The ecotoxicology of nanoparticles and nanomaterials: current status,knowledge gaps,challenges, and future needs

This paper introduces a special issue on the ecotoxicology and environmental chemistry of nanoparticles (NPs), and nanomaterials (NMs), in the journal Ecotoxicology. There are many types of NMs and the scientific community is making observations on NP ecotoxicity to inform the wider debate about the risks and benefits of these materials. Natural NPs have existed in the environment since the beginning of Earth's history, and natural sources can be found in volcanic dust, most natural waters, soils and sediments. Natural NPs are generated by a wide variety of geological and biological processes, and while there is evidence that some natural NPs can be toxic, organisms have also evolved in an environment containing natural NPs. There are concerns that natural nano-scale process could be influenced by the presence of pollution. Manufactured NPs show some complex colloid and aggregation chemistry, which is likely to be affected by particle shape, size, surface area and surface charge, as well as the adsorption properties of the material. Abiotic factors such as pH, ionic strength, water hardness and the presence of organic matter will alter aggregation chemistry; and are expected to influence toxicity. The physico-chemistry is essential to understanding of the fate and behaviour of NPs in the environment, as well as uptake and distribution within organisms, and the interactions of NPs with other pollutants. Data on biological effects show that NPs can be toxic to bacteria, algae, invertebrates and fish species, as well as mammals. However, much of the ecotoxicological data is limited to species used in regulatory testing and freshwater organism. Data on bacteria, terrestrial species, marine species and higher plants is particularly lacking. Detailed investigations of absorption, distribution, metabolism and excretion (ADME) remain to be performed on species from the major phyla, although there are some data on fish. The environmental risk assessment of NMs could be performed using the existing tiered approach and regulatory framework, but with modifications to methodology including chemical characterisation of the materials being used. There are many challenges ahead, and controversies (e.g., reference substances for ecotoxicology), but knowledge transfer from mammalian toxicology, colloid chemistry, as well as material and geological sciences, will enable ecotoxicology studies to move forward in this new multi-disciplinary field.     

The effects of serum on the toxicity of manufactured nanoparticles

The aim of the present study was to assess the effects of the presence and absence of serum in NP suspension media in relation to their cytotoxicity, as well as their potential to cause oxidative stress and stimulate pro-inflammatory cytokine release from J774.A1 murine ‘macrophage-like’ cells. Different sized (20 nm and 200 nm) carboxylated, fluorescent, model polystyrene beads (PBs) at concentrations from 12.5 μg ml⁻¹ to 100 μg ml⁻¹ were used. Both 20 nm and 200 nm PBs, independent of the suspension media, were observed to cause limited, yet significant (p < 0.05) cytotoxicity over 48 h up to 100 μg ml⁻¹. Significant differences (p > 0.05) were also found between NP size and serum content of the suspension media used. The smaller sized PBs were found to affect intracellular glutathione (GSH) levels, causing a significant loss (p < 0.05) in GSH when suspended in the presence of serum. Subsequent analysis also showed significant (p < 0.05) increases in tumour necrosis factor-α production after 48 h when the 20 nm PBs were suspended in both the presence and absence of serum, compared to the affects observed by the larger, 200 nm sized PBs. In conclusion, the results of the present study show that the interaction of NPs with serum can significantly affect their resultant toxicity in vitro.     

Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing

Based on a literature review and an overview of toxic effects of engineered nanoparticles in aquatic invertebrates, this paper proposes a number of recommendations for the developing field of nanoecotoxicology by highlighting the importance of invertebrates as sensitive and relevant test organisms. Results show that there is a pronounced lack of data in this field (less than 20 peer-reviewed papers are published so far), and the most frequently tested engineered nanoparticles in invertebrate tests are C(60), carbon nanotubes, and titanium dioxide. In addition, the majority of the studies have used Daphnia magna as the test organism. To date, the limited number of studies has indicated acute toxicity in the low mg l(-1) range and higher of engineered nanoparticles to aquatic invertebrates, although some indications of chronic toxicity and behavioral changes have also been described at concentrations in the high microg l(-1) range. Nanoparticles have also been found to act as contaminant carriers of co-existing contaminants and this interaction has altered the toxicity of specific chemicals towards D. magna. We recommend that invertebrate testing is used to advance the level of knowledge in nanoecotoxicology through standardized short-term (lethality) tests with invertebrates as a basis for investigating behaviour and bioavailability of engineered nanoparticles in the aquatic environment. Based on this literature review, we further recommend that research is directed towards invertebrate tests employing long-term low exposure with chronic endpoints along with more research in bioaccumulation of engineered nanoparticles in aquatic invertebrates.     

