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Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

Exosomes are cell-secreted nanovesicles present in biological fluids in normal and diseased conditions. Owing to their seminal role in cell-cell communication, emerging evidences suggest that exosomes are fundamental regulators of various diseases. Due to their potential usefulness in disease diagnosis, robust isolation and characterization of exosomes is critical in developing exosome-based assays. In the last few years, different exosome characterization methods, both biophysical and molecular, have been developed to characterize these tiny vesicles. Here, in this review we summarize: first, biophysical techniques based on spectroscopy (e.g., Raman spectroscopy, dynamic light scattering) and other principles, for example, scanning electron microscopy, atomic force microscopy; second, antibody-based molecular techniques including flow cytometry, transmission electron microscopy and third, nanotechnology-dependent exosome characterization methodologies.

PurposeCharacterizing submicron protein particles (approximately 0.1–1μm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS).MethodsResistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment.ResultsA method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4 × 107-1 × 1011 particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering.ConclusionRPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

Scattering techniques represent non-invasive experimental approaches and powerful tools for the investigation of structure and conformation of biomaterial systems in a wide range of distances, ranging from the nanometric to micrometric scale. More specifically, small-angle X-rays and neutron scattering and light scattering techniques represent well-established experimental techniques for the investigation of the structural properties of biomaterials and, through the use of suitable models, they allow to study and mimic various biological systems under physiologically relevant conditions. They provide the ensemble averaged (and then statistically relevant) information under in situ and operando conditions, and represent useful tools complementary to the various traditional imaging techniques that, on the contrary, reveal more local structural information. Together with the classical structure characterization approaches, we introduce the basic concepts that make it possible to examine inter-particles interactions, and to study the growth processes and conformational changes in nanostructures, which have become increasingly relevant for an accurate understanding and prediction of various mechanisms in the fields of biotechnology and nanotechnology. The upgrade of the various scattering techniques, such as the contrast variation or time resolved experiments, offers unique opportunities to study the nano- and mesoscopic structure and their evolution with time in a way not accessible by other techniques. For this reason, highly performant instruments are installed at most of the facility research centers worldwide. These new insights allow to largely ameliorate the control of (chemico-physical and biologic) processes of complex (bio-)materials at the molecular length scales, and open a full potential for the development and engineering of a variety of nano-scale biomaterials for advanced applications.

Focusing light deep inside living tissue has not been achieved despite its promise to play a central role in biomedical imaging, optical manipulation and therapy. To address this challenge, internal-guide-star-based wavefront engineering techniques—for example, time-reversed ultrasonically encoded (TRUE) optical focusing—were developed. The speeds of these techniques, however, were limited to no greater than 1 Hz, preventing them from in vivo applications. Here we improve the speed of optical focusing deep inside scattering media by two orders of magnitude, and focus diffuse light inside a dynamic scattering medium having a speckle correlation time as short as 5.6 ms, typical of living tissue. By imaging a target, we demonstrate the first focusing of diffuse light inside a dynamic scattering medium containing living tissue. Since the achieved focusing speed approaches the tissue decorrelation rate, this work is an important step towards in vivo deep tissue noninvasive optical imaging, optogenetics and photodynamic therapy. Shaping the incident wavefront allows optical focusing deep inside scattering media, but its application in dynamic media is hindered by its low speed. Here, Liu et al . improve the speed by two orders of magnitude and demonstrate in vivo light focusing inside dynamic scattering media.

We present the use of Multi-angle Dynamic Light Scattering (MADLS®) for the measurement of nanoparticle concentration. We describe the theory of the method and its application to nanoparticles made of gold, silica and polystyrene, with diameters ranging from 30 to 400 nm, and demonstrate some of the limitations with particles of sizes 500 nm and above. We evaluate the method accuracy, linearity and reproducibility, as well as the operational nanoparticle concentration and size range. We show that the concentration working range depends on the material’s optical properties, size and concentration. Here it is shown that the level of accuracy that can be expected for the concentration of particles is typically within 50% of the nominal value across a range of materials and sizes and, for some samples, within 20%. The repeatability of the measurements, in terms of relative standard deviation, is typically below 30%. A linearity of within 40% across a concentration range of 3·108 to 2·1011 mL−1 for concentration measurements was also demonstrated by using gold nanoparticles and gravimetric dilutions for method validation. Overall, we show that MADLS® is a rapid and straightforward method for the reproducible measurement of particle concentration, as well as size, requiring minimal sample preparation, without the need to calibrate using a pre-determined concentration series, and applicable to a broad range of materials. These features make it an ideal tool to support both development and quality control of particle materials for a broad range of applications.

