludens-1 (ZO-1), but treatment with xenon microbubbles attenuates this effect. Xenon treatment protects cerebrovasculature and reduces astroglial reactivity after TBI. Further, these data support the future use of localized delivery of various therapeutic agents for brain injury using microbubbles in order to limit systemic side effects and reduce costs.

Recombinant Protein Micelles to Block Transduction by SARS-CoV-2 Pseudovirus.

The continuing emergence of variants of the SARS-CoV-2 virus requires the development of modular molecular therapies. Here, we engineered a recombinant amphiphilic protein, oleosin, to spontaneously self-assemble into multivalent micellar nanostructures which can block the Spike S1 protein of SARS-CoV-2 pseudoviruses (PVs). Short recombinant proteins like oleosin can be formulated more easily than antibodies and can be functionalized with precision through genetic engineering. We cloned S1-binding mini-protein genes called LCBx, previously designed by David Baker's laboratory (UW Seattle), to the N-terminus of oleosin, expressing Oleo-LCBx proteins in E. coli. These proteins largely formed 10-100 nm micelles as verified by dynamic light scattering. Two proteins, Oleo-LCB1 and Oleo-LCB3, were seen to completely and irreversibly block transduction by both wild-type and delta variant PVs into 293T-hsACE2 cells at 10 µM. Presented in multivalent micelles, these proteins reduced transduction by PVs down to a functional protein concentration of 5 nM. Additionally, Oleo-LCB1 micelles outperformed corresponding synthetic LCB1 mini-proteins in reducing transduction by PVs. Tunable aqueous solubility of recombinant oleosin allowed incorporation of peptides/mini-proteins at high concentrations within micelles, thus enhancing drug loading. To validate the potential multifunctionality of the micelles, we showed that certain combinations of Oleo-LCB1 and Oleo-LCB3 performed much better than the individual proteins at the same concentration. These micelles, which we showed to be non-toxic to human cells, are thus a promising step toward the design of modular, multifunctional therapeutics that could bind to and inactivate multiple receptors and proteins necessary for the infection of the SARS-CoV-2 virus.

Can Ultrasound-Guided Xenon Delivery Provide Neuroprotection in Traumatic Brain Injury?

Traumatic brain injury (TBI) is associated with high mortality and morbidity in children and adults. Unfortunately, there is no effective management for TBI in the acute setting. Rodent studies have shown that xenon, a well-known anesthetic gas, can be neuroprotective when administered post-TBI. Gas inhalation therapy, however, the approach typically used for administering xenon, is expensive, inconvenient, and fraught with systemic side effects. Therapeutic delivery to the brain is minimal, with much of the inhaled gas cleared by the lungs. To bridge major gaps in clinical care and enhance cerebral delivery of xenon, this study introduces a novel xenon delivery technique, utilizing microbubbles, in which a high impulse ultrasound signal is used for targeted cerebral release of xenon. Briefly, an ultrasound pulse is applied along the carotid artery at the level of the neck on intravenous injection of xenon microbubbles (XeMBs) resulting in release of xenon from microbubbles into the brain. This delivery technique employs a hand-held, portable ultrasound system that could be adopted in resource-limited environments. Using a high-fidelity porcine model, this study demonstrates the neuroprotective efficacy of xenon microbubbles in TBI for the first time.

Multivariable Dependence of Acoustic Contrast of Fluorocarbon and Xenon Microbubbles under Flow.