Ecotoxicity test methods and environmental hazard assessment for engineered nanoparticles

This paper considers whether current standard ecotoxicity methods are fit for purpose for assessing the hazards of engineered nanoparticles. We conclude that the types of test species and biological endpoints used within standard environmental hazard assessment frameworks are generally appropriate. However, there are areas of considerable uncertainty associated with characterisation of nanoparticle exposure in test systems that apply to all ecotoxicity testing guidelines, except those in which dosing of nanoparticles is oral. These include the way in which the substance is dosed into, and maintained within, the test medium; measurement and characterisation of nanoparticles in the test system; better understanding and reporting of abiotic factors that influence behaviour of nanoparticles in the test medium; and agreement on how dosimetric data should be reported.     

Acute toxicity assessment of perfluorinated carboxylic acids towards the Baltic microalgae

The presence of high-energy carbon–fluorine bonds in perfluoro compounds lends them great stability and causes them to be environmentally persistent. Relatively little is known about the acute toxicity of perfluorinated carboxylic acids (PFCAs) to ecotoxicological markers such as aquatic plants and animals. This study tested the toxicity of these compounds to the green alga Chlorella vulgaris, the diatom Skeletonema marinoi and the blue-green alga Geitlerinema amphibium, which are species representative of the algal flora of the Baltic Sea. The EC₅₀ values obtained range from 0.28 mM to 12.84 mM. A distinct relationship between hydrophobicity and toxicity is demonstrated. For every extra perfluoromethylene group in the alkyl chain, the toxicity increases twofold. Log EC₅₀ values are very well correlated linearly with both the number of carbon atoms in the perfluoroalkyl chain and the partition coefficients. The results also indicate that there are clear differences between the responses of particular taxonomic groups of algae: blue-green algae and diatoms are far more sensitive to PFCAs than green algae, probably because of differences in cell wall structure.     

Regulatory perspective on the importance of ADME assessment of nanoscale material containing drugs

The promise of nanoscale material containing drug products to treat complex diseases is mounting. According to the literature, in addition to the liposomes, micelles, emulsions, there are novel drug delivery systems such as dendrimers and metal colloids at different stages of pre-clinical and clinical development. With the anticipation that more nanoscale material containing drug products will be submitted to the Food and Drug Administration (FDA) for approval in the future, FDA formed a Nanotechnology Task Force in 2006 to determine the critical regulatory issues regarding nanomaterials. As a result, all centers within the FDA are considering the development of guidance documents to address nanomaterial specific issues.It is well established in the literature that physico-chemical characterization (PCC) studies are crucial for nanomaterial containing drug products. However, this paper addresses the equally important topic of Absorption, Distribution, Metabolism and Excretion (ADME) studies for nanomaterials and provides examples of how physical properties affect biodistribution (i.e. the state of agglomeration, or aggregation, surface characteristics, stability of PEG). This paper also attempts to highlight some of the ADME study design issues related to nanomaterials such as the need for conducting biodistribution studies on each moiety of the multifunctional nanoparticles, dual labeled pharmacokinetic (PK) studies, and comparative PK studies on the free versus encapsulated drugs. In addition, this paper underlines the importance of long-term biodistribution and mass balance studies to understand the nanoparticle accumulation profile which may help to assess the safety and efficacy of the nanomaterial containing drug products. This review also lists some of the pre-clinical guidance documents that may help sponsors get started in developing data for inclusion in an initial investigational new drug application package for nanoscale material containing drug products.     

Refinement of the ECETOC approach to identify endocrine disrupting properties of chemicals in ecotoxicology

To use and implement an assessment scheme for the evaluation of endocrine disrupting properties of chemicals in ecotoxicology, the types of effect need to be agreed. Effects that merit further consideration in this context should fulfil the following three criteria: caused by an endocrine mode of action, be adverse, and be relevant at the population level to reflect the protection goal of ecotoxicological assessments. Thereafter, a comparison of effect values, regardless of the causative mechanisms, should be made, firstly to determine if endocrine toxicity generates the lowest endpoint within a taxon, and secondly if it is the lowest endpoint compared to that of other taxa living in the same compartment. These comparisons inform on two levels of specificity and determine if endocrine-mediated side-effects determine the ecotoxicological profile of a chemical. Various quantitative measures for the assessment of potency are also presented, which could assist in determining how to handle substances in the risk assessment when a regulatory concern is identified. Finally, derogation criteria should be defined for compounds that were designed as endocrine disruptors for non-vertebrates and those for which there is ‘negligible exposure’. This paper discusses and provides proposals on how to apply these concepts for assessment of substances.     

