An Assessment of the Impact of SiO2 Nanoparticles of Different Sizes on the Rest/Wake Behavior and the Developmental Profile of Zebrafish Larvae

In this study, zebrafish larvae are introduced as an in vivo platform to examine the neurotoxicity and developmental toxicity associated with continuous exposure to a concentration gradient of different sizes of SiO₂ nanoparticles (15 nm and 50 nm diameter) to determine the dose effect and size effect of SiO₂ nanoparticle (NP)‐induced toxicity. Bovine serum albumin (BSA‐V) is utilized as a stabilizing agent to prevent coagulation of the SiO₂ nanoparticles. To the best of our knowledge, this study is the first to describe locomotor activity assays linking rest/wake behavioral profiles for the purpose of investigating the neurotoxicity of NPs. In addition, developmental toxicological endpoints including mortality, LC₅₀, malformation, and cartilaginous deformity are assessed. The results show a concentration‐dependent increase in behavioral neurotoxicity, mortality, and malformation among larvae treated with the SiO₂ nanoparticles of 15 nm and 50 nm. A comparison of the 15 nm and 50 nm NPs by K‐means clustering analysis demonstrates that the 15 nm NPs have a greater neurotoxic effect than the 50 nm NPs, with the 50 nm NPs exhibiting greater developmental toxicity on the zebrafish larvae than the 15 nm NPs.     

Toxicity of silver nanoparticles in zebrafish models

This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). Using starch and bovine serum albumin (BSA) as capping agents, silver nanoparticles were synthesized to study their deleterious effects and distribution pattern in zebrafish embryos (Danio rerio). Toxicological endpoints like mortality, hatching, pericardial edema and heart rate were recorded. A concentration-dependent increase in mortality and hatching delay was observed in Ag-np treated embryos. Additionally, nanoparticle treatments resulted in concentration-dependent toxicity, typified by phenotypes that had abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. Ag(+) ions and stabilizing agents showed no significant defects in developing embryos. Transmission electron microscopy (TEM) of the embryos demonstrated that nanoparticles were distributed in the brain, heart, yolk and blood of embryos as evident from the electron-dispersive x-ray analysis (EDS). Furthermore, the acridine orange staining showed an increased apoptosis in Ag-np treated embryos. These results suggest that silver nanoparticles induce a dose-dependent toxicity in embryos, which hinders normal development.     

Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle–Cell Interactions

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real‐time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.     

Intrinsically Fluorescent,Stealth Polypyrazoline Nanoparticles with Large Stokes Shift for In Vivo Imaging

Recent advances in super‐resolution microscopy and fluorescence bioimaging allow exploring previously inaccessible biological processes. To this end, there is a need for novel fluorescent probes with specific features in size, photophysical properties, colloidal and optical stabilities, as well as biocompatibility and ability to evade the reticuloendothelial system. Herein, novel fluorescent nanoparticles are introduced based on an inherently fluorescent polypyrazoline (PPy) core and a polyethylene glycol (PEG) shell, which address all aforementioned challenges. Synthesis of the PPy‐PEG amphiphilic block copolymer by phototriggered step‐growth polymerization is investigated by NMR spectroscopy, size‐exclusion chromatography, and mass spectrometry. The corresponding nanoparticles are characterized for their luminescent properties and hydrodynamic size in various aqueous environments (e.g., cell culture media). PPy nanoparticles particularly exhibit a large Stokes shift (Δλ = 160 nm or Δν > 7000 cm^?1) with visible light excitation and strong colloidal stability. While clearance by macrophages and endothelial cells is minimal, PPy displays good biocompatibility. Finally, PPy nanoparticles prove to be long circulating when injected in zebrafish embryos, as observed by in vivo time‐lapse fluorescence microscopy. In summary, PPy nanoparticles are highly promising to be further developed as fluorescent nanodelivery systems with low toxicity and exquisite retention in the blood stream.     

