Cellular uptake and cytotoxic impact of chemically functionalized and polymer-coated carbon nanotubes

The impact of nanomaterials such as carbon nanotubes on biological matter is a topic of increasing interest and concern and requires a multifaceted approach to be resolved. A modified cytotoxic (lactate dehydrogenase (LDH)) assay is developed in an attempt to offer a valid and reliable methodology for screening carbon nanotube toxicity in vitro. Two of the most widely used types of surface-modified multiwalled carbon nanotubes (MWNTs) are tested: ammonium-functionalized MWNTs (MWNT-NH3+ ) and Pluronic F127 coated MWNTs (MWNT:F127). Chemically functionalized MWNTs show significantly greater cellular uptake into lung epithelial A549 cells compared to the non-covalently Pluronic F127-coated MWNTs. In spite of this, MWNT:F127 exhibit enhanced cytotoxicity according to the modified LDH assay. The validity of the modified LDH assay is further validated by direct comparison with other less reliable or accurate cytotoxicity assays. These findings indicate the reliability of the modified LDH assay as a screening tool to assess carbon nanotube cytotoxicity and illustrate that high levels of carbon nanotube cellular internalization do not necessarily lead to adverse responses.     

丙戊酸钠/LDH复合材料的制备及缓释性能研究

以共沉淀法与离子交换法制备丙戊酸钠插层Mg/Al-NO3-LDH,结合XRD、FT-IR、TG-DTA分析表明,丙戊酸钠进入LDH层间与层板相互作用形成超分子结构,使丙戊酸钠的热稳定性提高近100℃。提出了以共沉淀法制备丙戊酸钠插层Mg/Al-NO3-LDH,在LDH形成层板结构的过程中,形成中间态——溶胶-凝胶态,与离子交换法相比更易于客体进入层间。缓释实验表明,共沉淀法制备的产物具有良好的缓释性。     

Magnesium and calcium organophyllosilicates: synthesis and in vitro cytotoxicity study

Synthesis of multifunctional hybrid nanomaterials for biomedical applications has received great attention. Herein, we examine the potential toxicity of organophyllosilicates on cells from different organs such as A549 (lung epithelial cancer), HT-29 (colon epithelial cancer), MRC-5 (lung fibroblast) and CCD-986sk (skin fibroblast) cells. For this, aminopropyl functionalized magnesium phyllosilicate (AMP clay) and aminopropyl functionalized calcium phyllosilicate (ACP clay) were prepared using one-pot direct sol-gel method. Toxic effects of these organoclays on normal fibroblast and tumor cells were examined under varying concentrations and exposure times. MTT and LDH assays indicated that both organoclays had little cytotoxicity in all of the cells tested at concentrations as high as 500 μg/mL. Even at high concentration (1000 μg/mL), the toxicity of both organoclays on cell viability and membrane damage was not severe and appeared to be cell type specific. In addition, organoclays did not induce apoptosis at concentrations as high as 1000 μg/mL.     

Luminescent passive-oxidized silicon quantum dots as biological staining labels and their cytotoxicity effects at high concentration

Semiconductor quantum dots (QDs) hold some advantages over conventional organic fluorescent dyes. Due to these advantages, they are becoming increasingly popular in the field of bioimaging. However, recent work suggests that cadmium based QDs affect cellular activity. As a substitute for cadmium based QDs, we have developed photoluminescent stable silicon quantum dots (Si-QDs) with a passive-oxidation technique. Si-QDs (size: 6.5 ± 1.5?nm) emit green light, and they have been used as biological labels for living cell imaging. In order to determine the minimum concentration for cytotoxicity, we investigated the response of HeLa cells. We have shown that the toxicity of Si-QDs was not observed at 112?μg?ml(-1) and that Si-QDs were less toxic than CdSe-QDs at high concentration in mitochondrial assays and with lactate dehydrogenase (LDH) assays. Especially under UV exposure, Si-QDs were more than ten times safer than CdSe-QDs. We suggest that one mechanism for the cytotoxicity is that Si-QDs can generate oxygen radicals and these radicals are associated with membrane damages. This work has demonstrated the suitability of Si-QDs for bioimaging in lower concentration, and their cytotoxicity and one toxicity mechanism at high concentration.     

