Analysis of the Influence of Cell Heterogeneity on Nanoparticle Dose Response

Understanding the effect of variability in the interaction of individual cells with nanoparticles on the overall response of the cell population to a nanoagent is a fundamental challenge in bionanotechnology. Here, we show that the technique of time-resolved, high-throughput microscopy can be used in this endeavor. Mass measurement with single-cell resolution provides statistically robust assessments of cell heterogeneity, while the addition of a temporal element allows assessment of separate processes leading to deconvolution of the effects of particle supply and biological response. We provide a specific demonstration of the approach, in vitro, through time-resolved measurement of fibroblast cell (HFF-1) death caused by exposure to cationic nanoparticles. The results show that heterogeneity in cell area is the major source of variability with area-dependent nanoparticle capture rates determining the time of cell death and hence the form of the exposure–response characteristic. Moreover, due to the particulate nature of the nanoparticle suspension, there is a reduction in the particle concentration over the course of the experiment, eventually causing saturation in the level of measured biological outcome. A generalized mathematical description of the system is proposed, based on a simple model of particle depletion from a finite supply reservoir. This captures the essential aspects of the nanoparticle–cell interaction dynamics and accurately predicts the population exposure–response curves from individual cell heterogeneity distributions

Inhibition of human APE1 and MTH1 DNA repair proteins by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles

open access articleAim: To quantitatively evaluate the inhibition of human DNA repair proteins APE1 and MTH1 by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles (dUSPIONs). Materials & methods: Liquid chromatography-tandem mass spectrometry with isotope-dilution was used to measure the expression levels of APE1 and MTH1 in MCL-5 cells exposed to increasing doses of dUSPIONs. The expression levels of APE1 and MTH1 were measured in cytoplasmic and nuclear fractions of cell extracts. Results: APE1 and MTH1 expression was significantly inhibited in both cell fractions at the highest dUSPION dose. The expression of MTH1 was linearly inhibited across the full dUSPION dose range in both fractions. Conclusion: These findings warrant further studies to characterize the capacity of dUSPIONs to inhibit other DNA repair proteins in vitro and in vivo

Prevention and amelioration of erythrocyte instability observed under deficiency of vitamin B12 alone or combined with micronutrient limitation through dietary supplementation with Chlorella and Spirulina

Micronutrient rich microalgae, Chlorella and Spirulina, could be natural food supplements to overcome the micronutrient deficiency, increasingly recognised as a global health issue. In two independent experiments, the Spirulina and Chlorella were evaluated as prophylactic and ameliorative dietary supplements of vitamin B12. Erythrocyte stability (relative osmotic fragility and haemolysis percentage), haematological parameters, micronutrient deficiency (serum levels of iron, zinc), plasma vitamin B12 and vitamin B12 biomarker (methylmalonic acid) were analysed. The deficient groups receiving Spirulina and Chlorella as prophylactic dietary supplements showed a 1.34 to 1.41 folds increase in serum iron and a 2.13 to 2.19 folds increase in plasma vitamin B12, compared to B12 deficient group. Supplementation of Spirulina to ameliorate vitamin B12 deficiency combined with micronutrient limitation showed an increase of 1.14 folds and 1.2 folds in serum iron and zinc respectively and 1.51 folds in plasma vitamin B12 compared to the deficient group. The relative osmotic fragility of erythrocytes in deficient experimental animals was 17 to 45% higher compared to the control. The osmotic fragility and deformation in the morphology of erythrocytes observed under vitamin B12 deficiency, alone or in combination with micronutrient limitation, were prevented and ameliorated on dietary supplementation with the microalgal biomass

Quality by design micro-engineering optimisation of NSAID-loaded electrospun fibrous patches

