Intracellular accumulation and cytotoxicity of doxorubicin with different pharmaceutical formulations in human cancer cell lines

The structure of both carrier and anticancer drug affects the intracellular fate of a transported drug. The study investigated in vitro intracellular accumulation and cytotoxic activity of doxorubicin-loaded solid lipid nanoparticles (SLN), doxorubicin in pegylated liposomes (Caelyx) and free doxorubicin. Intracellular doxorubicin levels and cytotoxic activity were determined by high performance liquid chromatography with fluorescence detection, and by the trypan blue dye exclusion assay, respectively. Doxorubicin-loaded SLN inhibited cell growth more strongly than either free or liposomal doxorubicin, in human colorectal adenocarcinoma, HT-29, retinoblastoma Y79, and glioblastoma U373 cell lines. The IC50 values for doxorubicin-loaded SLN were significantly lower after 24 h exposure than those for free doxorubicin in all cell lines; after 48 h exposure they were lower than those for liposomal doxorubicin in HT-29 and Y79 cells. The enhanced cytotoxic activity of doxorubicin-loaded SLN was associated with increased drug incorporation in cells: intracellular doxorubicin levels were significantly enhanced after exposure to drug-loaded SLN versus either free or liposomal drug. Rate of intracellular accumulation and cytotoxic activity also differed among different cell lines; in particular, cells of epithelial origin were found to be more sensitive to doxorubicin-loaded SLN. In conclusion, the greater sensitivity of HT-29, Y79, and U373 cells to doxorubicin-loaded SLN than to the other drug formulations may be due to the capability of the delivery system to enhance drug action, through a marked uptake and accumulation of SLN within the cell.     

Induction and repair of DNA damage in human cells at different stages of differentiation

Use of cellular systems capable of undergoing in vitro differentiation can give useful information on the basic mechanisms of cellular radiation sensitivity. During differentiation the cellular organisation, including the nuclear structure and the intracellular concentration of several compounds and enzymes change drastically. Accordingly, radiation response to ionising radiation is also expected to change. The human proerythroblastoid cell line K562 can be induced to pseudoerythroid differentiation. This process has been characterised and studies have been carried out on DNA single strand break and double strand break induction and repair before and after differentiation commitment. Rejoining studies have been performed for both types of damage and correct double strand break rejoining has been also measured in particular genomic locations. An overview is presented of these results together with preliminary data recently obtained on radiation induced DNA fragmentation as a function of radiation quality.     

Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines

We developed a miniaturized LC-MS system with a high-recovery phosphopeptide enrichment protocol that allows phosphoproteome analysis of 10(4) cells. In the enrichment protocol, the key step is to add sodium deoxycholate and sodium lauroyl sarcosinate to the buffer solution for protein extraction and digestion and to omit any subsequent desalt/desurfactant step before phosphopeptide enrichment. The phosphopeptides enriched by hydroxy acid-modified metal oxide chromatography (HAMMOC) are directly injected onto a miniaturized LC column using a nitrogen-pressure-driven cell, instead of switching valve-type injectors. The miniaturized analytical column of 25 μm diameter provided a 3.6-fold improvement in sensitivity over the conventional 100 μm diameter column. Overall, our analytical system provided approximately 80-fold improvement on average in the LC-MS response, and we identified 1011 unique phosphorylated sites based on 995 unique phosphopeptides from a single analysis of 10(4) HeLa cells (approximately 1 μg of proteins). This is the most sensitive phosphoproteomics system that has so far been reported for proteome-wide analysis of in vivo phosphorylation in mammalian cells.     

Shape-dependent cytotoxicity of polyaniline nanomaterials in human fibroblast cells

The toxicity of polyaniline (PANI) nanomaterials with four different aspect ratios on human lung fibroblast cells was investigated by cell viability assay, cytotoxicity assay, apoptosis/necrosis measurement, and reactive oxygen species production. The toxicity increased with decreasing aspect ratio of PANI nanomaterials. In contrast, the highest aspect ratio PANI nanomaterials showed similar results with bulk PANI materials. The adverse effect of PANI nanomaterials was also concentration- and time-dependent. Low aspect ratio PANI nanomaterials induced more necrosis and more reactive oxygen species than others. These results provide new understanding of shape-dependent toxicity of nanomaterials.     

