Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake.

BACKGROUND: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. METHODS: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. RESULTS: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. CONCLUSION: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.

Exosome- and extracellular vesicle-based approaches for the treatment of lysosomal storage disorders.

Cell-generated extracellular vesicles (EVs) are being engineered as biologically-inspired vehicles for targeted delivery of therapeutic agents to treat difficult-to-manage human diseases, including lysosomal storage disorders (LSDs). Engineered EVs offer distinct advantages for targeted delivery of therapeutics compared to existing synthetic and semi-synthetic nanoscale systems, for example with regard to their biocompatibility, circulation lifetime, efficiencies in delivery of drugs and biologics to target cells, and clearance from the body. Here, we review literature related to the design and preparation of EVs as therapeutic carriers for targeted delivery and therapy of drugs and biologics with a focus on LSDs. First, we introduce the basic pathophysiology of LDSs and summarize current approaches to diagnose and treat LSDs. Second, we provide specific details about EVs, including subtypes, biogenesis, biological properties and their potential to treat LSDs. Third, we review state-of-the-art approaches to engineer EVs for treatments of LSDs. Finally, we summarize explorative basic research and applied applications of engineered EVs for LSDs, and highlight current challenges, and identify new directions in developing EV-based therapies and their potential impact on clinical medicine.

Ferroelectric Sr3 Sn2 O7 :Nd3+ : A New Multipiezo Material with Ultrasensitive and Sustainable Near-Infrared Piezoluminescence.

Ultrasensitive and sustainable near-infrared (NIR)-emitting piezoluminescence is observed from noncentrosymmetric and ferroelectric-phase Sr3 Sn2 O7 doped with rare earth Nd3+ ions. Sr3 Sn2 O7 :Nd3+ (SSN) with polar A21 am structure is demonstrated to emit piezoluminescence of wavelength of 800-1500 nm at microstrain levels, which is enhanced by the ferroelectrically polarized charges in the multipiezo material. These discoveries provide new research opportunities to study luminescence properties of multipiezo and piezo-photonic materials, and to explore their potential as novel ultrasensitive probes for deep-imaging of stress distributions in diverse materials and structures including artificial bone and other implanted structures (in vivo, in situ, etc).

Sequential deletion of CD63 identifies topologically distinct scaffolds for surface engineering of exosomes in living human cells.

Exosomes are cell-derived extracellular vesicles that have great potential in the field of nano-medicine. However, a fundamental challenge in the engineering of exosomes is the design of biocompatible molecular scaffolds on their surface to enable cell targeting and therapeutic functions. CD63 is a hallmark protein of natural exosomes that is highly enriched on the external surface of the membrane. We have previously described engineering of CD63 for use as a molecular scaffold in order to introduce cell-targeting features to the exosome surface. Despite this initial success, the restrictive M-shaped topology of full-length CD63 may hinder specific applications that require N- or C-terminal display of cell-targeting moieties on the outer surface of the exosome. In this study, we describe new and topologically distinct CD63 scaffolds that enable robust and flexible surface engineering of exosomes. In particular, we conducted sequential deletions of the transmembrane helix of CD63 to generate a series of CD63 truncates, each genetically-fused to a fluorescent protein. Molecular and cellular characterization studies showed truncates of CD63 harboring the transmembrane helix 3 (TM3) correctly targeted and anchored to the exosome membrane and exhibited distinct n-, N-, Ω-, or I-shaped membrane topologies in the exosomal membrane. We further established that these truncates retained robust membrane-anchoring and exosome-targeting activities when stably expressed in the HEK293 cells. Moreover, HEK293 cells produced engineered exosomes in similar quantities to cells expressing full-length CD63. Based on the results of our systematic sequential deletion studies, we propose a model to understand molecular mechanisms that underlie membrane-anchoring and exosome targeting features of CD63. In summary, we have established new and topologically distinct scaffolds based on engineering of CD63 that enables flexible engineering of the exosome surface for applications in disease-targeted drug delivery and therapy.

Bead-Based Immunocomplex Entrapment Assays for Rapid, Sensitive, and Multiplexed Detection of Disease Biomarkers with Minimal User Intervention.