Effect of starch-based biomaterials on the <Emphasis Type="Italic">in vitro</Emphasis> proliferation and viability of osteoblast-like cells

The cytotoxicity of starch-based polymers was investigated using different methodologies. Poly-L-lactic acid (PLLA) was used as a control for comparison purposes. Extracts of four different starch-based blends (corn starch and ethylene vinyl alcohol (SEVA-C), corn starch and cellulose acetate (SCA), corn starch and polycaprolactone (SPCL) and starch and poly-lactic acid (SPLA70) were prepared in culture medium and their toxicity was analysed. Osteoblast-like cells (SaOs-2) were incubated with the extracts and cell viability was assessed using the MTT test and a lactate dehydrogenase (LDH) assay. In addition DNA and total protein were quantified in order to evaluate cell proliferation. Cells were also cultured in direct contact with the polymers for 3 and 7 days and observed in light and scanning electron microscopy (SEM). LDH and DNA quantification revealed to be the most sensitive tests to assess respectively cell viability and cell proliferation after incubation with starch-based materials and PLLA. SCA was the starch blend with higher cytotoxicity index although similar to PLLA polymer. Cell adhesion tests confirmed the worst performance of the blend of starch with cellulose acetate but also showed that SPCL does not perform as well as it could be expected. All the other materials were shown to present a comparable behaviour in terms of cell adhesion showing slight differences in morphology that seem to disappear for longer culture times.The results of this study suggest that not only the extract of the materials but also their three-dimensional form has to be biologically tested in order to analyse material-associated parameters that are not possible to consider within the degradation extract. In this study, the majority of the starch-based biomaterials presented very promising results in terms of cytotoxicity, comparable to the currently used biodegradable PLLA which might lead the biocompatibility evaluation of those novel biomaterials to other studies.     

Aging of trivalent metal hydroxide/oxide gels in divalent metal salt solutions: Mechanism of formation of layered double hydroxides (LDHs)

While the aging of freshly precipitated Al(OH)₃ gels in solutions of Mg and Ni salts leads to LDH formation at high (> 12) pH, aging of ‘Fe(OH)₃“ leads to LDH formation in Mg salt solutions but not in Ni salt₃’ gels do not form LDHs on aging in any of the divalent metal salts. In general, conditions that promote the redissolution of the trivalent hydroxide also promote LDH formation showing that oxoanionic species such as AlO _- ² have a role in LDH formation.     

Biocompatibility study of theophylline/chitosan/β-cyclodextrin microspheres as pulmonary delivery carriers

To evaluate the biocompatibility of the theophylline/chitosan/β-cyclodextrin microspheres, which has a potential application in pulmonary delivery system. The detection of LDH and protein in BALF was examined acute cell toxicity, hemolysis test was carried out to estimate blood toxicity; Micronucleus Test was reckoned to identify genotoxicity, MTT assay was used to evaluate in vitro cytotoxicity, and muscle implantation investigated the tissue biocompatibility. The results demonstrated that the total contents of protein and LDH in BALF were not significantly different from that of normal group. The experiments showed that the cytotoxicity was depended on the concentration and had no cytoxicity at low concentration and no hemolysis activity. The micronucleus frequency of MS B was 0.99‰, which showed no genotoxic effects either. The results of implantation showed that the microspheres had no effect on hemoglobin and no toxicity in the liver and kidney. The inflammations of muscle tissue were not significantly different from that of operative suture, therefore, the MS B possess high good biocompatibility and can be applied in pulmonary sustained release systems.     

Toxicity of nano gamma alumina to neural stem cells

Nano alumina, one of the most important nanomaterials, is widely used in diverse areas. It was reported that nano alumina could cross the blood brain barrier to enter the brain. Considering aluminum accumulation in brain is closely related to many neural diseases. We studied the neural toxicity of four nano gamma-alumina samples by using neural stem cells (NSCs) C17.2 as a model. We find that the toxicity of nano gamma-alumina is pretty low, though these alumina particles are easily internalized by cells. The loss of cell viability and membrane integrity are dose-dependent and sample-dependent after alumina exposure. At concentrations lower than 100 microg/mL, no significant toxicity is observed for all alumina samples. When the concentration reaches 200 microg/mL, alumina treated cells begin to loss their activities. No culture period effect (up to 3 days) is observed. Very tiny soluble aluminum and the absorption of culture medium ingredients onto alumina particles do not affect the cell viability. Intracellular reactive oxygen species generation may contribute to the cytotoxicity of alumina particles at high concentration, but it does not induce the apoptosis of NSCs.     

