Nanoparticle-protein corona formation and immunotoxicity of zinc oxide nanoparticles

Saptarshi, Shruti R. (2015) Nanoparticle-protein corona formation and immunotoxicity of zinc oxide nanoparticles. PhD thesis, James Cook University.

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Engineered zinc oxide nanoparticles (ZnO-NPs) offer versatility and properties that have a vast array of applications, and are widely used in cosmetics and sunscreens, because of their excellent UV filtering properties and aesthetic appeal. Interestingly, despite high production volumes and a broad application base, there is always a possibility of unintentional side-effects of NPs due to their increased reactivity at the biological level. Bio-reactivity of nanoparticles can be influenced by their physical characteristics such as size, surface coating and propensity to interact with proteins. The main objective of this PhD thesis was to investigate and characterise the phenomenon of protein corona formation on ZnO NP surface in a protein rich environment such as cell culture medium, evaluate the nanotoxicological potential of these NPs using in vitro as well as in vivo test systems and furthermore relate this information to their physico-chemical properties.

NP-protein interactions as reviewed in chapter II can be affected by the primary size and agglomeration state of the NPs. Therefore accurate characterisation of the NP physico-chemical properties is vital. ZnO NP solutions of three different sizes, 30 nm, 80 nm and 200 nm in pristine and surfactant dispersed forms were characterised for their size, agglomeration and solubility in chapter III. Analytical disc centrifugation technique was chosen to determine the agglomeration state of the ZnO NPs in a protein enriched cell culture medium. The pristine ZnO NPs formed micron-sized agglomerates whereas; surfactant-treated material remained relatively monodispersed. Extracellular dissolution of ZnO NPs has been shown to affect their cellular interaction. Using ICP-AES technique I confirmed that the ZnO NPs used in this study have a low dissolution rate in cell culture medium.

Chapter IV of this thesis explored the identification and characterisation of the proteins adsorbed on the surface of ZnO NPs upon interaction with protein fortified cell culture medium which forms the foundation of good nanotoxicological studies. NPs due to their unique sizes interact with protein molecules, forming the NP-protein corona (NP-PC) that may influence their bio-reactivity. The adsorption of proteins on ZnO NP surface was instantaneous and did not change with time. Subsequent mass spectrometric analysis of the isolated ZnO NP-PC revealed enrichment of proteins with definite physiological roles, on the two 30 nm ZnO NP surfaces analysed. Interestingly, pristine and surfactant-dispersed ZnO-NP formed specific protein coronas by selectively binding low molecular weight proteins, derived from larger proteins such as haemoglobin, histones, fibrinogen etc. Interestingly, ZnO-NP induced protein conformational change depended on the type of protein interacting with the NP surface. ZnO NPs were also able to retain their BSA pre-coating after transfer into a new solution of cell lysate proteins, which demonstrated for the first time that, the protein corona formation of ZnO NPs is a constantly evolving process. Flow cytometry based side and forward scatter (measures of cytoplasmic granularity and size of cells respectively) analysis of human lung epithelial cells (A549) was largely affected when they were exposed to the 30 nm ZnO NP solutions dispersed in the absence of proteins vs 10% fetal bovine serum proteins. Furthermore, ZnO NP induced cytotoxicity at 100 μg/mL concentration was also significantly affected in the presence of increasing amounts of FBS proteins (10 or 40%). This clearly demonstrated that the adsorbed protein layer onto the NP surface influenced ZnO NP uptake and cytotoxicity.

