Browsing by Author "Singh, Neenu"
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Item Open Access Analysis of the influence of cell heterogeneity on nanoparticle dose response.(ACS Publications, 2014-07-13) Ware, Matthew J.; Godin, Biana; Singh, Neenu; Majithia, Ravish; Shamsudeen, Sabeel; Serda, Rita E.; Meissner, Kenith E.; Rees, Paul; Summers, Huw D.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.Item Metadata only Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity(Oxford University Press, 2016-11-04) Evans, S. J.; Clifton, M. J.; Singh, Neenu; de Oliveira Mallia, J.; Burgum, M.; Wills, J. W.; Wilkinson, T. S.; Doak, Shareen H.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.Item Open Access Electrosprayed mesoporous particles for improved aqueous solubility of a poorly water soluble anticancer agent: in vitro and ex vivo evaluation(Elsevier, 2018-03-30) Ahmad, Z.; Sayed, E.; Karavasili, Chirstina; Ruparelia, K. C.; Haj-Ahmad, R.; Charalambopoulou, G; Giasafaki, D.; Cox, Paul; Singh, Neenu; Giassafaki, Lefki-Pavlina N.; Mpenekou, A.; Steriotis, T.; Markopoulou, C. K.; Vizirianakis, I. S.; Chang, Ming-Wei; Fatoutos, D. G.Encapsulation of poorly water-soluble drugs into mesoporous materials (e.g. silica) has evolved as a favorable strategy to improve drug solubility and bioavailability. Several techniques (e.g. spray drying, solvent evaporation, microwave irradiation) have been utilized for the encapsulation of active pharmaceutical ingredients (APIs) into inorganic porous matrices. In the present work, a novel chalcone (KAZ3) with anticancer properties was successfully synthesized by Claisen-Schmidt condensation. KAZ3 was loaded into mesoporous (SBA-15 and MCM-41) and non-porous (fumed silica, FS) materials via two techniques; electrohydrodynamic atomization (EHDA) and solvent impregnation. The effect of both loading methods on the physicochemical properties of the particles (e.g. size, charge, entrapment efficiency, crystallinity, dissolution and permeability) was investigated. Results indicated that EHDA technique can load the active in a complete amorphous form within the pores of the silica particles. In contrast, reduced crystallinity (~79%) was obtained for the solvent impregnated formulations. EHDA engineered formulations significantly improved drug dissolution up to 30-fold, compared to the crystalline drug. Ex vivo studies showed EHDA formulations to exhibit higher permeability across rat intestine than their solvent impregnated counterparts. Cytocompatibility studies on Caco-2 cells demonstrated moderate toxicity at high concentrations of the anticancer agent. The findings of the present study clearly show the immense potential of EHDA as a loading technique for mesoporous materials to produce poorly water-soluble API carriers of high payload at ambient conditions. Furthermore, the scale up potential in EHDA technologies indicate a viable route to enhance drug encapsulation and dissolution rate of loaded porous inorganic materials.Item Metadata only Emerging metrology for high-throughput nanomaterial genotoxicology(Oxford Journals, 2016-08-27) Nelson, Bryant C.; Wright, Christa W.; Ibuki, Yuko; Moreno-Villanueva, Maria; Karlsson, Hanna L.; Hendriks, Giel; Sims, Christopher M.; Singh, Neenu; Doak, Shareen H.The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ- H2AX assay, automated ‘Fluorimetric Detection of Alkaline DNA Unwinding’ (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also providedItem Open Access Engineering and development of chitosan-based Nanocoatings for Ocular Contact Lenses(2018-12-01) Mehta, P.; Al-Kinani, Ali A.; Arshad, Muhammad Sohail; Singh, Neenu; van der Merwe, Susanna M.; Chang, Ming-Wei; Alany, Raid G.; Ahmad, Z.