Vitis vinifera peel polyphenols stabilized gold nanoparticles induce cytotoxicity and apoptotic cell death in A431 skin cancer cell lines

Gold nanoparticles (AuNPs) are considered beneficial in the field of biomedicine and in the development of therapeutic nanomedicine products. In the present study, Vitis vinifera. L (grapes) peel polyphenols were utilized as reducing and stabilizing agents for the biosynthesis of gold nanoparticles, and their cytotoxicity and apoptotic effects were assessed. The synthesized gold nanoparticles were characterized using UV-Visible spectroscopy, Transmission electron microscopy (TEM), X-ray diffraction (XRD), Particle size distribution, Fourier transform infrared spectroscopy (FTIR) and zeta potential analysis. TEM analysis confirmed that the nanoparticles were spherical with ～20–40 nm in size. Particle size distribution revealed ～50 ± 5 nm nanoparticles and FTIR confirmed the presence of polyphenols capped onto the peel gold nanoparticles. The V. vinifera peel gold nanoparticles were studied for their antiproliferative activities and induction of apoptosis at the inhibitory concentration (IC₅₀) of 23.6 µM. A431 cell lines incubated with V. vinifera peel gold nanoparticles for 24 h exhibited cytotoxicity activity mediated by increased reactive oxygen species (ROS) production, apoptotic morphological changes and loss of membrane potential significantly (p < 0.01). Thus, the cytotoxicity of the gold nanoparticles could be attributed to the synergistic effects of the phenolic moieties of the V. vinifera peels and the efficiency of the bioconjugated gold nanoparticles causing apoptosis and secondary necrosis.     

Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles

Nanoparticles are increasingly being recognized for their potential utility in biological applications including nanomedicine. Here we examine the response of normal human cells to ZnO nanoparticles under different signaling environments and compare it to the response of cancerous cells. ZnO nanoparticles exhibit a strong preferential ability to kill cancerous T cells ( approximately 28-35x) compared to normal cells. Interestingly, the activation state of the cell contributes toward nanoparticle toxicity, as resting T cells display a relative resistance while cells stimulated through the T cell receptor and CD28 costimulatory pathway show greater toxicity in direct relation to the level of activation. Mechanisms of toxicity appear to involve the generation of reactive oxygen species, with cancerous T cells producing higher inducible levels than normal T cells. In addition, nanoparticles were found to induce apoptosis and the inhibition of reactive oxygen species was found to be protective against nanoparticle induced cell death. The novel findings of cell selective toxicity, towards potential disease causing cells, indicate a potential utility of ZnO nanoparticles in the treatment of cancer and/or autoimmunity.     

Single-walled carbon nanotubes induces oxidative stress in rat lung epithelial cells

Single-walled carbon nanotubes (SWCNT) show unique properties find applications in micro devices; electronics to biological systems specially drug delivery and gene therapy. However the manufacture and extensive use of nanotubes raises concern about its safe use and human health. Very few studies have been carried out on toxicity of carbon nanotubes in experimental animals and humans, thus resulted in limiting their use. The extensive toxicological studies using in vitro and in vivo models are necessary and are required to establish safe manufacturing guidelines and also the use of SWCNT. These studies also help the chemists to prepare derivative of SWCNT with less or no toxicity. The present study was undertaken to determine the toxicity exhibited by SWCNT in rat lung epithelial cells as a model system. Lung epithelial cells (LE cells) were cultured with or without SWCNT and reactive oxygen species (ROS) produced were measured by change in fluorescence using dichloro fluorescein (DCF). The results show increased ROS on exposure to SWCNT in a dose and time dependent manner. The decrease in glutathione content suggested the depletion and loss of protective mechanism against ROS in SWCNT treated cells. Use of rotenone, the inhibitor of mitochondrial function have no effect on ROS levels suggested that mitochondria is not involved in SWCNT induced ROS production. Studies carried out on the effect of SWCNT on superoxide dismutase (SOD-1 and SOD-2) levels in LE cells, indicates that these enzyme levels decreased by 24 hours. The increased ROS induced by SWCNT on LE cells decreased by treating the cells with 1 mM of glutathione, N-Acetyl Cysteine, and Vitamin C. These results further prove that SWCNT induces oxidative stress in LE cells and shows loss of antioxidants.     

