Particle Metrology Approach to Understanding How Storage Conditions Affect Long-Term Liposome Stability.

Lipid nanoparticles are a generic type of nanomaterial with broad applicability in medicine as drug delivery vehicles. Liposomes are a subtype of lipid nanoparticles and, as a therapeutic platform, can be loaded with a genetic material or pharmaceutical agents for use as drug treatments. An open question for these types of lipid nanoparticles is what factor(s) affect the long-term stability of the particles. The stability of the particle is of great interest to understand and predict the effective shelf-life and storage requirements. In this report, we detail a one-year study of liposome stability as a function of lipid composition, buffer composition/pH, and storage temperature. This was done in aqueous solution without freezing. The effect of lipid composition is shown to be a critical factor when evaluating stability of the measured particle size and number concentration. Other factors (i.e., storage temperature and buffer pH/composition) were shown to be less critical but still have some effect. The stability of these particles informs formulation and optimal storage requirements and assists with future developmental planning of a NIST liposome-based reference material. This work also highlights the complex nature of long-term soft particle storage in biopharmaceutical applications.

Number Concentration Measurements of Polystyrene Submicrometer Particles.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques. Diameter values were also measured where possible. The diameter values were found to agree within 20%, but the number concentration values differed by as much as a factor of two. Accuracy and reproducibility related with the different techniques are discussed with the goal of using number concentration standards for instrument calibration. Three of the techniques were used to determine SI-traceable number concentration values, and the three independent values were averaged to give consensus values. This consensus approach is proposed as a protocol for certifying SI-traceable number concentration standards.

Effect of Azide Preservative on Thermomechanical Aggregation of Purified Reference Protein Materials.

Protein aggregation can affect the quality of protein-based therapeutics. Attempting to unravel factors influencing protein aggregation involves systematic studies. These studies often include sodium azide or similar preservatives in the aggregation buffer. This work shows effects of azide on aggregation of two highly purified reference proteins, both a bovine serum albumin (BSA) as well as a monoclonal antibody (NISTmAb). The proteins were aggregated by thermomechanical stress, consisting of simultaneous heating of the solution with gentle agitation. Protein aggregates were characterized by asymmetric flow field flow fractionation (AF4) with light scattering measurements along with quantification by UV spectroscopy, revealing strong time-dependent generation of aggregated protein and an increase in aggregate molar mass. Gel electrophoresis was used to probe the reversibility of the aggregation and demonstrated complete reversibility for the NISTmAb, but not so for the BSA. Kinetic fitting to a commonly implemented nucleated polymerization model was also employed to provide mechanistic details into the kinetic process. The model suggests that the aggregation of the NISTmAb proceeds via nucleated growth and aggregate-aggregate condensation in a way that is dependent on the concentration (and presence) of the azide anion. This work overall implicates azide preservatives as having demonstrable effects on thermomechanical stress and aggregation of proteins undergoing systematic aggregation and stability studies.

Biocompliant Composite Au/pHEMA Plasmonic Scaffolds for 3D Cell Culture and Noninvasive Sensing of Cellular Metabolites.

The field of 3D printing is an area of active research, with a substantial focus given to the design and construction of customized tools for applications in technology. There exists a particular need in these developing areas of opportunity for new multi-functional soft materials that are biologically compatible for the growth and directed culturing of cells. Herein, a composite material consisting of gold nanoparticles with useful plasmonic properties embedded within a highly hydrophilic poly-2-hydroxyethylmethacrylate matrix is described and characterized. This composite material serves dual functions as both host framework scaffold for cell lines such as pre-osteoblasts as well as a plasmonic biosensor for in situ measurements of living cells. The plasmonic properties of this system are characterized as a function of the material properties and related to compositional features of the material through a proposed light-directed mechanism. This chemistry provides a tunable, 3D printable plasmonic composite material of encapsulated gold nanoparticles in a biologically-compliant, acrylate-based hydrogel matrix. Surface-enhanced Raman scattering studies of 3D-microcultures supported by the scaffolds are carried out and the strong influence of perm-selective molecular diffusion in its analytical responses is established. Most notably, specific, largely hydrophilic, cellular metabolites are detected within the supported live cultures.

Refractive index sensing and surface-enhanced Raman spectroscopy using silver-gold layered bimetallic plasmonic crystals.

Herein we describe the fabrication and characterization of Ag and Au bimetallic plasmonic crystals as a system that exhibits improved capabilities for quantitative, bulk refractive index (RI) sensing and surface-enhanced Raman spectroscopy (SERS) as compared to monometallic plasmonic crystals of similar form. The sensing optics, which are bimetallic plasmonic crystals consisting of sequential nanoscale layers of Ag coated by Au, are chemically stable and useful for quantitative, multispectral, refractive index and spectroscopic chemical sensing. Compared to previously reported homometallic devices, the results presented herein illustrate improvements in performance that stem from the distinctive plasmonic features and strong localized electric fields produced by the Ag and Au layers, which are optimized in terms of metal thickness and geometric features. Finite-difference time-domain (FDTD) simulations theoretically verify the nature of the multimode plasmonic resonances generated by the devices and allow for a better understanding of the enhancements in multispectral refractive index and SERS-based sensing. Taken together, these results demonstrate a robust and potentially useful new platform for chemical/spectroscopic sensing.