Microbubbles (MBs) are 1 to 10 µm gas particles stabilized by an amphiphilic shell capable of responding to biomedical ultrasound with strong acoustic signals, allowing them to be commonly used in ultrasound imaging and therapy. The composition of both the shell and the core determines their stability and acoustic properties. While there has been extensive characterization of the dissolution, oscillation, cavitation, collapse and therefore, ultrasound contrast of MBs under static conditions, few reports have examined such behavior under hydrodynamic flow. In this study, we evaluate the interplay of ultrasound parameters (five different mechanical indices [MIs]), MB shell parameter (shell stiffness), type of gas (perfluorocarbon for diagnostic imaging and xenon as a therapeutic gas), and a flow parameter (flow rate) on the ultrasound signal of phospholipid-stabilized MBs flowing through a latex tube embedded in a tissue-mimicking phantom. We find that the contrast gradient (CG), a metric of the rate of decay of contrast along the length of the tube, and the contrast peak (CP), the location where the maximum contrast is reached, depend on the conditions of flow, imaging, and MB material. For instance, while the contrast near the flow inlet of the field of view is highest for a softer shell (dipalmitoylphosphatidylcholine [DPPC], C16) than for stiffer shells (distearoylphosphatidylcholine [DSPC], C18, and dibehenoylphosphatidylcholine [DBPC], C22), the contrast decay is also faster; stiffer shells provide more resistance and hence lead to slower MB dissolution/destruction. At higher flow rates, the CG is low for a fixed length of time because each MB is exposed to ultrasound for a shorter period. The CG becomes high for low flow rates, especially at high incident pressures (high MI), causing more MB destruction closer to the inlet of the field of view. Also, the CP shifts toward the inlet at low flow rates, high MIs, and low shell stiffness. We also report the first demonstration of sustained ultrasound flow imaging of a water-soluble, therapeutic gas MB (xenon). We find that an increased MB concentration is necessary for obtaining the same signal magnitude for xenon MBs. In summary, this study builds a framework depicting how multiple variables simultaneously affect the evolution of MB ultrasound contrast under flow. Depending on the MB composition, imaging conditions, transducer positioning, and image processing, building on such a framework could potentially allow for extraction of additional diagnostic information than is commonly analyzed for physiological flow.

Ultrasound Responsive Noble Gas Microbubbles for Applications in Image-Guided Gas Delivery.

Noble gases, especially xenon (Xe), have been shown to have antiapoptotic effects in treating hypoxia ischemia related injuries. Currently, in vivo gas delivery is systemic and performed through inhalation, leading to reduced efficacy at the injury site. This report provides a first demonstration of the encapsulation of pure Xe, Ar, or He in phospholipid-coated sub-10 µm microbubbles, without the necessity of stabilizing perfluorocarbon additives. Optimization of shell compositions and preparation techniques show that distearoylphosphatidylcholine (DSPC) with DSPE-PEG5000 can produce stable microbubbles upon shaking, while dibehenoylphosphatidylcholine (DBPC) blended with either DSPE-PEG2000 or DSPE-PEG5000 produces a high yield of microbubbles via a sonication/centrifugation method. Xe and Ar concentrations released into the microbubble suspension headspace are measured using GC-MS, while Xe released directly in solution is detected by the fluorescence quenching of a Xe-sensitive cryptophane molecule. Bubble production is found to be amenable to scale-up while maintaining their size distribution and stability. Excellent ultrasound contrast is observed in a phantom for several minutes under physiological conditions, while an intravenous administration of a bolus of pure Xe microbubbles provides significant contrast in a mouse in pre- and post-lung settings (heart and kidney, respectively), paving the way for image-guided, localized gas delivery for theranostic applications.

Photoacoustic and Ultrasound Dual-Mode Imaging via Functionalization of Recombinant Protein-Stabilized Microbubbles with Methylene Blue.

Contrast-enhanced photoacoustics and ultrasonics are complementary methods of bioimaging. In this study, a flow-focusing junction microfluidic device is used for the generation of uniform microbubbles (<5 µm) for simultaneous enhancement of photoacoustic and ultrasound imaging. Microbubbles stabilized with a mixture of a recombinant protein and a synthetic amphiphilic block copolymer are functionalized with an FDA-approved photoacoustic dye, methylene blue (MetB). These microbubbles are uniform in size and stable. We show that the ultrasound and photoacoustic signals can be independently controlled by changing the concentration of MetB during microbubble preparation and the concentration of MetB-functionalized microbubbles in the probe suspension. We also perform animal tests to demonstrate the enhancement of ultrasound and acoustic signals upon injection of MetB-functionalized microbubbles in mice. The increase in the sonographic and photoacoustic signals is visibly obvious in the images. Taken together, MetB-functionalized microbubbles represent promising dual-mode ultrasound and photoacoustic imaging contrast agents for theranostic applications.

Temperature-Responsive Hydrophobic Silica Nanoparticle Ultrasound Contrast Agents Directed by Phospholipid Phase Behavior.

