Investigating the Effect of Silver and Silica Nanoparticles on the Hemolysis of Human Blood Cells and the Survival of Jurkat Lymphocyte Cells in a Cell Culture Model

Assessment of cytotoxic and genotoxic potential of refinery waste effluent using plant, animal and bacterial systems

Aspergillus sydowii is a mesophilic soil saprobe that is a food contaminant as well as a human pathogen in immune-compromised patients. The biological fabrication of silica and silver nanoparticles provides advancements over the chemical approach, as it is eco-friendly and cost-effective. In the present study, Aspergillus sydowii isolates were collected from the soil fields of six different sites in the western area of Saudi Arabia and then identified using the PCR technique following sequencing analysis by BLAST and phylogenetic analysis. Then, applied silica and silver nanoparticles were synthesized by biological methods, using Aspergillus niger as a reducer. Silver and silica nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The antifungal activity of silver and silica nanoparticles against Aspergillus sydowii isolates was evaluated using the disc diffusion method and the minimum inhibitory concentration (MIC). The physiochemical results emphasized the fabrication of silver and silica nanoparticles in spherical shapes with a diameter in the range of 15 and 40 nm, respectively, without any aggregation. MIC of Ag-NPs and Si-NPs against Aspergillus sydowii isolates were 31.25 and 62.5 µg/mL, respectively. Finally, the aim of the study is the use of silver as well as silica nanoparticles as antifungal agents against Aspergillus sydowii.

A novel specifically targeted antimicrobial peptide (STAMP) that was especially effective against methicillin-resistant Staphylococcus aureus (MRSA) was designed by fusing the AgrD1 pheromone to the N-terminal end of plectasin. This STAMP was named Agplectasin, and its gene was synthesized and expressed in Pichia pastoris X-33 via pPICZαA. The highest amount of total secreted protein reached 1,285.5 mg/l at 108 h during the 120-h induction. The recombinant Agplectasin (rAgP) was purified by cation exchange chromatography and hydrophobic exchange chromatography; its yield reached 150 mg/l with 94 % purity. The rAgP exhibited strong bactericidal activity against S. aureus but not Staphylococcus epidermidis or other types of tested bacteria. A bactericidal kinetics assay showed that the rAgP killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in both Mueller-Hinton medium and human blood within 10 h when treated with 4× minimal inhibitory concentration. The rAgP caused only approximately 1 % hemolysis of human blood cells, even when the concentration reached 512 μg/ml, making it potentially feasible as a clinical injection agent. In addition, it maintained a high activity over a wide range of pH values (2.0-10.0) and demonstrated a high thermal stability at 100 °C for 1 h. These results suggested that this STAMP has the potential to eliminate MRSA strains without disrupting the normal flora.

Incorporating zinc into biocompatible materials has been identified as a potential strategy for promoting bone regeneration and osteogenic activity during hard tissue regeneration. This work aimed to investigate the impact of zinc doping on the structure of akermanite, which was synthesized using the sol-gel combustion method, with the goal of improving the biological response. Powder XRD and FT-IR analysis confirmed the phase purity and the respective functional groups associated with Zn-doped akermanite. Further XPS analysis confirmed the presence of zinc with the respective binding energies in the akermanite matrix. According to the results obtained from the analysis, the apatite-forming ability of Zn-doped akermanite demonstrated enhanced apatite deposition on the surface of the pellet after 9 days of immersion in the SBF medium. The measured mechanical parameters, including compressive strength (140-189 MPa) and Young's modulus (2505-3599 MPa), fall within the range of human cortical bone. Antimicrobial results showed an improved inhibition rate of the doped ceramics compared to pure akermanite with an inhibition percentage of 87% even at lower concentrations. The hemocompatibility of the materials showed hemolysis of human blood cells within the acceptable range without exhibiting toxicity. Cytotoxicity results demonstrate the biocompatibility of the materials with the MG-63 cell line. Based on the results, akermanite doped with zinc at optimal concentrations was found to be compatible and nontoxic promoting it as a potential alternative for bone regeneration in orthopedic applications.

