Mitochondrial dysfunction in titanium dioxide nanoparticle-induced neurotoxicity

Abstract

Nanotechnology has emerged as a field of scientific innovation which has opened up a plethora of concerns for the potential impact on human and environment. Various toxicological studies have confirmed that nanoparticles (NPs) can be potentially hazardous because of their unique small size and physico-chemical properties. With the wide applications of titanium dioxide nanoparticles (TNPs) in day-to-day life in form of cosmetics, paints, sterilization and so on, there is growing concern regarding the deleterious effects of TNPs on central nervous system. Mitochondria is an important origin for generation of energy as well as free radicals and these free radicals can lead to mitochondrial damage and finally lead to apoptosis. The objective of our study was to elucidate the potential neurotoxic effect of TNPs in anatase form. Oxidative stress was determined by measuring lipid peroxidation and protein carbonyl content which was found to be significantly increased. Reduced glutathione content and major glutathione metabolizing enzymes were also modulated signifying the role of glutathione redox cycle in the pathophysiology of TNPs. Mitochondrial complexes were also modulated from the exposure to TNPs. The present study indicates that nanosize TNPs may pose a health risk to mitochondrial brain with the generation of reactive oxygen species, and thus NPs should be carefully used.     

Application of Bayesian networks for hazard ranking of nanomaterials to support human health risk assessment

In this study, a Bayesian Network (BN) was developed for the prediction of the hazard potential and biological effects with the focus on metal- and metal-oxide nanomaterials to support human health risk assessment. The developed BN captures the (inter) relationships between the exposure route, the nanomaterials physicochemical properties and the ultimate biological effects in a holistic manner and was based on international expert consultation and the scientific literature (e.g., in vitro/in vivo data). The BN was validated with independent data extracted from published studies and the accuracy of the prediction of the nanomaterials hazard potential was 72% and for the biological effect 71%, respectively. The application of the BN is shown with scenario studies for TiO₂, SiO₂, Ag, CeO₂, ZnO nanomaterials. It is demonstrated that the BN may be used by different stakeholders at several stages in the risk assessment to predict certain properties of a nanomaterials of which little information is available or to prioritize nanomaterials for further screening.     

Formulation,characterization, and cellular toxicity assessment of tamoxifen-loaded silk fibroin nanoparticles in breast cancer

Silk fibroin (SF) is a natural polymeric biomaterial that is widely adopted for the preparation of drug delivery systems. Herein, we aimed to fabricate and characterize SF nanoparticles loaded with the selective estrogen receptor modulator; tamoxifen citrate (TC-SF-NPs) and to assess their in vitro efficacy against breast cancer cell lines (MCF-7 and MDA-MB-231). TC-loaded SF-NPs were characterized for particle size, morphology, entrapment efficiency, and release profile. In addition, we examined the in vitro cytotoxicity of TC-SF-NPs against human breast cancer cell lines and evaluated the anticancer potential of TC-SF-NPs through apoptosis assay and cell cycle analysis. Drug-loaded SF-NPs showed an average particle size of 186.1 ± 5.9 nm and entrapment efficiency of 79.08%. Scanning electron microscopy (SEM) showed the nanoparticles had a spherical morphology with smooth surface. Tamoxifen release from SF-NPs exhibited a biphasic release profile with an initial burst release within the first 6 h and sustained release for 48 h. TC-SF-NPs exerted a dose-dependent cytotoxic effect against breast cancer cell lines. In addition, flow cytometry analysis revealed that cells accumulate in G₀/G₁ phase, with a concomitant reduction of S- and G₂-M-phase cells upon treatment with TC-SF-NPs. Consequently, the potent anticancer activities of TC-SF-NPs against breast cancer cells were mainly attributed to the induction of apoptosis and cell cycle arrest. Our results indicate that SF nanoparticles may represent an attractive nontoxic nanocarrier for the delivery of anticancer drugs.     

The Application of Machine Learning Techniques as an Adjunct to Clinical Decision Making in Alcohol Dependence Treatment

With few exceptions, research in the addictive sciences has relied on linear statistics and methodologies. Addiction involves a complex array of nonlinear behaviors. This study applies two machine learning techniques, Bayesian and decision tree classifiers, in the assessment of outcome of an alcohol dependence treatment program. These nonlinear approaches are compared to a standard linear analysis. Seventy-three alcohol-dependent subjects undertaking a 12-week cognitive-behavioral therapy (CBT) program and 66 subjects undertaking an identical program but also prescribed the relapse prevention agent Acamprosate were employed in this study. Demographic, alcohol use, dependence severity, craving, health-related quality of life, and psychological measures at baseline were used to predict abstinence at 12 weeks. Decision trees had a 77% predictive accuracy across both data sets, Bayesian networks 73%, and discriminant analysis 42%. Combined with clinical experience, machine learning approaches offer promise in understanding the complex relationships that underlie treatment outcome for abstinence-based alcohol treatment programs.     

