Project ID Assignment: HTS-4: Property-activity analysis of silica nanoparticles, including the
relationship of surface chemistry to toxicological potential
Haiyuan Zhang, Tian Xia, Andre Nel
Silica nanoparticles can exist in crystalline as well as amorphous form. Although crystalline silica in the
form of quartz is capable of inducing silicosis, lung cancer and autoimmune diseases, there is a
considerable debate about the relative toxicity of amorphous, fumed, mesoporous and other crystalline
silica polymorphs, particularly as it pertains to nanoparticles. Due to the widespread use of silica
nanoparticles in consumer products (e.g., as desiccants), it is important to determine what the basis for
silica toxicity is and to explain material hazard in terms of the physicochemical properties of the
different silica types. Project ENM-3 describes the acquisition and physicochemical characterization of a
library of silica nanoparticles that includes amorphous colloidal (Stober) silica, fumed silica, mesoporous
silica, silicalite and Min-U Sil (quartz) for conducting in vitro and in vivo property-activity analysis. Quartz
toxicity in the human lung has been hypothesized to be dependent on particle properties that include
the surface display of silanols groups and surface defects capable of generating adverse biological effects
such as oxidative stress and cell membrane damage. Moreover, the presence of strained and unstrained
siloxane rings in the particle structure could determine the surface display of silanols and hydroxyl
groups that contribute to the biological activity of the material. Project ENM-3 describes the
physicochemical characterization of the silica nanoparticles in the library, including the assessment of
their crystalline phases, primary sizes, hydrodynamic sizes, display of various silanols groups and the
presence of strained and unstrained siloxane rings. These materials were dispersed in tissue culture
media and used for conducting MTS, LDH and ATP assays in BEAS-2B and RAW 264.7 cell lines. The
classic single parameter assays showed increased cytotoxicity for fumed silica and quartz compared to
amorphous colloidal silica, mesoporous nanoparticles and silicalite that showed little or no toxicity. This
hazard ranking was confirmed by in the multi-parameter HTS assay showing that fumed silica and quartz
could induce decreased plasma membrane integrity, increased intracellular calcium flux and increased
oxygen radical generation. We demonstrated that the inclusion of N-acetylcysteine (NAC, a thiol
antioxidant) could reduce the ROS production and cytotoxicity in response to Min-U-Sil but not change
fumed silica toxicity, indicating that the latter material type could be operating by a different mechanism
than Min-U-Sil. Confocal microscopy revealed most FITC-labeled fumed silica were bound to the cell
membrane rather than being taken up into cells in the same way as Stober silica. Since previous work
has suggested that high temperature calcination and the state of hydration affects the surface properties
of silica, we were interested in determining whether the synthesis of fumed silica under high
temperature flame spray conditions would change the toxic potential of this material. Fumed silica
nanoparticles calcined at 600 and 800oC showed a progressive decline of cytotoxic potential compared
to non-calcined particles. The reduced toxicity was accompanied by decreased density of vicinal silanols
groups as shown by the IR analysis (see Project ENM-3). Not only did the decrease in these closely
spaced silanols groups lead to decreased hydroxyl radical generation, but also decreased red cell
membrane disruption by fumed silica. Moreover, these changes were accompanied by an increase in the
number of strained three-membered siloxane rings as demonstrated by the Raman spectroscopy (see
Project ENM-3). In contrast, rehydration of calcined particles increased their cytotoxic potential in
parallel with increased surface vicinal silanol display, increased hydroxyl radical generation and increased
lytic potential of red cell membranes. Project ENM-3 further demonstrates that water reflux decreased
the number of three-membered siloxane rings in the calcined particles, suggesting that the formation of
the vicinal silanols is dependent on reconstruction of strained rings. In summary, the toxicity of fumed
silica nanoparticles, which are produced in large quantities for industrial use, is related to the surface
display of vicinal silanol groups through reconstruction of strained siloxane rings. We now need to
determine whether fumed silica pose environmental hazard by studying bacteria, environmental
organisms and rodents. We are currently characterizing other silica chemical constructs to probe their
structure-activity relationships. To date, we have not seen any toxicity related to mesoporous silica
nanoparticles that are being used in CEIN as stealth delivery particles (e.g., for delivery of metal and
metal oxides intracellular) or for studying the effect of long aspect ratio nanomaterials on the cellular
response (Project HTS-8).