Doxorubicin-conjugated mesoporous magnetic colloidal nanocrystal clusters stabilized by polysaccharide as a smart anticancer drug vehicle

Fabrication of magnetic nanocarriers that demonstrate enhanced biocompatibility and excellent colloidal stability is critical for the application of magnetic-motored drug delivery, and it remains a challenge. Herein, a novel approach to synthesize mesoporous magnetic colloidal nanocrystal clusters (MMCNCs) that are stabilized by agarose is described; these clusters demonstrate high magnetization, large surface area and pore volume, excellent colloidal stability, enhanced biocompatibility, and acid degradability. The hydroxyl groups of agarose, which cover the surface of the magnetic nanocrystals, are modified with vinyl groups, followed by click reaction with mercaptoacetyl hydrazine to form the terminal hydrazide (-CONHNH(2) ). The anticancer agent doxorubicin (DOX) is then conjugated to MMCNCs through a hydrazone bond. The resulting hydrazone is acid cleavable, thereby providing a pH-sensitive drug release capability. This novel carrier provides an important step towards the construction of a new family of magnetic-motored drug-delivery systems. The experimental results show that the release rate of DOX from the DOX-conjugated MMCNCs (MMCNCs-DOX) is dramatically improved at low pH (tumor cell: pH 4-5 in the late stage of endolysosome and pH 5-6 from the early to late endosome), while almost no DOX is released at neutral pH (blood plasma). The cell cytotoxicity of the MMCNCs-DOX measured by MTT assay exhibits a comparable antitumor efficacy but lower cytotoxicity for normal cell lines, when measured against the free drug, thus achieving the aim of reducing side effects to normal tissues associated with controlled drug release.     

Multifunctional Upconversion Mesoporous Silica Nanostructures for Dual Modal Imaging and In Vivo Drug Delivery

Incorporating the agents for magnetic resonance imaging (MRI), optical imaging, and therapy in one nanostructured matrix to construct multifunctional nanomedical platform has attracted great attention for simultaneous diagnostic and therapeutic applications. In this work, a facile methodology is developed to construct a multifunctional anticancer drug nanocarrier by combining the special advantages of upconversion nanoparticles and mesoporous silica. β‐NaYF₄:Yb³⁺, Er³⁺@β‐NaGdF₄:Yb³⁺ is chosen as it can provide the dual modality of upconversion luminescence and MRI. Then mesoporous silica is directly coated onto the upconversion nanoparticles to form discrete, monodisperse, highly uniform, and core–shell structured nanospheres (labeled as UCNPs@mSiO₂), which are subsequently functionalized with hydrophilic polymer poly(ethylene glycol) (PEG) to improve the colloidal stability and biocompatibility. The obtained multifunctional nanocomposites can be used as an anticancer drug delivery carrier and applied for imaging. The anticancer drug doxorubicin (DOX) is absorbed into UCNPs@mSiO₂‐PEG nanospheres and released in a pH‐sensitive pattern. In vitro cell cytotoxicity tests on cancer cells verify that the DOX‐loaded UCNPs@mSiO₂‐PEG has comparable cytotoxicity with free DOX at the same concentration of DOX. In addition, the T₁‐weighted MRI that measures in aqueous solutions reveals that the contrast brightening increases with the concentration of Gd³⁺ component. Upconversion luminescence images of UCNPs@mSiO₂‐PEG uptaken by cells show green emission under 980 nm infrared laser excitation. Finally, the nanocomposites show low systematic toxicity and high in vivo antitumor therapy efficacy. These findings highlight the fascinating features of upconversion‐mesoporous nanocomposites as multimodality imaging contrast agents and nanocarrier for drug molecules.     

