Cryo-XPS for Surface Characterization of Nanomedicines.

Nanoparticles used for medical applications commonly possess coatings or surface functionalities intended to provide specific behavior in vivo, for example, the use of PEG to provide stealth properties. Direct, quantitative measurement of the surface chemistry and composition of such systems in a hydrated environment has thus far not been demonstrated, yet such measurements are of great importance for the development of nanomedicine systems. Here we demonstrate the first use of cryo-XPS for the measurement of two PEG-functionalized nanomedicines: a polymeric drug delivery system and a lipid nanoparticle mRNA carrier. The observed differences between cryo-XPS and standard XPS measurements indicate the potential of cryo-XPS for providing quantitative measurements of such nanoparticle systems in hydrated conditions.

Sticky Measurement Problem: Number Concentration of Agglomerated Nanoparticles.

Measuring the number concentration of colloidal nanoparticles (NPs) is critical for assessing reproducibility, enabling compliance with regulation, and performing risk assessments of NP-enabled products. For nanomedicines, their number concentration directly relates to their dose. However, the lack of relevant reference materials and established traceable measurement approaches make the validation of methods for NP number concentration difficult. Furthermore, commercial products often exhibit agglomeration, but guidelines for dealing with nonideal samples are scarce. We have compared the performance of five benchtop measurement methods for the measurement of colloidal number concentration in the presence of different levels of agglomeration. The methods are UV-visible spectroscopy, differential centrifugal sedimentation, dynamic light scattering, particle tracking analysis, and single-particle inductively coupled plasma mass spectrometry. We find that both ensemble and particle-by-particle methods are in close agreement for monodisperse NP samples and three methods are within 20% agreement for agglomerated samples. We discuss the sources of measurement uncertainties, including how particle agglomeration affects measurement results. This work is a first step toward validation and expansion of the toolbox of methods available for the measurement of real-world NP products.

Improved Metrological Traceability of Particle Size Values Measured with Line-Start Incremental Centrifugal Liquid Sedimentation.

Line-start incremental centrifugal liquid sedimentation (disc-CLS) is a powerful method to determine particle size based on the principles of Stokes' law. Because several of the input quantities of the Stokes equation cannot be easily determined for this case of a rotating disc, the disc-CLS approach relies on calibrating the sedimentation time scale with reference particles. To use these calibrant particles for establishing metrological traceability, they must fulfill the same requirements as those imposed on a certified reference material, i.e., their certified Stokes diameter and density value must come with a realistic measurement uncertainty and with a traceability statement. As is the case for several other techniques, the calibrants do not always come with uncertainties for the assigned modal diameter and effective particle density. The lack of such information and the absence of a traceability statement make it difficult for the end-user to estimate the uncertainty of the measurement results and to compare them with results obtained by others. We present the results of a collaborative study that aimed at demonstrating the traceability of particle size results obtained with disc-CLS. For this purpose, the particle size and effective particle density of polyvinyl chloride calibrants were measured using different validated methods, and measurement uncertainties were estimated according to the Guide to the Expression of Uncertainty in Measurement. The results indicate that the modal Stokes diameter and effective particle density that are assigned to the calibrants are accurate within 5% and 3.5%, respectively, and that they can be used to establish traceability of particle size results obtained with disc-CLS. This conclusion has a great impact on the traceability statement of certified particle size reference materials, for which the traceability is limited to the size and density values of the calibrant particles.

Size and ζ-Potential Measurement of Silica Nanoparticles in Serum Using Tunable Resistive Pulse Sensing.

