The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Microcapsules and Nanoliposomes Based Strategies to Improve the Stability of Blueberry Anthocyanins.

Blueberry anthocyanins are water-soluble natural pigments that can be used as both natural antioxidants and natural colorants. However, their structural instability greatly limits their application in the food, pharmaceutical, and cosmetic industries. In this study, blueberry anthocyanin microcapsules (BAM) and blueberry anthocyanin liposomes (BAL) were fabricated based on blueberry anthocyanins. Film dispersion methods were used to prepare the BAL. Their preparation processes were optimized and compared to improve the stability of the blueberry anthocyanins following exposure to light and high temperatures. The BAM were prepared through complex phase emulsification. The blueberry anthocyanins were protected by the shell materials composed of sodium alginate after being formed into BAM. Under the optimal conditions, the embedding rate of BAM and BAL can reach as high as 96.14% and 81.26%, respectively. In addition, the particle size, zeta potential, microtopography, and structure feature information of the BAM and BAL were compared. The average particle sizes of the BAM and BAL were 9.78 µm and 290.2 nm, respectively, measured using a laser particle size analyzer, and the zeta potentials of the BAM and BAL were 34.46 mV and 43.0 mV, respectively. In addition, the optimal preparation processes were determined through single-factor and response surface optimization experiments. The most important factors in the single-factor experiment for the preparation of microcapsules and liposomes were the content of CaCl2 and the amount of anthocyanin. The preservation rates in the light and dark were also compared, and the thermal stabilities of the BAM and BAL were characterized through differential thermal scanning. The results showed that both the BAM and BAL maintained the stability of blueberry anthocyanins, and no significant difference was found between the indices used to evaluate their stability. The results of this study provide theoretical support for the development of effective systems to maintain the stability of anthocyanins, thereby improving their bioavailability after ingestion by humans.

Single Molecule-Level Detection via Liposome-Based Signal Amplification Mass Spectrometry Counting Assay.

Because of the low atomization and/or ionization efficiencies of many biological macromolecules, the application of mass spectrometry to the direct quantitative detection of low-abundance proteins and nucleic acids remains a significant challenge. Herein, we report mass spectrum tags (MS-tags) based upon gold nanoparticle (AuNP)-templated phosphatidylcholine phospholipid (DSPC) liposomes, which exhibit high and reliable signals via electrospray ionization (ESI). Using these MS-tags, we constructed a liposome signal amplification-based mass spectrometric (LSAMS) "digital" counting assay to enable ultrasensitive detection of target nucleic acids. The LSAMS system consists of liposomes modified with a gold nanoparticle core and surface-anchored photocleavable DNA. In the presence of target nucleic acids, the modified liposome and a magnetic bead simultaneously hybridize with the target nucleic acid. After magnetic separation and photolysis, the MS-tag is released and can be analyzed by ESI-MS. At very low target concentrations, one liposome particle corresponds to one target molecule; thus, the concentration of the target can be estimated by counting the number of liposomes. With this assay, hepatitis C (HCV) virus RNA was successfully analyzed in clinical samples.

Biophysical analysis of lipidic nanoparticles.

Biological nanoparticles include liposomes, extracellular vesicle and lipid-based discoidal systems. When studying such particles, there are several key parameters of interest, including particle size and concentration. Measuring these characteristics can be of particular importance in the research laboratory or when producing such particles as biotherapeutics. This article briefly describes the major types of lipid-containing nanoparticles and the techniques that can be used to study them. Such methodologies include electron microscopy, atomic force microscopy, dynamic light scattering, nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing and microfluidic resistive pulse sensing. Whilst no technique is perfect for the analysis of all nanoparticles, this article provides advantages and disadvantages of each, highlighting the latest developments in the field. Finally, we demonstrate the use of microfluidic resistive pulse sensing for the analysis of biological nanoparticles.

Interferometric plasmonic imaging and detection of single exosomes.

