Christian Gollwitzer

Towards traceable size determination of extracellular vesicles

Background Extracellular vesicles (EVs) have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. Methods In this manuscript, the size distribution of an erythrocyte-derived EV sample is determined using state-of-the-art techniques such as nanoparticle tracking analysis, resistive pulse sensing, and electron microscopy, and novel techniques in the field, such as small-angle X-ray scattering (SAXS) and size exclusion chromatography coupled with dynamic light scattering detection. Results The mode values of the size distributions of the studied erythrocyte EVs reported by the different methods show only small deviations around 130 nm, but there are differences in the widths of the size distributions. Conclusion SAXS is a promising technique with respect to traceability, as this technique was already applied for traceable size determination of solid nanoparticles in suspension. To reach the traceable measurement of EVs, monodisperse and highly concentrated samples are required.

Characterization of an in-vacuum PILATUS 1M detector.

A dedicated in-vacuum X-ray detector based on the hybrid pixel PILATUS 1M detector has been installed at the four-crystal monochromator beamline of the PTB at the electron storage ring BESSY II in Berlin, Germany. Owing to its windowless operation, the detector can be used in the entire photon energy range of the beamline from 10 keV down to 1.75 keV for small-angle X-ray scattering (SAXS) experiments and anomalous SAXS at absorption edges of light elements. The radiometric and geometric properties of the detector such as quantum efficiency, pixel pitch and module alignment have been determined with low uncertainties. The first grazing-incidence SAXS results demonstrate the superior resolution in momentum transfer achievable at low photon energies.

Reference materials and representative test materials to develop nanoparticle characterization methods: the NanoChOp project case

This paper describes the production and characteristics of the nanoparticle test materials prepared for common use in the collaborative research project NanoChOp (Chemical and optical characterization of nanomaterials in biological systems), in casu suspensions of silica nanoparticles and CdSe/CdS/ZnS quantum dots (QDs). This paper is the first to illustrate how to assess whether nanoparticle test materials meet the requirements of a “reference material” (ISO Guide 30, 2015) or rather those of the recently defined category of “representative test material (RTM)” (ISO/TS 16195, 2013). The NanoChOp test materials were investigated with small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and centrifugal liquid sedimentation (CLS) to establish whether they complied with the required monomodal particle size distribution. The presence of impurities, aggregates, agglomerates, and viable microorganisms in the suspensions was investigated with DLS, CLS, optical and electron microscopy and via plating on nutrient agar. Suitability of surface functionalization was investigated with attenuated total reflection Fourier transform infrared spectrometry (ATR-FTIR) and via the capacity of the nanoparticles to be fluorescently labeled or to bind antibodies. Between-unit homogeneity and stability were investigated in terms of particle size and zeta potential. This paper shows that only based on the outcome of a detailed characterization process one can raise the status of a test material to RTM or reference material, and how this status depends on its intended use.

Total synthesis of isotopically enriched Si-29 silica NPs as potential spikes for isotope dilution quantification of natural silica NPs.

A new method was developed for the preparation of highly monodisperse isotopically enriched Si-29 silica nanoparticles ((29)Si-silica NPs) with the purpose of using them as spikes for isotope dilution mass spectrometry (IDMS) quantification of silica NPs with natural isotopic distribution. Si-29 tetraethyl orthosilicate ((29)Si-TEOS), the silica precursor was prepared in two steps starting from elementary silicon-29 pellets. In the first step Si-29 silicon tetrachloride ((29)SiCl4) was prepared by heating elementary silicon-29 in chlorine gas stream. By using a multistep cooling system and the dilution of the volatile and moisture-sensitive (29)SiCl4 in carbon tetrachloride as inert medium we managed to reduce product loss caused by evaporation. (29)Si-TEOS was obtained by treating (29)SiCl4 with absolute ethanol. Structural characterisation of (29)Si-TEOS was performed by using (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy and Fourier-transform infrared (FTIR) spectroscopy. For the NP preparation, a basic amino acid catalysis route was used and the resulting NPs were analysed using transmission electron microscopy (TEM), small angle X-ray scattering (SAXS), dynamic light scattering (DLS) and zeta potential measurements. Finally, the feasibility of using enriched NPs for on-line field-flow fractionation coupled with multi-angle light scattering and inductively coupled plasma mass spectrometry (FFF/MALS/ICP-MS) has been demonstrated.

A comparison of techniques for size measurement of nanoparticles in cell culture medium

Plain and aminated silica nanoparticles dispersed in purified water, in 50 mM Tris–HCl buffer and in cell culture medium were measured using dynamic light scattering (DLS), centrifugal liquid sedimentation (CLS), small-angle X-ray scattering (SAXS), and particle tracking analysis (PTA). The test samples were measured by all methods immediately after dispersion and after incubation at room temperature for 24 h. The effect of the biological dispersion medium on the modal value of the particle size distribution was compared for each method taking into account the estimated uncertainty. For the methods based on light scattering, DLS and PTA, the size distributions obtained were significantly altered due to the formation of a protein corona and induced agglomeration effects. With SAXS and CLS, the measured size of the primary particles was mostly unchanged. While SAXS offers excellent precision and traceability to the SI unit system if the model fitting approach is used for data analysis, CLS provides detailed size distributions from which additional information on the agglomeration state can be deduced.