Current interest in at-home diagnostic devices derives from their potential to disrupt expensive and time-consuming hospital-based diagnostic practices. Conventional immunoassays are often touted for use in at-home diagnostic devices, although in practice they are slow, labor-intensive and require expensive equipment. Here, we introduce bead-based sensors as alternative biomarker detection platforms for at-home diagnostic devices. The immunocomplex entrapment assay (ICEA), and the related enzyme-linked ICEA (ELICEA) offer enhancements over conventional immunoassays in terms of their speed, and minimal requirements for user intervention and instrumentation. In particular, we designed bead-based sensors to entrap large molecular weight complexes between target molecules and signal-generating immunoconjugates while allowing any unbound conjugates to escape from the bead. Confocal fluorescence microscopy was used to demonstrate the sensitivity, robustness, and reproducibility of the ICEA and ELICEA platforms. For example, we showed the intensity of signals generated by entrapped immunoconjugate complexes correlate linearly with the concentration of target molecule in the sample. We employed ICEA, and ELICEA platforms to detect human forms of immunoglobulins, albumin, and κ light chain (KLC). For example, we used ICEA to detect KLC at 5 µg·mL-1 in urine, which would allow for earlier diagnosis of Bence-Jones disease compared to conventional assays. In addition, we showed bead-entrapped phosphatases (AP) in immunocomplexes generate insoluble, blue-colored dyes from AP-substrates that accumulate in beads and allow for visual and cellphone camera-based detection of IgG to 10 ng·mL-1 within 20 min. Finally, we described ICEA and ELICEA platforms to analyze multiple target proteins within individual beads.

Targeted delivery of lysosomal enzymes to the endocytic compartment in human cells using engineered extracellular vesicles.

Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal ß-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications.

Decoy exosomes as a novel biologic reagent to antagonize inflammation.

Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.

Daylight-Mediated, Passive, and Sustained Release of the Glaucoma Drug Timolol from a Contact Lens.

Timolol, a potent inhibitor of ß-adrenergic receptors (ßARs), is a first-line drug for decreasing the intraocular pressure (IOP) of patients with glaucoma. Timolol is administered using 0.5% eye-drop solutions at >3 × 107 times the inhibitory concentration (k i) for ßARs. This high dose is wasteful and triggers off-target effects that increase medication noncompliance. Here, we introduce contact lenses that release timolol to the eye throughout the day during passive exposures to natural daylight at a more therapeutically relevant concentration (>3000 k i). Timolol is coupled to the polymer of the contact lens via a photocleavable caged cross-linker and is released exclusively to the surrounding fluid after the 400-430 nm mediated cleavage of the cross-linking group. Studies conducted in a preclinical mouse model of glaucoma show photoreleased timolol is effective as authentic timolol in reducing IOP. Our studies highlight several advantages of daylight-mediated release of timolol from lenses compared to eye-drops. First, fitted contact lenses exposed to natural daylight release sufficient timolol to sustain the inhibition of ßARs over a 10 h period. Second, the contact lenses inhibit ßARs in the eye using only 5.7% of the timolol within a single eye-drop. Third, the lenses allow the patient to passively control the amount of timolol released from the lens-for example, early morning exposure to outdoor sunlight would release enough timolol to maximally reduce the IOP, whereas subsequent periodic exposures to indoor daylight would release sufficient timolol to overcome the effects of its spontaneous dissociation from ßARs. Fourth, our lenses are disposable, designed for single day use, and manufactured at a low cost.

Pseudotyping exosomes for enhanced protein delivery in mammalian cells.

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells.

High-contrast grating resonators for label-free detection of disease biomarkers.

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI.

Human platelets repurposed as vehicles for in vivo imaging of myeloma xenotransplants.