槲皮素PLGA-TPGS纳米粒在肝癌细胞中的摄取及其细胞抑制率的研究

目的：考察槲皮素（quercetin,QT）/香豆素-6（coumarin-6,C6）乙交酯丙交酯共聚物-维生素E聚乙二醇1000琥珀酸酯（polylactide-co-glycolide-D-α-tocopheryl polyethylene glycol 1000 succinate,PLGA-TPGS）纳米粒（QT/C6-loaded PLGA-TPGS nanoparticles,QCPTN）体外分别被人肝癌细胞株HepG2和小鼠腹水型高淋巴道转移肝癌细胞HCa-F的摄取情况,以及研究槲皮素PLGA-TPGS纳米粒（QT-loaded PLGA-TPGS nanoparticles,QPTN）体外对HepG2细胞的生长抑制率。方法：用激光共聚焦显微镜考察2种肝癌细胞HepG2和HCa-F对QCPTN的体外细胞摄取情况。采用WST-1法体外研究QPTN和槲皮素PLGA纳米粒（QT-loaded PLGA nanoparticles,QPN）对HepG2细胞生长的抑制性。试验分为6组,分别为阴性对照组、空白纳米粒（empty PLGA-TPGS nanoparticles,EPTN）组、氟尿嘧啶溶液（fluorouracil solutions,FS）组、槲皮素溶液（quercetin solutions,QTS）组、QPN组、QPTN组,分别在培育24、48、72 h加入WST-1后在酶标仪上450 nm下测定吸光度值,计算对HepG2细胞的生长抑制率。结果：在2种不同类型肝癌细胞摄取试验中,激光共聚焦显微镜镜下均可见显绿色荧光的QCPTN分布在2种肝癌细胞的细胞核周围,表明QCPTN可被HepG2和HCa-F细胞分别摄取进入细胞内。体外肝癌细胞生长抑制率试验结果表明,自制材料PLGA-TPGS对HepG2细胞生长无明显的抑制性;QPN和QPTN的体外对HepG2细胞生长抑制率有浓度依赖性和时间依赖性;QPTN对HepG2细胞的抑制率大于QPN、QTS和FS。结论： QCPTN通过细胞摄取作用可将模型药物QT和荧光标记物C6同时带入HepG2和HCa-F细胞内,QPTN体外显示对HepG2细胞生长具有较强的抑制性,与QPN、QTS和FS相比,具有较好的抑制肝癌细胞生长的作用。     

汉黄芩素固体脂质纳米粒的制备及体外释放度考察

目的:制备汉黄芩素固体脂质纳米粒并对其体外释放度进行考察.方法:采用乳化分散-超声法制备汉黄芩素固体脂质纳米粒,以包封率和载药量为评价指标,进行正交试验筛选最优处方,并对最优处方的外观、粒径和体外释放度进行考察.结果:制得的纳米粒为均一球形,平均粒径为(153 ±34)nm,其平均载药量为(60.53±2.17)％,平均包封率为(85.54±4.16)％,48 h累积释放达80％.结论:本试验获得了较理想的汉黄芩素固体脂质纳米粒,其体外释放具有缓释作用.     

Three-dimensional spheroid cell culture of human MSC-derived neuron-like cells: New in vitro model to assess magnetite nanoparticle-induced neurotoxicity effects

As nanoparticles (NPs) can access the brain and impact on CNS function, novel in vitro models for the evaluation of NPs-induced neurotoxicity are advocated. Three-dimensional spheroids of primary neuron-like cells (hNLCs) of human origin have been generated, from differentiation of human umbilical cord mesenchymal stem cells (MSCs). The study evaluated Fe₃O₄NP impact on the differentiation process by applying the challenge at complete 3D hNLC spheroid formation (after 4 days, T4) or at beginning of neurogenic induction/simultaneously 3D forming (T0). Different endpoints were monitored over time (up to 10 days): spheroid growth, size, morphology, ATP, cell death, neuronal markers (β-Tub III, MAP-2, and NSE), NP uptake. At T0 application, a marked concentration- and time-dependent cell mortality occurred: effect started early (day 2) and low concentration (1 μg/ml) and exacerbated (80% mortality) after prolonged time (day 6) and increased concentrations (50 μg/ml). ATP was strikingly affected. All neuronal markers were downregulated, and spheroid morphology altered in a concentration-dependent manner (from ≥5 μg/ml) after day 2. Fe₃O₄NPs applied at complete 3D formation (T4) still induced adverse effects although less severe: cell mortality (20–60%) and ATP content decrease (10–40%) were observed in a concentration-dependent manner (from ≥ 5 μg/ml). A neuronal-specific marker effect and spheroid size reduction from 25 μg/ml without morphology alteration were evidenced. This finding provides additional information on neurotoxic effects of Fe₃O₄NPs in a new 3D hNLC spheroid model derived from MSCs that could find a consistent application as in a testing strategy serving in first step hazard identification for correct risk assessment.     