Exosomes play an important role in numerous cellular processes. Fundamental study and practical use of exosomes are significantly constrained by the lack of analytical tools capable of physical and biochemical characterization. In this paper, we present an optical approach capable of imaging single exosomes in a label-free manner, using interferometric plasmonic microscopy. We demonstrate monitoring of the real-time adsorption of exosomes onto a chemically modified Au surface, calculating the image intensity, and determining the size distribution. The sizing capability enables us to quantitatively measure the membrane fusion activity between exosomes and liposomes. We also report the recording of the dynamic interaction between exosomes and antibodies at the single-exosome level, and the tracking of hit-stay-run behavior of exosomes on an antibody-coated surface. We anticipate that the proposed method will contribute to clinical exosome analysis and to the exploration of fundamental issues such as the exosome-antibody binding kinetics.

Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays.

We have designed and fabricated a microwell array chip (MWAC) to trap and detect the entire content of individual vesicles after disruption of the vesicular membrane by an applied electrical potential. To understand the mechanism of vesicle impact electrochemical cytometry (VIEC) in microwells, we simulated the rupture of the vesicles and subsequent diffusion of entrapped analytes. Two possibilities were tested: (i) the vesicle opens toward the electrode, and (ii) the vesicle opens away from the electrode. These two possibilities were simulated in the different microwells with varied depth and width. Experimental VIEC measurements of the number of molecules for each vesicle in the MWAC were compared to VIEC on a gold microdisk electrode as a control, and the quantified catecholamines between these two techniques was the same. We observed a prespike foot in a significant number of events (∼20%) and argue this supports the hypothesis that the vesicles rupture toward the electrode surface with a more complex mechanism including the formation of a stable pore intermediate. This study not only confirms that in standard VIEC experiments the whole content of the vesicle is oxidized and quantified at the surface of the microdisk electrode but actively verifies that the adsorbed vesicle on the surface of the electrode forms a pore in the vicinity of the electrode rather than away from it. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in bioanalysis of the vesicles.

Fast Detection of Single Liposomes Using a Combined Nanopore Microelectrode Sensor.

Here we report the development and characterization of a high throughput sensing device for single liposome detection. The device incorporates a quartz nanopipette positioned near a carbon-fiber microelectrode (CFE). Liposomes (∼200 nm diameter) loaded with Fe(CN)64- are driven out of the nanopipette orifice where they are sensed as a transient decrease in the measured ionic current (resistive-pulse analysis). Simultaneously, a redox signal is collected at the CFE due to the release of internalized redox molecules from translocating liposomes to the CFE surface. Interestingly, we observed that the redox signals arise coincidently with resistive-pulses, suggesting that leakage of liposome contents occurs during translocation. Further investigation suggested that liposome disruption occurs at the nanopore orifice and is not dependent on the nanopore electric field. The probability of this disruption appears to rely on the velocity of fluid flow in the nanopore as well as the nanopore geometry. The high-throughput nature of our technique may prove useful for rapid analysis of liposomal drug formulations or rapid, robust, direct measurement of neurotransmitter concentration in isolated vesicles from neurons and neuroendocrine cells.

Multimodal Positron Emission Tomography Imaging to Quantify Uptake of 89Zr-Labeled Liposomes in the Atherosclerotic Vessel Wall.

Nanotherapy has recently emerged as an experimental treatment option for atherosclerosis. To fulfill its promise, robust noninvasive imaging approaches for subject selection and treatment evaluation are warranted. To that end, we present here a positron emission tomography (PET)-based method for quantification of liposomal nanoparticle uptake in the atherosclerotic vessel wall. We evaluated a modular procedure to label liposomal nanoparticles with the radioisotope zirconium-89 (89Zr). Their biodistribution and vessel wall targeting in a rabbit atherosclerosis model was evaluated up to 15 days after intravenous injection by PET/computed tomography (CT) and PET/magnetic resonance imaging (PET/MRI). Vascular permeability was assessed in vivo using three-dimensional dynamic contrast-enhanced MRI (3D DCE-MRI) and ex vivo using near-infrared fluorescence (NIRF) imaging. The 89Zr-radiolabeled liposomes displayed a biodistribution pattern typical of long-circulating nanoparticles. Importantly, they markedly accumulated in atherosclerotic lesions in the abdominal aorta, as evident on PET/MRI and confirmed by autoradiography, and this uptake moderately correlated with vascular permeability. The method presented herein facilitates the development of nanotherapy for atherosclerotic disease as it provides a tool to screen for nanoparticle targeting in individual subjects' plaques.

