Study of uptake and loss of silica nanoparticles in living human lung epithelial cells at single cell level

The toxicology of nanomaterials is a blooming field of study, yet it is difficult to keep pace with the innovations in new materials and material applications. Those applications are quickly being introduced in research, industrial, and consumer settings. Even though the cytotoxicity of many types of nanoparticles has been demonstrated, the behavior of those particles in a biological environment is not yet fully known. This work characterized the following over time: protein adsorption on silica particle surfaces, the internalization of particles in human lung carcinoma (A549) cells when coated with different specific proteins or no proteins at all, and the cellular loss of particles following the removal of extracellular particles. Proteins were shown to quickly saturate the particle surface, followed by a competitive process of particle agglomeration and protein adsorption. Uptake of particles peaked at 8–10 h, and it was determined that, in this system, the charge of the protein-coated particles changed the rate of uptake if the charge difference was great enough. Cells internalized particles lacking any adsorbed proteins with approximately 3 times the rate of protein-coated particles with the same charge. Although particles exited cells over time, the process was slower than uptake and did not near completion within 24 h. Finally, analysis at the single cell level afforded observations of particle agglomerates loosely associated with cell membranes when serum was present in the culture medium, but in the absence of serum, particles adhered to the dish floor and formed smaller agglomerates on cell surfaces. Although data trends were easily distinguished, all samples showed considerable variation from cell to cell. Figure Silica-capped fluorescent semiconductor nanoparticles as internalized by human lung epithelial cells and adsorbed to a glass substrate in protein-free culture medium.     

The toxicity outcome of silica nanoparticles (Ludox?) is influenced by testing techniques and treatment modalities

We analyzed the influence of the kind of cytotoxicity test and its application modality in defining the level of hazard of the in vitro exposures to nanostructures. We assessed the cytotoxicity induced by two different Ludox? silica nanoparticles (NPs), AS30 and SM30, on three human cell lines, CCD-34Lu, A549, and HT-1080. Dynamic light scattering measurements showed particle agglomeration when NPs are diluted in culture medium supplemented with fetal calf serum. We examined the impact of such particle aggregation on the cytotoxicity by exposing the cells to NPs under different treatment modalities: short incubation (2?h) in serum-free medium or long incubation (24–72?h) in serum-containing medium. Under this last modality, NP suspensions tended to form aggregates and were toxic at concentrations five- to tenfold higher than in serum-free medium. The results of cell survival varied considerably when the long-term clonogenic assay was performed to validate the data of the short-term MTS assay. Indeed, the half maximum effective concentrations (EC₅₀) in all the three cell lines were four- to fivefold lower when calculated from the data of clonogenic assay than of MTS. Moreover, the mechanisms of NP toxicity were cell-type-specific, showing that CCD-34Lu are prone to the induction of plasma membrane damages and HT-1080 are prone to DNA double-strand break and apoptosis induction. Taken together, our results demonstrate that the choice of testing strategy and treatment conditions plays an important role in assessing the in vitro toxicity of NPs.

Ultrastable, redispersible, small, and highly organomodified mesoporous silica nanotherapeutics

Practical biomedical application of mesoporous silica nanoparticles is limited by poor particle dispersity and stability due to serious irreversible aggregation in biological media. To solve this problem, hydrothermally treated mesoporous silica nanoparticles of small size with dual-organosilane (hydrophilic and hydrophobic silane) surface modification have been synthesized. These highly organomodified mesoporous silica nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, N(2) adsorption-desorption, dynamic light scattering, zeta potential, and solid-state (29)Si NMR, and they prove to be very stable in simulated body fluid at physiological temperature. Additionally, they can be dried to a powdered solid and easily redispersed in biological media, maintaining their small size for a period of at least 15 days. Furthermore, this preparation method can be expanded to synthesize redispersible fluorescent and magnetic mesoporous silica nanoparticles. The highly stable and redispersible mesoporous silica NPs show minimal toxicity during in vitro cellular assays. Most importantly, two types of doxorubicin, water-soluble doxorubicin and poorly water-soluble doxorubicin, can be loaded into these highly stable mesoporous silica nanoparticles, and these drug-loaded nanoparticles can also be well-redispersed in aqueous solution. Enhanced cytotoxicity to cervical cancer (HeLa) cells was found upon treatment with water-soluble doxorubicin-loaded nanoparticles compared to free water-soluble doxorubicin. These results suggest that highly stable, redispersible, and small mesoporous silica nanoparticles are promising agents for in vivo biomedical applications.     