Risk characterization of vapor intrusion in former manufactured gas plant sites

Soil vapor intrusion (SVI) has recently garnered much interest as a potential exposure route for occupants of properties overlying and surrounding former Manufactured Gas Plants (MGPs). This investigation evaluates SVI at 10 commercial buildings and 26 single family and multi-family residential properties overlying and/or adjacent to three former MGPs. SVI was evaluated in three categories according to thickness of the vadose zones: no vadose zone; 0-6 feet thick, and 6-25 feet thick. Indoor and outdoor air, and soil vapor samples were analyzed for volatile organic compounds (VOCs). Comparative risks were evaluated based on maximum and mean concentrations for benzene, toluene, ethylbenzene, and xylenes relative to background levels. All calculated Hazard Indices were less than 1 or were comparable to mean and maximum background levels. Cancer risks for exposure to benzene ranged from 9.75×10(-6) to 4.52×10(-4). Comparative background cancer risk from benzene exposure not related to former MGP sites ranged from 9.9×10(-6) to 3.59×10(-3). The results did not identify evidence of MGP-related soil vapor intrusion from any of the 36 sites. No increased public health risks were associated with occupied residential or commercial properties overlying or surrounding MGPs.     

The immunomodulatory effects of titanium dioxide and silver nanoparticles

Due to their characteristic physical, chemical and optical properties, titanium dioxide and silver nanoparticles are attractive tools for use in a wide range of applications. The use of nanoparticles for biological applications is, however, dependent upon their biocompatibility with living cells. Because of the importance of inflammation as a modulator of human health, the safe and efficacious in vivo use of titanium dioxide and silver nanoparticles is inherently linked to a favorable interaction with immune system cells. However, both titanium dioxide and silver nanoparticles have demonstrated potential to exert immunomodulatory and immunotoxic effects. Titanium dioxide and silver nanoparticles are readily internalized by immune system cells, may accumulate in peripheral lymphoid organs, and can influence multiple manifestations of immune cell activity. Although the factors influencing the biocompatibility of titanium dioxide and silver nanoparticles with immune system cells have not been fully elucidated, nanoparticle core composition, size, concentration and the duration of cell exposure seem to be important. Because titanium dioxide and silver nanoparticles are widely utilized in pharmaceutical, commercial and industrial products, it is vital that their effects on human health and immune system function be more thoroughly evaluated.     

Developmental stability and its applications in ecotoxicology

Developmental stability refers to the ability of a developing organism to produce a consistent phenotype in a given environment. It provides a simple, reliable method of detecting stressed populations and monitoring their recovery. The most common measure of developmental instability, fluctuating asymmetry, assesses minor deviations from perfect bilateral symmetry in traits that are normally symmetrical. Measures of developmental instability are based upon the concept of developmental invariance. The biotest approach consists of the simultaneous analysis of developmental instability (and related physiological instability) in a variety of species.     

Production and physico-chemical characterisation of nanoparticles

Synthetic nanoscaled metal oxides are mainly produced by pyrogenic decomposition of precursors in the gas phase using a hot-wall or plasma reactor. Due to their low production rate and limited scalability, these processes are of minor technical relevance in manufacturing commercial quantities of nanoparticles.     

The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays

We report results of noncellular tests, revealing the occurrence of photocatalytic interactions between titanium dioxide (TiO₂, titania) nanoparticles and the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] cytotoxicity indicator. These interactions induce the reduction of MTT and formation of purple formazan under biologically relevant conditions. Classical MTT assays have been performed to evaluate the production of formazan in DMEM-F12 and RPMI-1640 cell culture media (containing 10% fetal bovine serum-FBS) treated with Degussa-P25 TiO₂ nanoparticles, in the absence of cells. The colorimetric determinations revealed the noncellular MTT to formazan transformation induced by TiO₂ nanoparticles, under conditions commonly used for in vitro cytotoxicity testing of nanomaterials. The formazan precipitation was found to be proportional to the TiO₂ concentration, being enhanced under laboratory daylight exposure. The photocatalytic nature of the studied effect was assessed under UV irradiation at 365 nm. The biological significance of the reported reaction was established with respect to cellular reference experiments performed on V79-4, HeLa and B16 cell lines. The results show false viability increases with up to 14% (for TiO₂ concentrations generally higher than 50 μg/ml), induced by the TiO₂–MTT reaction. This type of artifacts may lead to underestimated toxicity or false proliferation results.     