Instrument-Dependent Factors Affecting the Precision in the Atomic Force Microscopy Stiffness Measurement of Nanoscale Liposomes.

The mechanical strength (stiffness) of liposomes affects their cellular uptake efficiency and drug release in drug delivery processes. We recently developed a tip shape evaluation method for improving the precision of liposome stiffness measurement by quantitative imaging (QI)-mode atomic force microscopy (AFM). The present study applied our method to the widely-used AFM instruments equipped for intermittent contact (IC)-mode force curve measurements, and examined instrument-dependent factors that affect the liposome stiffness measurements. We demonstrated that the evaluation of the tip shape for cantilever selection can be applicable to the IC mode as well as the QI mode. With the cantilever selection, the improved precision of the liposome stiffness was obtained when the stiffness of each liposome was determined from the slope in the force-deformation curve by the IC-mode force curve measurement. Further, the stiffness values were found to be similar to that measured by QI-mode measurements. These results indicate that our developed method can be widely used via IC-mode force curve measurements as well as via QI mode. It was also revealed that spatial drift of the cantilever position was instrument-dependent factors which could affect the precision of liposome stiffness measurements in the case of IC-mode force curve measurement. Therefore, in case of stiffness measurement by IC-mode force curve measurement, it is vital to obtain force-deformation curves immediately after imaging a liposome for the precise stiffness measurement of liposomes. These findings will promote the usage of the AFM stiffness measurement method for the characterization of lipid nanoparticle-based drug delivery systems.

Identification of Abies sibirica L. Polyprenols and Characterisation of Polyprenol-Containing Liposomes.

The needles of conifer trees are one of the richest sources of natural polyprenols. Polyprenol homologs from Abies sibirica L. lipophilic 80% purified extract were analyzed and quantified. In total, 10 peaks (Prenol-11 to Prenol-20) were observed in the ultra-high-performance liquid chromatography-diode array detector (UHPLC-DAD) chromatogram of Siberian fir with the most abundant compound being Prenol-15 (relative amount 37.23 + 0.56% of the total polyprenol yield). Abies sibirica L. polyprenol solubility and incorporation efficiency into liposomes were studied in various commercially available lecithin mixtures (Phosal IP40, Phosal 75SA, and Lipoid P45). The resulting multilamellar polyprenol liposomes were morphologically characterized by Light and Transmission Electron Microscopy, and the liposome size was discovered to be polymodal with the main peak at 1360 nm (90% of the volume). As polyprenols are fully soluble only in lipids, a liposomal formulation based upon co-solubilization and a modified ethanol injection method of polyprenols into the ethanol-phospholipid system was developed for the entrapment and delivery of polyprenols for potential commercial applications in food supplement and cosmetic industries.

Fast and Purification-Free Characterization of Bio-Nanoparticles in Biological Media by Electrical Asymmetrical Flow Field-Flow Fractionation Hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis Detection.

Accurate physico-chemical characterization of exosomes and liposomes in biological media is challenging due to the inherent complexity of the sample matrix. An appropriate purification step can significantly reduce matrix interferences, and thus facilitate analysis of such demanding samples. Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4) provides online sample purification while simultaneously enabling access to size and Zeta potential of sample constituents in the size range of approx. 1-1000 nm. Hyphenation of EAF4 with Multi-Angle Light Scattering (MALS) and Nanoparticle Tracking Analysis (NTA) detection adds high resolution size and number concentration information turning this setup into a powerful analytical platform for the comprehensive physico-chemical characterization of such challenging samples. We here present EAF4-MALS hyphenated with NTA for the analysis of liposomes and exosomes in complex, biological media. Coupling of the two systems was realized using a flow splitter to deliver the sample at an appropriate flow speed for the NTA measurement. After a proof-of-concept study using polystyrene nanoparticles, the combined setup was successfully applied to analyze liposomes and exosomes spiked into cell culture medium and rabbit serum, respectively. Obtained results highlight the benefits of the EAF4-MALS-NTA platform to study the behavior of these promising drug delivery vesicles under in vivo like conditions.