Persistency of Enlarged Autolysosomes Underscores Nanoparticle‐Induced Autophagy in Hepatocytes

The diverse biological effects of nanomaterials form the basis for their applications in biomedicine but also cause safety issues. Induction of autophagy is a cellular response after nanoparticles exposure. It may be beneficial in some circumstances, yet autophagy‐mediated toxicity raises an alarming concern. Previously, it has been reported that upconversion nanoparticles (UCNs) elicit liver damage, with autophagy contributing most of this toxicity. However, the detailed mechanism is unclear. This study reveals persistent presence of enlarged autolysosomes in hepatocytes after exposure to UCNs and SiO₂ nanoparticles both in vitro and in vivo. This phenomenon is due to anomaly in the autophagy termination process named autophagic lysosome reformation (ALR). Phosphatidylinositol 4‐phosphate (PI(4)P) relocates onto autolysosome membrane, which is a key event of ALR. PI(4)P is then converted into phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) by phosphatidylinositol‐4‐phosphate 5‐kinase. Clathrin is subsequently recruited by PI(4,5)P₂ and leads to tubule budding of ALR. Yet it is observed that PI(4)P cannot be converted in nanoparticle‐treated hepatocytes cells. Exogenous supplement of PI(4,5)P₂ suppresses the enlarged autolysosomes in vitro. Abolishment of these enlarged autolysosomes by autophagy inhibitor relieves the hepatotoxicity of UCNs in vivo. The results provide evidence for disrupted ALR in nanoparticle‐treated hepatocytes, suggesting that the termination of nanoparticle‐induced autophagy is of equal importance as the initiation.     

In vitro and in vivo toxicity of CdTe nanoparticles

Cadmium telluride (CdTe) nanoparticles exhibit strong and stable fluorescence that is attractive for many applications such as biological probing and solid state lighting. The evaluation of nanoparticle toxicity is important for realizing these practical applications. However, no systematic studies of CdTe nanoparticle toxicity have been reported. We investigated and compared the size- and concentration-dependent cytotoxicity of CdTe nanoparticles in human hepatoma HepG2 cells using the MTT assay. CdTe nanoparticles elicited cytotoxicity in a concentration- and size-dependent manner, with smaller-sized particles exhibiting somewhat higher potency. Lesser cytotoxicity of partially purified CdTe-Red particles (following methanol precipitation and resuspension) suggested that free cadmium ions may contribute to cytotoxicity. We also evaluated the acute toxicity of CdTe-Red particles following intravenous exposure in male rats (2 micromol/kg). Few signs of functional toxicity or clinical (urinary or blood) changes were noted. Interestingly, motor activity was transiently reduced (2 hours after treatment) and then significantly increased at a later timepoint (24 hours after dosing). These studies provide a framework for further characterizing the in vitro and in vivo toxic potential of different types of CdTe nanoparticles and suggest that the nervous system may be targeted by these nanoparticles under some conditions.     

Effects of copper nanoparticles on the development of zebrafish embryos

The environmental behavior and the potential toxicity of copper nanoparticles (nano-Cu) in water are major concerns for assessing their environmental safety. The present study was undertaken to characterize the properties of nano-Cu in E3 medium, such as size changes, solubility, zeta-potential and pH, and to test the toxicity of nano-Cu suspension to zebrafish embryos. Dynamic light scattering and solubility experiments showed that three components coexisted in the nano-Cu exposure system, including small nano-Cu aggregates still suspended in E3 medium, large nano-Cu aggregates deposited on the container bottom and dissolved copper species (Cu(dis)). Both the zeta-potential of nano-Cu particles in E3 medium and the pH of the nano-Cu suspension showed no change during a 24 hour period. It is found that nano-Cu retarded the hatching of zebrafish embryos and caused morphological malformation of the larvae, and high concentrations (>0.1 mg/L) of nano-Cu even killed the gastrula-stage zebrafish embryos. Cu2+ ions were used to study the toxicity caused by nano-Cu dissolution. The embryo toxicity of nano-Cu at 0.01 and 0.05 mg/L showed no significant difference from Cu2+ at the corresponding concentrations (0.006 and 0.03 mg/L), but 0.1 mg/L nano-Cu had a greater toxicity than 0.06 mg/L Cu2+.     