The contact of nanoparticles with biological fluids such as serum results in rapid adsorption of proteins at the nanoparticle surface in a layer known as the "protein corona". Protein coatings modify and control the behavior of the nanoparticles potentially altering the aggregation state and cellular response, which may influence their fate and hazard to human health. Cells are likely to interact with the protein interface rather than with bare surface; therefore it is important to study the protein layer and develop appropriate measurement tools. In this study we investigate how adsorbed proteins from serum affect the size and the surface charge of plain and aminated silica nanoparticles. Particle size and size distributions in buffer and serum-based biological media were studied using tunable resistive pulse sensing (TRPS), as well as differential centrifugal sedimentation (DCS) and dynamic light scattering (DLS). Average and single particle ζ-potentials (related to surface charge) were also measured by electrophoretic light scattering (ELS) and TRPS, respectively. Size measurements showed an increase in size of the nanoparticles upon acquisition of a protein layer, thus allowing an estimation of its thickness. DLS proved incapable of providing an accurate measurement of the nanoparticles' size in serum due to the presence of agglomerates. The ability of TRPS to measure sample agglomeration was investigated by comparison with the high resolution technique of DCS. Particle-by-particle ζ-potential measurements by TRPS were consistent with those performed with ELS and allowed a description of the ζ-potential distribution within the samples.

Advanced Chemical and Imaging Methods for Studying Structure Morphology and Excipients Solid State Transformations in Pharmaceutical Multiparticulate Formulations.

The formulation of paediatric medicines faces significant challenges to meet the requirements for safe and accurate administration, while maintaining a suitable taste. Multiparticulate formulations have a strong potential to address these challenges because they combine dose flexibility with ease of administration. Understanding the stability of multiparticulate formulations over storage as a function of time and environmental parameters, such as humidity and temperature, is important to manage their commercialisation and use. In this work, we have expanded the toolkit of available techniques for studying multiparticulates beyond those such as scanning electron microscopy (SEM) and confocal laser scanning microscopy. We include advanced methods of environmentally-controlled SEM to monitor temperature- and humidity-induced changes in-situ, and a variety of Raman spectroscopies including stimulated Raman scattering microscopy to identify and localise the different ingredients at the surface and inside the multiparticulates. These techniques allowed unprecedented monitoring of specific changes to the particulate structure and distribution of individual ingredients due to product aging. These methods should be considered as valuable novel tools for in-depth characterisation of multiparticulate formulations to further understand chemical changes occurring during their development, manufacturing and long-term storage. We envisage these techniques to be useful in furthering the development of future medicine formulations.

Toward an international standardisation roadmap for nanomedicine.

The French National Metrology Institute (LNE) initiated a series of events to identify priorities for test methods and their harmonisation that directly address regulatory needs in Nanomedicine. One of these workshops entitled "The International Standardisation Roadmap for Nanomedicine" held in October 2023 (Paris, France) brought together key experts in the characterisation of nanomedicines and medical products containing nanomaterials, including the Joint Research Centre of the European Commission, SINTEF Industry and the metrology institutes of France, the UK, the USA and Canada, two flagship initiatives of the European Commission (PHOENIX and SAFE-n-MEDTECH Open Innovation Test Beds), representatives of a working party on mRNA vaccines at the European Directorate for the Quality of Medicines (EDQM) and members of international standardisation and pre-normative organisations (including CEN, ISO, ASTM, VAMAS). Two take-home message came out from the discussion. First, developing standard test methods and Reference Materials (RMs) for nanomedicines is a key priority for the European Commission and various stakeholders. Furthermore, there was a unanimous recognition of the need for a unified approach between standardisation committees, regulators and the nanomedicine community. At the USA, Canadian and European level, examples of success stories and of future initiative have been discussed. Future perspectives include the creation of a dedicated Working Group under CEN/TC 352 to consolidate efforts and develop a nanomedicine standardisation roadmap.

Combining orthogonal measurements to unveil diclofenac encapsulation into polymeric and lipid nanocarriers.