In this paper, we report ultrasonically active nanoscale contrast agents that behave as thermometric sensors through phase change in their stabilizing phospholipid monolayer. Phospholipid-stabilized, hydrophobic mesoporous silica nanoparticles (P@hMSNs) are known to interact with high-intensity focused ultrasound (HIFU) to promote cavitation at their surfaces, which can be used for both imaging and therapy. We show that the lateral lipid phase behavior of the phosphocholine lipid dictates the acoustic contrast of the P@hMSNs. When the lipids are in the gel phase below their melting temperature, the P@hMSNs generate detectable microbubbles when exposed to HIFU. However, if the lipids exhibit a liquid expanded phase, the P@hMSNs cease to generate bubbles in response to HIFU insonation. We verify that the heating and subsequent transition of lipid coating the hMSN are associated with the loss of acoustic response by doping laurdan dye into the lipid monolayer and imaging lipid phase through red shifts in emission spectra. Similarly, cessation of cavitation was also induced by adding a fluidizing surfactant such as Triton X, which could be reversed upon washing away the excess surfactant. Finally, by controlling for the partial fluidization caused by the adsorption of protein, P@hMSNs may be used as thermometric sensors of the bulk fluid temperature. These findings not only impact the utilization of nanoscale agents as stimulus-responsive ultrasound contrast agents but also have broader implications for how cavitation may be initiated at surfaces coated by a surfactant.

Nanoparticle-Mediated Acoustic Cavitation Enables High Intensity Focused Ultrasound Ablation Without Tissue Heating.

While thermal ablation of various solid tumors has been demonstrated using high intensity focused ultrasound (HIFU), the therapeutic outcomes of this technique are still unsatisfactory because of common recurrence of thermally ablated cancers and treatment side effects due to the high ultrasound intensity and acoustic pressure requirements. More precise ablation of tumors can be achieved by generating cavitating bubbles in the tissue using shorter pulses with higher acoustic pressures, which induce mechanical damage rather than thermal. However, it has remained as a challenge to safely deliver the acoustic pressures required for mechanical ablation of solid tumors. Here, we report a method to achieve mechanical ablation at lower acoustic pressures by utilizing phospholipid-stabilized hydrophobic mesoporous silica nanoparticles (PL-hMSN). The PL-hMSNs act as seeds for nucleation of cavitation events and thus significantly reduce the peak negative pressures and spatial-average temporal-average HIFU intensities needed to achieve mechanical ablation. Substantial mechanical damage was observed in the red blood cell or tumor spheroid containing tissue mimicking phantoms at PL-hMSN concentrations as low as 10 µg mL-1, after only 5 s of HIFU treatment with peak negative pressures ∼11 MPa and duty cycles ∼0.01%. Even the application of HIFU (peak negative pressure of 16.8 MPa and duty cycle of 0.017%) for 1 min in the presence of PL-hMSN (200 µg mL-1) did not cause any detectable temperature increase in tissue-mimicking phantoms. In addition, the mechanical effects of cavitation promoted by PL-hMSNs were observed up to 0.5 mm from the center of the cavitation events. This method may thus also improve delivery of therapeutics or nanoparticles to tumor environments with limited macromolecular transport.

Nanoparticles Formed by Acoustic Destruction of Microbubbles and Their Utilization for Imaging and Effects on Therapy by High Intensity Focused Ultrasound.

This work reports that when PEG-lipid-shelled microbubbles with fluorocarbon interior (C4F10, C5F12, or C6F14) are subjected to ultrasound pulses, they produce metastable, fluid-filled nanoparticles that can be re-imaged upon administration of HIFU. The nanoparticles produced by destruction of the microbubbles (MBNPs) are of 150 nm average diameter and can be re-imaged for up to an hour after creation for C 4F10, and for at least one day for C5F12. The active species were found to be fluid (gas or liquid) filled nanoparticles rather than lipid debris. The acoustic droplet vaporization threshold of the nanoparticles was found to vary with the vapor pressure of the encapsulated fluorocarbon, and integrated image brightness was found to increase dramatically when the temperature was raised above the normal boiling point of the fluorocarbon. Finally, the vaporization threshold decreases in serum as compared to buffer, and administration of HIFU to the nanoparticles caused breast cancer cells to completely detach from their culture substrate. This work demonstrates a new functionality of microbubbles that could serve as a platform technology for ultrasound-based theranostics.

Phospholipid Capped Mesoporous Nanoparticles for Targeted High Intensity Focused Ultrasound Ablation.