Indirect cytotoxicity evaluation of pseudowollastonite

This study aimed to evaluate the cytotoxicity of substances leached by pseudowollastonite (CaSiO₃). It has been previously shown that calcium (Ca²⁺) and silicate (SiO₃ ^–) ions are released from pseudowollastonite into biological solutions. Both of these ions are known to influence the biological metabolism of osteoblastic cells essential in the mineralization process and bone-bonding mechanism. The indirect toxicity evaluation was performed by extraction method, according to International Standard Organization (ISO). Pseudowollastonite pellets obtained by solid-state reaction were incubated, in culture medium, during 24, 48, 72 or 168 h at different concentrations (5, 10, 15, 50, 100, 200 mg/ml). The cytotoxicity of each extract in presence of human osteoblastic cell line (SaOS-2) was quantitatively assessed by measuring the viability (succinate dehydrogenase activity, MTT), the membrane integrity (the uptake of the neutral red by viable cells, NR) as well as the cell necrosis by measuring the lactate dehydrogenase (LDH) released in the culture medium. No significant alteration of membrane integrity or cell suffering was detectable. However, increased cell metabolism was observed for cells exposed to pseudowollastonite extract with longest extraction time (168 h). In conclusion, mineral elements leached by pseudowollastonite do not significantly affect the metabolism of osteoblastic cells.     

Influence of shape,adhension and simulated lung mechanics on amorphous silica nanoparticle toxicity

Prevailing theories suggest that acicular, or fiber-like, particles induce enhanced toxicity over isotropic material through hindrance of phagocyte-mediated clearance mechanisms and through the aggravation of proximal cells via mechanical interactions. Currently, the degree to which either of these mechanisms operates is not well understood. To gain a more fundamental understanding of acicular particle toxicity, we have synthesized submicron and nanoscale amorphous silica spheres and rods as model materials for shape-driven toxicological experimentation. To accentuate contributions from mechanical damage in vitro, exposure studies were performed in the presence and absence of simulated lung mechanics. To promote and mitigate cell–particle contact-mediated mechanical interactions, the adhesion of the particles to the cell membrane was respectively modified by the physisorption of fibronectin and chemisorption of the polyethylene glycol to the silica particle surface. Lactic acid dehydrogense (LDH) and interleukin (IL)-8 release were used as endpoints for cytotoxicity and inflammation, respectively. The results indicate that particle exposures in the presence of physiological stretch induce increased LDH release and IL-8 expression regardless of shape. Moreover, it is evident that shape-induced aggregation may play a significant role in mitigating particle clearance pathways.     

Cytoprotective activities of water-soluble fullerenes in zebrafish models

Using zebrafish (Danio rerio) embryos as a model system, we compared the antioxidant and cytoprotective effects of a series of new water-soluble fullerenes 1–12. Since zebrafish embryos are transparent during the first week of life, the effects of fullerenes on multiple organ systems, including CNS, PNS, and heart, could be assessed in situ. Both positively charged and negatively charged water-soluble fullerenes were added at concentrations between 1 and 500?µM to 96 well plates containing zebrafish embryos at 24–120 hours post fertilization (hpf). Direct toxicity of each fullerene compound was assessed by LC50. In addition, we assessed the ability of each fullerene to protect against toxicity induced by known chemical toxins in this system. Four different drug/chemical toxicity models were used in our study: (i) protection of neuromast hair cells from gentamicin-induced toxicity; (ii) protection of neuromast hair cells from cisplatinum-induced toxicity; (iii) protection of tyrosine hydroxylase-containing dopaminergic CNS neurons from 6-hydroxydopamine toxicity; and (iv) protection of total CNS neurons from 6-hydroxydopamine toxicity. Our results indicate that (i) positively charged water-soluble fullerenes tend to exhibit greater toxicity than negatively charged fullerenes with similar structures; (ii) toxicity varies considerably among negatively charged fullerenes from very low to moderate, depending on structural features; (iii) dendrofullerenes 2–7 (monoadducts of C₆₀) show stronger protection against cisplatinum toxicity in neuromast hair cells while then the e,e,e-trismalonic acid 1 (so called C3) shows stronger protection against gentamicin-induced cytotoxicity in the same cells; (iv) C3 (1) is relatively unstable in all aqueous solvents tested and breaks down mainly through decarboxylation reactions to form penta, tetra and tris carboxylated forms, which exhibit increased toxicity in vivo compared with C3 (1). Our findings indicate that water-soluble fullerenes can protect against chemical toxin-induced apoptotic cell death in a vertebrate, whole-animal model that may be useful for predicting the efficacy and toxicity of these compounds in mammals. Furthermore, the relative potential for pharmacologic use of these compounds varies considerably with respect to stability.     