BSA adsorption on differently charged polystyrene nanoparticles using isothermal titration calorimetry and the influence on cellular uptake

BSA adsorption onto negatively and positively charged polystyrene nanoparticles was investigated. The nanoparticles were characterized in terms of particle size, zeta potential, surface group density, and morphology. The adsorption behavior of BSA on the particle surface, as a function of pH and overall charge of the particle, was studied using ITC. Different thermodynamic data such as enthalpy changes upon binding and stoichiometry of the systems were determined and discussed. The degree of surface coverage with BSA was calculated using the thermodynamic data. The cellular uptake of particles before and after BSA adsorption was studied using HeLa cells in the presence and absence of supplemented FCS in the cell culture medium.     

Nanomaterial characterization: considerations and needs for hazard assessment and safety evaluation

Nanotechnology is a rapidly emerging field of great interest and promise. As new materials are developed and commercialized, hazard information also needs to be generated to reassure regulators, workers, and consumers that these materials can be used safely. The biological properties of nanomaterials are closely tied to the physical characteristics, including size, shape, dissolution rate, agglomeration state, and surface chemistry, to name a few. Furthermore, these properties can be altered by the medium used to suspend or disperse these water-insoluble particles. However, the current toxicology literature lacks much of the characterization information that allows toxicologists and regulators to develop “rules of thumb” that could be used to assess potential hazards. To effectively develop these rules, toxicologists need to know the characteristics of the particle that interacts with the biological system. This void leaves the scientific community with no options other than to evaluate all materials for all potential hazards. Lack of characterization could also lead to different laboratories reporting discordant results on seemingly the same test material because of subtle differences in the particle or differences in the dispersion medium used that resulted in altered properties and toxicity of the particle. For these reasons, good characterization using a minimal characterization data set should accompany and be required of all scientific publications on nanomaterials.     

Challenge in understanding size and shape dependent toxicity of gold nanomaterials in human skin keratinocytes

As the nanotechnology field continues to develop, assessing nanoparticle toxicity is very important for advancing nanoparticles for biomedical application. Here we report cytotoxicity of gold nanomaterial of different size and shape using MTT test, absorption spectroscopy and TEM. Spherical gold nanoparticles of different sizes are not inherently toxic to human skin cells, but gold nanorods are highly toxic due to the presence of CTAB as coating material. Due to toxicity of CTAB, and aggregation of gold nanomaterials in the presence of cell media, we have demonstrated that it is difficult to understand the cytotoxicity of gold nanomaterials individually.     

Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium

The physicochemical characterization of nanoparticles in suspension is a prerequisite for the adequate assessment of their potential biological effect. Little is known to date about the colloidal stability of TiO2 nanoparticles in cell culture medium. This study investigates the effect of particle concentration, ionic strength, pH, and the presence of fetal bovine serum (FBS) and human serum albumin (HSA) on the colloidal stability of TiO2 nanoparticles in RPMI cell culture medium, by sedimentation measurements, dynamic light scattering, and electrokinetic measurements (zeta-potential). TEM revealed that the particles were polydisperse, with diameters ranging from approximately 15 to approximately 350 nm. The agglomeration rate and sedimentation rate increased with particles' concentration. The size of the agglomerates at 100 mg/L TiO2 was significantly reduced, from 1620+/-160 to 348+/-13 and 378+/-15 nm, upon the addition of 10% (v/v) FBS and 1% (w/w) HSA, respectively. The isoelectric point of TiO2 in water was 2.9 and the measured zeta-potential in RPMI was -16+/-2 mV at pH 7.4. A slight increase in the zeta-potential of TiO2 in RPMI was observed upon the addition of FBS and HSA. The addition of FBS and HSA prevented high agglomeration, leading to a stable dispersion of TiO2 nanoparticles for at least 24 h, possibly due to steric stabilization of the particles.     

