Combinatorial toxicity of ZnO and TiO2 nanoparticles in human primary epidermal keratinocytes

Nanotoxicology encapsulates studying the interactions nanomaterials can have with biological systems and the environment. Recently, this field has gained momentum due to the influx of new nanomaterials, many of which have already become popular components of consumer products like cosmetics. Some of these applications use combinations of nanomaterials; dual nanoparticle systems. However, almost all the studies to date have focused on evaluating the toxicology of single nanomaterials. Since dual nanoparticle systems are often used due to a synergistic or antagonistic relationship between the two materials, it is only logical to expect such an interplay to trickle into their combinatorial toxic influence. This study is focused on investigating the toxicity of the dual nanoparticle system of ZnO and TiO2, which is most common in sunscreens. The combined effect of the two nanoparticles was studied using skin-mimicking human primary epidermal keratinocytes as the in vitro model. Various cellular processes were evaluated including cell death, ROS generation and DNA damage. These were linked to nanoparticle properties which were heavily characterized in terms of size, shape, solubility, zeta potential, etc. It was found that individual TiO2 nanoparticles induced low cytotoxicity even at higher concentrations while ZnO nanoparticles caused a high degree of cell death even at lower concentrations. The reason behind this difference was the solubility of ZnO nanoparticles which allowed intracellular Zn2+ ion concentration to be imbalanced inducing cell death, ROS generation and sharp DNA damage. Interesting, TiO2 nanoparticles demonstrated an ability to adsorb Zn2+ ions onto their negative surface and caused a reduction of intracellular Zn2+ ion concentration when treated alongside ZnO nanoparticles. This antagonism caused a substantial decrease in cytotoxicity and genotoxicity of the dual nanoparticle system. Intracellular TiO2 nanoparticles were singled out as the “vigilante” causing this reduction in toxicity.DOCTOR OF PHILOSOPHY (MSE

TiO2 nanoparticles alleviate toxicity by reducing free Zn2+ ion in human primary epidermal keratinocytes exposed to ZnO nanoparticles

Nanoparticles have been a subject of intense safety screenings due to their influx in various applications. Although recent studies have reported on the plausible cytotoxicity of nanoparticles, many of these focused only on single-material nanoparticles, while the cytotoxicity of dual-nanoparticle systems (e.g., ZnO with TiO2) has remained unexplored. For example, commercial products like sunscreens and cosmetics contain both nano-sized ZnO and TiO2, but cytotoxicity studies of such systems are meager. In this paper, the cytotoxicity of this dual-nanoparticle system comprising both ZnO and TiO2 was evaluated in vitro on skin-mimicking human primary epidermal keratinocytes (HPEKs). Inductively coupled plasma mass spectrometry, flow cytometry, and confocal microscopy were used to investigate the uptake of nanoparticles and free ions. Results revealed that ZnO nanoparticles were partially soluble (up to 20 μg ml−1 after 1 day) and could induce strong cytotoxicity as compared to the insoluble TiO2 nanoparticles which remained non-toxic until very high concentrations. It was found that TiO2 nanoparticles could play “vigilante” by protecting keratinocytes from acute toxicity of ZnO nanoparticles. This is in agreement with the observation that TiO2 nanoparticles caused an attenuation of free intracellular Zn2+ ions concentration, by adsorbing and immobilizing free Zn2+ ions. This study reveals a unique dual-nanoparticle observation in vitro on HPEKs, and highlights the importance of dual-nanoparticulate toxicity studies, especially in applications where more than one nanoparticle material-type is present.MOH (Min. of Health, S’pore

TiO2-nanoparticles shield HPEKs against ZnO-induced genotoxicity

Usage of sunscreens has become commonplace amongst outdoor sports. Recently, nanomaterials have gained increasing market share as ingredients in sunscreens (as well as other topically applied products). In particular ZnO and TiO2 nanoparticles (ZNP and TNP) have found their niche in this application. This study investigated the safety aspects of these nanoparticles from a combinatorial exposure point of view. Focus was on investigating generation of oxidative stress and induction of DNA damage which the two nanoparticles caused. It was found that TNPs triggered stronger oxidative stress than ZNPs but ZNPs remained more potent at causing DNA damage. The individual mechanisms of DNA damage were found to be through oxidative stress for TNPs (indirect genotoxicity) and through Zn2 + ion nuclear uptake resulting in DNA damage for ZNP (direct genotoxicity). Interesting, it was found that intracellular TNPs could adsorb Zn2 + ions and lower their nuclear uptake in turn shielding the HPEKs from ZNP-induced genotoxicity. Toxicological assessments of dual nanoparticle systems remain an unstudied area and based on the results obtained deserves further consideration

Composite hydrogels in three-dimensional in vitro models

3-dimensional (3D) in vitro models were developed in order to mimic the complexity of real organ/tissue in a dish. They offer new possibilities to model biological processes in more physiologically relevant ways which can be applied to a myriad of applications including drug development, toxicity screening and regenerative medicine. Hydrogels are the most relevant tissue-like matrices to support the development of 3D in vitro models since they are in many ways akin to the native extracellular matrix (ECM). For the purpose of further improving matrix relevance or to impart specific functionalities, composite hydrogels have attracted increasing attention. These could incorporate drugs to control cell fates, additional ECM elements to improve mechanical properties, biomolecules to improve biological activities or any combinations of the above. In this Review, recent developments in using composite hydrogels laden with cells as biomimetic tissue- or organ-like constructs, and as matrices for multi-cell type organoid cultures are highlighted. The latest composite hydrogel systems that contain nanomaterials, biological factors, and combinations of biopolymers (e.g., proteins and polysaccharide), such as Interpenetrating Networks (IPNs) and Soft Network Composites (SNCs) are also presented. While promising, challenges remain. These will be discussed in light of future perspectives toward encompassing diverse composite hydrogel platforms for an improved organ environment in vitro.Agency for Science, Technology and Research (A*STAR)Published versionThis research was supported by the Agency for Science, Technology and Research (A*STAR) under its Acne and Sebaceous Gland Program & Wound Care Innovation for the Tropics IAF-PP (H17/01/a0/008 and H17/01/a0/0L9)

