The Origin of the Intracellular Silver in Bacteria: A Comprehensive Study using Targeting Gold-Silver Alloy Nanoparticles.

The bactericidal effects of silver nanoparticles (Ag NPs) against infectious strains of multiresistant bacteria is a well-studied phenomenon, highly relevant for many researchers and clinicians battling bacterial infections. However, little is known about the uptake of the Ag NPs into the bacteria, the related uptake mechanisms, and how they are connected to antimicrobial activity. Even less information is available on AgAu alloy NPs uptake. In this work, the interactions between colloidal silver-gold alloy nanoparticles (AgAu NPs) and Staphylococcus aureus (S. aureus) using advanced electron microscopy methods are studied. The localization of the nanoparticles is monitored on the membrane and inside the bacterial cells and the elemental compositions of intra- and extracellular nanoparticle species. The findings reveal the formation of pure silver nanoparticles with diameters smaller than 10 nm inside the bacteria, even though those particles are not present in the original colloid. This finding is explained by a local RElease PEnetration Reduction (REPER) mechanism of silver cations emitted from the AgAu nanoparticles, emphasized by the localization of the AgAu nanoparticles on the bacterial membrane by aptamer targeting ligands. These findings can deepen the understanding of the antimicrobial effect of nanosilver, where the microbes are defusing the attacking silver ions via their reduction, and aid in the development of suitable therapeutic approaches.

Hollow silver-gold alloy nanoparticles for enhanced photothermal/photodynamic synergetic therapy against bacterial infection and acceleration of wound healing.

Bacterial infection seriously restricts the wound healing process due to severe inflammation and delayed wound healing. Unfortunately, the overuse or improper use of antibiotics leads to the advent of multidrug-resistant strains and intractable biofilms, severely affecting the therapeutic effect. Therefore, there is an urgent need to develop antibiotic-free strategies to accelerate the healing process of wounds with bacterial infection. Considering that single photothermal therapy (PTT) or photodynamic therapy (PDT) cannot fully meet the requirements of clinical sterilization and accelerating wound healing, herein, hollow silver-gold alloy nanoparticles immobilized with the photosensitizer molecule Ce6 (Ag@Au-Ce6 NPs) integrated with PTT and PDT are proposed for killing bacteria and accelerating wound healing. The photothermal conversion properties of Ag@Au-Ce6 NPs are obtained using an infrared thermal imager, and the generation of singlet oxygen (1O2) is verified with an 1O2 fluorescent probe DCFH-DA. Manipulated by near-infrared laser triggered mild hyperthermia and limited ROS amount, Ag@Au-Ce6 NPs could effectively kill bacteria that are free and colonized on the surface of wounded skin, promoting epithelium migration and vascularization, further accelerating wound healing, which showed great promise for biomedical application.

Atomic-engineered gold@silvergold alloy nanoflowers for in vivo inhibition of bacteria.

The problems of multidrug-resistant bacteria and environmental pollution associated with the abuse of antibiotics call for effective antibiotic alternatives. Here, gold@silvergold alloy nanoflowers (Au@AgAu ANFs) with distinct atomic structures are first fabricated and then demonstrated for in vivo inhibition of bacteria. The Au@AgAu ANFs display high antibacterial activity against the model Gram-negative bacterium Escherichia coli, with a minimum inhibitory concentration value of 4.8 µg mL-1, which is 3.1 times lower than that of silver nanoparticles. The alloy structure with a rough surface enables Au@AgAu ANFs to firmly adhere to the bacterial surface and damage the cell membrane, resulting in long-term (48 h) and highly stable (30 days) antibacterial activity. Meanwhile, the Au@AgAu ANFs show remarkable biocompatibility with human cells even at a high concentration of 40 µg mL-1. Application of Au@AgAu ANFs in the treatment of bacterial infections in the mouse intestine significantly reduces the reproduction of bacteria compared to an untreated mouse, giving results similar to those of the current antibiotic treatment, with no cytotoxicity. Our study opens up a new avenue for the rational design of safe and highly efficient antibacterial materials.

A facile and green strategy for the synthesis of Au, Ag and Au-Ag alloy nanoparticles using aerial parts of R. hypocrateriformis extract and their biological evaluation.