In vivo biodistribution and toxicity depends on nanomaterial composition,size, surface functionalisation and route of exposure

Anticipated growth of the nanotechnology industry has motivated the development of rapid, relevant and efficient testing strategies to evaluate the biological activity and toxic potential of the growing number of novel nanoparticles. Since nanoparticles may interact with biological systems in unforeseen ways, it is important that evaluation of nanomaterial–biological interactions cover a broad range of cell types, tissues, organs and systems. Here, we use the embryonic zebrafish as a dynamic whole animal (in vivo) assay to investigate the importance of chemical composition, size, surface functionalisation and route of exposure on nanomaterial–biological interactions and delineate nanomaterials that are biologically active from those that are not. Information gained using model systems, such as the embryonic zebrafish, can be used to direct the rational development of safer, less hazardous nanoparticles. Our results demonstrate the utility of this model as an effective and accurate tool to assess the biological activity and toxic potential of nanomaterials in a short period of time with minimal cost.     

Comparative Evaluation of Intestinal Nitric Oxide in Embryonic Zebrafish Exposed to Metal Oxide Nanoparticles

Nanoparticle (NP) exposure may induce oxidative stress through generation of reactive oxygen and nitrogen species, which can lead to cellular and tissue damage. The digestive system is one of the initial organs affected by NP exposure. Here, it is demonstrated that exposure to metal oxide NPs induces differential changes in zebrafish intestinal NO concentrations. Intestinal NO concentrations are quantified electrochemically with a carbon fiber microelectrode inserted in the intestine of live embryos. Specificity of the electrochemical signals is demonstrated by NO‐specific pharmacological manipulations and the results are correlated with the 4,5‐diaminofluorescein‐diacetate (DAF‐FM‐DA). NPs are demonstrated to either induce or reduce physiological NO levels depending on their redox reactivity, type and dose. NO level is altered following exposure of zebrafish embryos to CuO and CeO₂ NPs at various stages and concentrations. CuO NPs increase NO concentration, suggesting an intestinal oxidative damage. In contrast, low CeO₂ NP concentration exposure significantly reduces NO levels, suggesting NO scavenging activity. However, high concentration exposure results in increased NO. Alterations in NO concentration suggest changes in intestinal physiology and oxidative stress, which will ultimately correspond to NPs toxicity. This work also demonstrates the use of electrochemistry to monitor in vivo changes of NO within zebrafish organs.     

Tracking the Fate of Porous Silicon Nanoparticles Delivering a Peptide Payload by Intrinsic Photoluminescence Lifetime

A nanoparticle system for systemic delivery of therapeutics is described, which incorporates a means of tracking the fate of the nanocarrier and its residual drug payload in vivo by photoluminescence (PL). Porous silicon nanoparticles (PSiNPs) containing the proapoptotic antimicrobial peptide payload, _D[KLAKLAK]₂, are monitored by measurement of the intrinsic PL intensity and the PL lifetime of the nanoparticles. The PL lifetime of the PSiNPs is on the order of microseconds, substantially longer than the nanosecond lifetimes typically exhibited by conventional fluorescent tags or by autofluorescence from cells and tissues; thus, emission from the nanoparticles is readily discerned in the time‐resolved PL spectrum. It is found that the luminescence lifetime of the PSiNP host decreases as the nanoparticle dissolves in phosphate‐buffered saline solution (37 °C), and this correlates with the extent of release of the peptide payload. The time‐resolved PL measurement allows tracking of the in vivo fate of PSiNPs injected (via tail vein) into mice. Clearance of the nanoparticles through the liver, kidneys, and lungs of the animals is observed. The luminescence lifetime of the PSiNPs decreases with increasing residence time in the mice, providing a measure of half‐life for degradation of the drug nanocarriers.     