The quantification of the drug associated to nanoparticle carriers, often expressed in terms of encapsulation efficiency, is a regulatory requirement. The establishment of independent methods to evaluate this parameter provides a means for measurement validation, which is critical in providing confidence in the methods and enabling the robust characterization of nanomedicines. Chromatography is traditionally used to measure drug encapsulation into nanoparticles. Here, we describe an additional independent strategy based on analytical centrifugation. The encapsulation of diclofenac into nanocarriers was quantified based on the mass difference between placebo (i.e. unloaded) and loaded nanoparticles. This difference was estimated using particle densities measured by differential centrifugal sedimentation (DCS) and size and concentration values measured by particle tracking analysis (PTA). The proposed strategy was applied to two types of formulations, namely poly(lactic-co-glycolic acid) (PLGA) nanoparticles and nanostructured lipid carriers, which were analysed by DCS operated in sedimentation and flotation modes, respectively. The results were compared to those from high performance liquid chromatography (HPLC) measurements. Additionally, X-ray photoelectron spectroscopy analysis was used to elucidate the surface chemical composition of the placebo and loaded nanoparticles. The proposed approach enables the monitoring of batch-to-batch consistency and the quantification of diclofenac association to PLGA nanoparticles from 0.7 ng to 5 ng of drug per 1 µg of PLGA, with good linear correlation between DCS and HPLC results (R2 = 0.975). Using the same approach, similar quantification in lipid nanocarriers was possible for a loading of diclofenac ≥1.1 ng per 1 µg of lipids, with results in agreement with the HPLC method (R2 = 0.971). Hence, the strategy proposed here expands the analytical tools available for evaluating nanoparticles encapsulation efficiency, being thus significant for increasing the robustness of drug-delivery nanocarriers characterization.

NMR proton relaxation for measuring the relative concentration of nanoparticles in liquids.

The measurement of relative concentration of nanoparticles in liquids has been investigated using NMR proton relaxation, addressing a gap in analytical capabilities for highly concentrated dispersions. This technique has a limited footprint, short measurement time and ease of operation making it a promising quality control method to support the development and manufacture of nanomaterials.

Nanoparticle Isolation from Biological Media for Protein Corona Analysis: The Impact of Incubation and Recovery Protocols on Nanoparticle Properties.

Nanoparticles are increasingly implemented in biomedical applications, including the diagnosis and treatment of disease. When exposed to complex biological media, nanoparticles spontaneously interact with their surrounding environment, leading to the surface-adsorption of small and bio- macromolecules- termed the "corona". Corona composition is governed by nanoparticle properties and incubation parameters. While the focus of most studies is on the protein signature of the nanoparticle corona, the impact of experimental protocols on nanoparticle size in the presence of complex biological media, and the impact of nanoparticle recovery from biological media has not yet been reported. Here using a non-degradable robust model, we show how centrifugation-resuspension protocols used for the isolation of nanoparticles from incubation media, incubation duration and shear flow conditions alter nanoparticle parameters including particle size, zeta potential and total protein content. Our results show significant changes in nanoparticle size following exposure to media containing protein under different flow conditions, which also altered the composition of surface-adsorbed proteins profiled by SDS-PAGE. Our in situ analysis of nanoparticle size in media containing protein using particle tracking analysis highlights that centrifugation-resuspension is disruptive to agglomerates that are spontaneously formed in protein containing media, highlighting the need for in situ analytical methods that do not alter the intermediates formed following nanoparticle exposure to biological media. Nanomedicines are mostly intended for parenteral administration, and our findings show that parameters such as shear flow can significantly alter nanoparticle physicochemical parameters. Overall, we show that the centrifugation-resuspension isolation of nanoparticles from media significantly alters particle parameters in addition to the overall protein composition of surface-adsorbed proteins. We recommend that nanoparticle characterization pipelines studying bio-nano interactions during early nanomedicine development consider biologically-relevant shear flow conditions and media composition that can significantly alter particle physical parameters and subsequent conclusions from these studies.

Emerging techniques for submicrometer particle sizing applied to Stöber silica.