Integrated Nanogap Platform for Sub-Volt Dielectrophoretic Trapping and Real-Time Raman Imaging of Biological Nanoparticles.

A rapid, label-free, and broadly applicable chemical analysis platform for nanovesicles and subcellular components is highly desirable for diagnostic assays. We demonstrate an integrated nanogap plasmonic sensing platform that combines subvolt dielectrophoresis (DEP) trapping, gold nanoparticles (AuNPs), and a lineated illumination scheme for real-time, surface-enhanced Raman spectroscopy (SERS) imaging of biological nanoparticles. Our system is capable of isolating suspended sub-100 nm vesicles and imaging the Raman spectra of their cargo within seconds, 100 times faster than conventional point-scan Raman systems. Bare AuNPs are spiked into solution and simultaneously trapped with the nanovesicles along the gap to boost local optical fields. In addition, our platform offers simultaneous and delay-free spatial and temporal multiplexing functionality. These nanogap devices can be mass-produced via atomic layer lithography and provide a practical platform for high-speed SERS analysis of biological nanoparticles.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

A lyophilized sterically stabilized liposome-containing docetaxel: in vitro and in vivo evaluation.

OBJECTIVES: In this study, an improved lyophilized PEGylated liposomal formulation of docetaxel (DOC) has been developed. METHODS: PEGylated docetaxel liposome (PL-DOC) was prepared by thin-film evaporation method and lyophilization. The effect of various components of the lipids and their compatibility with DOC on the entrapment efficiency (EE) of liposome was investigated. The lyophilized PL-DOC was characterized by morphology, particle size, zeta potential, EE, release in vitro and stability. Pharmacokinetics and biodistribution in vivo of lyophilized PL-DOC were also investigated. RESULTS: The optimal liposome formulation was egg phosphatidylcholine (EPC):cholesterol (CH):DSPE-PEG2000:DOC = 56:40:4:4 (molar ratio). Sucrose and mannitol were chosen as cryoprotectant in the lyophilization (cryoprotectant-to-lipid (C/L) mass ratio = 8:1). The size of lyophilized PL-DOC was 152.3 ± 1.0 nm with negative charge and the EE was 89.75 ± 1.79%. Compared with nonlyophilized PL-DOC, the lyophilized PL-DOC was more stable at 4 °C for six months. The lyophilized PL-DOC also showed the good stability after reconstituted by 5% glucose injection. In vitro release study of PL-DOC showed that PL-DOC had a sustained release effect. After i.v. administration at the dose of 10 mg/kg in rats, a significant increase in the AUC0-∞, MRT0-∞ and t1/2 was observed in PL-DOC group compared with conventional docetaxel liposome (CL-DOC) and DOC injection (DOC-I) group. Biodistribution studies in mice showed that PL-DOC significantly decreased the uptake by the organs of mononuclear phagocytic system (MPS), such as liver and spleen, while prolonging the retention time of DOC in the plasma. CONCLUSION: Our PEGylated liposome formulation reported in this study could potentially produce viable clinical strategies for improved delivery of DOC for the treatment of human cancer.

Integrated Microfluidic System for Size-Based Selection and Trapping of Giant Vesicles.

Vesicles composed of phospholipids (liposomes) have attracted interest as artificial cell models and have been widely studied to explore lipid-lipid and lipid-protein interactions. However, the size dispersity of liposomes prepared by conventional methods was a major problem that inhibited their use in high-throughput analyses based on monodisperse liposomes. In this study, we developed an integrative microfluidic device that enables both the size-based selection and trapping of liposomes. This device consists of hydrodynamic selection and trapping channels in series, which made it possible to successfully produce an array of more than 60 monodisperse liposomes from a polydisperse liposome suspension with a narrow size distribution (the coefficient of variation was less than 12%). We successfully observed a size-dependent response of the liposomes to sequential osmotic stimuli, which had not clarified so far, by using this device. Our device will be a powerful tool to facilitate the statistical analysis of liposome dynamics.