Preparation and properties of ionically cross‐linked chitosan nanoparticles

Chitosan nanoparticles were fabricated by a method of tripolyphosphate (TPP) cross‐linking. The influence of fabrication conditions on the physical properties and drug loading and release properties was investigated by transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV–vis spectroscopy. The nanoparticles could be prepared only within a zone of appropriate chitosan and TPP concentrations. The particle size and surface zeta potential can be manipulated by variation of the fabrication conditions such as chitosan/TPP ratio and concentration, solution pH and salt addition. TEM observation revealed a core–shell structure for the as‐prepared nanoparticles, but a filled structure for the ciprofloxacin (CH) loaded particles. Results show that the chitosan nanoparticles were rather stable and no cytotoxicity of the chitosan nanoparticles was found in an in vitro cell culture experiment. Loading and release of CH can be modulated by the environmental factors such as solution pH and medium quality. Copyright © 2008 John Wiley & Sons, Ltd.     

Dynamic light scattering as an efficient tool to study glyconanoparticle-lectin interactions

Glyconanomaterials, an emerging class of bio-functional nanomaterials, have shown promise in detecting, imaging and targeting proteins, bacteria, and cells. In this article, we report that dynamic light scattering (DLS) can be used as an efficient tool to study glyconanoparticle (GNP)--lectin interactions. Silica and Au nanoparticles (NPs) conjugated with D-mannose (Man) and D-galactose (Gal) were treated with the lectins Concanavalin A (Con A) and Ricinus communis agglutinin (RCA(120)), and the hydrodynamic volumes of the resulting aggregates were measured by DLS. The results showed that the particle size grew with increasing lectin concentration. The limit of detection (LOD) was determined to be 2.9 nM for Con A with Man-conjugated and 6.6 nM for RCA(120) with Gal-conjugated silica NPs (35 nm), respectively. The binding affinity was also determined by DLS and the results showed 3-4 orders of magnitude higher affinity of GNPs than the free ligands with lectins. The assay sensitivity and affinity were particle size dependent and decreased with increasing particle diameter. Because the method relies on the particle size growth, it is therefore general and can be applied to nanomaterials of different compositions.     

硅壳纳米颗粒对COS-7细胞的生物效应

从硅壳纳米颗粒对细胞存活率、细胞周期及细胞生长曲线的影响等方面系统地考察了包裹RuBpy染料的硅壳荧光纳米颗粒(FSiNPs)对美洲绿猴肾细胞(COS-7)的生物效应. 结果表明, FSiNPs对COS-7细胞的影响是浓度依赖性的, 低浓度(<0.2 μg/μL)的FSiNPs对细胞的存活率、细胞周期及整个生长过程均无负面影响, 但随着与COS-7细胞作用的FSiNPs浓度的增大, FSiNPs对COS-7细胞的毒性也逐渐增大, 尤其是对细胞周期及细胞生长曲线的影响更为敏感. 同时, 利用FSiNPs的荧光信号同步指示作用, 考察了COS-7细胞对FSiNPs的吞噬作用, 发现 FSiNPs通过细胞膜的吞噬作用随机地进入到细胞内, 一部分FSiNPs被细胞当成异物外排到细胞培养基中, 另一部分则进入到下一代细胞中. 随着细胞传代次数的增多和新生胞质的产生, FSiNPs在细胞内的含量逐渐减少, 最后消失. 在这一过程中, 细胞的形态和生长状况依然良好. 上述研究结果有望为FSiNPs在细胞生物学的研究和应用提供一定的安全标准, 并为开展基于新型纳米颗粒的纳米颗粒器件的研究与应用打下了基础.     