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.Item Embargo Engineering of tetanus toxoid-loaded polymeric microneedle patches(Springer, 2022-11-17) Arshad, Muhammad Sohail; Gulfam, Shafaq; Zafar, Saman; Jalil, Najmusama Abdul; Ahmad, Nadia; Qutachi, Omar; Chang, Ming-Wei; Singh, Neenu; Ahmad, ZeeshanThis study is aimed to fabricate tetanus toxoid laden microneedle patches by using a polymeric blend comprising of polyvinyl pyrrolidone and sodium carboxymethyl cellulose as base materials and sorbitol as a plasticizer. The tetanus toxoid was mixed with polymeric blend and patches were prepared by using vacuum micromolding technique. Microneedle patches were evaluated for physical attributes such as uniformity of thickness, folding endurance, and swelling profile. Morphological features were assessed by optical and scanning electron microscopy. In vitro performance of fabricated patches was studied by using bicinchoninic acid assay (BCA). Insertion ability of microstructures was studied in vitro on model skin parafilm and in vivo in albino rat. In vivo immunogenic activity of the formulation was assessed by recording immunoglobulin G (IgG) levels, interferon gamma (IFN-γ) levels, and T-cell (CD4+ and CD8+) count following the application of dosage forms. Prepared patches, displaying sharp-tipped and smooth-surfaced microstructures, remained intact after 350 ± 36 foldings. Optimized microneedle patch formulation showed ~ 74% swelling and ~ 85.6% vaccine release within an hour. The microneedles successfully pierced parafilm. Histological examination of microneedle-treated rat skin confirmed disruption of epidermis without damaging the underneath vasculature. A significant increase in IgG levels (~ 21%), IFN-γ levels (~ 30%), CD4+ (~ 41.5%), and CD8+ (~ 48.5%) cell count was observed in tetanus vaccine-loaded microneedle patches treated albino rats with respect to control (untreated) group at 42nd day of immunization. In conclusion, tetanus toxoid-loaded microneedle patches can be considered as an efficient choice for transdermal delivery of vaccine without inducing pain commonly experienced with hypodermic needles.Item Metadata only Exposure to Engineered Nanomaterials: Impact on DNA Repair Pathways(MDPI, 2017-07-13) Singh, Neenu; Nelson, Bryant C.; Scanlan, Leona D.; Coskun, Erdem; Jaruga, Pawel; Doak, Shareen H.Some engineered nanomaterials (ENMs) may have the potential to cause damage to the genetic material in living systems. The mechanistic machinery functioning at the cellular/molecular level, in the form of DNA repair processes, has evolved to help circumvent DNA damage caused by exposure to a variety of foreign substances. Recent studies have contributed to our understanding of the various DNA damage repair pathways involved in the processing of DNA damage. However, the vast array of ENMs may present a relatively new challenge to the integrity of the human genome; therefore, the potential hazard posed by some ENMs necessitates the evaluation and understanding of ENM-induced DNA damage repair pathways. This review focuses on recent studies highlighting the differential regulation of DNA repair pathways, in response to a variety of ENMs, and discusses the various factors that dictate aberrant repair processes, including intracellular signalling, spatial interactions and ENM-specific responses.Item Open Access In vitro detection of in vitro secondary mechanisms of genotoxicity induced by engineered nanomaterials(BMC, 2019-02-13) Singh, Neenu; Evans, S. J.; Clift, M.J.D.; Wills, J. W.; Hondow, Nicole; Wilkinson, T. S.; Burgum, M.J.; Brown, A.P.; Jenkins, Gareth J. S.; Doak, Shareen H.Abstract 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.Item Open Access Influence of Critical Parameters on Cytotoxicity Induced by Mesoporous Silica Nanoparticles(MDPI, 2022-06-11) Amirsadra, Ahmadi; Sokunbi, Moses; Patel, Trisha; Ahmad, Zeeshan; Singh, NeenuMesoporous Silica Nanoparticles (MSNs) have received increasing attention in biomedical applications due to their tuneable pore size, surface area, size, surface chemistry, and thermal stability. The biocompatibility of MSNs, although generally believed to be satisfactory, is unclear. Physicochemical properties of MSNs, such as diameter size, morphology, and surface charge, control their biological interactions and toxicity. Experimental conditions also play an essential role in influencing toxicological results. Therefore, the