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.     

In vitro release characteristics and cellular uptake of poly(D,L-lactic-co-glycolic acid) nanoparticles for topical delivery of antisense oligodeoxynucleotides

The efficacy of antisense oligodeoxynucleotides (AsODNs) is compromised by their poor stability in biological fluids and the inefficient cellular uptake due to their size and negative charge. Since chemical modifications of these molecules have resulted in a number of non-antisense activities, incorporation into particulate delivery systems has offered a promising alternative. The aim of this study was to evaluate various poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles for AsODN entrapment and delivery. PLGA nanoparticles were prepared using the double emulsion solvent evaporation method. The influence of formulation parameters such as PLGA concentration and volume ratio of internal aqueous phase volume (Va1) to organic phase volume (Vo) to external aqueous phase volume (Va2) on particle size, polydispersity index (PDI) and zeta potential (ZP) was investigated using a full factorial study. The particle size increased with increasing PLGA concentrations and volume ratios, with an interaction detectable between the two factors. AsODN entrapment efficiencies ranged between 49.97% and 54.95% with no significant difference between various formulations. By fitting the in vitro release profiles to a dual first order release model it was shown that the AsODN release occurred via two processes: a diffusion controlled process in the early phase (25 to 32% within one day) and a PLGA degradation process in the latter (39 to 70% after 14 days). Cellular uptake studies using primary corneal epithelial cells suggested active transport of nanoparticles via endocytosis. PLGA nanoparticles therefore show potential to successfully entrap AsODNs, transport them into cells and release them over time due to polymer erosion.     

The in Vitro Cell Association of Invasin Coated Polylactide-Co-glycolide Nanoparticles

Purpose. To determine the effect of particle size and ligand surface density on the cellular association of poly lactide-co-glycolide nanoparticles covalently coated with bacterial invasin. Methods. Poly lactide-co-glycolide nanoparticles containing a flourescent probe were prepared at four diameters 155nm, 200nm, 375nm and 600nm using standard techniques. Bacterial invasin was covalently coupled to the particles surface at varying surface concentrations using a water soluble carbodiimide. The modified particle's cellular association with HEp2 2B cells in tissue culture was determined using flow cytometry. Results. Cellular association of modified nanoparticles was time dependent, abolished at low temperature, competitively inhibited by free invasin or the RGD peptide ligand and saturable. Increased cell association was produced by increasing the particle's surface invasin concentration however, this effect was size dependent. Small particles (155 nm and 200 nm) exhibiting a maximal association with increasing invasin concentration whilst the larger particles (375 nm and 600 nm) provide a minimum at low invasin concentrations. Conclusions. Modified particle cell association provided results commensurate with a receptor dependent uptake mechanism related to the presence of invasin. The size and surface concentration dependency however illustrate that application of these ligands to particulate drug delivery or targeting systems will be controlled by their natural cellular association properties.     

N-琥珀酰壳聚糖纳米粒的制备及体外评价

目的制备N-琥珀酰壳聚糖纳米粒并对其进行体外评价。方法采用乳化溶剂挥发法制备N-琥珀酰壳聚糖纳米粒;以包封率、载药量及粒径为指标,采用正交设计法对处方进行优化;考察其理化特征及体外释药行为。结果纳米粒包封率及载药量分别为62.36%和18.98%,平均粒径及zeta电位分别为(206.6±64.7)nm和(-27.2±0.2)mV;1 h药物释放达到45%,随后药物的释药行为是一个缓释过程。结论作者采用乳化溶剂挥发法成功制得N-琥珀酰壳聚糖纳米粒。该方法制得纳米粒包封率较高,制备工艺简单。     

