Project ID assignment: HTS-11: Toxicological assessment of cationic nanoparticles with different surface
charge densities in differentiated and undifferentiated bronchial epithelial cells
Haiyuan Zhang, Tian Xia, Andre Nel
One of the key physicochemical characteristics that determine ENM interactions with biological surfaces is the
surface charge of the materials. Previous research has demonstrated that the cationic surface charge could
generate cytotoxicity, including to environmental life forms such as bacteria and yeasts as well as the human
lung. The goal of this project is to fully understand the biological effects of charged nanoparticles with different
charge densities, including the possibility that the elucidation of a cationic threshold could be used to design
safer materials. In order to test this hypothesis, Theme 1 synthesized a library of used mesoporous silica
nanoparticles (MSNPs) in which there negatively charged surfaces were used for the electrostatic attachment
of a series of polyethylenimine (PEI) polymers that vary in length from 0.6-25 kD. These materials were
extensively characterized to obtain the zeta potential, primary size and hydrodynamic size by the performance
of TEM and DLS in the Nanoparticle Core. For comparison to the effects of the cationic and phosphonate
functionalized MSNP, we also generated a particle with non-charged surface through PEG attachment.
In Aim 1, the cytotoxicity and cellular uptake of anionic, neutral and cationic MSNPs were compared in a
variety of different cell types. While MSNP-PEI 25 kD exhibited high cytotoxicity ,MSNP-Phos and MSNP-PEG
showed little or non-toxicity. The increased toxicity of the PEI coated particles was associated with high cellular
uptake as determined by flow cytometry and confocal microscopy. This demonstrated that the relative
increase in fluorescent particle uptake was 2 orders of magnitude higher with MSNP-PEI than MSNP-Phos or
MSNP-PEG. This finding was corroborated by confocal studies, which showed a significant increase in the
number of MSNP-PEI 25 kD in all cell types looked at. Decrease of the in PEI polymer length below 10 KD had a
definitive effect on reducing cytotoxicity, which was absent at polymer lengths of 5 and 1.2 kD. The
contribution of polymer length to cytotoxicity was demonstrated in the multi-parametric HTS assay, which
confirmed that clear difference in their rates of sub-lethal and lethal toxicity when polymer length decreases
below 10 KD. Interestingly, the particles coated with polymers < 10 kD maintained a high level of cellular
uptake and this finding became the basis for the development of MSNP carrier that are capable of nucleic acid
delivery to cancer cells. This demonstrates the dynamic use of discovery at the nano/bio interface, allowing
toxicological screening to prestage the design of safer nanomaterials.
In Aim 2, the mechanistic effects of the cationic MSNP library were further explored in differentiated and
undifferentiated normal human bronchial epithelial cells (NHBE). The necessity to perform this comparison
became obvious in preliminary studies demonstrating that the state of NHBE differentiation determine their
susceptibility to cationic particles. Using the multi-parametric assay, cationic MSNPs with higher charge density
(MSNP-PEI 10 kD or 25 kD) showed increased intracellular calcium influx, increased cell membrane leakage,
increased superoxide generation and decreased mitochondrial membrane potential compared to MSNPs
coated with polymer lengths of 0.6-1.8 kD. Moreover, differentiated cells showed more toxicity than
undifferentiated cells, which could be explained by the fact that cellular differentiation leads to increased
expression of anionic proteoglycans on the cell surface, including the heparin sulfate glycoprotein, syndecan-1.
Syndecan-1 contains abundant heparin sulfate groups that are capable of binding to the cationic MSNPs.
Pretreatment with heparinase lowered particle association as well as cytotoxicity in differentiated cells. In
addition, cationic MSNPs lowered the membrane potential of differentiated NHBE cells and affected the
structural integrity of the RBC membrane, suggesting the surface-bound cationic particles can lead to surface
membrane damage as the most immediate injury effect.
In summary, cationic nanoparticles with high charge density is associated with higher cellular uptake and
cytotoxicity than anionic or neutral nanoparticles. For cationic particles we could define a definitive threshold
above which positive charge contributes to toxicity. Detailed analysis of this threshold demonstrated that both
the number of cationic groups as well as their placement on the particle surface determines their membrane
damaging potential. Thus, longer length polymers exhibit an asymmetric distribution of cationic charge that is
capable of making contact with the surface membrane, compared to shorter length polymers where these
cationic groups are screened by culture medium proteins that interact with the particle surface. Differentiated
NHBE is more susceptible to cationic injury due to the expression of anionic heparin sulfate proteoglycans.