A facile and green strategy is reported here to synthesize gold (Au), silver (Ag) and gold-silver (Au-Ag) alloy nanoparticles (NPs) through bio-reduction reactions of aqueous corresponding metal precursors mediated by extracts of aerial parts of R. hypocrateriformis, which act as both reducing and stabilizing agents, under microwave irradiation. UV-vis spectrophotometer, XRD, FT-IR, FESEM/TEM, TGA and EDAX analysis were used to characterize the obtained NPs. The formation of NPs is evident from their surface plasmon resonance peak observed at λmax=∼550, 450 and 500nm for Au, Ag and Au-Ag alloy NPs respectively. XRD pattern revealed that fcc structure, while FT-IR spectra signify the presence of phytochemicals adsorbed on NPs. Such a biofunctionalized NPs were characterized by their weight loss, 30% due to thermal degradation of plant phytochemicals observed in TG analysis. The spherical shape of Au, Ag and Au-Ag alloy NPs (∼10-50nm) is observed by FE-SEM/TEM images. EDAX analysis confirms the expected elemental composition. Moreover, these NPs showed enhanced antimicrobial, antioxidant, and anticancer activities, though it is more pronounced for Au-Ag alloy NPs, which is due to the combining effect of phytochemicals, Au and Ag metals. Thus, the biosynthesized NPs could be applied as effective growth inhibitors for various biomedical applications.

A new route for synthesis of silver:gold alloy nanoparticles loaded within phosphatidylcholine liposome structure as an effective antibacterial agent against Pseudomonas aeruginosa.

Ag:Au alloy nanoparticles were successfully synthesized through the new route using co-reduction method with silver nitrate, chloroauric acid, cetyl trimethyl ammonium bromide (CTAB) and sodium borohydride at room temperature. The Ag:Au alloy nanoparticles were then loaded within the phosphatidylcholine (97%) liposome structure. Various molar ratios of phosphotidylcholine and CTAB to the total metals were investigated showing its importance on the stability of nanocomposites suspension. The size distribution and morphology of encapsulated nanoparticles within the liposome structure were studied via ultraviolet (UV)-visible spectrum, transmission electron microscope (TEM) micrographs, and dynamic light scattering data. The synthesis of alloy nanoparticles were confirmed with formation of single band at 430, 465 and 500 nm for 75:25, 50:50 and 25:75 Ag:Au mole ratios, respectively. The TEM micrographs of different samples indicated formation of three various nanocomposite structures with size of 82-300 nm. The antibacterial activities of Ag:Au nanocomposites were studied against Pseudomonas aeruginosa through well-diffusion agar. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by Broth microdilution method. The results showed that 10 ppm nanocomposite reasonably killed the above bacteria.

Interfacial properties and in vitro cytotoxic effects of surface-modified near infrared absorbing Au-Au(2)S nanoparticles.

Near infrared (NIR) absorbing Au-Au(2)S nanoparticles were modified with surfactants of different hydrocarbon chain lengths to allow loading of anticancer drug, cisplatin. The interfacial interactions and surfactant chain length effects on drug loading, optical properties and cytotoxicity were discussed in this work. Short-chain surfactants were oriented closer to the surface normal and were adsorbed at higher densities. Surface modification also changed the optical properties of the particles. Notably, particles modified with short-chain surfactants exhibited a red shift, whereas particles modified with long-chain surfactants showed a blue shift. The in vitro cytotoxicity of drug-loaded surface-modified particles was dependent on the surfactants' chain length. Significant cytotoxicity was observed for 1 mg/ml of drug-loaded particles using surfactants with the shortest chain length. After NIR triggered drug release, the released Pt compounds were observed to be cytotoxic, while remaining nanoparticles did not exhibit any cytotoxicity. Also, the released Pt compounds upon NIR irradiation of drug-loaded particles were observed to be more toxic and had a different molecular structure from cisplatin.

Experimental assessment of biological compatibility of gold alloys by functional indices of salivary glands.

The effect of implanted gold alloys on composition and rate of excretion of oral fluid was studied on rats. It was found that gold alloy 900 modifies the function of the major salivary glands.

The effect of dental restorative materials on dental biofilm.

To investigate the arrangement of biofilms formed in vivo, volunteers wore splints with slabs of six different dental materials inserted to collect smooth surface plaque. After 5 d of undisturbed plaque accumulation, the specimens were vital stained and analyzed by the confocal laser scanning microscopy (CLSM) to evaluate the percentage of vital biofilm microflora (VF percentage). Further parameters were the area of the specimens covered by plaque (surface coating; SC, %) and the height of the biofilms (BH, pm). The metals amalgam and gold, the compomer, as well as the glass-ionomer cement harboured an almost entirely dead biofilm (VF <8%). Resin composite led to vitality values between 4 and 21%, while a very thin biofilm on ceramic revealed the highest vitality values (34-86%). SC varied from 6% on glass-ionomer cement to 100% on amalgam. BH reached its highest value on amalgam and gold of 17 and 11 microm, respectively, while heights of between 1 and 6 microm were found on the ceramic, resin composite, compomer and the glass-ionomer cement. Within their limits, the present findings indicate that amalgam, gold, compomer and glass-ionomer cement exert an influence against the adhering biofilm. No general relationship could be established between the different parameters VF percentage, SC percentage and BH (microm).