Zinc oxide nanoparticles have been classified in to the category of highly reactive metal oxide NPs as outlined in chapter V. Exposure to ZnO NPs can occur not only via skin contact, but also via inhalation especially in the manufacturing sector and is an important occupational safety concern. Chapter VI of this thesis evaluates the capacity ZnO NPs in eliciting cytotoxicity and immunomodulation in human lung epithelial (A549) cells. The A549 cells represent the type II alveolar cells which form the first line of defence against inhaled particulate matter and were therefore chosen as suitable targets for this study. Direct exposure to ZnO NPs resulted in a size and dose-dependent cytotoxic response, indicated by decreased cell viability and increased lactate dehydrogenase release. DAPI staining of nuclear material in exposed cells revealed that cytotoxicity was mediated by apoptosis. The main objective of this section was to further investigate the immunomodulatory potential of ZnO NPS at the sub-cytotoxic level. The sub-cytotoxic ZnO NP concentration (20 μg/mL) induced significant up-regulation of the key pro-inflammatory cytokine IL-8 and redox stress marker heme oxygenase-1 at the mRNA level along with increased release of IL-8 protein in a time-dependent manner. The low doses of ZnO NPs also demonstrated that the increased expression of IL-8 involved transcriptional activation of NFκB, followed by further stabilisation of IL-8 mRNA by p38 mitogen activated protein kinase pathway. Pre-treatment of A549 cells with the sulfhydryl antioxidant Nacetyl cysteine resulted in significant reduction in the up-regulation of inflammatory markers, thus confirming the role of reactive oxygen species in the observed reactivity. This data highlights the inflammatory potential of ZnO NP at sub-cytotoxic doses, possibly due to a redox imbalance generated in exposed cells. This chapter increases our understanding of the kinetics and mechanisms underlying the observed immune modulatory and cytotoxic effects of ZnO NPs.

The last module of this thesis, chapter VII analysed the reactivity of ZnO NPs-in vivo system. 30nm ZnO NPs not only demonstrated differential protein binding but also higher cytotoxicity and pro-inflammatory potential in vitro and were consequently selected for the murine model investigation. Intranasal instillation of ZnO NPs and their ability to cause systemic inflammatory response in an acute high-dosage exposure animal model has not been investigated before. In this study, administration of a single dose (5 mg/kg BW) of either 30nm ZnO NP solutions resulted in substantial inflammatory infiltration into the alveoli and peri-bronchial regions of exposed mice. 24hr after the NP challenge. Significant up-regulation of eotaxin mRNA was also observed in the lung tissue obtained from mice treated with pristine 30nm. Cytokine profiling of pooled mouse serum from each NP treated or control mice groups revealed the presence of pro-inflammatory chemokines such as MCP-1, RANTES, IP-10 which known to recruit leukocytes to inflammatory sites. The preliminary results of the in vivo study highlight the inflammatory potential of inhaled ZnO NPs.

In conclusion, this thesis describes the dynamic interaction of serum proteins with sunscreen ZnO NPs and their strong immunomodulatory potential at a sub-cytotoxic dose. Moreover exposure to a single but relatively high dose of ZnO NPs via intranasal instillation causes acute pulmonary inflammatory reactions in vivo. The outcome of this thesis assists towards development of bio-compatible ZnO NPs for future use.

Item ID: 43787
Item Type: Thesis (PhD)
Keywords: characteristics; cytotoxicity; immune response; immunotoxicity; inflammation; inflammatory reactions; nanoparticles; NPs; properties; proteins; reactivity; zinc oxide; zinc-oxide nanoparticles; ZnO; ZnO-NPs
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter II: Saptarshi, Shruti R., Duschl, Albert, and Lopata, Andreas L. (2013) Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle. Journal of Nanobiotechnology, 11. pp. 1-12.

Chapter V: Saptarshi, Shruti R., Duschl, Albert, and Lopata, Andreas L. (2015) Biological reactivity of zinc oxide nanoparticles with mammalian test systems: an overview. Nanomedicine, 10 (13). pp. 2075-2092.

Chapter VI: Saptarshi, Shruti R., Feltis, Bryce N., Wright, Paul F.A., and Lopata, Andreas L. (2015) Investigating the immunomodulatory nature of zinc oxide nanoparticles at sub-cytotoxic levels in vitro and after intranasal instillation in vivo. Journal of Nanobiotechnology, 13 (6). pp. 1-11.

Date Deposited: 18 May 2016 02:50
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 50%
11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110107 Metabolic Medicine @ 50%
SEO Codes: 92 HEALTH > 9202 Health and Support Services > 920203 Diagnostic Methods @ 50%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 50%
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