Silica Nanoparticle-Generated ROS as a Predictor of Cellular Toxicity: Mechanistic Insights and Safety by Design.

Evaluating toxicological responses of engineered nanomaterials such as silica nanoparticles is critical in assessing health risks and exposure limits. Biological assays can be used to evaluate cytotoxicity of individual materials, but specific nano-bio interactions-which govern its physiological response-cannot currently be predicted from materials characterization and physicochemical properties. Understanding the role of free radical generation from nanomaterial surfaces facilitates understanding of a potential toxicity mechanism and provides insight into how toxic effects can be assessed. Size-matched mesoporous and nonporous silica nanoparticles in aminopropyl-functionalized and native forms were investigated to analyze the effects of porosity and surface functionalization on the observed cytotoxicity. In vitro cell viability data in a murine macrophage cell line (RAW 264.7) provides a model for what might be observed in terms of cellular toxicity upon an environmental or industrial exposure to silica nanoparticles. Electron paramagnetic resonance spectroscopy was implemented to study free radical species generated from the surface of these nanomaterials and the signal intensity was correlated with cellular toxicity. In addition, in vitro assay of intracellular reactive oxygen species (ROS) matched well with both the EPR and cell viability data. Overall, spectroscopic and in vitro studies correlate well and implicate production of ROS from a surface-catalyzed reaction as a predictor of cellular toxicity. The data demonstrate that mesoporous materials are intrinsically less toxic than nonporous materials, and that surface functionalization can mitigate toxicity in nonporous materials by reducing free radical production. The broader implications are in terms of safety by design of nanomaterials, which can only be extracted by mechanistic studies such as the ones reported here.

Amine modification of nonporous silica nanoparticles reduces inflammatory response following intratracheal instillation in murine lungs.

Amorphous silica nanoparticles (NPs) possess unique material properties that make them ideal for many different applications. However, the impact of these materials on human and environmental health needs to be established. We investigated nonporous silica NPs both bare and modified with amine functional groups (3-aminopropyltriethoxysilane (APTES)) in order to evaluate the effect of surface chemistry on biocompatibility. In vitro data showed there to be little to no cytotoxicity in a human lung cancer epithelial cell line (A549) for bare silica NPs and amine-functionalized NPs using doses based on both mass concentration (below 200µg/mL) and exposed total surface area (below 14m(2)/L). To assess lung inflammation, C57BL/6 mice were administered bare or amine-functionalized silica NPs via intra-tracheal instillation. Two doses (0.1 and 0.5mg NPs/mouse) were tested using the in vivo model. At the higher dose used, bare silica NPs elicited a significantly higher inflammatory response, as evidence by increased neutrophils and total protein in bronchoalveolar lavage (BAL) fluid compared to amine-functionalized NPs. From this study, we conclude that functionalization of nonporous silica NPs with APTES molecules reduces murine lung inflammation and improves the overall biocompatibility of the nanomaterial.

Nano-Bio Interactions of Porous and Nonporous Silica Nanoparticles of Varied Surface Chemistry: A Structural, Kinetic, and Thermodynamic Study of Protein Adsorption from RPMI Culture Medium.

Understanding complex chemical changes that take place at nano-bio interfaces is of great concern for being able to sustainably implement nanomaterials in key applications such as drug delivery, imaging, and environmental remediation. Typical in vitro assays use cell viability as a proxy to understanding nanotoxicity but often neglect how the nanomaterial surface can be altered by adsorption of solution-phase components in the medium. Protein coronas form on the nanomaterial surface when incubated in proteinaceous solutions. Herein, we apply a broad array of techniques to characterize and quantify protein corona formation on silica nanoparticle surfaces. The porosity and surface chemistry of the silica nanoparticles have been systematically varied. Using spectroscopic tools such as FTIR and circular dichroism, structural changes and kinetic processes involved in protein adsorption were evaluated. Additionally, by implementing thermogravimetric analysis, quantitative protein adsorption measurements allowed for the direct comparison between samples. Taken together, these measurements enabled the extraction of useful chemical information on protein binding onto nanoparticles in solution. Overall, we demonstrate that small alkylamines can increase protein adsorption and that even large polymeric molecules such as poly(ethylene glycol) (PEG) cannot prevent protein adsorption in these systems. The implications of these results as they relate to further understanding nano-bio interactions are discussed.