Human platelets were identified in tumors by Trousseau in 1865, although their roles in tumor microenvironments have only recently attracted the attention of cancer researchers. In this study we exploit and enhance platelet interactions in tumor microenvironments by introducing tumor-targeting and imaging functions. The first step in repurposing human platelets as vehicles for tumor-targeting was to inhibit platelet-aggregation by cytoplasmic-loading of kabiramide (KabC), a potent inhibitor of actin polymerization and membrane protrusion. KabC-Platelets can accumulate high levels of other membrane-permeable cytoxins and probes, including epidoxorubicin, carboxyfluorescein di-ester and chlorin-e6. Finally, mild reaction conditions were developed to couple tumor-targeting proteins and antibodies to KabC-platelets. Fluorescence microscopy studies showed KabC-platelets, surface-coupled with transferrin and Cy5, bind specifically to RPMI8226 and K562 cells, both of which over-express the transferrin receptor. Repurposed platelets circulate for upto 9-days a feature that increases their chance of interacting with target cells. KabC-platelets, surface-coupled with transferrin and Cy7, or chlorin-e6, and injected in immuno-compromised mice were shown to accumulate specifically in sub-cutaneous and intra-cranial myeloma xenotransplants. The high-contrast, in vivo fluorescence images recorded from repurposed platelets within early-stage myeloma is a consequence in part of their large size (φ~2µm), which allows them to transport 100 to 1000-times more targeting-protein and probe molecules respectively. Human platelets can be configured with a plurality of therapeutic and targeting antibodies to help stage tumor environments for an immunotherapy, or with combinations of therapeutic antibodies and therapeutic agents to target and treat cardiovascular and neurologic diseases.

Genetically encoded sensors of protein hydrodynamics and molecular proximity.

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

Structural and biochemical studies of actin in complex with synthetic macrolide tail analogues.

The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. Structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis.

Silver nanoparticle-embedded microbubble as a dual-mode ultrasound and optical imaging probe.

Microbubbles (MBs) coupled with nanoparticles represent a new class of multifunctional probe for multiscale biomedical imaging and drug delivery. In this study, we describe the development of multifunctional, microscale microbubble probes that are composed of a nitrogen gas core and a biocompatible polymer shell harboring silver nanoparticles (AgNPs). Ultrasound imaging studies show that the presence of AgNPs in the MB significantly improves the contrast of ultrasound images. The AgNPs within individual MB can be also imaged by using dark-field microscopy (DFM), which suggests that AgNPs in the polymer shell adopt multiple structural forms. AgNPs are released from the polymer shell following a brief exposure to an ultrasonic field and are subsequently taken up by living cells. AgNPs within labeled cells are imaged by DFM, while surface-enhanced Raman scattering is used to identify specific cytoplasmic biomolecules that bind to the surface of the AgNP. Collectively, these studies demonstrate the application of multifunctional MBs for micrometer scale contrast-enhanced ultrasound imaging, as vehicles for the ultrasound-based delivery of optical probes and drugs to cells, and for imaging of chemical sensing of individual nanopartiles within cells and tissue.

High-contrast fluorescence imaging in fixed and living cells using optimized optical switches.

We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C12-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy.

Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens.

Structural dynamics of troponin I during Ca2+-activation of cardiac thin filaments: a multi-site Förster resonance energy transfer study.

A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca(2+)-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm. Unlike earlier FRET analyses of protein dynamics within the thin filament, this study considered the effects of non-random distribution of dipoles for the donor and acceptor probes. The major conclusion drawn from this study is that Ca(2+) and myosin S1-binding to the thin filament results in movement of the C-terminal domain of cTnI from the outer domain of F-actin towards the inner domain, which is associated with the myosin-binding. A hinge-linkage model is used to best-describe the finding of a Ca(2+)-induced movement of the C-terminus of cTnI with a stationary N-terminus. This dynamic model of the activation of the thin filament is discussed in the context of other structural and biochemical studies on normal and mutant cTnI found in hypertrophic cardiomyopathies.

Optical control of calcium affinity in a spiroamido-rhodamine based calcium chelator.

An optically controlled Ca(2+)-chelator 1 was developed to mimic natural calcium oscillations. Compound 1, a spiroamido-rhodamine derivative of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), underwent cycles of reversible transitions between a colorless closed state and a fluorescent open form. The closed-state exhibited a high affinity for Ca(2+) (K(d): 509 nM) with excellent selectivity over Mg(2+) (K(d): 19 mM). The open isomer had a 350-fold lower Ca(2+) affinity (K(d): 181 µM), while the Mg(2+) affinity was not significantly affected (K(d): 14 mM).