Dual contribution of surface charge and protein-binding affinity to the cytotoxicity of polystyrene nanoparticles in nonphagocytic A549 cells and phagocytic THP-1 cells.

Knowledge that links the physicochemical properties of nanoparticles (NP) to their toxicity is key to evaluating and understanding mechanisms underlying toxicity and developing appropriate testing methods for NP; however, this is currently limited since only a small set of NP have been used, with typically poor control of their physical properties. In this study, eight types of polystyrene NP (PLNP) were synthesized with different functional groups, but all based on an identical core. In vitro cell-based assays were performed to determine the influence of changes in physicochemical properties, such as charge, hydrodynamic size, and protein binding potential, in relation to NP-mediated toxicity. The PLNP were incubated with nonphagocytic A549 cells or phagocytic differentiated THP-1 cells for 4 h with/without fetal bovine serum (FBS), followed by incubation for 20 h in FBS-supplemented medium with/without a washing step, to assess cell-type specificity and impact of protein corona formation. The effect of surface charge on cytotoxicity differed between A549 cells and THP-1 cells. In nonphagocytic A549 cells, the zeta potential of PLNP exhibited a negative correlation with cytotoxicity, partly due to the level of coronated protein that might affect cellular uptake. In phagocytic THP-1 cells, the zeta potential of PLNP showed a positive correlation with cytotoxicity but coronated protein levels displayed no marked association with cytotoxicity, owing to the professional uptake efficacy of phagocytic cells. The consistency of our data with THP-1 cells with the surface charge paradigm in nanotoxicology suggests that phagocytic cells are the predominant targets for lung inflammatory reactions induced by PLNP.

Surface charge determines the lung inflammogenicity: A study with polystyrene nanoparticles.

Surface functionalization is a routine process to improve the behavior of nanoparticles (NPs), but the induced surface properties, such as surface charge, can produce differential toxicity profiles. Here, we synthesized a library of covalently functionalized fluorescent polymeric NPs (F-PLNPs) to evaluate the role of surface charge on the acute inflammation and the localization in the lung. Guanidinium-, acetylated-, zwitterionic-, hydroxylated-, PEGylated-, carboxylated- and sulfated-F-PLNPs were synthesized from aminated-F-PLNP. The primary particle sizes were identical, but the hydrodynamic sizes ranged from 210 to 345 nm. Following surface functionalization, the F-PLNPs showed diverse zeta potentials from -41.2 to 31.0 mV, and each F-PLNP showed a single, narrow peak. Pharyngeal aspiration with these eight types of F-PLNPs into rats produced diverse acute lung inflammation, with zeta potentials of the F-PLNPs showing excellent correlation with acute pulmonary inflammation parameters including the percentage of polymorphonuclear leukocytes (R(2) = 0.90, p < 0.0001) and the levels of interleukin-1ß (R(2) = 0.83, p < 0.0001) and of cytokine-induced neutrophil chemoattractant-3 (R(2) = 0.86, p < 0.0001). These results imply that surface charge is a key factor influencing lung inflammation by functionalized polymeric NPs, which further confirms and extends the surface charge paradigm that we reported for pristine metal oxide NPs. This demonstrates that the surface charge paradigm is a valuable tool to predict the toxicity of NPs.

Surface functionalization affects the zeta potential, coronal stability and membranolytic activity of polymeric nanoparticles.

Nano materials are commonly functionalized to boost their physicochemical properties. However, there is little known about the impact of these modifications on cellular systems. Herein, we synthesized eight types of polymeric nanoparticles (NPs) bearing different functional groups, and investigated their effects on interactions with cellular membranes. As models for particle membrane interactions, hemolysis assays using human red blood cells and culture with A549 cells were utilized. Under protein-free conditions, the NPs showed a wide distribution of zeta potentials (ζPs) which showed a good correlation with their hemolytic potential. However, in the presence of serum or lung lining fluid, the ζPs of all NPs coalesced towards a single common negative value and showed neither hemolytic activity nor cytotoxicity to A549 cells. Lipase and protease treatment of the coronated particles did not restore their reactivity. These result simply proves that particle functionalization influences the stability of the particle corona which, if intact, prevents hemolytic activity and membrane disrupture.