present study includes studies from the last five years to statistically analyse the effect of various physicochemical features on MSN-induced in-vitro cytotoxicity profiles. Due to non-normally distributed data and the presence of outliers, a Kruskal-Wallis H test was conducted on different physicochemical characteristics, including diameter sizes, zeta-potential measurements, and functionalisation of MSNs, based on the viability results, and statistical differences were obtained. Subsequently, pairwise comparisons were performed using Dunn's procedure with a Bonferroni correction for multiple comparisons. Other experimental parameters, such as type of cell line used, cell viability measurement assay, and incubation time, were also explored and analysed for statistically significant results.Item Open Access Inhibition of human APE1 and MTH1 DNA repair proteins by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles(Future Medicine, 2023-02-28) Singh, Neenu; Erdem, Coskun; Leona, Scanlan; Pawel, Jaruga; Shareen, Doak; Miral, Dizdaroglu; Bryant, NelsonAim: 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.Item Open Access Quantum Dot induced Cellular Perturbations Involving Varying Toxicity Pathways.(Royal Society of Chemistry, 2015-01-06) Al Ali, A.; Singh, Neenu; Manshian, Bella; Wilkinson, Tom; Jenkins, Gareth J. S.; Doak, Shareen H.The unique optical and electronic properties of quantum dots (QD) have led to rapid progress in their development and application, particularly in innovative therapeutic and diagnostic products. Along with the great pace at which QD are being developed, research is being focussed on fabricating less toxic QD with novel surface functionalities. The present study was therefore focused on assessing the impact of varying QD surface chemistry on cellular uptake and a range of indicators for cell perturbation following exposure. The study demonstrated that despite a low intrinsic cytotoxicity of three QD with different surface functional groups, they were all capable of inducing an acute inflammatory response and alterations in transcriptional gene activity, without affecting cell cycle regulation. Further, this investigation demonstrated that although the QD were capable of inducing an inflammatory and oxidative stress response, there was clearly variation in the degree of molecular change according to surface chemistry, which correlated with the degree of cellular uptake. These findings therefore highlight the potential for chronic inflammatory responses following exposure to QD, but in addition, they also demonstrate the importance of studying a wide range of toxicity pathways to generate a comprehensive picture of biological response to nanomaterials.Item Open Access Reprint of: A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.(Elsevier, 2018-08-18) Shah, U.K.; Mallia, J.O.; Singh, Neenu; Doak, Shareen H.; Jenkins, Gareth J. S.The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.Item Open Access A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.(Elsevier, 2017-12-15) Shah, U.K.; Mallia, J.O.; Chapman, K.E.; Doak, Shareen H.; Jenkins, Gareth J. S.; Singh, Neenuhe liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.Item Embargo Three-dimensional Models for In vitro Nanotoxicity Testing(Royal Society of Chemistry, 2017-11-13) Doak, Shareen H.; Wills, J. W.; Singh, NeenuThe intrinsic limitations and pitfalls exhibited by two-dimensional (2D) in vitro studies in conjunction with the ethical issues surrounding in vivo (nano)toxicology testing on animals have prompted the need to develop three-dimensional (3D) models. This chapter discusses the problems faced when using monolayer cell cultures for safety assessment, which led to the rationale behind the development of 3D models for safety evaluation purposes. The additional benefits of 3D models for nanosafety are presented, coupled with an overview of some of the 3D in vitro models that have been developed to mimic the physiological environment of certain tissues/organs including liver, lung, colon, intestine and gastrointestinal tract.