Rationally designed oxaliplatin-nanoparticle for enhanced antitumor efficacy

Nanoscale drug delivery vehicles have been extensively studied as carriers for cancer chemotherapeutics. However, the formulation of platinum chemotherapeutics in nanoparticles has been a challenge arising from their physicochemical properties. There are only a few reports describing oxaliplatin nanoparticles. In this study, we derivatized the monomeric units of a polyisobutylene maleic acid copolymer with glucosamine, which chelates trans-1,2-diaminocyclohexane (DACH) platinum (II) through a novel monocarboxylato and O --> Pt coordination linkage. At a specific polymer to platinum ratio, the complex self-assembled into a nanoparticle, where the polymeric units act as the leaving group, releasing DACH-platinum in a sustained pH-dependent manner. Sizing was done using dynamic light scatter and electron microscopy. The nanoparticles were evaluated for efficacy in vitro and in vivo. Biodistribution was quantified using inductively coupled plasma atomic absorption spectroscopy (ICP-AAS). The PIMA-GA-DACH-platinum nanoparticle was found to be more active than free oxaliplatin in vitro. In vivo, the nanoparticles resulted in greater tumor inhibition than oxaliplatin (equivalent to 5 mg kg?1 platinum dose) with minimal nephrotoxicity or body weight loss. ICP-AAS revealed significant preferential tumor accumulation of platinum with reduced biodistribution to the kidney or liver following PIMA-GA-DACH-platinum nanoparticle administration as compared with free oxaliplatin. These results indicate that the rational engineering of a novel polymeric nanoparticle inspired by the bioactivation of oxaliplatin results in increased antitumor potency with reduced systemic toxicity compared with the parent cytotoxic. Rational design can emerge as an exciting strategy in the synthesis of nanomedicines for cancer chemotherapy.     

Cationic silica nanoparticles as gene carriers: synthesis, characterization and transfection efficiency in vitro and in vivo

The potential of cationic SiO2 nanoparticles was investigated for in vivo gene transfer in this study. Cationic SiO2 nanoparticles with surface modification were generated using amino-hexyl-amino-propyltri-methoxysilane (AHAPS). The zeta potential of the nanoparticles at pH = 7.4 varied from -31.4 mV (unmodified particles; 10 nm) to +9.6 mV (modified by AHAPS). Complete immobilization of DNA at the nanoparticle surface was achieved at a particle ratio of 80 (w/w nanoparticle/DNA ratio). The surface modified nanoparticle had a size of 42 nm with a distribution from 10-100 nm. The ability of these particles to transfect pCMVbeta reporter gene was tested in Cos-1 cells, and optimum results were obtained in the presence of FCS and chloroquine at a particle ratio of 80. These nanoparticles were tested for their ability to transfer genes in vivo in the mouse lung, and a two-times increase in the expression levels was found with silica particles in comparison to EGFP alone. Very low or no cell toxicity was observed, suggesting silica nanoparticles as potential alternatives for gene transfection.     

Studying nanoparticles in-flight: applications of size-selected free nanoparticles

Experimental investigation of nanoparticles in form of free particles in an inert gas is advantageous due to absence of substrate effects. Gas-phase synthesis techniques offer many possibilities for producing and manipulating nanoparticles. The most advanced technique produces well-defined nanoparticles, by means of inert-gas evaporation in a flowing gas, size-selection on the basis of the electrical mobility and subsequent sintering into monocrystalline and quasispherical nanoparticles. The electrical charge obtained by nanoparticles allows further manipulation, such as the generation of well-defined nanoparticle pairs, in which the sizes of both the particles can be selected. The application of these well-defined nanoparticles for studying the sintering of metallic nanoparticles is demonstrated. The ability to deliver size-selected nanoparticle aerosols at larger flowrates, in this study up to 30 l/min, allows to deliver larger quantities of well-defined nanoparticles. The size-selection necessitates the charging of the nanoparticle aerosol. We show that photoionization by means of UV irradiation is able to charge nanoparticles with a high efficiency and that by optimizing the radiation intensity the amount of multiple charges can be brought to a minimum, resulting in a geometric standard deviation of the size distribution of 1.05.     

