Deagglomeration of DNA nanomedicine carriers using controlled ultrasonication.

Control over the agglomeration state of manufactured particle systems for drug and oligonucleotide intracellular delivery is paramount to ensure reproducible and scalable therapeutic efficacy. Ultrasonication is a well-established mechanism for the deagglomeration of bulk powders in dispersion. Its use in manufacturing requires strict control of the uniformity and reproducibility of the cavitation field within the sample volume to minimise within-batch and batch-to-batch variability. In this work, we demonstrate the use of a reference cavitating vessel which provides stable and reproducible cavitation fields over litre-scale volumes to assist the controlled deagglomeration of a novel non-viral particle-based plasmid delivery system. The system is the Nuvec delivery platform, comprising polyethylenimine-coated spiky silica particles with diameters of âˆ¼ 200 nm. We evaluated the use of controlled cavitation at different input powers and stages of preparation, for example before and after plasmid loading. Plasmid loading was confirmed by X-ray photoelectron spectroscopy and gel electrophoresis. The latter was also used to assess plasmid integrity and the ability of the particles to protect plasmid from potential degradation caused by the deagglomeration process. We show the utility of laser diffraction and differential centrifugal sedimentation in quantifying the efficacy of product de-agglomeration in the microscale and nanoscale size range respectively. Transmission electron microscopy was used to assess potential damages to the silica particle structure due to the sonication process.

Composition, thickness, and homogeneity of the coating of core-shell nanoparticles-possibilities, limits, and challenges of X-ray photoelectron spectroscopy.

Core-shell nanoparticles have attracted much attention in recent years due to their unique properties and their increasing importance in many technological and consumer products. However, the chemistry of nanoparticles is still rarely investigated in comparison to their size and morphology. In this review, the possibilities, limits, and challenges of X-ray photoelectron spectroscopy (XPS) for obtaining more insights into the composition, thickness, and homogeneity of nanoparticle coatings are discussed with four examples: CdSe/CdS quantum dots with a thick coating and a small core; NaYF4-based upconverting nanoparticles with a large Yb-doped core and a thin Er-doped coating; and two types of polymer nanoparticles with a poly(tetrafluoroethylene) core with either a poly(methyl methacrylate) or polystyrene coating. Different approaches for calculating the thickness of the coating are presented, like a simple numerical modelling or a more complex simulation of the photoelectron peaks. Additionally, modelling of the XPS background for the investigation of coating is discussed. Furthermore, the new possibilities to measure with varying excitation energies or with hard-energy X-ray sources (hard-energy X-ray photoelectron spectroscopy) are described. A discussion about the sources of uncertainty for the determination of the thickness of the coating completes this review. Graphical abstract.

Exposure of mass-selected bimetallic Pt-Ti nanoalloys to oxygen explored using scanning transmission electron microscopy and density functional theory.

The response of nanoparticles to exposure to ambient conditions and especially oxidation is fundamental to the application of nanotechnology. Bimetallic platinum-titanium nanoparticles of selected mass, 30 kDa and 90 kDa, were produced using a magnetron sputtering gas condensation cluster source and deposited onto amorphous carbon TEM grids. The nanoparticles were analysed with a Cs-corrected Scanning Transmission Electron Microscope (STEM) in High Angle Annular Dark Field (HAADF) mode. It was observed that prior to full Ti oxidation, Pt atoms were dispersed within a Ti shell. However, after full oxidation by prolonged exposure to ambient conditions prior to STEM, the smaller size 30 kDa particles form a single Pt core and the larger size 90 kDa particles exhibit a multi-core structure. Electron beam annealing induced a single core morphology in the larger particles. First principles density functional theory (DFT) calculations were employed to calculate the lowest energy structure of the Pt-Ti nanoparticles with and without the presence of oxygen. It was demonstrated that, as the concentration of oxygen increases, the lowest energy structure changes from dispersed Pt to multiple Pt cores and finally a single Pt core, which is in good agreement with the experimental observations.

Analysis of protein coatings on gold nanoparticles by XPS and liquid-based particle sizing techniques.

The precise use of nanoparticles in technological applications requires control over their surface properties. This implies the ability to quantitatively describe, for example, molecular coatings in terms of their thickness, areal mass, or number of molecules. Here, the authors describe two different approaches to the measurement of these parameters by using gold nanoparticles ranging in diameter from 10 to 80 nm and coated with three different proteins: immunoglobulin G, bovine serum albumin, and a peptide. One approach utilizes ultraviolet-visible spectroscopy, dynamic light scattering, and differential centrifugal sedimentation to measure the protein shell refractive indices and thicknesses, from which the number of molecules in the protein shell can be derived. The other approach employs x-ray photoelectron spectroscopy to measure the thickness of the dry molecular coatings and also to derive the number of molecules in the protein shell. The authors demonstrate that the two approaches, although very different, produce consistent measurement results. This finding is important to extend the quantitative analysis of nanoparticle molecular coatings to a wide range of materials.

Quantification of variable functional-group densities of mixed-silane monolayers on surfaces via a dual-mode fluorescence and XPS label.

