In vitro cytotoxicity of binary TI alloys for bone implants

Interest in using titanium (Ti) alloys as load-bearing implant materials has increased due to their high strength to weight ratio, lower elastic modulus, and superior biocompatibility and enhanced corrosion resistance compared to conventional metals such as stainless steel and Co-Cr alloys. In the present study, the in vitro cytotoxicity of five binary titanium alloys, Ti15Ta, Ti15Nb, Ti15Zr, Ti15Sn and Ti15Mo, was assessed using human osteosarcoma cell line, SaOS-2 cells. The Cell proliferation and viability were determined, and cell adhesion and morphology on the surfaces of the binary Ti alloys after cell culture were observed by SEM. Results indicated that the Ti binary alloys of Ti15Ta, Ti15Nb and Ti15Zr exhibited the same level of excellent biocompatibility; Ti15Sn alloy exhibited a moderate biocompatibility while Ti15Mo alloy exhibited a moderate cytotoxicity. The SaOS-2 osteoblast-like cells had flattened and spread across the surfaces of the Ti15Ta, Ti15Nb, Ti15Zr and Ti15Sn groups; however, the cell shapes on the Ti15Mo alloy was shrinking and unhealthy. These results indicated that the Mo contents should be limited to a certain level in the design and development of new Ti alloys for implant material applications.

An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8+ T cells

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8 T cell responses to H2Db complexed with peptides from the viral nucleoprotein (NP366, ASNENMETM) and acid polymerase (PA224, SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of DbNP366- and DbPA224-specific acute and memory CD8 T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8 T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8DbNP366 T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.

Cytotoxicity of titanium and titanium alloying elements

It is commonly accepted that titanium and the titanium alloying elements of tantalum, niobium, zirconium, molybdenum, tin, and silicon are biocompatible. However, our research in the development of new titanium alloys for biomedical applications indicated that some titanium alloys containing molybdenum, niobium, and silicon produced by powder metallurgy show a certain degree of cytotoxicity. We hypothesized that the cytotoxicity is linked to the ion release from the metals. To prove this hypothesis, we assessed the cytotoxicity of titanium and titanium alloying elements in both forms of powder and bulk, using osteoblast-like SaOS₂ cells. Results indicated that the metal powders of titanium, niobium, molybdenum, and silicon are cytotoxic, and the bulk metals of silicon and molybdenum also showed cytotoxicity. Meanwhile, we established that the safe ion concentrations (below which the ion concentration is non-toxic) are 8.5, 15.5, 172.0, and 37,000.0 μg/L for molybdenum, titanium, niobium, and silicon, respectively.

Dynamic analysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immobilization technology

Quantification of programmed and accidental cell death provides useful end-points for the anticancer drug efficacy assessment. Cell death is, however, a stochastic process. Therefore, the opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. In this work, we describe the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilization platform for the realtime analysis of cancer drug-induced cytotoxicity. Microelectrode arrays were designed to generate weak electro-thermal vortices that support efficient drug mixing and rapid cell immobilization at the delta-shape regions of strong electric field formed between the opposite microelectrodes. We applied this technology to the dynamic analysis of hematopoietic tumor cells that represent a particular challenge for real-time imaging due to their dislodgement during image acquisition. The present study was designed to provide a comprehensive mechanistic rationale for accelerated cell-based assays on DEP chips using real-time labeling with cell permeability markers. In this context, we provide data on the complex behavior of viable vs dying cells in the DEP fields and probe the effects of DEP fields upon cell responses to anticancer drugs and overall bioassay performance. Results indicate that simple DEP cell immobilization technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells. This ability is of particular importance in studying the outcome of patient derived cancer cells, when exposed to therapeutic drugs, as these cells are often rare and difficult to collect, purify and immobilize.

An in vitro comparative assessment with a series of new triphenyltin(IV) 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates endowed with anticancer activities: Structural modifications, analysis of efficacy and cytotoxicity involving human tumor cell lines

Four new triphenyltin(IV) complexes of composition Ph₃SnLH (where LH = 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1–4) were synthesized and characterized by spectroscopic (¹H, ¹³C and ¹¹⁹Sn NMR, IR, ¹¹⁹Sn Mössbauer) techniques in combination with elemental analysis. The ¹¹⁹Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph₃SnL¹H (1), Ph₃SnL³H (3), Ph₃SnL⁴H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ¹¹⁹Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1–4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6–7) and tributyltin(IV) complexes (8–11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1–3 are also comparable to those of its o-analogs i.e. 4–7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID₅₀ values in the range 41–53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1–3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8–11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1–7 and tributyltin(IV) complexes 8–11 is also discussed against a panel of human tumor cell lines.