Immobilization of type-I collagen and basic fibroblast growth factor (bFGF) onto poly (HEMA-co-MMA) hydrogel surface and its cytotoxicity study

Type-I collagen and bFGF were immobilized onto the surface of poly (HEMA-co-MMA) hydrogel by grafting and coating methods to improve its cytotoxicity. The multi-layered structure of the biocompatible layer was confirmed by FTIR, AFM and static water contact angles. The layers were stable in body-like environment (pH 7.4). Human skin fibroblast cells (HSFC) were seeded onto Col/bFGF-poly (HEMA-co-MMA), Col-poly (HEMA-co-MMA) and poly (HEMA-co-MMA) films for 1, 3 and 5 day. MTT assay was performed to evaluate the extraction toxicity of the materials. Results showed that the cell attachment, proliferation and differentiation on Col/bFGF-poly (HEMA-co-MMA) film were higher than those of the control group, which indicated the improvement of cell-material interaction. The extraction toxicity of the modified materials was also lower than that of the unmodified group. The protein and bFGF immobilized poly (HEMA-co-MMA) hydrogel might hold great promise to be a biocompatible material.     

Cytotoxicity of zinc oxide nanoparticles: importance of microenvironment

While ZnO particles are widely used in many fields, including personal care products, the high toxicity of ZnO nanoparticles has been reported and aroused great health concerns. In this study, the cytotoxicity of ZnO nanoparticles was evaluated and, in particular, the role of microenvironment in their toxicity was investigated. Our results show that ZnO nanoparticles are highly toxic to NIH/3T3 cells, inducing viability loss, membrane leakage and morphology changes. The microenviroment, here the CO2 atmosphere under cell culture condition, promoted the solubilization of ZnO nanoparticles. Then the released Zn from ZnO nanoparticles induces the cytotoxicity. The importance of microenvironment on the ZnO nanotoxicity is presented and the implications to future nanotoxicology studies are discussed.     

Safety assessment of nanoparamagnetic contrast agents with different coatings for molecular MRI

Despite the wide application of gadolinium as a contrast agent for magnetic resonance imaging (MRI), there is a serious lack of information on its toxicity. Gadolinium and gadolinium oxide (Gd-oxide) are used as contrast agents for magnetic resonance imaging (MRI). There are methods for reducing toxicity of these materials, such as core nanoparticles coating or conjugating. Therefore, for toxicity evaluation, we compared the viability of commercial contrast agents in MRI (Gd-DTPA) and three nanoparticles with the same core Gd₂O₃ and small particulate gadolinium oxide or SPGO (< 40 nm) but different coatings of diethyleneglycol (DEG) as Gd₂O₃-DEG and methoxy polyethylene glycol-silane (mPEG-silane: 550 and 2000 Dalton) as SPGO-mPEG-silane550 and SPGO-mPEG-silane2000, respectively, in the SK-MEL3 cell line, by light microscopy, MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity. The viability values were not statistically different between the three nanoparticles and Gd-DTPA. The MTT and LDH assay results showed that Gd₂O₃-DEG nanoparticles were more toxic than Gd-DTPA and other nanoparticles. Also, SPGO-mPEG-silane2000 was more biocompatible than other nanoparticles. The obtained results did not show any significant increase in cytotoxicity of the nanoparticles and Gd-DTPA, neither dose-dependent nor time-dependent. Therefore, DEG and PEG, due to their considerable properties and irregular sizes (different molecular weights), were selected as the useful surface covering materials of nanomagnetic particles that could reveal noticeable relaxivity and biocompatibility characteristics.     