Polyaspartamide derivative nanoparticles with tunable surface charge achieve highly efficient cellular uptake and low cytotoxicity

Cationic nanocarrier mediated intracellular therapeutic agent delivery acts as a double-edged sword: the carriers promote cellular uptake, but interact nonspecifically and strongly with negatively charged endogenic proteins and cell membranes, which results in aggregates and high cytotoxicity. The present study was aimed at exploring zwitterionic polyaspartamide derivative nanoparticles for efficient intracellular delivery with low cytotoxicity. Poly(aspartic acid) partially grafted tetraethylenepentamine (PASP-pg-TEPA) with different isoelectric points (IEPs) was synthesized. The PASP-pg-TEPA formed zwitterionic nanoparticles with an irregular core and a well-defined shell structure in aqueous medium. Their particle size decreased from about 300 to 80 nm with an increase of the IEP from 7.5 to 9.1. The surface charge of the PASP-pg-TEPA nanoparticles could be tuned from positive to negative with a change of the pH of the medium. The nanoparticles with an IEP above 8.5 exhibited good stability under simulated physiological conditions. It was noted that the zwitterionic PASP-pg-TEPA nanoparticles displayed highly efficient cellular uptake in HeLa cells (approximately 99%) in serum-containing medium and did not adversely affect the cell viability at concentrations up to 1 mg/mL. Furthermore, thermodynamic analysis using isothermal titration calorimetry provided direct evidence that these zwitterionic nanoparticles had low binding affinities for serum protein. Therefore, the zwitterionic PASP-pg-TEPA nanoparticles could overcome limitations of cationic nanocarriers and achieve efficient intracellular delivery with low cytotoxicity.     

FePt@CoS(2) yolk-shell nanocrystals as a potent agent to kill HeLa cells

We report the evaluation of cytotoxicity of a new type of engineered nanomaterials, FePt@CoS(2) yolk-shell nanocrystals, synthesized by the mechanism of the Kirkendall effect when FePt nanoparticles serve as the seeds. The cytotoxicity of FePt@CoS(2) yolk-shell nanocrystals, evaluated by MTT assay, shows a much lower IC(50) (35.5 +/- 4.7 ng of Pt/mL for HeLa cell) than that of cisplatin (230 ng of Pt/mL). In the control experiment, cysteine-modified FePt nanoparticles exhibit IC50 at 12.0 +/- 0.9 microg of Pt/mL. Transmission electron microscopy confirms the cellular uptake of FePt@CoS(2) nanocrystals, and the magnetic properties analysis (SQUID) proves the release of FePt nanoparticles from the yolk-shell nanostructures after cellular uptake. These results are significant because almost none of the platinum-based complexes produced for clinical trials in the past 3 decades have shown higher activity than that of the parent drug, cisplatin. The exceptionally high toxicity of FePt@CoS(2) yolk-shell nanocrystals (about 7 times higher than that of cisplatin in terms of Pt) may lead to a new design of an anticancer nanomedicine.     

Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species

The rapid advancement of nanotechnology has created a vast array of engineered nanomaterials (ENMs) which have unique physical (size, shape, crystallinity, surface charge) and chemical (surface coating, elemental composition and solubility) attributes. These physicochemical properties of ENMs can produce chemical conditions to induce a pro-oxidant environment in the cells, causing an imbalanced cellular energy system dependent on redox potential and thereby leading to adverse biological consequences, ranging from the initiation of inflammatory pathways through to cell death. The present study was designed to evaluate size-dependent cellular interactions of known biologically active silver nanoparticles (NPs, Ag-15 nm, Ag-30 nm, and Ag-55 nm). Alveolar macrophages provide the first defense and were studied for their potential role in initiating oxidative stress. Cell exposure produced morphologically abnormal sizes and adherence characteristics with significant NP uptake at high doses after 24 h. Toxicity evaluations using mitochondrial and cell membrane viability along with reactive oxygen species (ROS) were performed. After 24 h of exposure, viability metrics significantly decreased with increasing dose (10-75 microg/mL) of Ag-15 nm and Ag-30 nm NPs. A more than 10-fold increase of ROS levels in cells exposed to 50 microg/mL Ag-15 nm suggests that the cytotoxicity of Ag-15 nm is likely to be mediated through oxidative stress. In addition, activation of the release of traditional inflammatory mediators were examined by measuring levels of cytokines/chemokines, including tumor necrosis factor (TNF-alpha), macrophage inhibitory protein (MIP-2), and interleukin-6 (IL-6), released into the culture media. After 24 h of exposure to Ag-15 nm nanoparticles, a significant inflammatory response was observed by the release of TNF-alpha, MIP-2, and IL-1beta. However, there was no detectable level of IL-6 upon exposure to silver nanoparticles. In summary, a size-dependent toxicity was produced by silver nanoparticles, and one predominant mechanism of toxicity was found to be largely mediated through oxidative stress.     