Development of drug-loaded chitosan-vanillin nanoparticles and its cytotoxicity against HT-29 cells

Chitosan as a natural polysaccharide derived from chitin of arthropods like shrimp and crab, attracts much interest due to its inherent properties, especially for application in biomedical materials. Presently, biodegradable and biocompatible chitosan nanoparticles are attractive for drug delivery. However, some physicochemical characteristics of chitosan nanoparticles still need to be further improved in practice. In this work, chitosan nanoparticles were produced by crosslinking chitosan with 3-methoxy-4-hydroxybenzaldehyde (vanillin) through a Schiff reaction. Chitosan nanoparticles were 200-250 nm in diameter with smooth surface and were negatively charged with a zeta potential of - 17.4 mV in neutral solution. Efficient drug loading and drug encapsulation were achieved using 5-fluorouracil as a model of hydrophilic drug. Drug release from the nanoparticles was constant and controllable. The in vitro cytotoxicity against HT-29 cells and cellular uptake of the chitosan nanoparticles were evaluated by methyl thiazolyl tetrazolium method, confocal laser scanning microscope and flow cytometer, respectively. The results indicate that the chitosan nanoparticles crosslinked with vanillin are a promising vehicle for the delivery of anticancer drugs.

Evaluation of the cytotoxicity, cell-cycle arrest, and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells

CONTEXT: Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments. OBJECTIVE: The current study evaluates the cytotoxicity, cell-cycle arrest, and apoptotic induction by E. hirta in MCF-7 breast cancer cells. MATERIALS AND METHODS: Cytotoxic activity of methanol extract of whole part of E. hirta was determined by the MTT assay at various concentrations ranging from 1.96 to 250.00 µg/mL in MCF-7 cells. Cell morphology was assessed by light and fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by annexin V staining and flow cytometry. DNA fragmentation, caspase activity, and reactive oxygen species (ROS) assays were performed using the commercially available kits. To identify the cytotoxic fraction, E. hirta extract was subjected to bioassay-guided fractionation. RESULTS: Euphorbia hirta exhibited significant inhibition of the survival of MCF-7 cells and the half inhibitory concentration (IC50) values was 25.26 µg/mL at 24 h. Microscopic studies showed that E. hirta-treated cells exhibited marked morphological features characteristic of apoptosis. Euphorbia hirta extract also had an ignorable influence on the LDH leakage and generating intracellular ROS. The flow cytometry study confirmed that E. hirta extract induced apoptosis in MCF-7 cells. Euphorbia hirta also resulted in DNA fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase activity study revealed that E. hirta extract induced apoptosis through the caspase-3-independent pathway by the activation of caspase-2, 6, 8, and 9. Euphorbia hirta hexane fraction, namely HFsub4 fraction, demonstrated highest activity among all the fractions tested with an IC50 value of 10.01 µg/mL at 24 h. DISCUSSION AND CONCLUSION: This study revealed that E. hirta induced apoptotic cell death and suggests that E. hirta could be used as an apoptosis-inducing anticancer agent for breast cancer treatment with further detailed studies.

Doxorubicin conjugated to immunomodulatory anticancer lactoferrin displays improved cytotoxicity overcoming prostate cancer chemo resistance and Inhibits tumour development in TRAMP mice

Advanced, metastatic, castration resistant and chemo-resistant prostate cancer has triggered change in the drug development landscape against prostate cancer. Bovine lactoferrin (bLf) is currently attracting attention in clinics for its anti-cancer properties and proven safety profile. bLf internalises into cancer cells via receptor mediated endocytosis, boosts immunity and complements chemotherapy. We employed bLf as an excellent functional carrier protein for delivering doxorubicin (Dox) into DU145 cells, CD44+/EpCAM+ double positive enriched DU145 3D prostaspheres and drug resistant ADR1000-DU145 cells, thus circumventing Dox efflux, to overcome chemo-resistance. Successful bLf-Dox conjugation with iron free or iron saturated bLf forms did not affect the integrity and functionality of bLf and Dox. bLf-Dox internalised into DU145 cells within 6 h, enhanced nuclear Dox retention up to 24 h, and proved significantly effective (p < 0.001) in reducing LC50 value of Dox from 5.3 μM to 1.3 μM (4 fold). Orally fed iron saturated bLf-Dox inhibited tumour development, prolonged survival, reduced Dox induced general toxicity, cardiotoxicity, neurotoxicity in TRAMP mice and upregulated serum levels of anti-cancer molecules TNF-α, IFN-γ, CCL4 and CCL17. The study identifies promising potential of a novel and safer bLf-Dox conjugate containing a conventional cytotoxic drug along with bLf protein to target drug resistance.

Acetylacetonato chelated ruthenium organometallics incorporating imine-phenol function: spectroscopic, structural, electrochemical and cytotoxicity studies