UV-enhanced toxicity of water-stable quantum dots in human pancreatic carcinoma cells

Zn-based (capped with thioglycolic acid (TGA) or 3-mercaptopropionic acid (MPA)) and Cd-based quantum dots (QDs) (capped with TGA or L-glutathione), were synthesised and used to investigate their cytotoxicity to human pancreatic carcinoma cells (PANC-1) in absence and presence of UV irradiation. Zn-based QDs exhibited less intrinsic cytotoxicity than the Cd-based QDs, however, the excitation of 50?µg/mL-QDs using UV lamp significantly enhanced the cytotoxicity of both QDs. After 15?min of UV irradiation, the viability for cells exposed to Cd-based QDs capped with TGA or glutathione was 49%?±?6% or 56%?±?3%, respectively. The corresponding cell viability in the control test was 83%?±?8% after 15?min of UV irradiation. In turn, the viability for cells exposed to Zn-based QDs capped with 3-MPA or TGA was 64%?±?3% and 52%?±?3%, respectively, after 30?min of UV irradiation; the cell viability in the control test was 80%?±?7% for the same UV irradiation time. Laser scanning confocal analyses evidenced that QDs can be easily ingested by PANC-1. Based on their good compositional stability, Zn-based QDs capped with 3-MPA can be considered a promising material for nanomedicine applications until concentrations of 200?µg/mL.     

Cytotoxicity of single-walled carbon nanotubes suspended in various surfactants

The cytotoxicity of single-walled carbon nanotubes (SWCNTs) suspended in various surfactants was investigated by phase contrast light microscopy characterization in combination with an absorbance spectroscopy cytotoxicity analysis. Our data indicate that individual SWCNTs suspended in the surfactants, sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS), were toxic to 1321N1 human astrocytoma cells due to the toxicity of SDS and SDBS on the nanotube surfaces. This toxicity was observed when cells were exposed to an SDS or SDBS solution having a concentration as low as 0.05?mg?ml(-1) for 30?min. The proliferation and viability of the cells were not affected by SWCNTs alone or by conjugates of SWCNTs with various concentrations of sodium cholate (SC) or single-stranded DNA. The cells proliferated similarly to untreated cells when surrounded by SWCNTs as they grow, which indicated that the nanotubes did not affect cells adversely. The cytotoxicity of the nanotube-surfactant conjugates was controlled in these experiments by the toxicity of the surfactants. Consequently, when evaluating a surfactant to be used for the dispersion of nanoscale materials in applications such as nanoscale electronics or non-viral biomolecular transporters, the cytotoxicity needs to be evaluated. The methodology proposed in this study can be used to investigate the cytotoxicity of other nanoscale materials suspended in a variety?of?surfactants.     

锌铝层状复合氢氧化物的合成研究

该研究以硝酸锌、硝酸铝为原料,氢氧化钠为沉淀剂,采用恒定PH值法和变化pH值法进行共沉淀合成锌铝层状复合氢氧化物[ZnAl－LDH].全面探讨了各种因素在合成过程中的影响,以XRD分析确定其结构类型,通过TG－DTA考察其热行为．结果表明,在该研究的实验条件下,无需氮气保护,即可在较宽范围内合成结构单一、结晶度高的复合层状物．首次提出以115℃、2h的短时间水热处理代替传统的80℃、20h的回流处理,大大简化操作过程,缩短反应时间.实验结果表明,合成体系中pH值不同,合成LDH相应的Zn／Al比范围不同,而土板层间水含量又与合成物中Zn／Al比有关．在诸多的影响因素中,pH值是最关键的影响因素．     

Confined Synthesis of Carbon Nitride in a Layered Host Matrix with Unprecedented Solid‐State Quantum Yield and Stability

Fluorescent carbon nanomaterials have drawn tremendous attention for their intriguing optical performances, but their employment in solid‐state luminescent devices is rather limited as a result of aggregation‐induced photoluminescence quenching. Herein, ultrathin carbon nitride (CN) is synthesized within the 2D confined region of layered double hydroxide (LDH) via triggering the interlayer condensation reaction of citric acid and urea. The resulting CN/LDH phosphor emits strong cyan light under UV‐light irradiation with an absolute solid‐state quantum yield (SSQY) of 95.9 ± 2.2%, which is, to the best of our knowledge, the highest value of carbon‐based fluorescent materials ever reported. Furthermore, it exhibits a strong luminescence stability toward temperature, environmental pH, and photocorrosion. Both experimental studies and theoretical calculations reveal that the host–guest interactions between the rigid LDH matrix and interlayer carbon nitride give the predominant contribution to the unprecedented SSQY and stability. In addition, prospective applications of the CN/LDH material are demonstrated in both white light‐emitting diodes and upconversion fluorescence imaging of cancer cells.