Tamoxifen-encapsulated vesicular systems: cytotoxicity evaluation in human epidermal keratinocyte cell line

Aim: Tamoxifen is a nonsteroidal estrogen receptor modulator indicated in the treatment of breast cancer. Apoptosis has been reported to be a major mechanism for its antitumor effect. Tamoxifen has also shown significant potential in treating various dermatological disorders including psoriasis, characterized by hyperproliferation of epidermal keratinocytes. An endeavor was made in the current studies to investigate the potency of vesicle-encapsulated tamoxifen on human epidermal keratinocyte cell lines. Methods: Drug was encapsulated in the phospholipid-based vesicular systems, namely, conventional liposomes and flexible-membrane liposomes. In vitro cytotoxicity evaluation of the formulations was carried out employing MTT cell proliferation assay. Results: A composition-dependent strong inhibition in the viability of epidermal keratinocyte cells was observed. Conclusion: The encouraging findings of this work construe immense potential of the tamoxifen-encapsulated vesicular systems in the management of psoriasis.     

Use of DNA repair enzymes in electrochemical detection of damage to DNA bases in vitro and in cells

Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells.     

Silica nanoparticle induces oxidative stress and provokes inflammation in human lung cells

Amorphous silica has been used in a wide variety of industrial and consumer applications. Nanosized silica is being considered a potentially ideal nanomaterial for biomedical and biotechnological applications. In the present study, hierarchical oxidative stress paradigms of nanoparticle toxicity were assumed to evaluate the toxicity of amorphous silica nanoparticle in human healthy lung cells, L-132. The cells were exposed to varied concentrations viz. 0, 100, 200, 300, 400 and 500 μg/ml of silica nanoparticle for 24 h. Different parameters of cytotoxicity, oxidative stress and pro-inflammatory markers were assessed. Silica nanoparticle exposure showed concentration-dependent decrease in cell viability, increase in reactive oxygen species with depletion of antioxidant enzyme activity, induction of inteleukin-8 and cyclooxygenase-2 expression and subsequent DNA damage in L-132 cells. The results indicated that amorphous silica nanoparticle followed hierarchical oxidative stress model and generated oxidative stress which provoked the pro-inflammatory response and caused necrosis in human lung cells.     

Laser-induced damage and recovery of plasmonically targeted human endothelial cells

Laser-induced techniques that employ the surface plasmon resonances of nanoparticles have recently been introduced as an effective therapeutic tool for destroying tumor cells. Here, we adopt a low-intensity laser-induced technique to manipulate the damage and repair of a vital category of noncancerous cells, human endothelial cells. Endothelial cells construct the interior of blood vessels and play a pivotal role in angiogenesis. The degree of damage and repair of the cells is shown to be influenced by laser illumination in the presence of gold nanoparticles of different morphologies, which either target the cellular membrane or are endocytosed. A pronounced influence of the plasmonic nanoparticle laser treatment on the expression of critical angiogenic genes is shown. Our results show that plasmon-mediated mild laser treatment, combined with specific targeting of cellular membranes, enables new routes for controlling cell permeability and gene regulation in endothelial cells.     

DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells

Carbon nanotubes (CNTs) have shown promise as an important new class of multifunctional building blocks and innovative tools in a large variety of applications, ranging from nanocomposite materials through nanoelectronics to biomedical devices. Because of their unusual one-dimensional hollow nanostructure and unique physicochemical properties, CNTs are particularly useful as novel drug delivery tools and imaging agents. However, such biomedical applications will not be realized if there is no proper assessment of the potential hazards of CNTs to humans and other biological systems. Although a few reports on the cytotoxicity of CNTs have been published, very little is known about the toxicity at the molecular level, or genotoxicity, of CNTs in mammalian cells. We have for the first time assessed the DNA damage response to multiwalled carbon nanotubes (MWNTs) in mouse embryonic stem (ES) cells. We found that MWNTs can accumulate and induce apoptosis in mouse ES cells and activate the tumor suppressor protein p53 within 2 h of exposure. Furthermore, we also observed increased expression of two isoforms of base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1), double strand break repair protein Rad 51, phosphorylation of H2AX histone at serine 139, and SUMO modification of XRCC4 following the treatment with MWNTs. A mutagenesis study using an endogenous molecular marker, adenine phosphoribosyltransferase (Aprt), showed that MWNTs increased the mutation frequency by 2-fold compared with the spontaneous mutation frequency in mouse ES cells. These results suggest that careful scrutiny of the genotoxicity of nanomaterials is needed even for those materials, like multiwalled carbon nanotubes, that have been previously demonstrated to have limited or no toxicity at the cellular level.     