Aim:This study was designed to describe a series of in vitro tests that may aid the discovery of probiotic strains from actinomycetes.Materials and Methods:Actinomycetes were isolated from marine sediments using four different isolation media, followed by antimicrobial activity and toxicity assessment by the agar diffusion method and the hemolysis of human blood cells, respectively. Extracellular enzymatic production was monitored by the hydrolysis of proteins, lipids and carbohydrates. Tolerance to different pH values and salt concentrations was also determined, followed by hydrophobicity analysis and genetic identification of the most promising strains.Results:Five out of 31 isolated strains showed antimicrobial activity against three Vibrio species. Three non-hemolytic strains (N7, RL8 and V4) among these active isolates yielded positive results in hydrophobicity tests and exhibited good growth at salt concentrations ranging from 0% to 10%, except strain RL8, which required a salt concentration >0.6%. Although these strains did not grow at pH<3, they showed different enzymatic activities. Phylogenetic analysis revealed that strains N7 and V4 have more than 99% identity with several Streptomyces species, whereas the closest matches to strain RL8 are Streptomyces panacagri and Streptomyces flocculus, with 98% and 98.2% similarity, respectively.Conclusion:Three actinomycetes strains showing probiotic-like properties were discovered using several in vitro tests that can be easily implemented in different institutions around the world.

Function improvement of wool fabric based on surface assembly of silica and silver nanoparticles

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely – a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16INK4a, p21, p27 and p57), p14ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies.

BackgroundAs nanoparticles (NPs) become more prevalent in the pharmaceutical industry, questions have arisen from both industry and regulatory stakeholders about the long term effects of these materials. This study was designed to evaluate whether gold (10 nm), silver (50 nm), or silica (10 nm) nanoparticles administered intravenously to mice for up to 8 weeks at doses known to be sub-toxic (non-toxic at single acute or repeat dosing levels) and clinically relevant could produce significant bioaccumulation in liver and spleen macrophages.ResultsRepeated dosing with gold, silver, and silica nanoparticles did not saturate bioaccumulation in liver or spleen macrophages. While no toxicity was observed with gold and silver nanoparticles throughout the 8 week experiment, some effects including histopathological and serum chemistry changes were observed with silica nanoparticles starting at week 3. No major changes in the splenocyte population were observed during the study for any of the nanoparticles tested.ConclusionsThe clinical impact of these changes is unclear but suggests that the mononuclear phagocytic system is able to handle repeated doses of nanoparticles.

Introduction Up to 40% of patients with gastro-oesophageal reflux disease remain symptomatic despite PPI therapy. In addition to acid the refluxate contains pepsin and bile acids, which may be important in symptom perception. A topical therapy could protect against all components of the refluxate. A reliable model for testing could be used to screen compounds for clinical evaluation. We aimed to evaluate and compare the topical protective effect of an alginate solution in a cell culture and human biopsy model. Method Primary human oesophageal epithelial cells were established into multilayer stratified squamous epithelia using an air-liquid interface technique. After placing in Ussing chambers the apical surface was “protected” with 200 µl of either alginate, viscous control, or was unprotected. After 15 min washing in neutral solution the tissue was exposed to pH 3 + bile acid solution for 30 min and TER change was calculated. Distal human oesophageal biopsies were taken, placed in Ussing chambers and “protected” as above. After 15 min or 60 min washing in neutral solution, change in TER on 30 min exposure to pH 1 + bile acid + pepsin solution was measured. Thickness of a fluorescent-labelled alginate coating on the biopsy luminal surface was measured after 15 min (5 biopsies) and 60 min (5 biopsies) washing in pH 7.4 solution. Results Acid-induced change in TER was significantly less in alginate-protected epithelium than in epithelium protected with viscous control or unprotected tissue. This was seen in both the cell culture (–1.2 ± 5.1%, n = 5 vs. –38.4 ± 6.2%, n = 4 vs. –41.9 ± 8.1%, n = 3) and human biopsy models (−2.7 ± 1.8% vs. −15.26 ± 3.7% vs. −21.1 ± 4.4%, all n = 12) after 15 min washing. Statistically significant protection was still seen after 60 min washing (–8.3 ± 2.2% vs. –25.1 ± 4.5% vs. –26.4 ± 5.3%, n = 12). After 15 min washing the mean alginate layer thickness was 5.7 ± 0.3 µm, and was 4.3 ± 0.3 µm after 60 min washing. Conclusion Alginate topical protection of human oesophageal epithelium against acid-injury is demonstrated in both cell culture and human biopsy models. Epithelial adherence persists for up to 60 min. Topical alginate mucosal protection may be promising as an add-on/alternative therapy for gastro-oesophageal reflux disease. Since the cell culture model was able to reproduce ex vivobiopsy results, it could be an effective tool to screen candidate products prior to clinical evaluation. Disclosure of interest F. Batista-Lima: None Declared, C. Lee: None Declared, D. Sifrim Grant/ Research Support from: Research grant from Reckitt-Benckiser, P. Woodland: None Declared.

