Interlaboratory Measurement of Adeno-Associated Virus: Comparative Quantification of Full and Empty Capsids.

Recombinant adeno-associated virus (AAV) is one of the main viral vector-based gene therapy platforms. AAV is a virus consisting of a ≈25 nm diameter capsid with a ≈4.7 kb cargo capacity.

Particle Metrology Approach to Understanding How Storage Conditions Affect Long-Term Liposome Stability.

Lipid nanoparticles are a generic type of nanomaterial with broad applicability in medicine as drug delivery vehicles. Liposomes are a subtype of lipid nanoparticles and, as a therapeutic platform, can be loaded with a genetic material or pharmaceutical agents for use as drug treatments. An open question for these types of lipid nanoparticles is what factor(s) affect the long-term stability of the particles.

Number Concentration Measurements of Polystyrene Submicrometer Particles.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques.

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database.

Distribution of Average Aggregate Density from Stir-Stressed NISTmAb Protein.

Quantifying the heterogeneous nature of protein aggregates is important to understanding the impact aggregates may have on the performance of antibody therapeutics. The spatially averaged density ρ of aggregates, defined as the total mass, including water, divided by the volume, is a parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of aggregated protein in a sample. We report measurements by two methods on the distribution of density values for different aggregate sizes, where the aggregates were produced by stir-stressing fluorescently labeled monoclonal antibody (NISTmAb).

Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB).

Effect of Azide Preservative on Thermomechanical Aggregation of Purified Reference Protein Materials.

Protein aggregation can affect the quality of protein-based therapeutics. Attempting to unravel factors influencing protein aggregation involves systematic studies. These studies often include sodium azide or similar preservatives in the aggregation buffer.

Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This article provides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic.

Using Image Attributes to Assure Accurate Particle Size and Count Using Nanoparticle Tracking Analysis.

Nanoparticle tracking analysis (NTA) obtains particle size by analysis of particle diffusion through a time series of micrographs and particle count by a count of imaged particles. The number of observed particles imaged is controlled by the scattering cross-section of the particles and by camera settings such as sensitivity and shutter speed. Appropriate camera settings are defined as those that image, track, and analyze a sufficient number of particles for statistical repeatability.

Asymmetric flow field flow fractionation with light scattering detection - an orthogonal sensitivity analysis.

Asymmetric flow field flow fractionation (AF) has several instrumental factors that may have a direct effect on separation performance. A sensitivity analysis was applied to ascertain the relative importance of AF primary instrument factor settings for the separation of a complex environmental sample. The analysis evaluated the impact of instrumental factors namely, cross flow, ramp time, focus flow, injection volume, and run buffer concentration on the multi-angle light scattering measurement of natural organic matter (NOM) molar mass (MM).

Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules.

Intercellular adhesion molecule 1 (ICAM-1) is a cell-surface protein overexpressed in many diseases and explored for endocytosis and transcytosis of drug delivery systems. All previous evidence demonstrating ICAM-1-mediated transport of therapeutics into or across cells was obtained using nanocarriers or conjugates coupled to multiple copies of anti-ICAM antibodies or peptides. Yet, transport of therapeutics linked to non-multivalent anti-ICAM ligands has never been shown, since multivalency was believed to be necessary to induce transport.

Using light scattering to evaluate the separation of polydisperse nanoparticles.

The analysis of natural and otherwise complex samples is challenging and yields uncertainty about the accuracy and precision of measurements. Here we present a practical tool to assess relative accuracy among separation protocols for techniques using light scattering detection. Due to the highly non-linear relationship between particle size and the intensity of scattered light, a few large particles may obfuscate greater numbers of small particles.

A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array.

A novel microscale device has been developed to enable the one-step continuous flow assembly of monodisperse nanoscale liposomes using three-dimensional microfluidic hydrodynamic focusing (3D-MHF) in a concentric capillary array. The 3D-MHF flow technique displays patent advantages over conventional methods for nanoscale liposome manufacture (i.e.

Microfluidic-enabled liposomes elucidate size-dependent transdermal transport.

Microfluidic synthesis of small and nearly-monodisperse liposomes is used to investigate the size-dependent passive transdermal transport of nanoscale lipid vesicles. While large liposomes with diameters above 105 nm are found to be excluded from deeper skin layers past the stratum corneum, the primary barrier to nanoparticle transport, liposomes with mean diameters between 31-41 nm exhibit significantly enhanced penetration. Furthermore, multicolor fluorescence imaging reveals that the smaller liposomes pass rapidly through the stratum corneum without vesicle rupture.

Liposome-based delivery of a boron-containing cholesteryl ester for high-LET particle-induced damage of prostate cancer cells: a boron neutron capture therapy study.

Purpose: The efficacy of a boron-containing cholesteryl ester compound (BCH) as a boron neutron capture therapy (BNCT) agent for the targeted irradiation of PC-3 human prostate cancer cells was examined.

Materials And Methods: Liposome-based delivery of BCH was quantified with inductively coupled plasma-mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC). Cytotoxicity of the BCH-containing liposomes was evaluated with neutral red, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and lactate dehydrogenase assays.

