Abstract: At-line static light scattering and fluorescence monitoring allows direct in-process tracking of fluorescent virus-like particles. We have demonstrated this by coupling at-line multi-angle light scattering and fluorescence detectors to the downstream processing of enveloped virus-like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus-like particles containing the Human Immunodeficiency Virus-1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single-column anion-exchange chromatography method was used for direct capture and purification. The majority of host-cell protein impurities passed through the column without binding. Virus-like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi-angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus-like particles were concentrated 17-fold to final a concentration of 4.45 × 1010 particles/mL. Simple buffers and operation make this process suitable for large scale purposes.

Abstract: The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry.

Abstract: The detailed characterization of biological nanoparticles is of paramount importance for various industrial sectors, as for production of viral therapeutics. More recently, technologies that allow real-time quantification with simultaneous sizing and determination of surface potentials of virus particles in solution have been developed. In this study, nanoparticle tracking analysis (NTA) was applied to determine the size and the zeta potential of human adenovirus type 5 (AdV5), one the most frequently used therapeutic/oncolytic agents and viral vectors. Virus aggregation was detected, and the kinetics of the dissolution of virus aggregates were studied in real time. In addition, advanced fluorescence detection of AdV5 was performed enabling the measurements in matrices and discrimination of viral subpopulations. It was shown that NTA is an efficient approach for investigating infectious viruses in a live viewing mode. Consequently, NTA provides a promising methodology for virus particle detection and analysis in real time beyond assays requiring nucleic acids or infectivity.

Abstract: Additional file 3: Figure S2. EVs characterization. (A) Box-plot representing the average mode of EVs isolated from the peritoneal lavage and ascitic fluid of CRC and control patients, respectively (Mean ± SD); measured by Nanoparticle Tracking Analysis. (B) Size distribution and concentration of isolated EVs of a peritoneal lavage of a CRC patient (left) and a ascitic fluid of a control patient (right), measured by Nanoparticle Tracking Analysis.

Abstract: Figure S4. FBS enhances exosome production. (a) Fold changes in number of particles per mL media and per final cell count in the P100 fractions when HEK293 and THP-1 cells were cultured in 10% FBS compared to SF conditions for 48 h. (b,c) These bar graphs show the number of exosomes (b) per mL media and (c) per final cell count when P100 exosomes were isolated from HEK293 CM grown in 10% FBS (blue) and in SF (red) for 48 h. (d) The number of particles per mL media were measured before (shaded) and 48 h after (solid) supplied to HEK293 cells. Exosome samples were isolated by ultracentrifugation (P100). Media were prepared in three types: SF (gray), 10% ED-FBS (exosome depleted FBS) (blue), and 10% FBS (red). (e,f) Time-series analysis of extracellular exosomes of (e) THP-1 cells and (f) HEK293 cells in either SF media (blue), SF media supplemented with the liposomes (orange), and P100 extracted from FBS (red). (g) Histograms showing the size distribution and concentrations of liposomes. (h) The graph shows the number of particles per mL in each ultracentrifugal sub-fraction from 10% FBS media. The exosomes were isolated by Exo-quick-TC. Experiments were repeated three times, each including six technical repeats. The results are displayed as mean ± S.E.M. *Significant difference analyzed by t-test (**p

Abstract: The design of a simple platform to target the delivery of notably hydrophobic drugs into cancer cells is an ultimate goal. Here, three strategies were combined in the same nanovector, in limiting the use of excipients: cell-penetrating peptides, an amphiphilic prodrug, and self-assembly. Light scattering and cryogenic transmission electron microscopy revealed one size population of objects around 100 nm with a narrow size distribution. However, in-depth analysis of the suspension by nanoparticle tracking analysis, small-angle X-ray scattering, and nuclear magnetic resonance (NMR) diffusometry demonstrated the presence of another population of small objects (<2 nm). It has been shown that these small self-assemblies represented >99% of the matter! This presence was clearly and unambiguously demonstrated by NMR diffusometry experiments. The study highlights the importance and the complementary contribution of each characterization method to reflect the reality of the studied nanoassembly.

Abstract: Figure S4. FBS enhances exosome production. (a) Fold changes in number of particles per mL media and per final cell count in the P100 fractions when HEK293 and THP-1 cells were cultured in 10% FBS compared to SF conditions for 48 h. (b,c) These bar graphs show the number of exosomes (b) per mL media and (c) per final cell count when P100 exosomes were isolated from HEK293 CM grown in 10% FBS (blue) and in SF (red) for 48 h. (d) The number of particles per mL media were measured before (shaded) and 48 h after (solid) supplied to HEK293 cells. Exosome samples were isolated by ultracentrifugation (P100). Media were prepared in three types: SF (gray), 10% ED-FBS (exosome depleted FBS) (blue), and 10% FBS (red). (e,f) Time-series analysis of extracellular exosomes of (e) THP-1 cells and (f) HEK293 cells in either SF media (blue), SF media supplemented with the liposomes (orange), and P100 extracted from FBS (red). (g) Histograms showing the size distribution and concentrations of liposomes. (h) The graph shows the number of particles per mL in each ultracentrifugal sub-fraction from 10% FBS media. The exosomes were isolated by Exo-quick-TC. Experiments were repeated three times, each including six technical repeats. The results are displayed as mean ± S.E.M. *Significant difference analyzed by t-test (**p

Abstract: Additional file 3: Figure S2. EVs characterization. (A) Box-plot representing the average mode of EVs isolated from the peritoneal lavage and ascitic fluid of CRC and control patients, respectively (Mean ± SD); measured by Nanoparticle Tracking Analysis. (B) Size distribution and concentration of isolated EVs of a peritoneal lavage of a CRC patient (left) and a ascitic fluid of a control patient (right), measured by Nanoparticle Tracking Analysis.

Abstract: Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration.

Abstract: In recent years, nanoparticle tracking analysis (NTA) has emerged as an alternative tool for particle size characterization. Especially when examining polydisperse systems, individual particle to particle tracking allows for higher peak resolution than dynamic light scattering techniques. However, NTA requires an experienced user with a good insight into how the different settings can affect the determination of particle size and size distributions. This chapter provides a guideline for protein aggregation studies using the example of temperature-induced aggregation of IgG at low concentration.