The use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics has increased significantly owing to their antibacterial and antifungal properties. As a consequence, the need for validated rapid screening methods to assess their toxicity is necessary to ensure consumer safety. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential cytotoxicity of food- and cosmetic-related nanoparticles. The two cell culture models were utilized to compare the potential cytotoxicity of 20-nm silver. The average size of the silver nanoparticle determined by our transmission electron microscopy (TEM) analysis was 20.4 nm. The dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The concentration of the 20-nm silver solution determined by our inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Our ICP-MS and TEM analysis demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Cytotoxicity, determined by the Alamar Blue reduction assay, was evaluated in the nanosilver concentration range of 0.1 to 20 µg ml(-1) . Significant concentration-dependent cytotoxicity of the nanosilver in HepG2 cells was observed in the concentration range of 1 to 20 µg ml(-1) and at a higher concentration range of 10 to 20 µg ml(-1) in Caco2 cells compared with the vehicle control. A concentration-dependent decrease in dsDNA content was observed in both cell types exposed to nanosilver but not controls, suggesting an increase in DNA damage. The DNA damage was observed in the concentration range of 1 to 20 µg ml(-1) . Nanosilver-exposed HepG2 and Caco2 cells showed no cellular oxidative stress, determined by the dichlorofluorescein assay, compared with the vehicle control in the concentration range used in this study. A concentration-dependent decrease in mitochondria membrane potential in both nanosilver exposed cell types suggested increased mitochondria injury compared with the vehicle control. The mitochondrial injury in HepG2 cells was significant in the concentration range of 1 to 20 µg ml(-1) , but in Caco2 cells it was significant at a higher concentration range of 10 to 20 µg ml(-1) . These results indicated that HepG2 cells were more sensitive to nanosilver exposure than Caco2 cells. It is generally believed that cellular oxidative stress induces cytotoxicity of nanoparticles. However, in this study we did not detect any nanosilver-induced oxidative stress in either cell type at the concentration range used in this study. Our results suggest that cellular oxidative stress did not play a major role in the observed cytotoxicity of nanosilver in HepG2 and Caco2 cells and that a different mechanism of nanosilver-induced mitochondrial injury leads to the cytotoxicity. The HepG2 and Caco2 cells used this study appear to be targets for silver nanoparticles. The results of this study suggest that the differences in the mechanisms of toxicity induced by nanosilver may be largely as a consequence of the type of cells used. This differential rather than universal response of different cell types exposed to nanoparticles may play an important role in the mechanism of their toxicity. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, are excellent systems for screening cytotoxicity of silver nanoparticles. These long established cell culture models and simple assays used in this study can provide useful toxicity and mechanistic information that can help to better inform safety assessments of food- and cosmetic-related silver nanoparticles.

Background:The ultimate goal of endodontic therapy is to eliminate all microorganisms present inside root canal and thereby sealing all the possible communicating pathways between pulpal and periradicular tissues, which prevents all the factors that cause recontamination and reinfection of the root canal system. If endodontic treatment fails, next approach is surgical endodontics. Bioceramics are recently introduced materials specifically designed for their potential use in medical field and dentistry.Aim:To evaluate and compare the push-out bond strength of mineral trioxide aggregate (MTA) by adding titanium dioxide (TiO2), silver, and silicon dioxide nanoparticles.Materials and Methods:Totally, 60 single-rooted human teeth were used. Middle third of the root was sectioned to obtain 2-mm thick root section. Acrylic was adapted to the section to obtain disks of 5 mm diameter and 2 mm thickness. Canal was prepared by GG Drill. Samples were divided into four groups of 15 each (n = 15): • Group I (control): MTA • Group II: MTA + TiO2 nanoparticles. • Group III: MTA + silver nanoparticles. • Group IV: MTA + silicon dioxide nanoparticles. The cement mixture was compacted into the canal. Samples were subjected to push-out bond strength using universal testing machine.Statistical Analysis Used:The data were analyzed statistically by analysis of variance and post hoc comparison by Tukey's t-test.Results:The highest push-out bond strength was shown by Group II (MTA with TiO2 nanoparticles), followed by Group III (MTA with silver nanoparticles) and Group I (MTA control group). The lowest push-out bond strength was shown by Group III (MTA with silicon dioxide nanoparticles).Conclusions:TiO2 and silver nanoparticles when added into MTA lead to an increase in push-out bond strength of MTA.