Graphene as a nanocarrier for tamoxifen induces apoptosis in transformed cancer cell lines of different origins

A cationic amphiphile, cholest-5en-3β-oxyethyl pyridinium bromide (PY(+) -Chol), is able to efficiently disperse exfoliated graphene (GR) in water by the physical adsorption of PY(+) -Chol on the surface of GR to form stable, dark aqueous suspensions at room temperature. The GR-PY(+) -Chol suspension can then be used to solubilize Tamoxifen Citrate (TmC), a breast cancer drug, in water. The resulting TmC-GR-PY(+) -Chol is stable for a long time without any precipitation. Fluorescence emission and UV absorption spectra indicate the existence of noncovalent interactions between TmC, GR, and PY(+) -Chol in these suspensions. Electron microscopy shows the existence of segregated GR sheets and TmC 'ribbons' in the composite suspensions. Atomic force microscopy indicates the presence of 'extended' structures of GR-PY(+) -Chol, which grows wider in the presence of TmC. The slow time-dependent release of TmC is noticed in a reconstituted cell culture medium, a property useful as a drug carrier. TmC-GR-PY(+) -Chol selectively enhanced the cell death (apoptosis) of the transformed cancer cells compared to normal cells. This potency is found to be true for a wide range of transformed cancer cells viz. HeLa, A549, ras oncogene-transformed NIH3T3, HepG2, MDA-MB231, MCF-7, and HEK293T compared to the normal cell HEK293 in vitro. Confocal microscopy confirmed the high efficiency of TmC-GR-PY(+) -Chol in delivering the drug to the cells, compared to the suspensions devoid of GR.     

Nano titanium dioxide induces the generation of ROS and potential damage in HaCaT cells under UVA irradiation

Nano titanium dioxide (nano-TiO2) is frequently used in cosmetics, especially in sunscreen. Nano-TiO2 has been reported to be an efficient photocatalyst, which is able to produce reactive oxygen species (ROS) under UVA irradiation. However, the effects and mechanisms of skin toxicity caused by nano-TiO2 remain unclear. In this study, we explored the cytotoxicity and oxidative stress induced by nano-TiO2 under UVA irradiation with different crystal forms (anatase, rutile and anatase/rutile) and sizes (4 nm, 10 nm, 21 nm, 25 nm, 60 nm) in human keratinocyte HaCaT cells. Intracellular distribution of nano-TiO2, cell viability, intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), super oxide dismutase (SOD) activity and Malondialdehyde (MDA) content were measured. The results showed that nano-TiO2 (10-200 microg/ml) significantly reduced cell viability in a dose-dependent manner in HaCaT cells. The cell viability was 76.9%, 60.2%, and 44.1% following nano-TiO2 (4 nm), nano-TiO2 (10 nm) and P25 treatment at the concentration of 200 microg/ml, respectively (P < 0.01). Nano-TiO2 induced ROS resulted in oxidative stress in these cells by reducing SOD and increasing MDA levels. The MMP of the cells was decreased significantly (P < 0.01) while the apoptosis rate was increased. Anatase and amorphous forms of nano-TiO2 showed higher cytotoxicity than the rutile form. The results indicated that nano-TiO2 could induce the generation of ROS and damage HaCaT cells under UVA irradiation.     

Cell cycle perturbations and cytogenetic damage induced by low energy protons in human primary fibroblasts

Endpoints related to cell cycle perturbations were investigated in human primary fibroblasts irradiated with 1-4 Gy of either protons (7.7 and 28.5 keV.micron-1) or X rays. Regardless of the quality of radiation, 8 h after irradiation cells accumulated in the G2 phase, but such accumulation was maintained at 24 h from treatment only in 28.5 keV.micron-1 protons. A marked G1/S-transition block was observed in proton as well as in X ray-treated cultures up to 24 h, with doses as low as 1 Gy. On the other hand, the number of cells positive to p21 antibody reaction was higher after proton than after X ray treatment, indicating that the 'complexity' of lesions may play a critical role in the induction of p21.     

Optical nanomanipulations of malignant cells: controlled cell damage and fusion

Specifically targeting and manipulating living cells is a key challenge in biomedicine and in cancer research in particular. Several studies have shown that nanoparticles irradiated by intense lasers are capable of conveying damage to nearby cells for various therapeutic and biological applications. In this work ultrashort laser pulses and gold nanospheres are used for the generation of localized, nanometric disruptions on the membranes of specifically targeted cells. The high structural stability of the nanospheres and the resonance pulse irradiation allow effective means for controlling the induced nanometric effects. The technique is demonstrated by inducing desired death mechanisms in epidermoid carcinoma and Burkitt lymphoma cells, and initiating efficient cell fusion between various cell types. Main advantages of the presented approach include low toxicity, high specificity, and high flexibility in the regulation of cell damage and cell fusion, which would allow it to play an important role in various future clinical and scientific applications.