载基因壳聚糖纳米粒的制备及其特性的研究

目的制备壳聚糖纳米粒,并连接上质粒,研究壳聚糖纳米粒的特性及其对DNA的结合及保护能力。方法采用离子交联法制备壳聚糖纳米粒,并用喷金扫描电子显微镜检测,了解粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基因);经琼脂糖凝胶电泳分析壳聚糖纳米载体与质粒DNA的结合能力,及不同pH值的壳聚糖纳米粒对质粒DNA的结合能力;并通过DnaseⅠ消化壳聚糖纳米-质粒结合物以观察壳聚糖纳米载体对质粒的保护作用。结果喷金扫描电镜检测证实壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5nm;琼脂糖凝胶电泳的结果显示壳聚糖纳米粒能有效地结合载体pGenesil-1质粒;不同pH值的壳聚糖纳米粒对质粒的保护作用不同,当pH值<7时壳聚糖纳米载体能100%结合质粒;DnaseⅠ消化试验证实壳聚糖纳米载体对质粒DNA有保护作用。结论采用离子交联法制备出粒径较小、均匀的壳聚糖纳米粒,并且壳聚糖纳米粒能有效地连接上质粒并对其有保护作用。     

Reactivity of engineered inorganic nanoparticles and carbon nanostructures in biological media

Biocompatibility, biodistribution, biodegradation, inflammation and interference with cell and normal functioning of tissues, among others, will determine the toxicity of engineered inorganic nanoparticles and carbon nanostructures, and therefore the extent of their use. Recent examples in the literature show that engineered inorganic nanoparticles and carbon nanostructures, which may incidentally or intendedly enter into contact with living organisms, normally do not cause acute toxic effects. However, their interaction with living organisms may disrupt normal activity leading to malfunctioning and diseases. Indeed, the observed nanoparticle-biology interactions, which can be used to detect and manipulate biological states and to heal damaged organs, could also lead to environmental and human health hazards. Therefore, there should be proper risk assessments to avoid the consequences of an uncontrolled release of massive amounts of nanoparticles in the environment. This review focuses on the particular physico-chemical properties of inorganic matter at the nanoscale in order to understand and track its evolution within living organisms, and thus monitor their interactions.     

Formulation and characterization of tobramycin-chitosan nanoparticles coated with zinc oxide nanoparticles

Herein we describe the preparation, characterization and the antibacterial effect of Tobramycin-chitosan nanoparticles (TOB-CS NPs) coated with zinc oxide nanoparticles (ZnO NPs). Four formulations of TOB-CS NPs (A-D) were prepared to study the effect of experimental variables on the NPs behavior. Two formulations of ZnO NPs were prepared using the solvothermal and the precipitation methods (ZnO₁ and ZnO₂), and then characterized. TOB-CS NPs (Formula d) was coated with the ZnO₁. Moreover, the antibacterial activity of TOB-CS NPs, ZnO NPs and the coated nanoparticles against S. aureus and E. coli was examined. Changing the variables in preparing TOB-CS NPs resulting in variabilities in sizes (297.6–1116.3 nm), charges (+8.29–+39.00 mV), entrapment (51.95–90.60%). Further, TOB release was sustained over four days. ZnO NPs have sizes of 47.44 and 394.4 nm and charges of ?62.3 and 89.4 mV when prepared by solvothermal and precipitation technique, respectively. Coated TOB-CS NPs had a size of 342 nm, a charge of +4.39 and released 100 µg/ mL of the drug after four days. The antimicrobial activity of TOB-CS NPs was lower than free TOB against S. aureus and E. coli. The coated NPs showed higher antimicrobial effect in comparison to formula D and ZnO₁. In conclusion, coating TOB-CS NPs with ZnO NPs exhibited a great antibacterial effect that may be sustained for days.     

含RGD修饰的丹参酮ⅡA多级靶向纳米粒的制备及工艺优化

目的：探讨丹参酮ⅡA多级靶向纳米粒的制备及其工艺优化。方法采用乳化溶剂蒸发法制备丹参酮ⅡA多级靶向纳米粒;考察单因素在纳米粒制备过程中的影响,包括TSⅡA载药纳米粒中的药物浓度、乳化剂浓度、有机相/外水相、超声时间和强度等的改变;并通过正交设计优化TSⅡA多级靶向纳米粒的制备工艺。结果本实验成功制备了含RGD修饰的丹参酮ⅡA多级靶向纳米粒,优选工艺参数为：药物浓度1mg/mL,载体材料浓度20mg/mL,有机相/外水相为1∶10,超声强度和时间分别为200W,20×5s。结论此丹参酮ⅡA多级靶向纳米粒的制备工艺切实可行。所制备的TNP包封率和载药量较高,为丹参酮ⅡA的临床应用提供了更广阔的前景。     