Dietary CdSe/ZnS quantum dot exposure in estuarine fish: bioavailability, oxidative stress responses, reproduction, and maternal transfer.

Continued development, use, and disposal of quantum dots (QDs) ensure their entrance into aquatic environments where they could pose a risk to biological organisms as whole nanoparticles or as degraded metal constituents. Reproductive Fundulus heteroclitus were fed a control diet with lecithin, diets containing 1 or 10 μg of lecithin-encapsulated CdSe/ZnS QD/day, or a diet containing 5.9 μg CdCl2/day for 85 days.

Microfluidic preparation of liposomes to determine particle size influence on cellular uptake mechanisms.

Purpose: This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40.6 nm to 276.6 nm.

Microfluidic synthesis of PEG- and folate-conjugated liposomes for one-step formation of targeted stealth nanocarriers.

Purpose: A microfluidic hydrodynamic flow focusing technique enabling the formation of small and nearly monodisperse liposomes is investigated for continuous-flow synthesis of poly(ethylene glycol) (PEG)-modified and PEG-folate-functionalized liposomes for targeted drug delivery.

Methods: Controlled laminar flow in thermoplastic microfluidic devices facilitated liposome self-assembly from initial lipid compositions including lipid/cholesterol mixtures containing PEG-lipid and folate-PEG-lipid conjugates. Relationships among flow conditions, lipid composition, and liposome size were evaluated; their impact on PEG and folate incorporation were determined through a combination of UV-vis absorbance measurements and characterization of liposome zeta potential.

Microfluidic mixing and the formation of nanoscale lipid vesicles.

We investigate the formation of unilamellar lipid vesicles (liposomes) with diameters of tens of nanometers by controlled microfluidic mixing and nanoparticle determination (COMMAND). Our study includes liposome synthesis experiments and numerical modeling of our microfluidic implementation of the batch solvent injection method. We consider microfluidic liposome formation from the perspective of fluid interfaces and convective-diffusive mixing, as we find that bulk fluid flow parameters including hydrodynamically focused alcohol stream width, final alcohol concentration, and shear stress do not primarily determine the vesicle formation process.

Controlled self-assembly of monodisperse niosomes by microfluidic hydrodynamic focusing.

Niosomes are synthetic membrane vesicles formed by self-assembly of nonionic surfactant, often in a mixture with cholesterol and dicetyl phosphate. Because of their inner aqueous core and bilayer membrane shell, niosomes are commonly used as carriers of treatment agents for pharmaceutical and cosmetic applications or contrast agents for clinical imaging applications. In those applications, niosomes are considered as a more economical and stable alternative to their biological counterpart (i.

Magnetic connectors for microfluidic applications.

We present a new type of microfluidic connector that employs a ring magnet on one side of the microfluidic chip and a disc magnet on the other side to produce a sealed connection between external tubing and inlets or outlets of microfluidic devices. The connectors are low-cost, simple to use and assemble, and reusable. We used numerical (finite element) simulations in order to optimize their geometry.

Accurate optical analysis of single-molecule entrapment in nanoscale vesicles.

We present a nondestructive method to accurately characterize low analyte concentrations (0-10 molecules) in nanometer-scale lipid vesicles. Our approach is based on the application of fluorescence fluctuation analysis (FFA) and multiangle laser light scattering (MALLS) in conjunction with asymmetric field flow fractionation (AFFF) to measure the entrapment efficiency (the ratio of the concentration of encapsulated dye to the initial bulk concentration) of an ensemble of liposomes with an average diameter less than 100 nm. Water-soluble sulforhodamine B (SRB) was loaded into the aqueous interior of nanoscale liposomes synthesized in a microfluidic device.

Liposome-templated supramolecular assembly of responsive alginate nanogels.

Nanosized gel particles (nanogels) are of interest for a variety of applications, including drug delivery and single-molecule encapsulation. Here, we employ the cores of nanoscale liposomes as reaction vessels to template the assembly of calcium alginate nanogels. For our experiments, a liposome formulation with a high bilayer melting temperature (Tm) is selected, and sodium alginate is encapsulated in the liposomal core.

Microfluidic directed formation of liposomes of controlled size.

A new method to tailor liposome size and size distribution in a microfluidic format is presented. Liposomes are spherical structures formed from lipid bilayers that are from tens of nanometers to several micrometers in diameter. Liposome size and size distribution are tailored for a particular application and are inherently important for in vivo applications such as drug delivery and transfection across nuclear membranes in gene therapy.

Scanning temperature gradient focusing.

Temperature gradient focusing (TGF) is a recently developed technique for the simultaneous concentration and electrophoretic separation of ionic analytes in microfluidic channels. One drawback to TGF as it has previously been described is the limited peak capacity; only a small number of analyte peaks (approximately 2-3) can be simultaneously focused and separated. In this paper, we report on a variation of the TGF method whereby the bulk flow rate is varied over time so that a large number of analytes can be sequentially focused, moved past a fixed detection point, and flushed to waste.