Healing of chronic wounds : an update of recent developments and future possibilities

Chronic wounds are the result of disruptions in the body's usual process of healing. They are not only a source of significant pain and discomfort but also, more importantly, an unguarded port of entry for pathogens into the body. While our current understanding of this phenomenon is far from complete, findings in physiological patterns and advancements in wound healing technologies have helped develop wound management and healing solutions to this long-standing medical challenge. This review presents an overview of known wound healing mechanics, abnormalities that lead to chronic wounds, and a summary of established and new wound healing technologies. Various approaches to heal wounds are discussed, from dermal replacements to advanced biomaterial-based treatments, from cell-, synthetic-, and composite-based approaches to preclinical approaches, which make developing such products possible. While tested breakthrough products are described, the authors focused more on recently developed innovations, which are at varying stages of maturity. The review concludes with a note on future perspectives and opinions on where the field and industry are headed and where they should be.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted versio

Understanding the implications of engineered nanoparticle induced autophagy in human epidermal keratinocytes in vitro

Engineered nanoparticles (NPs) such as TiO2 and ZnO are key UV-blocking ingredients in sunscreens. While toxicological risks of applying these materials are generally regarded as low due to minute levels of penetration across the skin, our understanding of the physiological influence of potential cell-nanoparticle interactions in the skin is limited. This study plugs the current knowledge gap by profiling TiO2 and ZnO NP interaction with primary human epidermal keratinocytes, based on potential levels of NP penetration across the skin. Specific attention was given to profiling real-life relevant levels of exposure, and accurate dosimetry measurements in vitro. ZnO was expectedly more cytotoxic than TiO2. Although both NPs generated Reactive Oxygen Species (ROS) and Mitochondrial Superoxide (MSO) within 4 h exposure to sub-lethal concentrations, ZnO induction of these oxidative stress markers increased much more significantly after 24 h exposure. Exposure to increasing NP concentrations increased autophagy induction along with activation of inflammatory responses in the keratinocytes, primarily through the TRAF6-mediated pathway. Sustained induction of autophagy led to degradation of TRAF6 and, only in the case of TiO2, reduced NF-κB activation. This pro-survival mode of autophagy induction provides further insights into the on-going debate on the use of these NPs in consumer products.Accepted versio

Inhaled nanomaterials and the respiratory microbiome: clinical, immunological and toxicological perspectives

Abstract Our development and usage of engineered nanomaterials has grown exponentially despite concerns about their unfavourable cardiorespiratory consequence, one that parallels ambient ultrafine particle exposure from vehicle emissions. Most research in the field has so far focused on airway inflammation in response to nanoparticle inhalation, however, little is known about nanoparticle-microbiome interaction in the human airway and the environment. Emerging evidence illustrates that the airway, even in its healthy state, is not sterile. The resident human airway microbiome is further altered in chronic inflammatory respiratory disease however little is known about the impact of nanoparticle inhalation on this airway microbiome. The composition of the airway microbiome, which is involved in the development and progression of respiratory disease is dynamic, adding further complexity to understanding microbiota-host interaction in the lung, particularly in the context of nanoparticle exposure. This article reviews the size-dependent properties of nanomaterials, their body deposition after inhalation and factors that influence their fate. We evaluate what is currently known about nanoparticle-microbiome interactions in the human airway and summarise the known clinical, immunological and toxicological consequences of this relationship. While associations between inhaled ambient ultrafine particles and host immune-inflammatory response are known, the airway and environmental microbiomes likely act as intermediaries and facilitate individual susceptibility to inhaled nanoparticles and toxicants. Characterising the precise interaction between the environment and airway microbiomes, inhaled nanoparticles and the host immune system is therefore critical and will provide insight into mechanisms promoting nanoparticle induced airway damage

Biomolecular interaction and kinematics differences between P25 and E171 TiO2 nanoparticles

Titanium dioxide (TiO2) nanoparticles (NPs) are used abundantly as food additives (E171). For the purpose of risk assessment, it is imperative to understand the behavior of these nanoparticles in a food relevant environment, and their consequent toxicology impacts. However, most of such studies use model TiO2 NPs (P25) as substitutes for E171. To understand the suitability of this approach, we investigated the functional behavior of E171 and P25 in solutions of bovine serum albumin (BSA) and sucrose as model food ingredients. Our data showed that E171 were better dispersed in BSA than P25. In sucrose, E171 displayed a reduction in agglomerated size while P25 agglomerated extensively. Adsorption studies showed that P25 attracted more pronounced corona formation per unit mass of material compared to E171. In vitro sedimentation, diffusion and dosimetry (ISDD) results demonstrated that the time-weighted dosage of E171 was more than two-folds higher than P25, implying that any test performed using P25 to model E171 would underestimate actual dosage and potential toxicity. Taken collectively, this study demonstrated the specificity of TiO2 nanoparticle interaction with food ingredients, and the importance of using food-grade E171 TiO2 for food-relevant toxicological assessments.Accepted versio