The mechanical effects of cavitation can be effective for therapy but difficult to control, thus potentially leading to off-target side effects in patients. While administration of ultrasound active agents such as fluorocarbon microbubbles and nanodroplets can locally enhance the effects of high intensity focused ultrasound (HIFU), it has been challenging to prepare ultrasound active agents that are small and stable enough to accumulate in tumors and internalize into cancer cells. Here, this paper reports the synthesis of 100 nm nanoparticle ultrasound agents based on phospholipid-coated, mesoporous, hydrophobically functionalized silica nanoparticles that can internalize into cancer cells and remain acoustically active. The ultrasound agents produce bubbles when subjected to short HIFU pulses (≈6 µs) with peak negative pressure as low as ≈7 MPa and at particle concentrations down to 12.5 µg mL-1 (7 × 109 particles mL-1 ). Importantly, ultrasound agents are effectively uptaken by cancer cells without cytotoxic effects, but HIFU insonation causes destruction of the cells by the acoustically generated bubbles, as demonstrated by (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and lactate dehydrogenase assays and flow cytometry. Finally, it is showed that the HIFU dose required to effectively eliminate cancer cells in the presence of ultrasound agents causes only a small temperature increase of ≈3.5 °C.

Phase Behavior of Mixed Lipid Monolayers on Perfluorocarbon Nanoemulsions and its Effect on Acoustic Contrast.

Lipid-stabilized nanoemulsions containing a volatile liquid perfluorocarbon core have been studied as ultrasound contrast agents owing to their ability to transform into high-contrast microbubbles when subjected to high intensity focused ultrasound (HIFU). However, while there have been several studies on the effect of acoustic parameters on contrast, the effect of the droplet's stabilizing shell has not been studied as extensively. Inspired by previous studies showing lateral phase separation in microbubbles and vesicles, nanodroplets were formulated with a perfluorohexane core and a shell composed of varying amounts of saturated (DPPC) phospholipids, unsaturated (DOPC) phospholipids, and cholesterol, which were fractionated to obtain nanodroplets of mean diameter 300-400 nm and were stable over one week. When the DOPC content was increased to 40 mol%, ultrasound contrast increased by about one order of magnitude over DPPC-only droplets. Based on fluorescence microscopy results of lateral lipid phase separation on the droplet surface, the various combinations of DPPC, DOPC, and cholesterol were assigned to three regimes on the ternary phase diagram: solid-liquid ordered (low contrast), liquid ordered-liquid disordered (medium contrast), and solid-liquid disordered (high contrast). These regimes were confirmed by TEM analysis of nanoscale droplets. Droplets containing mixed lipid monolayers were also found to produce a significantly greater yield than single-component droplets. The discovery of the dependence of acoustic response on lipid phase separation will help to understand the formulation, behavior, and vaporization mechanism of acoustically-responsive nanoemulsions.

Understanding Acoustic Cavitation Initiation by Porous Nanoparticles: Toward Nanoscale Agents for Ultrasound Imaging and Therapy.

Ultrasound is widely applied in medical diagnosis and therapy due to its safety, high penetration depth, and low cost. In order to improve the contrast of sonographs and efficiency of the ultrasound therapy, echogenic gas bodies or droplets (with diameters from 200 nm to 10 µm) are often used, which are not very stable in the bloodstream and unable to penetrate into target tissues. Recently, it was demonstrated that nanobubbles stabilized by nanoparticles can nucleate ultrasound responsive microbubbles under reduced acoustic pressures, which is very promising for the development of nanoscale (<100 nm) ultrasound agents. However, there is still very little understanding about the effects of nanoparticle properties on the stabilization of nanobubbles and nucleation of acoustic cavitation by these nanobubbles. Here, a series of mesoporous silica nanoparticles with sizes around 100 nm but with different morphologies were synthesized to understand the effects of nanoparticle porosity, surface roughness, hydrophobicity, and hydrophilic surface modification on acoustic cavitation inception by porous nanoparticles. The chemical analyses of the nanoparticles showed that, while the nanoparticles were prepared using the same silica precursor (TEOS) and surfactant (CTAB), they revealed varying amounts of carbon impurities, hydroxyl content, and degrees of silica crosslinking. Carbon impurities or hydrophobic modification with methyl groups is found to be essential for nanobubble stabilization by mesoporous silica nanoparticles. The acoustic cavitation experiments in the presence of ethanol and/or bovine serum albumin (BSA) demonstrated that acoustic cavitation is predominantly nucleated by the nanobubbles stabilized at the nanoparticle surface not inside the mesopores. Finally, acoustic cavitation experiments with rough and smooth nanoparticles were suggested that a rough nanoparticle surface is needed to largely preserve surface nanobubbles after coating the surface with hydrophilic macromolecules, which is required for in vivo applications of nanoparticles.

Stable Encapsulation of Air in Mesoporous Silica Nanoparticles: Fluorocarbon-Free Nanoscale Ultrasound Contrast Agents.