In spite of the vast nutritional and environmental benefits provided by fava bean (Vicia faba), the ingestion of vicine/convicine provokes an acute hemolytic anemia called favism in individuals with a glucose-6-phosphate dehydrogenase (G6PD) deficiency. The elimination of these glycosides is a goal that could be accomplished using different processing methods including bacteriological treatment. Laban as a good source of lactic acid bacteria was tested in an ex vivo assay on human blood samples in order to determine its capacity in decreasing the hemolysis crisis induced by the ingestion of fava beans. Results indicate a significant decrease in human blood cell hemolysis after the treatment of fava beans by Laban. This decrease in hemolysis was also correlated with the G6PD deficiency categorization. The highest hemolysis level (mean: 23.11 ± 0.76%) was observed in samples with G6PD activity between 10 and 30%, while the lowest hemolysis level (mean: 5.75 ± 0.64%) was observed in samples with G6PD activity more than 60%. This decrease was correlated with a high antioxidant capacity of Laban (51.61 ± 1.13% expressed by the percentage inhibition of DPPH radical). The counts of isolates from MRS and M17 culture plates were 6.75 ± 0.095 and 7.91 ± 0.061 log cfu ml–1, respectively. In conclusion, the synergy between the antioxidant properties of Laban and the possible decrease of vicine and convicine concentrations by lactobacillus found in the fermented dairy products could explain the ability of Laban to reduce the hemolysis crisis ex vivo.

Silver and silica nanoparticles stabilised by long chain alkanes have been prepared and characterised using 1H-NMR, transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS). Upon solvent evaporation, the alkane stabilised silica and silver nanoparticles self-assemble into close-packed two- and three-dimensional structures. Furthermore, it is shown that a mixture of large silica (90 A) and small silver (48 A) nanoparticles self-assemble into mixed well-defined close-packed structures. It has been demonstrated that particle size, core material and solvent composition can be employed to control the forces between individual nanoparticles and, consequently, the structure of the self-assembled arrays. These mixed structures have also been studied using UV-visible spectroscopy and their collective optical properties are shown to depend on their overall composition.

Shadoo (Sho) is a neuronally expressed glycoprotein of unknown function. Although there is no overall sequence homology to the cellular prion protein (PrP(C)), both proteins contain a highly conserved internal hydrophobic domain (HD) and are tethered to the outer leaflet of the plasma membrane via a C-terminal glycosylphosphatidylinositol anchor. A previous study revealed that Sho can reduce toxicity of a PrP mutant devoid of the HD (PrPΔHD). We have now studied the stress-protective activity of Sho in detail and identified domains involved in this activity. Like PrP(C), Sho protects cells against physiological stressors such as the excitotoxin glutamate. Moreover, both PrP(C) and Sho required the N-terminal domain for this activity; the stress-protective capacity of PrPΔN as well as ShoΔN was significantly impaired. In both proteins, the HD promoted homodimer formation; however, deletion of the HD had different effects. Although ShoΔHD lost its stress-protective activity, PrPΔHD acquired a neurotoxic potential. Finally, we could show that the N-terminal domain of PrP(C) could be functionally replaced by that of Sho, suggesting a similar function of the N termini of Sho and PrP(C). Our study reveals a conserved physiological activity between PrP(C) and Sho to protect cells from stress-induced toxicity and suggests that Sho and PrP(C) might act on similar signaling pathways.

Liver fibrosis in vitro: Cell culture models and precision-cut liver slices