肝靶向阿柔比星A聚氰基丙烯酸异丁酯毫微粒药物动力学研究

为提高阿柔比星（aclarubicin A,ACRA)体内抗肝癌效果,降低其对正常组织器官的毒副作用,以氰基丙烯酸异丁酯（isobutylcyanoacrylate,IBC)为载体材料,制备出肝靶向阿柔比星A聚氰基丙烯酸异丁酯毫微粒（aclarubicin A polyisobutylcyanoacrylate nonopaticles,ACR-IBC-NP),以3H－ACR A为示踪物,采用液体闪烁计数技术研究了ACR－IBC－NP灌胃与尾静脉注射两种给药途径在小鼠血液及靶器官肝脏中的药物动力学规律,结果表明,灌胃给药时ACR－IBC－NP在血液与肝脏中的药时数据均可以用血管外给药的二室模型描述：尾静脉注射给药时ACR－IBC－NP在血液与肝脏中的药时数据虽然可以分别用二室模型与血管外给药的二室模型进行较好地描述,但是,靶向至肝脏的ACR－IBC－NP作为药物储库,使血液中药时曲线出现双峰,常规药物动力学模型均不能很好地拟合,提示具靶向缓释特点的药物传输系统的药物动力学模型有待进一步研究,ACR－IBC－NP的体内过程表明,ACR－IBC－NP在尾静脉注与灌胃两种给药途径时均具有肝靶向作用与缓释作用,以靶向效率（targeting efficiency,TE;TE=AUC靶器官／AUC血液）为指标评价ACR0－IBC－NP的靶向效果,尾静脉注射与灌胃两种给药途径的TE分别是14．41与28．47,灌胃给药时,ACR－IBC－NP的绝对生物利用度为38．53％,由于靶向药物传输系统希望靶器官药物浓度水平与维持时间适宜,作者认为,血管外给药时,靶器官中药物的利用速度与程度应该作为靶向药物传输系统的重要质量参数,参考常规的药物生物利用度概念,作者首次定义靶器官生物利用率（F）按下式计算,F靶器官＝AUC靶器官,血管外给药／AUC靶器官,静脉给药×100％,得出ACR－IBC－NP灌胃给药的靶器官生物利用为76．01％。     

环孢菌素A-纳米乳输液剂的制备及鉴定

目的制备鉴定负载环孢菌素A(Cyclosporine A,CsA)的纳米乳(Cyclosporine A-nanopaticals e-mulsion,CsA-NP)并评估CsA-NP对体外干细胞增殖和分化的影响。方法采用乳化法制备油水(o/w)型CsA-NP。以蛋黄磷脂(Lipoid E 80)、泊洛沙姆188为水相,中链油(Lipoid MCT)为油相,采用高压均质法制备而成。高效液相色谱法(HPLC)评估产品的载药率,乳径的粒径经激光粒度分析仪测定。CsA-NP对干细胞增殖和分化影响采用细胞直接计数和MTT检测。结果本研究制备的CsA-NP工艺稳定性好,粒径均匀,50%的乳滴微粒直径在162 nm以内。CsA-NP浓度在(0.01～1 mg/mL)之间1～3 d对细胞增殖有明显促进作用(P<0.01);其中又以0.5 mg/mL的CsA-NP浓度组为最佳。结论CsA-NP是一种稳定性良好的静脉注射用纳米乳。     

Physicochemical characterization of protamine-phosphorothioate nanoparticles

Protamine-oligonucleotide nanoparticles represent effective colloidal drug carriers for antisense phosphorothioate oligonucleotides (PTO). This study describes improvements in particle preparation and the physicochemical properties of the complexes prepared. The influence of component concentrations, length of the PTO chain and the PTO/protamine weight ratio on particle formation and size, shape and surface charge of the particles were studied in detail. Nanoparticles with diameters of 90–200?nm were obtained, using protamine free base (PFB) and phosphorothioate in water. The chemical composition of the nanoparticles was analysed. More than 90% of the PTO could be assembled in the particle matrix using a ≥1?:?2 ratio (w/w) of PTO and PFB. About 53–68% of the PFB was incorporated in the particle matrix. The complexes had a zetapotential of ?19 up to +32?mV, depending on the PTO/PFB ratio. The kinetics of the assembly of this binary system were observed by dynamic light scattering (DLS) measurements and by sedimentation velocity analysis in the analytical ultracentrifuge (AUC). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied to verify the results of DLS and the ultracentrifuge measurements. According to sedimentation velocity analysis, the particles were only moderately stable in water and unstable in salt solutions. However, the colloidal solution in water could be stabilized by polyethylenglycol 20?000 (PEG), which also led to an increase of stability in cell medium.