A Novel Route to Organic–Inorganic Hybrid Nanomaterials

The in situ formation of functionalized silica nanoparticles is reported. The reactive stabilizers used in the study, [3‐(2‐bromoisobutyryl)propyl]triethoxysilane and [3‐(2‐bromoisobutyryl)propyl]ethoxydimethylsilane, have an atom transfer radical polymerization (ATRP) initiator at the noncondensable end. Condensation with tetraethoxysilane yields silica nanoparticles with a surface‐immobilized initiator. The size of these functionalized silica nanoparticles can be controlled by varying the time of initiator addition and initiator concentration. The silica particle sizes ranged from 10 to 300 nm. With the initiator functionalized silica nanoparticles, ATRP synthesis was performed with styrene, tert‐butyl acrylate, and methyl acrylate to produce organic–inorganic nanomaterials.

     

Cyborg cells: functionalisation of living cells with polymers and nanomaterials

Living cells interfaced with a range of polyelectrolyte coatings, magnetic and noble metal nanoparticles, hard mineral shells and other complex nanomaterials can perform functions often completely different from their original specialisation. Such "cyborg cells" are already finding a range of novel applications in areas like whole cell biosensors, bioelectronics, toxicity microscreening, tissue engineering, cell implant protection and bioanalytical chemistry. In this tutorial review, we describe the development of novel methods for functionalisation of cells with polymers and nanoparticles and comment on future advances in this technology in the light of other literature approaches. We review recent studies on the cell viability and function upon direct deposition of nanoparticles, coating with polyelectrolytes, polymer assisted assembly of nanomaterials and hard shells on the cell surface. The cell toxicity issues are considered for many practical applications in terms of possible adverse effects of the deposited polymers, polyelectrolytes and nanoparticles on the cell surface.     

Evaluating the antimicrobial activity and cytotoxicity of polydopamine capped silver and silver/polydopamine core-shell nanocomposites

Fabrication of bioactive nanomaterials with improved stability and low toxicity towards healthy mammalian cells have recently been a topic of interest. Bioactive metal nanomaterials such as silver nanoparticles (AgNPs) tend to lose their stability with time and become toxic to some extent, limiting their biological applications. AgNPs were separately encapsulated and loaded on the surface of a biocompatible polydopamine (PDA) to produce Ag-PDA and Ag@PDA nanocomposites to unravel the issue of agglomeration. PDA was coated through the self-polymerization of dopamine on the surface of AgNPs to produce Ag-PDA core-shells nanocomposites. For Ag@PDA, PDA spheres were first designed through self-polymerization of dopamine followed by in situ reduction of silver nitrate (AgNO3) without any reductant. AgNPs sizes were controlled by varying the concentration of AgNO₃. The TEM micrograms showed monodispersed PDA spheres with an average diameter of 238 nm for Ag-PDA and Ag@PDA nanocomposites. Compared to Ag@PDA, Ag-PDA nanocomposites have shown insignificant toxicity towards human embryonic kidney (HEK-293T) and human dermal fibroblasts (HDF) cells with cell viability of over 95% at concentration of 250 µg/mL. A excellent antimicrobial activity of the nanocomposites was observed; with Ag@PDA possessing bactericidal effect at concentration as low as 12.5 µg/mL. Ag-PDA on the other hand were only found to be bacteriostatic against gram-positive and gram-negative bacteria was also observed.     