Magnetically Engineered Semiconductor Quantum Dots as Multimodal Imaging Probes

Light‐emitting semiconductor quantum dots (QDs) combined with magnetic resonance imaging contrast agents within a single nanoparticle platform are considered to perform as multimodal imaging probes in biomedical research and related clinical applications. The principles of their rational design are outlined and contemporary synthetic strategies are reviewed (heterocrystalline growth; co‐encapsulation or assembly of preformed QDs and magnetic nanoparticles; conjugation of magnetic chelates onto QDs; and doping of QDs with transition metal ions), identifying the strengths and weaknesses of different approaches. Some of the opportunities and benefits that arise through in vivo imaging using these dual‐mode probes are highlighted where tumor location and delineation is demonstrated in both MRI and fluorescence modality. Work on the toxicological assessments of QD/magnetic nanoparticles is also reviewed, along with progress in reducing their toxicological side effects for eventual clinical use. The review concludes with an outlook for future biomedical imaging and the identification of key challenges in reaching clinical applications.     

In vitro cytotoxicity of surface modified bismuth nanoparticles

This paper describes in vitro cytotoxicity of bismuth nanoparticles revealed by three complementary assays (MTT, G6PD, and calcein AM/EthD-1). The results show that bismuth nanoparticles are more toxic than most previously reported bismuth compounds. Concentration dependent cytotoxicities have been observed for bismuth nanoparticles and surface modified bismuth nanoparticles. The bismuth nanoparticles are non-toxic at concentration of 0.5 nM. Nanoparticles at high concentration (50 nM) kill 45, 52, 41, 34 % HeLa cells for bare nanoparticles, amine terminated bismuth nanoparticles, silica coated bismuth nanoparticles, and polyethylene glycol (PEG) modified bismuth nanoparticles, respectively; which indicates cytotoxicity in terms of cell viability is in the descending order of amine terminated bismuth nanoparticles, bare bismuth nanoparticles, silica coated bismuth nanoparticles, and PEG modified bismuth nanoparticles. HeLa cells are more susceptible to toxicity from bismuth nanoparticles than MG-63 cells. The simultaneous use of three toxicity assays provides information on how nanoparticles interact with cells. Silica coated bismuth nanoparticles can damage cellular membrane yet keep mitochondria less influenced; while amine terminated bismuth nanoparticles can affect the metabolic functions of cells. The findings have important implications for caution of nanoparticle exposure and evaluating toxicity of bismuth nanoparticles.     

Shedding Light on Metal‐Based Nanoparticles in Zebrafish by Computed Tomography with Micrometer Resolution

Metal‐based nanoparticles are clinically used for diagnostic and therapeutic applications. After parenteral administration, they will distribute throughout different organs. Quantification of their distribution within tissues in the 3D space, however, remains a challenge owing to the small particle diameter. In this study, synchrotron radiation‐based hard X‐ray tomography (SRμCT) in absorption and phase contrast modes is evaluated for the localization of superparamagnetic iron oxide nanoparticles (SPIONs) in soft tissues based on their electron density and X‐ray attenuation. Biodistribution of SPIONs is studied using zebrafish embryos as a vertebrate screening model. This label‐free approach gives rise to an isotropic, 3D, direct space visualization of the entire 2.5 mm‐long animal with a spatial resolution of around 2 µm. High resolution image stacks are available on a dedicated internet page ( http://zebrafish.pharma-te.ch ). X‐ray tomography is combined with physico‐chemical characterization and cellular uptake studies to confirm the safety and effectiveness of protective SPION coatings. It is demonstrated that SRμCT provides unprecedented insights into the zebrafish embryo anatomy and tissue distribution of label‐free metal oxide nanoparticles.     