Capillary electrophoresis method to determine siRNA complexation with cationic liposomes.

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation in gel electrophoresis and RiboGreen® fluorescence-based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this study is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this study, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short-end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared with the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated with the CE approach since no carcinogenic product is used. Finally, this methodology can also be extended for the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles.

Nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) of liposomes: applicability of the technique for nano vesicle batch control.

Liposomes are biodegradable nanoparticle vesicles consisting of a lipid bilayer encapsulating an aqueous core. Entrapped cargo material is shielded from the extra-vesicular medium and sustained release of encapsulated material can be achieved. However, application of liposomes as nano-carriers demands their characterization concerning size and size distribution, particle-number concentration, occurrence of vesicle building blocks in solution and determination of the resulting vesicle encapsulation capacity. These questions can be targeted via gas-phase electrophoretic mobility molecular analysis (GEMMA) based on a nano electrospray (nES) charge-reduction source. This instrument separates single-charged nanoparticles in the gas-phase according to size in a high-laminar sheath-flow by means of an orthogonal, tunable electric field. nES GEMMA analysis enables to confirm liposome integrity after passage through the instrument (in combination with atomic force microscopy) as well as to exclude vesicle aggregation. Additionally, nanoparticle diameters at peak apexes and size distribution data are obtained. Differences of hydrodynamic and dry particle diameter values, as well as the effect of number- and mass-based concentration data analysis on obtained liposome diameters are shown. Furthermore, the repeatability of liposome preparation is studied, especially upon incorporation of PEGylated lipids in the bilayer. Finally, the instruments applicability to monitor mechanical stress applied to vesicles is demonstrated.

Synthesis and validation of novel cholesterol-based fluorescent lipids designed to observe the cellular trafficking of cationic liposomes.

Cholesterol-based fluorescent lipids with ether linker were synthesized using NBD (Chol-E-NBD) or Rhodamine B (Chol-E-Rh), and the usefulnesses as fluorescent probes for tracing cholesterol-based liposomes were validated. The fluorescent intensities of liposomes containing these modified lipids were measured and observed under a microscope. Neither compound interfered with the expression of GFP plasmid, and live cell images were obtained without interferences. Changes in the fluorescent intensity of liposomes containing Chol-E-NBD were followed by flow cytometry for up to 24h. These fluorescent lipids could be useful probes for trafficking of cationic liposome-mediated gene delivery.

NOrmalized MAgnetization Ratio (NOMAR) filtering for creation of tissue selective contrast maps.

An MRI segmentation technique based on collecting two additional saturation transfer images is proposed as an aid for improved detection of chemical exchange saturation transfer agents. In this approach, the additional images are acquired at saturation frequencies of -12.5 and -50 ppm. Use of the ratio of these images allows differentiation of voxels with low magnetization transfer contrast (such as fat, cerebrospinal fluid, edema, or blood) from target tissue voxels using a global threshold determined by histogram analysis. We demonstrate that this technique can reduce artifacts, in vitro, in a phantom containing tubes with chemical exchange saturation transfer contrast agent embedded in either crosslinked bovine serum albumin or buffer, and in vivo for detecting diamagnetic CEST (DIACEST) liposomes injected into mice.

Luminescence stability improvement in liposome-based homogeneous luminescence resonance energy transfer.

A stable liposome-based time-resolved luminescence resonance energy transfer (TR-LRET) assay was developed based on the interaction of biotinylated lipids and streptavidin. Eu(3+) ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide was incorporated into liposomes. Acceptor-labeled streptavidin bound to biotinylated lipids of the liposomes enables TR-LRET. A stable assay performance was achieved by optimization. High Eu(3+) signal and stability, low variation, and sensitivity below 100 pM for free biotin was achieved by incorporating the chelate into liposomes containing cholesterol in a carbonate buffer. Potentially, the stable assay compared with the assay without cholesterol offers an improved platform to liposome-based detection systems.