The accurate characterization of submicrometer and nanometer sized particles presents a major challenge in the diverse applications envisaged for them including cosmetics, biosensors, renewable energy, and electronics. Size is one of the principal parameters for classifying particles and understanding their behavior, with other particle characteristics usually only quantifiable when size is accounted for. We present a comparative study of emerging and established techniques to size submicrometer particles, evaluating their sizing precision and relative resolution, and demonstrating the variety of physical principles upon which they are based, with the aim of developing a framework in which they can be compared. We used in-house synthesized Stöber silica particles between 100 and 400 nm in diameter as reference materials for this study. The emerging techniques of scanning ion occlusion sensing (SIOS), differential centrifugal sedimentation (DCS), and nanoparticle tracking analysis (NTA) were compared to the established techniques of transmission electron microscopy (TEM), scanning mobility particle sizing (SMPS), and dynamic light scattering (DLS). The size distributions were described using the mode, arithmetic mean, and standard deviation. Uncertainties associated with the six techniques were evaluated, including the statistical uncertainties in the mean sizes measured by the single-particle counting techniques. Q-Q plots were used to analyze the shapes of the size distributions. Through the use of complementary techniques for particle sizing, a more complete characterization of the particles was achieved, with additional information on their density and porosity attained.

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.

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Measurement of the Size Distribution of Multimodal Colloidal Systems by Laser Diffraction.

Laser diffraction (LD) is a well-established tool for the measurement of particle size distribution. Recently, its demand and use for the measurement of complex biological systems have increased. Among the challenges that these types of samples present, there is the presence of multiple particle populations whose modal size may span across several orders of magnitude. In this study, we assessed the accuracy of LD for the measurement of the modal diameter of both single and mixed populations of polystyrene particles with diameters ranging from 60 nm to 40 µm. We discuss the application of different available algorithms to the analysis of the data and their impact on the measurement results. Independent methods were applied to guide the selection of the algorithms and validate the measured size distributions. We found that the modal diameters of the particle size distribution measured by LD for the mixed suspension was accurate within 2 % for particles larger than 1 µm and generally within 25 % for the particles tested. Method repeatability was found to be robust, with deviations below 1%. The method was also found to be useful for estimating the relative concentration of the particle populations in the mixed samples. This study provides confidence in the use of LD for the measurement of complex multimodal colloidal samples.

Measurement of Peptide Coating Thickness and Chemical Composition Using XPS.

X-ray photoelectron spectroscopy is a highly surface-sensitive analytical technique, capable of providing quantitative information on the chemical composition of materials within the top ∼10 nm of their surface. For samples consisting of distinct underlayer and overlayer materials, the thickness of the coating can also be determined if it falls within this ∼10 nm information depth, which is often the case for peptide layers. Such measurements are simple to perform for flat samples and can also be performed on nanoparticulate samples provided that either the core radius or total particle radius are known. Here, we describe a straightforward protocol for obtaining such measurements from peptide coatings on both flat surfaces and nanoparticles, including preparation of nanoparticle samples from suspension, data acquisition, and analysis.

Formulation and Characterization of Phytostanol Ester Solid Lipid Nanoparticles for the Management of Hypercholesterolemia: An ex vivo Study.

BACKGROUND: Phytostanols are naturally occurring compounds that reduce blood cholesterol levels significantly. However, their aqueous insolubility poses formulation challenges. AIM: To formulate and characterize solid lipid nanoparticle carriers for phytostanol esters to enhance the bioavailability of phytostanols. METHODS: Phytostanol ester solid lipid nanoparticles were formulated by the microemulsion method. They were characterized for particle size distribution, polydispersity index, shape, surface charge, entrapment efficiency, stability, chemical structure, and thermal properties. The uptake of the formulation by cell lines, HepG2 and HT-29, and its effect on cell viability were evaluated. RESULTS: The formulation of solid lipid nanoparticles was successfully optimised by varying the type of lipids and their concentration relative to that of surfactants in the present study. The optimised formulation had an average diameter of (171 ± 9) nm, a negative surface charge of (-23.0 ± 0.8) mV and was generally spherical in shape. We report high levels of drug entrapment at (89 ± 5)% in amorphous form, drug loading of (9.1 ± 0.5)%, nanoparticle yield of (67 ± 4)% and drug excipient compatibility. The biological safety and uptake of the formulations were demonstrated on hepatic and intestinal cell lines. CONCLUSION: Phytostanol ester solid lipid nanoparticles were successfully formulated and characterized. The formulation has the potential to provide an innovative drug delivery system for phytostanols which reduce cholesterol and have a potentially ideal safety profile. This can contribute to better management of one of the main risk factors of cardiovascular diseases.