The preparation of aminated monolayers with a controlled density of functional groups on silica surfaces through a simple vapor deposition process employing different ratios of two suitable monoalkoxysilanes, (3-aminopropyl)diisopropylethoxysilane (APDIPES) and (3-cyanopropyl)dimethylmethoxysilane (CPDMMS), and advances in the reliable quantification of such tailored surfaces are presented here. The one-step codeposition process was carried out with binary silane mixtures, rendering possible the control over a wide range of densities in a single step. In particular, APDIPES constitutes the functional silane and CPDMMS the inert component. The procedure requires only small amounts of silanes, several ratios can be produced in a single batch, the deposition can be carried out within a few hours and a dry atmosphere can easily be employed, limiting self-condensation of the silanes. Characterization of the ratio of silanes actually bound to the surface can then be performed in a facile manner through contact angle measurements using the Cassie equation. The reliable estimation of the number of surface functional groups was approached with a dual-mode BODIPY-type fluorescence label, which allows quantification by fluorescence and XPS on one and the same sample. We found that fluorescence and XPS signals correlate over at least 1 order of magnitude, allowing for a direct linking of quantitative fluorescence analysis to XPS quantification. Employment of synchrotron-based methods (XPS; reference-free total reflection X-ray fluorescence, TXRF) made the traceable quantification of surface functional groups possible, providing an absolute reference for quantitative fluorescence measurements through a traceable measurement chain.

Depth resolution, angle dependence, and the sputtering yield of Irganox 1010 by coronene primary ions.

A study is reported of the depth resolution and angle dependence of sputtering yields using the reference organic material, Irganox 1010, for a new coronene(+) depth profiling ion source at 8 and 16 keV beam energies. This source provides excellent depth profiles as shown by 8.5 nm marker layers of Irganox 3114. Damage occurs but may be ignored for angles of incidence above 70° from the surface normal, as shown by X-ray photoelectron spectroscopy (XPS) of the C 1s peak structure. Above 70°, XPS profiles of excellent depth resolution are obtained. The depth resolution, after removal of the thickness of the delta layers, shows a basic contribution of 5.7 nm together with a contribution of 0.043 times the depth sputtered. This is lower than generally reported for cluster sources. The coronene(+) source is thus found to be a useful and practical source for depth profiling organic materials. The angle dependencies of both the undamaged and damaged materials are described by a simple equation. The sputtering yields for the undamaged material are described by a universal equation and are consistent with those obtained for C60(+) sputtering. Comparison with the sputtering yields using an argon gas cluster ion source shows great similarities, but the yields for both the coronene(+) and C60(+) primary ion sources are slightly lower.

Chemical and biological characterisation of a sensor surface for bioprocess monitoring.

This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin™ (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 µg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest.

Simplifying the delivery of melanocytes and keratinocytes for the treatment of vitiligo using a chemically defined carrier dressing.

Obtaining pigmentary function in autologous skin grafts is a current challenge for burn surgeons as is developing reliable robust grafting strategies for patients with vitiligo and piebaldism. In this paper, we present the development of a simple methodology for delivering cultured keratinocytes and melanocytes to the patient that is of low risk for the patient but also user friendly for the surgeon. In this study, we examined the ability of keratinocytes and melanocytes to transfer from potential cell carriers under different media conditions to an in vitro human wound bed model. The number of melanocytes transferred, their location within the neoepidermis, and their ability to pigment were evaluated as preclinical end points. Two inert substrates (polyvinyl chloride and silicone sheets) and three candidate plasma-polymerized coatings with controlled surface chemistry deposited on these substrates were explored. Two media for expansion of cells, Greens, currently used clinically (but which contains fetal calf serum), and a serum-free alternative, M2 (melanocyte medium), were explored. Reproducible transfer of physiologically relevant numbers of melanocytes capable of pigmentation from the coculture of melanocytes and keratinocytes was obtained using either Greens medium or M2 medium, and a silicone carrier pretreated with 20% carboxylic acid deposited by plasma polymerization.

A chemically defined surface for the co-culture of melanocytes and keratinocytes.

Patients with stable vitiligo can be helped surgically using transplantation of autologous cultured melanocytes, but there is a need for a culture methodology that is free from xenobiotic agents and for a simple way of delivering cultured melanocytes to the patient to achieve pigmentation with good wound healing. The aim of this study was to develop a chemically defined surface, suitable for the co-culture of melanocytes and keratinocytes which could be used in the future for the treatment vitiligo patients to achieve both restoration of pigmentation and good wound healing. Two keratinocyte growth media and two melanocyte growth media were compared; two of these were serum free. Cells were seeded on a range of chemically defined substrates (produced by plasma polymerisation of acrylic acid, allylamine or a mixture of these monomers) either as mono- or co-cultures. Melanocytes and keratinocytes attached and proliferated on both acid and amine substrates (without significant preferences), and co-cultures of cells proliferated more successfully than individual cultures. One media, M2, which is serum free, supported expansion of melanocytes and to a lesser extent keratinocytes on several plasma polymer substrates. In conclusion, these data indicate that a combination of a chemically defined substrate with M2 media allows serum-free co-culture of melanocytes and keratinocytes.

The effect of positive ion energy on plasma polymerization: a comparison between acrylic and propionic acids.

Using a novel RF biasing technique, the energy of positive ions at a depositing substrate is controlled, independently of other parameters. Under bias conditions which gave the maximum and minimum ion energies, plasmas of propionic and acrylic acid were investigated using mass spectrometry, an ion flux probe, quartz crystal microbalance, and X-ray photoelectron spectroscopy (XPS). For both compounds investigated, the ion energy affects the deposition rate but leaves the neutral gas-phase chemistry and positive ion fluxes unchanged. The chemistry of the polymer deposit for acrylic acid is unaffected by the change in ion energy, but the chemistry of the propionic acid plasma polymer changes markedly. We argue that the results presented are consistent with the hypothesis that, under the plasma conditions explored, the carbon-carbon double bond present in acrylic acid plays a significant role in the formation of the polymer. Conversely, the absence of this bond in propionic acid leads us to conclude that positive ions contribute significantly to film formation for this compound.