Micro/Nanoparticle‐Augmented Sonodynamic Therapy (SDT): Breaking the Depth Shallow of Photoactivation

The fast development of photoactivation for cancer treatment provides an efficient photo‐therapeutic strategy for cancer treatment, but traditional photodynamic or photothermal therapy suffers from the critical issue of low in vivo penetration depth of tissues. As a non‐invasive therapeutic modality, sonodynamic therapy (SDT) can break the depth barrier of photoactivation because ultrasound has an intrinsically high tissue‐penetration performance. Micro/nanoparticles can efficiently augment the SDT efficiency based on nanobiotechnology. The state‐of‐art of the representative achievements on micro/nanoparticle‐enhanced SDT is summarized, and specific functions of micro/nanoparticles for SDT are discussed, from the different viewpoints of ultrasound medicine, material science and nanobiotechnology. Emphasis is put on the relationship of structure/composition‐SDT performance of micro/nanoparticle‐based sonosensitizers. Three types of micro/nanoparticle‐augmented SDT are discussed, including organic and inorganic sonosensitizers and micro/nanoparticle‐based but sonosensitizer‐free strategies to enhance the SDT outcome. SDT‐based synergistic cancer therapy augmented by micro/nanoparticles and their biosafety are also included. Some urgent critical issues and potential developments of micro/nanoparticle‐augmented SDT for efficient cancer treatment are addressed. It is highly expected that micro/nanoparticle‐augmented SDT will be quickly developed as a new and efficient therapeutic modality which will find practical applications in cancer treatment. At the same time, fundamental disciplines regarding materials science, chemistry, medicine and nanotechnology will be advanced.     

Time-Gated FRET Nanoprobes for Autofluorescence-Free Long-Term In Vivo Imaging of Developing Zebrafish

The zebrafish is an important vertebrate model for disease, drug discovery, toxicity, embryogenesis, and neuroscience. In vivo fluorescence microscopy can reveal cellular and subcellular details down to the molecular level with fluorescent proteins (FPs) currently the main tool for zebrafish imaging. However, long maturation times, low brightness, photobleaching, broad emission spectra, and sample autofluorescence are disadvantages that cannot be easily overcome by FPs. Here, a bright and photostable terbium-to-quantum dot (QD) Förster resonance energy transfer (FRET) nanoprobe with narrow and tunable emission bands for intracellular in vivo imaging is presented. The long photoluminescence (PL) lifetime enables time-gated (TG) detection without autofluorescence background. Intracellular four-color multiplexing with a single excitation wavelength and in situ assembly and FRET to mCherry demonstrate the versatility of the TG-FRET nanoprobes and the possibility of in vivo bioconjugation to FPs and combined nanoprobe-FP FRET sensing. Upon injection at the one-cell stage, FRET nanoprobes can be imaged in developing zebrafish embryos over seven days with toxicity similar to injected RNA and strongly improved signal-to-background ratios compared to non-TG imaging. This work provides a strategy for advancing in vivo fluorescence imaging applications beyond the capabilities of FPs.     

pH‐Responsive Isoniazid‐Loaded Nanoparticles Markedly Improve Tuberculosis Treatment in Mice

Tuberculosis is a major global health problem for which improved therapeutics are needed to shorten the course of treatment and combat emergence of drug resistance. Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of mononuclear phagocytes. As such, it is an ideal pathogen for nanotherapeutics because macrophages avidly ingest nanoparticles even without specific targeting molecules. Hence, a nanoparticle drug delivery system has the potential to target and deliver high concentrations of drug directly into M. tuberculosis‐infected cells—greatly enhancing efficacy while avoiding off‐target toxicities. Stimulus‐responsive mesoporous silica nanoparticles of two different sizes, 100 and 50 nm, are developed as carriers for the major anti‐tuberculosis drug isoniazid in a prodrug configuration. The drug is captured by the aldehyde‐functionalized nanoparticle via hydrazone bond formation and coated with poly(ethylene imine)–poly(ethylene glycol) (PEI–PEG). The drug is released from the nanoparticles in response to acidic pH at levels that naturally occur within acidified endolysosomes. It is demonstrated that isoniazid‐loaded PEI–PEG‐coated nanoparticles are avidly ingested by M. tuberculosis‐infected human macrophages and kill the intracellular bacteria in a dose‐dependent manner. It is further demonstrated in a mouse model of pulmonary tuberculosis that the nanoparticles are well tolerated and much more efficacious than an equivalent amount of free drug.