Influence of spacer length on the cellular uptake of polymeric nanoparticles.

Nanotechnology is finding ever increasing application in the life science arena where nanoparticles can be used to deliver cargoes in cells. However, a clear understanding of the relationship between the chemical properties of the particle and its uptake efficiency is lacking. Herein, the effects on particle cellular uptake following modification with a variety of spacers, all bearing a positive charge, but differing in length, and the influence on formation of the protein corona are investigated. Although no significant differences in the composition of the protein corona are detected, the spacer length influences the cellular uptake of the nanoparticles. These findings will allow the target-orientated functionalisation of particles to increase the specificity of cellular uptake.

Zeta potential and solubility to toxic ions as mechanisms of lung inflammation caused by metal/metal oxide nanoparticles.

The toxicology of nanoparticles (NPs) is an area of intense investigation that would be greatly aided by improved understanding of the relationship between NP structure and inflammogenicity. To evaluate how their physicochemical parameters influence toxicity, we assembled a panel of 15 metal/metal oxide NPs and attempted to relate various physicochemical parameters, including zeta potential (ζP) and solubility, to lung inflammogenicity. The acute pulmonary inflammogenicity of the 15 NPs showed a significant correlation with one of two structural parameters-ζP under acid conditions for low-solubility NPs and solubility to toxic species for high-solubility NPs. ζP is the electrical potential created between the surface of a particle, with its associated ions, and the medium it exists in and provides information concerning the particle surface charge. We suggest that inside the phagolysosome under acid conditions, a high positive ζP may allow NPs to damage the integrity of the phagolysosomal membrane leading to inflammation. In the case of high-solubility NPs, inflammogenicity depends on the ions that are produced during dissolution of NP inside the acidic phagolysosomes; if the ions are toxic, then phagolysosomes will be destabilized and cause inflammation. These two parameters may have utility in preliminary assessment of the potential lung inflammation hazard of the large number of NPs that require testing.

Polymerizable fluorescein derivatives: synthesis of fluorescent particles and their cellular uptake.

Fluorescent particles are used for a diverse number of biochemical assays including intracellular imaging, cellular tracking, as well as detection of a variety of biomolecules. They are typically prepared by postpolymerization conjugations of dyes onto preformed particles. Herein we report the synthesis of aminomethyl-functionalized fluorescent particles via the synthesis and application of polymerizable fluorescein monomers. These monomers allowed high and controllable fluorophore loading into the particles, resulting in enhanced fluorescence properties in comparison with more commonly used carboxyfluorescein conjugated particles. Furthermore, the particles were rapidly taken up by cells with enhanced fluorescence. The herein presented results demonstrate the advantages of dye polymerization in contrast with more conventional conjugation strategies for fluorescent particle generation with applications in the life sciences.

Zeta potential mediated reaction monitoring on nano and microparticles.

Nano and microparticles are widely used across the life science interface, with applications ranging from chemical probes of biological function to fluorescent particles for flow cytometry and cellular tracking. Increasingly, particles are modified with a variety of chemistries to boost their functionality and broaden their biological applicability. However, although particle modification has become standard laboratory practice, the ability to determine the extent and efficiency of chemical modification is often very limited and empirical in nature. Herein, we report the use of zeta potential analysis as a simple and rapid "direct-on-particle" approach allowing levels of bead modification and derivatization to be evaluated. As a proof-of-concept, aminomethyl-functionalized nano and microparticles were derivatized to display a variety of surface functionalities and their zeta potentials measured, allowing verification of the applicability of the approach for particle analysis. We demonstrate that zeta potential measurement is a convenient approach which allows multistep reaction sequences to be followed, and show that this method can be used to verify and validate successful particle modification.