While gas-filled micrometer-sized ultrasound contrast agents vastly improve signal-to-noise ratios, microbubbles have short circulation lifetimes and poor extravasation from the blood. Previously reported fluorocarbon-based nanoscale contrast agents are more stable but their contrast is generally lower owing to their size and dispersity. The contrast agents reported here are composed of silica nanoparticles of ≈100 nm diameter that are filled with ≈3 nm columnar mesopores. Functionalization of the silica surface with octyl groups and resuspension with Pluronic F127 create particles with pores that remain filled with air but are stable in buffer and serum. Administration of high intensity focused ultrasound (HIFU) allows sensitive imaging of the silica nanoparticles down to 10(10) particles mL(-1) , with continuous imaging for at least 20 min. Control experiments with different silica particles supported the hypothesis that entrapped air could be pulled into bubble nuclei, which can then in turn act as acoustic scatterers. This process results in very little hemolysis in whole blood, indicating potential for nontoxic blood pool imaging. Finally, the particles are lyophilized and reconstituted or stored in PBS (phosphate-buffered saline, at least for four months) with no loss in contrast, indicating stability to storage and reformulation.

Mutually-Reactive, Fluorogenic Hydrocyanine/Quinone Reporter Pairs for In-Solution Biosensing via Nanodroplet Association.

Mutually reactive, fluorogenic molecules are presented as a simple and novel technique for in-solution biosensing. The hypothesis behind this work was that aggregating droplets into close proximity would cause rapid mixing of their contents. To take advantage of this effect, a novel pair of fluorogenic redox molecules were designed to remain in lipid-stabilized oil droplets but mix once aggregated. First, the hydrophobic cyanine dye 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) was reduced with sodium borohydride to form a nonfluorescent analog (HDiI). Hydrophobic quinone derivatives were then screened as oxidizing agents, and it was found that p-fluoranil oxidized nonfluorescent HDiI back to fluorescent DiI. Next, HDiI and p-fluoranil were loaded into NEOBEE oil nanodroplets of average diameter 600 nm that were stabilized by a monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol (PEG), and DSPE-PEG-biotin. Addition of streptavidin caused aggregation of droplets and the appearance of red fluorescent aggregates within 30 min. Next, Nanoparticle Tracking Analysis was used to record the fluorescence of the droplets and their aggregates. By integrating the fluorescence emission of the tracked droplets, streptavidin could be detected down to 100 fM. Finally, the droplets were reformulated to sense for vascular endothelial growth factor (VEGF), a biomarker for tumor metastasis. Using anti-VEGF aptamers attached to DSPE-PEG incorporated into the nanodroplet monolayer, VEGF could also be detected down to 100 fM.

Self-assembled gold nanostar-NaYF4:Yb/Er clusters for multimodal imaging, photothermal and photodynamic therapy.

A grand challenge for medicine is to develop tools to selectively image and treat diseased cells. Rare earth doped upconverting nanoparticles (UCNPs) have been extensively studied for imaging applications because of their ability to absorb near infrared radiation (NIR) and emit visible light, but these particles cannot induce therapy alone. Recently, we developed methods to couple the UCNPs to visible and NIR-absorbing gold nanostructures through nucleic acid interactions. Here, we show that gold-UCNP clusters with optimized plasmon resonance and particle compositions provide both in vitro imaging contrast and combination cell killing through simultaneous photothermal (PTT) and photodynamic (PDT) therapy. PDT was induced by embedding singlet oxygen photosensitizers in silica shells on the UCNPs. Upon photoexcitation with 980 nm light, the NIR absorbing gold-UCNP clusters both increased the local temperature and generated singlet oxygen, increasing cell killing relative to either modality alone. The multifunctional polyethylene glycol (PEG) coated gold-NaYF4:Yb/Er clusters exhibited high biocompatibility without irradiation but synergistic cell killing of MCF-7 cancer cells under light excitation. Finally, we also demonstrate that an optimal gold plasmon resonance is critical for minimizing absorbance overlap with the photosensitizers.

Selective Vaporization of Superheated Nanodroplets for Rapid, Sensitive, Acoustic Biosensing.

Superheated perfluorocarbon nano-droplets exhibit promise as sensitive acoustic biosensors. Aggregation of biotin-decorated lipid-shelled droplets by streptavidin greatly increases the yield of bubbles formed by ultrasound-induced vaporization. Streptavidin is sensed down to 1 × 10(-13) m, with differentiable signal appearing in as little as two minutes, using a scalable assay without washing, processing, or development steps.