Simple in vitro models can predict pulmonary toxicity of silver nanoparticles

To study the effects of nanomaterials after inhalation, a large number of in vitro lung models have been reported in literature. Although the in vitro models contribute to the reduction of animal studies, insufficient data exists to determine the predictive value of these in vitro models for the in vivo situation. The aim of this study was to determine the correlation between in vitro and in vivo data by comparing the dose metrics of silver nanoparticles in an in vitro lung model of increasing complexity to our previously published in vivo inhalation study. In vivo, the previously published study showed that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation. The results of the present study show that particle surface area is a suitable dose metric to describe the effects of silver nanoparticles when using a simple monolayer of lung epithelial cells. The dose metric shifted from particle surface area to particle mass when adding an increasing number of macrophages. In addition, a co-culture of endothelial cells, epithelial cells and macrophages on a Transwell® insert correlated less well to the in vivo results compared to the epithelial monolayer. We conclude that for studying the acute pulmonary toxicity of nanoparticles simple in vitro models using an epithelial monolayer better predict the in vivo response compared to complex co-culture models.     

胰岛素壳聚糖胶态纳米粒的制备及体外释药性能

采用离子趋向凝胶化法制备了胰岛素壳聚糖胶态微粒,并考察了外观、粒径和体外释药性能.所得产品呈球形,表面光滑圆整,平均粒径为276nm,多分散系数为0.08.体外释药呈pH依赖性,释药速度受载药量及泊洛沙姆188含量的影响.     

功能化氧化石墨烯负载氟尿嘧啶制备及体外释放研究

目的 通过β环糊精(β-CD)功能化氧化石墨烯(graphene oxide,GO)负载氟尿嘧啶(5-Fu),以增加5-Fu释放时间。方法 采用改进Hummers方法制备GO,使GO与β-CD酯化得到功能化GO-β-CD负载5-Fu。采用高效液相色谱法(HPLC)测定GO-β-CD-5-Fu的体外释放度。利用紫外、傅立叶变换红外、透射电镜、扫描电镜和拉曼光谱分别对GO,GO-β-CD,GO-β-CD-5-Fu表征测定。结果 GO功能化、负载均成功,负载率为105%。HPLC测定结果表明GO-β-CD-5-Fu突释效应减弱,呈梯度释放。T_1/2为(4.5±0.08)d。结论 GO-β-CD负载5-Fu成功,体外释放时间增加,T1/2提高了324倍。     

Bayesian反馈法研究左氧氟沙星在呼吸系统感染患者中药动学及药效学

目的研究两种给药方案中左氧氟沙星在呼吸系统感染患者中药动学特性。方法患者静脉输注左氧氟沙星200m g,一天二次(组1)或400m g,一天一次(组2),高效液相色谱法测定血中左氧氟沙星浓度;B ayes ian反馈法拟合药动学参数。结果根据药-时数据得到组1和组2中左氧氟沙星峰浓度(Cm ax)分别为(2.80±0.29)和(5.27±0.68)m g/L;AUC0~24分别为(30.17±4.06)和(37.81±7.50)(m g.h)/L;消除半衰期t1/2β为(7.10±0.36)和(7.37±0.21)h;表观分布容积(V d)为(96.75±16.82)和(101.20±26.01)L;中央室分布容积(V c)为(66.45±6.76)和(64.63±7.41)L;血清清除率(C l)为(148.47±41.43)和(142.04±25.40)m l/h。统计学处理结果表明两组间的Cm ax和AUC0~24值有显著性差异(P<0.001),t1/2β、V d、V c和C l无差异(P>0.05)。药动学和药效学研究结果显示,两种给药方案中LVLX对于肺炎克雷伯菌、肠杆菌、变形菌、β-溶血链球菌、不动杆菌、嗜血杆菌、肺炎链球菌和厌氧菌的AUC0~24/M IC90值均大于30;对肺炎克雷伯菌和变形菌的AUC0~24/M IC90值大于240。结论组2的Cm ax和AUC0~24/M IC90值均明显高于组1,且两组均未发生不良反应,组2的给药方案更方便。     

Assessment of the in vitro genotoxicity of TiO2 nanoparticles in a regulatory context

A review of in vitro genotoxicity studies on titanium dioxide nanoparticles (TiO₂-NPs) published between 2010 and 2016 was performed by France in the framework of the CLP Regulation 1272/2008/EC. Neither the few in vivo studies of low quality nor the larger number of acceptable in vitro studies available for genotoxicity allowed France to conclude on the genotoxicity of TiO₂-NPs. Based on this work, it was decided to compare the acceptable in vitro studies to understand the reasons for the diverging results observed, such as the materials tested or of the protocols used and their inherent interferences. The systematic review performed on in vitro genotoxicity data for TiO₂-NPs was then restricted to studies with the highest level of confidence among studies following OECD guidelines and the largely applied comet assay. Indeed, the aim of this article is to understand why, even if judged of good quality, the 36 publications selected and analyzed did not lead to a clear picture. Some recommendations to be taken into account before performing new in vitro genotoxicity assays for insoluble particles such as TiO₂-NPs are proposed. Although secondary genotoxic effects consequent to oxidative stress seem to be the major mechanism responsible for the genotoxicity of TiO₂-NPs reported in some studies, primary genotoxic effects cannot be excluded. Further studies are needed to clarify the exact mode of action of TiO₂-NPs and to highlight which physicochemical properties lead to their genotoxicity in vitro to ultimately identify a specific combination of parameters that could represent a risk in vivo.     