The purpose of this study was to apply the Quality by Design (QbD) approach to the electrospinning of fibres loaded with the nonsteroidal anti-inflammatory drugs (NSAIDs) indomethacin (INDO) and diclofenac sodium (DICLO). A Quality Target Product Profile (QTPP) was made, and risk assessments (preliminary hazard analysis) were conducted to identify the impact of material attributes and process parameters on the critical quality attributes (CQAs) of the fibres. A full factorial design of experiments (DoE) of 20 runs was built, which was used to carry out experiments. The following factors were assessed: Drugs, voltage, flow rate, and the distance between the processing needle and collector. Release studies exhibited INDO fibres had greater total release of active drug compared to DICLO fibres. Voltage and distance were found to be the most significant factors of the experiment. Multivariate statistical analytical software helped to build six feasible design spaces and two flexible, universal design spaces for both drugs, at distances of 5 cm and 12.5 cm, along with a flexible control strategy. The current findings and their analysis confirm that QbD is a viable and invaluable tool to enhance product and process understanding of electrospinning for the assurance of high-quality fibres

Prevention and amelioration of erythrocyte instability observed under deficiency of vitamin B12 alone or combined with micronutrient limitation through dietary supplementation with Chlorella and Spirulina

7-16Micronutrient rich microalgae, Chlorella and Spirulina, could be natural food supplements to overcome the micronutrient deficiency, increasingly recognised as a global health issue. In two independent experiments, the Spirulina and Chlorella were evaluated as prophylactic and ameliorative dietary supplements of vitamin B12. Erythrocyte stability (relative osmotic fragility and haemolysis percentage), haematological parameters, micronutrient deficiency (serum levels of iron, zinc), plasma vitamin B12 and vitamin B12 biomarker (methylmalonic acid) were analysed. The deficient groups receiving Spirulina and Chlorella as prophylactic dietary supplements showed a 1.34 to 1.41 folds increase in serum iron and a 2.13 to 2.19 folds increase in plasma vitamin B12, compared to B12 deficient group. Supplementation of Spirulina to ameliorate vitamin B12 deficiency combined with micronutrient limitation showed an increase of 1.14 folds and 1.2 folds in serum iron and zinc respectively and 1.51 folds in plasma vitamin B12 compared to the deficient group. The relative osmotic fragility of erythrocytes in deficient experimental animals was 17 to 45% higher compared to the control. The osmotic fragility and deformation in the morphology of erythrocytes observed under vitamin B12 deficiency, alone or in combination with micronutrient limitation, were prevented and ameliorated on dietary supplementation with the microalgal biomass

Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity.

With the need to understand the potential biological impact of the plethora of nanoparticles (NPs) being manufactured for a wide range of potential human applications, due to their inevitable human exposure, research activities in the field of NP toxicology has grown exponentially over the last decade. Whilst such increased research efforts have elucidated an increasingly significant knowledge base pertaining to the potential human health hazard posed by NPs, understanding regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to adequately discriminate between the specific modes of action associated with the onset of genotoxicity. Additionally, in line with the recent European directives, there is an inherent need to move away from invasive animal testing strategies. Thus, in vitro systems are an important tool for expanding our mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their validity and specificity for this purpose due to the unique challenges presented when correlating NP behaviour in vitro and in vivo This review therefore highlights the current state of the art in advanced in vitro systems and their specific advantages and disadvantages from a NP genotoxicity testing perspective. Key indicators will be given related to how these systems might be used or improved to enhance understanding of NP genotoxicity

ACS Nano

open access articleUnderstanding the effect of variability in the interaction of individual cells with nanoparticles on the overall response of the cell population to a nanoagent is a fundamental challenge in bionanotechnology. Here, we show that the technique of time-resolved, high-throughput microscopy can be used in this endeavor. Mass measurement with single-cell resolution provides statistically robust assessments of cell heterogeneity, while the addition of a temporal element allows assessment of separate processes leading to deconvolution of the effects of particle supply and biological response. We provide a specific demonstration of the approach, in vitro, through time-resolved measurement of fibroblast cell (HFF-1) death caused by exposure to cationic nanoparticles. The results show that heterogeneity in cell area is the major source of variability with area-dependent nanoparticle capture rates determining the time of cell death and hence the form of the exposure–response characteristic. Moreover, due to the particulate nature of the nanoparticle suspension, there is a reduction in the particle concentration over the course of the experiment, eventually causing saturation in the level of measured biological outcome. A generalized mathematical description of the system is proposed, based on a simple model of particle depletion from a finite supply reservoir. This captures the essential aspects of the nanoparticle–cell interaction dynamics and accurately predicts the population exposure–response curves from individual cell heterogeneity distributions

Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity

The Publisher's final version can be found by following the DOI link. open access articleWith the need to understand the potential biological impact of the plethora of nanoparticles (NPs) being manufactured for a wide range of potential human applications, due to their inevitable human exposure, research activities in the field of NP toxicology has grown exponentially over the last decade. Whilst such increased research efforts have elucidated an increasingly significant knowledge base pertaining to the potential human health hazard posed by NPs, understanding regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to adequately discriminate between the specific modes of action associated with the onset of genotoxicity. Additionally, in line with the recent European directives, there is an inherent need to move away from invasive animal testing strategies. Thus, in vitro systems are an important tool for expanding our mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their validity and specificity for this purpose due to the unique challenges presented when correlating NP behaviour in vitro and in vivo This review therefore highlights the current state of the art in advanced in vitro systems and their specific advantages and disadvantages from a NP genotoxicity testing perspective. Key indicators will be given related to how these systems might be used or improved to enhance understanding of NP genotoxicity

Engineering and development of chitosan-based nanocoatings for ocular contact lenses

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.The research manuscript reports on Electrohydrodynamic Atomisation (EHDA) to engineer on-demand novel coatings for ocular contact lenses. A formulation approach was adopted to modulate the release of timolol maleate (TM) using chitosan and borneol. Polymers polyvinylpyrrolidone (PVP) and poly (N-isopropylacrylamide) (PNIPAM) were utilised to encapsulate TM and were electrically atomised to produce optimised, stationary contact lens coatings. The particle and fibre diameter, thermal stability, material compatibility of the formed coatings along with their in vitro release-modulating effect and ocular tolerability were investigated. The results demonstrated highly stable nano-matrices with advantageous morphology and size. All formulations yielded coatings with high TM encapsulation (>88%); with excellent ocular biocompatibility. The coatings presented biphasic and triphasic release profiles; depending on composition. Kinetic modelling revealed a noticeable effect of chitosan; the higher the concentration, the more the release of TM due to chitosan swelling; with the release mechanism changing from Fickian diffusion (1% w/v; n = 0.5) to non-Fickian (5% w/v, 0.45 < n < 0.89). The use of EHDA has not yet been explored in depth within the ocular research remit; engineering on demand lens coatings capable of sustaining TM release. This is likely to offer an alternative dosage form for management of glaucoma with particular emphasis on improving poor patient compliance

In vitro detection of in vitro secondary mechanisms of genotoxicity induced by engineered nanomaterials.

BACKGROUND: It is well established that toxicological evaluation of engineered nanomaterials (NMs) is vital to ensure the health and safety of those exposed to them. Further, there is a distinct need for the development of advanced physiologically relevant in vitro techniques for NM hazard prediction due to the limited predictive power of current in vitro models and the unsustainability of conducting nano-safety evaluations in vivo. Thus, the purpose of this study was to develop alternative in vitro approaches to assess the potential of NMs to induce genotoxicity by secondary mechanisms. RESULTS: This was first undertaken by a conditioned media-based technique, whereby cell culture media was transferred from differentiated THP-1 (dTHP-1) macrophages treated with γ-Fe2O3 or Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) to the bronchial cell line 16HBE14o-. Secondly construction and SPION treatment of a co-culture model comprising of 16HBE14o- cells and dTHP-1 macrophages. For both of these approaches no cytotoxicity was detected and chromosomal damage was evaluated by the in vitro micronucleus assay. Genotoxicity assessment was also performed using 16HBE14o- monocultures, which demonstrated only γ-Fe2O3 nanoparticles to be capable of inducing chromosomal damage. In contrast, immune cell conditioned media and dual cell co-culture SPION treatments showed both SPION types to be genotoxic to 16HBE14o- cells due to secondary genotoxicity promoted by SPION-immune cell interaction. CONCLUSIONS: The findings of the present study demonstrate that the approach of using single in vitro cell test systems precludes the ability to consider secondary genotoxic mechanisms. Consequently, the use of multi-cell type models is preferable as they better mimic the in vivo environment and thus offer the potential to enhance understanding and detection of a wider breadth of potential damage induced by NMs