A multi-angle light scattering (MALS) system, combined with chromatographic separation, directly measures the absolute molar mass, size and concentration of the eluate species. The measurement of these crucial properties in solution is essential in basic macromolecular characterization and all research and production stages of bio-therapeutic products. We developed a new MALS methodology that has overcome the long-standing, stubborn barrier to microliter-scale peak volumes and achieved the highest resolution and signal-to-noise performance of any MALS measurement. The novel design simultaneously facilitates online dynamic light scattering (DLS) measurements. As National Institute of Standards and Technology (NIST) new protein standard reference material (SRM 8671) is becoming the benchmark molecule against which many biomolecular analytical techniques are assessed and evaluated, we present its measurement results as a demonstration of the unique capability of our system to swiftly resolve and measure sharp (20~25 µL full-width-half-maximum) chromatography peaks. Precise measurements of protein mass and size can be accomplished 10 times faster than before with improved resolution. In the meantime the sample amount required for such measurements is reduced commensurately. These abilities will have far-reaching impacts at every stage of the development and production of biologics and bio-therapeutic formulations.

•Scattered Light Imaging (SLI) reveals individual nerve fiber directions in the brain.•The simple prototypic setup contains only a standard LED light source and a camera.•SLI reconstructs multiple crossing nerve fiber directions within each image pixel.•We measured various brain sections (rodent/monkey/human) with micrometer resolution.•We validated our results against simulated/measured scattering patterns and 3D-PLI. [Display omitted] For developing a detailed network model of the brain based on image reconstructions, it is necessary to spatially resolve crossing nerve fibers. The accuracy hereby depends on many factors, including the spatial resolution of the imaging technique. 3D Polarized Light Imaging (3D-PLI) allows the three-dimensional reconstruction of nerve fiber tracts in whole brain sections with micrometer in-plane resolution, but leaves uncertainties in pixels containing crossing fibers. Here we introduce Scattered Light Imaging (SLI) to resolve the substructure of nerve fiber crossings. The measurement is performed on the same unstained histological brain sections as in 3D-PLI. By illuminating the brain sections from different angles and measuring the transmitted (scattered) light under normal incidence, light intensity profiles are obtained that are characteristic for the underlying brain tissue structure. We have developed a fully automated evaluation of the intensity profiles, allowing the user to extract various characteristics, like the individual directions of in-plane crossing nerve fibers, for each image pixel at once. We validate the reconstructed nerve fiber directions against results from previous simulation studies, scatterometry measurements, and fiber directions obtained from 3D-PLI. We demonstrate in different brain samples (human optic tracts, vervet monkey brain, rat brain) that the 2D fiber directions can be reliably reconstructed for up to three crossing nerve fiber bundles in each image pixel with an in-plane resolution of up to 6.5 μm. We show that SLI also yields reliable fiber directions in brain regions with low 3D-PLI signals coming from regions with a low density of myelinated nerve fibers or out-of-plane fibers. This makes Scattered Light Imaging a promising new imaging technique, providing crucial information about the organization of crossing nerve fibers in the brain.

Comparative analytical assessment (CAA) between the reference product and the proposed product forms the basis of the biosimilarity demonstration. Though Dynamic Light Scattering (DLS) has been implemented for CAA, its capability beyond signature peak for similarity assessments has remained unexplored. Herein, we have developed a innovative forced degradation based sweet spot method consisting of signature peak, temperature range, increment and hold time using high throughput-DLS (HT-DLS) to show similarity in hydrodynamic size between products. In our study, we used rituximab, its biosimilars, and insulin analogs as model products to demonstrate product similarity in hydrodynamic size (D h ) size through the HT-DLS sweet spot approach. Our data indicate that temperature range, temperature increment, hold time, and regularization algorithm, all play a role in showing analytical similarity in D h size. Our data also indicate that establishing DLS signature peaks of the products is insufficient to show analytical similarity in D h size distribution. Additionally, the temperature range (sweet spot) varies from product to product. Principal component analysis modeling was used for detailed data interpretation. Overall, our HT-DLS based sweet spot method provided informative data to support similarity in D h size distribution.

Prostate-specific antigen (PSA) is a key biomarker for the early detection of prostate cancer recurrence following surgical treatment. In this study, we present a PSA-responsive, aptamer-based switchable aggregate system, named AS2-US-AuNP-Aggregate, composed of ultrasmall gold nanoparticles (US-AuNPs) linked by (partially) pairing oligomers that selectively disassemble in the presence of PSA. The system was optimised also using a previously developed in silico routine and is designed for enhanced detection capabilities and for supporting in vivo applicability. We measured the sizes of the nanosystems by dynamic light scattering (DLS) and their extinction spectra, also in the presence of PSA in simple buffers, in the presence of DNaseI, and under blood-mimicking conditions (filtered plasma), obtaining a response down to 10 fM PSA in buffers and to 1 pM in filtered plasma. Our findings highlight the potential of aptamer-based nanoparticle aggregates as a basis for user-friendly diagnostic tools. Additionally, we discuss key optimisation strategies to further advance their development for in vivo diagnostic applications.