Size Determination of a Liposomal Drug by Small-Angle X-ray Scattering Using Continuous Contrast Variation.

The continuously growing complexity of nanodrugs urges for complementary characterization techniques which can elude the current limitations. In this paper, the applicability of continuous contrast variation in small-angle X-ray scattering (SAXS) for the accurate size determination of a complex nanocarrier is demonstrated on the example of PEGylated liposomal doxorubicin (Caelyx). The mean size and average electron density of Caelyx was determined by SAXS using a gradient of aqueous iodixanol (Optiprep), an iso-osmolar suspending medium. The study is focused on the isoscattering point position and the analysis of the Guinier region of the scattering curves recorded at different solvent densities. An average diameter of (69 ± 5) nm and electron density of (346.2 ± 1.2) nm(-3) were determined for the liposomal formulation of doxorubicin. The response of the liposomal nanocarrier to increasing solvent osmolality and the structure of the liposome-encapsulated doxorubicin after the osmotic shrinkage of the liposome are evaluated with sucrose contrast variation in SAXS and wide-angle X-ray scattering (WAXS). In the case of using sucrose as contrast agent, a clear osmolality threshold at 670 mOsm kg(-1) was observed, above which the liposomal drug carriers start to shrink, though preserving the intraliposomal doxorubicin structure. The average size obtained by this technique is smaller than the value measured by dynamic light scattering (DLS), though this difference is expected due to the hydrodynamic size of the PEG moieties attached to the liposomal surface, which are not probed with solvent contrast variation in SAXS. The advantages and drawbacks of the proposed technique are discussed in comparison to DLS, the most frequently used sizing method in nanomedicine.

Nanoparticle characterization by continuous contrast variation in small-angle X-ray scattering with a solvent density gradient

Many low-density nanoparticles show a radial inner structure. This work proposes a novel approach to contrast variation with small-angle X-ray scattering based on the constitution of a solvent density gradient in a glass capillary in order to resolve this internal morphology. Scattering curves of a polymeric core–shell colloid were recorded at different suspending medium contrasts at the four-crystal monochromator beamline of PTB at the synchrotron radiation facility BESSY II. The mean size and size distribution of the particles as well as an insight into the colloid electron density composition were determined using the position of the isoscattering points in the Fourier region of the scattering curves and by examining the Guinier region in detail. These results were corroborated with a model fit to the experimental data, which provided complementary information about the inner electron density distribution of the suspended nanoparticles.

Effect of fluorescent staining on size measurements of polymeric nanoparticles using DLS and SAXS

The influence of fluorescence on nanoparticle size measurements using dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) was investigated. For this purpose, two series of 100 nm-sized polymer nanoparticles stained with different concentrations of the fluorescent dyes DY555 and DY680 were prepared, absorbing/emitting at around 560 nm/590 nm and 695 nm/715 nm, respectively. SAXS measurements of these particle series and a corresponding blank control (without dye) revealed similar sizes of all particles within an uncertainty of 1 nm. DLS measurements carried out at three different laboratories using four different DLS instruments and two different laser wavelengths, i.e., 532 nm and 633 nm, revealed also no significant changes in size (intensity-weighted harmonic mean diameter, Z-Average) and size distribution (polydispersity index, PI) within and between the two dye-stained particle series and the blank sample. Nevertheless, a significant decrease of the detected correlation coefficients was observed with increasing dye concentration, due to the increased absorption of the incident light and thus, less coherent light scattering. This effect was wavelength dependent, i.e. only measurable for the dye-stained particles that absorb at the laser wavelength used for the DLS measurements.

Hollow organosilica beads as reference particles for optical detection of extracellular vesicles

Essentials Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement. Hollow organosilica beads were prepared, characterized, and tested as reference particles. Light scattering properties of hollow beads resemble that of platelet‐derived EVs. Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs.

A diffraction effect in X-ray area detectors

When an X-ray area detector based on a single-crystalline material, for instance a state-of-the-art hybrid pixel detector, is illuminated from a point source by monochromatic radiation, a pattern of lines appears which overlays the detected image. These lines are easily seen in scattering experiments with smooth patterns, such as small-angle X-ray scattering. The origin of this effect is Bragg reflection within the sensor layer of the detector. Experimental images are presented over a photon energy range from 3.4 to 10 keV, together with a theoretical analysis. The intensity of the detected signal is decreased by up to 20% on this pattern, which can affect the evaluation of scattering and diffraction experiments. The patterns can be exploited to check the alignment of the detector surface with the direct beam, and the alignment of individual detector modules with each other in the case of modular detectors, as well as for energy calibration of the radiation.