Solid lipid nanoparticles for transdermal delivery of diclofenac sodium: preparation,characterization and in vitro studies

The aim of this study was to prepare diclofenac sodium (DNa) solid lipid nanoparticles (SLNs) by a modified emulsion/solvent evaporation method for transdermal delivery. Five independent processing parameters including the lipid matrix, emulsifiers, co-emulsifiers, water-dispersed phase and organic phase were assessed systematically to enhance the entrapment of DNa. The SLNs produced by optimal formulation were submicrometre size with low polydispersity index, the entrapment efficiency was about 89% and the drug loading was about 9.5%. Shape and surface morphology were determined by transmission electron microscopy, which revealed the fairly spherical and core-shell shapes of the SLNs. The in vitro release of SLNs showed a two-step release pattern: one initial burst release followed by a second slow-release phase. In the in vitro cutaneous permeation studies, value of flux obtained for DNa solution was higher than that of SLNs suspension. SLNs had also been shown to improve the dermal localization of DNa.     

Probabilistic hazard assessment for skin sensitization potency by dose–response modeling using feature elimination instead of quantitative structure–activity relationships

Supervised learning methods promise to improve integrated testing strategies (ITS), but must be adjusted to handle high dimensionality and dose–response data. ITS approaches are currently fueled by the increasing mechanistic understanding of adverse outcome pathways (AOP) and the development of tests reflecting these mechanisms. Simple approaches to combine skin sensitization data sets, such as weight of evidence, fail due to problems in information redundancy and high dimensionality. The problem is further amplified when potency information (dose/response) of hazards would be estimated. Skin sensitization currently serves as the foster child for AOP and ITS development, as legislative pressures combined with a very good mechanistic understanding of contact dermatitis have led to test development and relatively large high‐quality data sets. We curated such a data set and combined a recursive variable selection algorithm to evaluate the information available through in silico, in chemico and in vitro assays. Chemical similarity alone could not cluster chemicals' potency, and in vitro models consistently ranked high in recursive feature elimination. This allows reducing the number of tests included in an ITS. Next, we analyzed with a hidden Markov model that takes advantage of an intrinsic inter‐relationship among the local lymph node assay classes, i.e. the monotonous connection between local lymph node assay and dose. The dose‐informed random forest/hidden Markov model was superior to the dose‐naive random forest model on all data sets. Although balanced accuracy improvement may seem small, this obscures the actual improvement in misclassifications as the dose‐informed hidden Markov model strongly reduced " false‐negatives" (i.e. extreme sensitizers as non‐sensitizer) on all data sets. Copyright © 2015 John Wiley & Sons, Ltd.     

Optimization of paclitaxel-loaded poly (d,l-lactide-co-glycolide-N-p-maleimido benzoic hydrazide) nanoparticles size using artificial neural networks

Abstract

The aim of this study was to find a model using artificial neural networks (ANNs) to predict PLGA-PMBH nanoparticles (NPs) size in preparation by modified nanoprecipitation. The input variables were polymer content, drug content, power of sonication and ratio of organic/aqueous phase (i.e. acetone/water), while the NPs size of PLGA-PMBH was assumed as the output variable. Forty samples of PLGA-PMBH NPs containing anticancer drug (i.e. paclitaxel) were synthesized by changing the variable factors in the experiments. The data modeling were performed using ANNs. The effects of input variables (namely, polymer content, drug content, power of sonication and ratio of acetone/water) on the output variables were evaluated using the 3D graphs obtained after modeling. Contrasting the 3D graphs from the generated model revealed that the amount of polymer (PLGA-PMBH) and drug content (PTX) have direct relation with the size of polymeric NPs in the process. In addition, it was illustrated that the ratio of acetone/water was the most important factor affecting the particle size of PLGA-PMBH NPs provided by solvent evaporation technique. Also, it was found that increasing the sonication power (up to a certain amount) indirectly affects the polymeric NPs size however it was directly affected in higher values.