Disruption of supported lipid bilayers by semihydrophobic nanoparticles

Understanding the interaction between functional nanoparticles and cell membranes is critical to use nanomaterials for broad biomedical applications with minimal cytotoxicity. In this work, we have investigated the effect of adsorbed semihydrophobic nanoparticles (NPs) on the dynamics and morphology of model cell membranes. We have systematically varied the degree of surface hydrophobicity of carboxyl end-functionalized polystyrene NPs of varied size in buffer solutions with varied ionic strength. It is observed that semihydrophobic NPs can readily adsorb on neutral SLBs and drag lipids from SLBs to NP surfaces. Above a critical NP concentration, the disruption of SLBs is observed, accompanied with the formation and rapid growth of lipid-poor regions on NP-adsorbed SLBs. In the study of the effect of solution ionic strength on NP surface hydrophobic degree and the growth of lipid-poor regions, we have concluded that the hydrophobic interaction enhanced by screened electrostatic interaction underlies the envelopment of NPs by lipids that are attracted from SLBs to the surface of NPs or their aggregates. Hence, the formation and growth of lipid-poor regions, or vaguely referred as "pores" or "holes" in the literature, can be controlled by NP concentration, size, and surface hydrophobicity, which is critical to design functional nanomaterials for effective nanomedicine while minimizing possible cytotoxicity.     

Fungus-mediated biotransformation of amorphous silica in rice husk to nanocrystalline silica

Rice husk is a cheap agro-based waste material, which harbors a substantial amount of silica in the form of amorphous hydrated silica grains. However, there have been no attempts at harnessing the enormous amount of amorphous silica present in rice husk and its room-temperature biotransformation into crystalline silica nanoparticles. In this study, we address this issue and describe how naturally deposited amorphous biosilica in rice husk can be bioleached and simultaneously biotransformed into high value crystalline silica nanoparticles. We show here that the fungus Fusarium oxysporum rapidly biotransforms the naturally occurring amorphous plant biosilica into crystalline silica and leach out silica extracellularly at room temperature in the form of 2-6 nm quasi-spherical, highly crystalline silica nanoparticles capped by stabilizing proteins; that the nanoparticles are released into solution is an advantage of this process with significant application and commercial potential. Calcination of the silica nanoparticles leads to loss of occluded protein and to an apparently porous structure often of cubic morphology. The room-temperature synthesis of oxide nanomaterials using microorganisms starting from potential cheap agro-industrial waste materials is an exciting possibility and could lead to an energy-conserving and economically viable green approach toward the large-scale synthesis of oxide nanomaterials.     

Preparation of hybrid mesoporous silica luminescent nanoparticles with lanthanide(III) complexes and their exhibition of white emission

We chose dipicolinic acid as a tridentate chelating unit featuring ONO donors to react with lanthanide(III) ions to yield tight and protective N(3)O(6) environments around the lanthanide(III) ions. We immobilized the lanthanide(III)-dipicolinic acid complexes on colloidal mesoporous silica with diameter smaller than 100 nm by a covalent bond grafting technique and obtained nearly monodisperse luminescent Eu-dpa-Si and Tb-dpa-Si functionalized hybrid mesoporous silica nanomaterials. These hybrid nanomaterials were characterized by powder X-ray diffraction, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, nitrogen adsorption-desorption, and photoluminescence spectroscopic techniques. The hybrid mesoporous silica nanoparticles exhibit intense emission lines upon UV-light irradiation, owing to the effective intramolecular energy transfer from the chromophore to the central lanthanide Eu(3+) and Tb(3+) ions. Furthermore, the functionalized nanomaterials can be turned to white light materials after annealing at high temperature.     

Manipulating microparticles with single surface-immobilized nanoparticles

This experimental study explores the capture and manipulation of micrometer-scale particles by single surface-immobilized nanoparticles. The nanoparticles, approximately 10 nm in diameter, are cationic and therefore attract the micrometer-scale silica particles in an analyte suspension. The supporting surface on which the nanoparticles reside is negative (also silica) and repulsive toward approaching microparticles. In the limit where there are as few as 9 nanoparticles per square micrometer of collector, it becomes possible to capture and hold micrometer-scale silica particles with single nanoparticles. The strong nanoparticle-microparticle attractions, their nanometer-scale protrusion forward of the supporting surface, and their controlled density on the supporting surface facilitate microparticle-surface contact occurring through a single nanoelement. This behavior differs from most particle-particle, cell-cell, or particle (or cell)-surface interactions that involve multiple ligand-receptor bonds or much larger contact areas. Despite the limited contact of microparticles with surface-immobilized nanoparticles, microparticles resist shear forces of 9 pN or more but can be released through an increase in the ionic strength. The ability of nanoparticles to reversibly trap and hold much larger targets has implications in materials self-assembly, cell capture, and sorting applications, whereas the single point of contact affords precision in particle manipulation.