Engineering nanocomposite materials for cancer therapy.

Cancer accounted for 13% of all deaths worldwide in 2005. Although early detection is critical for the successful treatment of many cancers, there are sensitivity limitations associated with current detection methodologies. Furthermore, many traditional anticancer drug treatments exhibit limited efficacy and cause high morbidity. The unique physical properties of nanoscale materials can be utilized to produce novel and effective sensors for cancer diagnosis, agents for tumor imaging, and therapeutics for cancer treatment. Functionalizing inorganic nanoparticles with biocompatible polymers and natural or rationally designed biomolecules offers a route towards engineering responsive and multifunctional composite systems. Although only a few such innovations have reached human clinical trial to date, nanocomposite materials based on functionalized metal and semiconductor nanoparticles promise to transform the way cancer is diagnosed and treated. This review summarizes the current state-of-the-art in the development of inorganic nanocomposites for cancer-related applications.

Evaluation of Particle Size Techniques to Support the Development of Manufacturing Scale Nanoparticles for Application in Pharmaceuticals.

The measurement of nanoparticle size, and size distribution, is important to the development of pharmaceutical nanoparticle products and their manufacturing processes. In this work we report on the use of 4 widely-used liquid-phase techniques, dynamic light scattering, differential centrifugal sedimentation, particle tracking analysis, and tuneable resistive pulse sensing to measure 4 different batches of AZD2811NPs. The techniques rely on different physical principles to measure nanoparticle size. The batches cover a range of different manufacturing scales and different sites of manufacture, and were made to support toxicity, clinical, and engineering studies. The results from the different techniques and different batches are compared in terms of the average size, and size distribution, measured. In addition, we discuss the suitability of techniques for different applications, for example, QC and process understanding.

Emerging Standards and Analytical Science for Nanoenabled Medical Products.

Development and application of nanotechnology-enabled medical products, including drugs, devices, and in vitro diagnostics, are rapidly expanding in the global marketplace. In this review, the focus is on providing the reader with an introduction to the landscape of commercially available nanotechnology-enabled medical products as well as an overview of the international documentary standards and reference materials that support and facilitate efficient regulatory evaluation and reliable manufacturing of this diverse group of medical products. We describe the materials, test methods, and standards development needs for emerging medical products. Scientific and measurement challenges involved in the development and application of innovative nanoenabled medical products motivate discussion throughout this review.

Substrate stiffness affects early differentiation events in embryonic stem cells.

Embryonic stem cells (ESC) are both a potential source of cells for tissue replacement therapies and an accessible tool to model early embryonic development. Chemical factors such as soluble growth factors and insoluble components of the extracellular matrix are known to affect the differentiation of murine ESCs. However, there is also evidence to suggest that undifferentiated cells can both sense the mechanical properties of their environment and differentiate accordingly. By growing ESCs on flexible polydimethylsiloxane substrates with varying stiffness, we tested the hypothesis that substrate stiffness can influence ESC differentiation. While cell attachment was unaffected by the stiffness of the growth substrate, cell spreading and cell growth were all increased as a function of substrate stiffness. Similarly, several genes expressed in the primitive streak during gastrulation and implicated in early mesendoderm differentiation, such as Brachyury, Mixl1 and Eomes, were upregulated in cell cultures on stiffer compared to softer substrates. Finally, we demonstrated that osteogenic differentiation of ESCs was enhanced on stiff substrates compared to soft substrates, illustrating that the mechanical environment can play a role in both early and terminal ESC differentiation. Our results suggest a fundamental role for mechanosensing in mammalian development and illustrate that the mechanical environment should be taken into consideration when engineering implantable scaffolds or when producing therapeutically relevant cell populations in vitro.

Correction: Schavkan, A., et al. Number Concentration of Gold Nanoparticles in Suspension: SAXS and spICPMS as Traceable Methods Compared to Laboratory Methods. Nanomaterials 2019, 9, 502.

The authors wish to make the following corrections to this paper [...].