The heterogeneous phase reaction of Ru(η²-RL)(PPh₃)₂(CO)Cl, 1 with lithium acetylacetonate (Liacac) afforded the complexes of the type Ru(η¹-RL)(PPh₃)₂(CO)(acac), 2 in excellent yield where η²-RL is C₆H₂O-2-CHNHC₆H₄R(p)-3-Me-5 and η¹-RL is C₆H₂OH-2-CHNC₆H₄R(p)-3-Me-5 and R is H, Me, Cl. The chelation of acac is attended with the cleavage of Ru-O and Ru-Cl bonds and iminium-phenolato → imine-phenol prototropic shift. A sterically controlled change in rotational conformation is involved in the 1 → 2 conversion. The conversion is irreversible and the type 2 species are thermodynamically more stable than the carboxylate, nitrite and nitrate complexes of 1. The crystal structures of Ru(η¹-MeL)(PPh₃)₂(CO)(acac), 2(Me) and Ru(η¹-ClL)(PPh₃)₂(CO)(acac), 2(Cl) are reported. Spectral (UV-Vis, IR, ¹H NMR) and electrochemical data of the complexes are also reported. The electronic structure and the absorption spectra of the complexes are scrutinized by the density functional theory (DFT) and time-dependent density functional theory (TD-DFT) analyses. The complexes were also screened in vitro for their antiproliferative properties against the MCF-7 breast cancer cell lines by using the MTT assay. Flow cytometric analysis showed that the complexes arrested the cell cycle in the sub G0 phase.

SEPS1 protects RAW264.7 cells from pharmacological ER stress agent-induced apoptosis

Selenoprotein S (SEPS1) is a novel endoplasmic reticulum (ER) resident protein and it is known to play an important role in production of inflammatory cytokines. Here, we show evidence that SEPS1 is stimulated by pharmacological ER stress agents in RAW264.7 macrophages as well as other cell types. Overexpression studies reveal a protective action of SEPS1 in macrophages against ER stress-induced cytotoxicity and apoptosis, resulting in promoting cell survival during ER stress. The protective action of SEPS1 is largely dependent on ER stress-mediated cell death signal with less effect on non-ER stress component cell death signals. Conversely, suppression of SEPS1 in macrophages results in sensitization of cells to ER stress-induced cell death. These findings suggest that SEPS1 could be a new ER stress-dependent survival factor that protects macrophage against ER stress-induced cellular dysfunction.

Degradation of phenanthrene and pyrene in soil slurry reactors with immobilized bacteria Zoogloea sp.

The environmental fate of polycyclic aromatic hydrocarbons (PAHs) in soils is motivated by their wide distribution, high persistence, and potentially deleterious effect on human health. Polycyclic aromatic hydrocarbons constitute the largest group of environmental contaminants released in the environment. Therefore, the potential biodegradation of these compounds is of vital importance. A biocarrier suitable for the colonization by micro-organisms for the purpose of purifying soil contaminated by polycyclic aromatic hydrocarbons was developed. The optimized composition of the biocarrier was polyvinyl alcohol (PVA) 10%, sodium alginate (SA) 0.5%, and powdered activated carbon (PAC) 5%. There was no observable cytotoxicity of biocarriers on immobilized cells and a viable cell population of 1.86 × 10¹⁰ g^–1 was maintained for immobilized bacterium. Biocarriers made from chemical methods had a higher biodegradation but lower mechanical strengths. Immobilized bacterium Zoogloea sp. had an ideal capability of biodegradation for phenanthrene and pyrene over a relative wide concentration range. The study results showed that the biodegradation of phenanthrene and pyrene reached 87.0 and 75.4%, respectively, by using the optimal immobilized method of Zoogloea sp. cultivated in a sterilized soil. Immobilized Zoogloea sp. was found to be effective for biodegrading the soil contaminated with phenanthrene and pyrene. Even in "natural" (unsterilized) soil, the biodegradation of phenanthrene and pyrene using immobilized Zoogloea sp. reached 85.0 and 67.1%, respectively, after 168 h of cultivation, more than twice that achieved if the cells were not immobilized on the biocarrier. Therefore, the immobilization technology enhanced the competitive ability of introduced micro-organisms and represents an effective method for the biotreatment of soil contaminated with phenanthrene and pyrene.

Intracellular drug delivery by sulfatide-mediated liposomes to gliomas

We described here a liposomal carrier system in which the targeting ligand was sulfatide, a glycosphingolipid known to bind several extracellular matrix (ECM) glycoproteins whose expression was highly up-regulated in many tumors. In vitro experiments with human glioma cell lines demonstrated that robust intracellular uptake of the liposomes depended specifically on the presence of sulfatide as the key liposomal component. Significant amount of the liposomes remained largely intact in the cytoplasm for hours following their internalization. When anticancer drug doxorubicin (DOX) was encapsulated in such liposomes, most of the drug was preferably delivered into the cell nuclei to exert its cytotoxicity. Use of this drug delivery system to deliver DOX for treatment of tumor-bearing nude mice displayed much improved therapeutic effects over the free drug or the drug carried by polyethylene glycol (PEG)-grafted liposomes. Our results demonstrate a close link between effective intracellular uptake of the drug delivery system and its therapeutic outcome. Moreover, the sulfatide-containing liposomes (SCL) may represent an interesting ligand-targeted drug carrier for a wide spectrum of cancers in which sulfatide-binding ECM glycoproteins are expressed.

Small interfering RNA-mediated silencing of cytochromeP450 3A4 gene

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC₅₀ increased from 55 to 210 µM to >1000 µM. Nifedipine at 5.78, 14.44, and 28.88 µM was significantly (P < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study demonstrated, for the first time, the specific suppression of CYP3A4 expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.