Effects of dental materials on insulin release from isolated islets of Langerhans.

OBJECTIVES: To investigate the possibility of using a whole organ model for evaluating the biological effects of dental restoration materials in vitro. METHODS: The effect on insulin release of isolated Langerhans islets of a series of dental materials was examined. The islets were incubated for 1h with extracts obtained from various dental materials and insulin was assayed radioimmunologically with crystalline mouse insulin. The results were analysed statistically using analysis of variance (ANOVA) and Scheffe's test at a significance level of p < 0.05. RESULTS: One dental ceramic, Vita VMK 95, significantly (p < 0.01) decreased the insulin release, whereas another dental ceramic, Empress (p < 0.01), a partly re-cast high-noble gold alloy (p < 0.001), a modified high-noble gold alloy (p < 0,05), and unalloyed copper (p < 0.001) significantly increased the release of insulin. SIGNIFICANCE: The results demonstrate a new instance of examining the biological effects of dental restoration materials. The method provides information about the effect of different materials on organ level in vitro that can complement other in vitro tests.

Cell proliferation rates and fibronectin arrangement as parameters for biocompatibility evaluation of dental metal alloys in vitro.

A short-term (72-96 hours) biocompatibility evaluation in vitro of four single phase dental metal alloys was conducted by determining cell proliferation rates correlated to the organization of the extracellular matrix protein fibronectin in human fibroblast cultures. Immunocytochemical methods were performed to detect both cell proliferation rates by 5-bromodeoxyuridine (BrdU) incorporation, and fibronectin arrangement, i.e., diffuse in the extracellular matrix, organized in fibrils or in focal adhesions. We showed that cell proliferation rates were related to fibronectin expression. In particular, a higher percentage of cells in the S-phase were related to a predominance of fibronectin organized both in fibrils and in focal adhesions. The alloy with the highest Au content seemed the most biocompatible among those tested, since it behaved in a very similar manner to the controls. On the contrary, fibroblasts exposed to the alloy with the highest percentage of Ag had the most different behavior as compared to the controls. We can assume that a correlation exists between fibronectin organization and the percentage of BrdU-positive cells and that these parameters are varying with the different metal composition of the alloys. The observation of fibronectin arrangement together with cell proliferation rates could be considered a useful tool to determine the biocompatibility of these biomaterials.

Cytotoxicity of dental casting alloys pretreated with biologic solutions.

STATEMENT OF PROBLEM: Short-term (72-168 hours) in vitro tests are used to evaluate the cytotoxicity of dental casting alloys. The ability of these short-term tests to predict long-term in vivo cytotoxicity has been questioned. A procedure to accelerate the testing of casting alloys would be useful in predicting longer-term alloy cytotoxicity. PURPOSE: This study hypothesized that preconditioning casting alloys by soaking in a biologic liquid would change subsequent cytotoxicity by removing some elements. Preconditioning may be 1 method of accelerating short-term in vitro tests. MATERIAL AND METHODS: Dental casting alloys were exposed to either saline, cell culture medium, or a saline/bovine serum albumin (BSA) solution for 72 hours before standard in vitro cytotoxicity testing. Six types of alloys were tested (n = 6): 5 Au-Ag-Cu-Pd alloys (single phase) and 1 Ag-Pd-Cu alloy (multiple phase). Teflon (Tf) samples served as a control. After preconditioning, alloys were placed in direct contact with Balb/c fibroblasts for 72 hours, after which cell viability was measured by succinic dehydrogenase activity (MTT method) relative to Tf controls (100% = no toxicity). Elements released into the preconditioning solutions were measured by atomic absorption spectroscopy. Cytotoxicities of preconditioned alloys and amounts of elemental release were compared with unconditioned alloys. RESULTS: A preconditioning time of 72 hours was sufficient to change the cytotoxicity of the tested alloys. The alloys that were more cytotoxic initially became less cytotoxic after preconditioning. For all the alloys tested, except the Ag-Pd-Cu multiphase alloy, preconditioning with either the saline or the saline/BSA solution caused an increase in cellular activity, therefore the preconditioned alloys were less cytotoxic. The cell culture medium preconditioning solution had a variable effect, causing increased or decreased cellular activity depending on the alloy treated. CONCLUSION: Preconditioning of casting alloys decreased subsequent cytotoxicity. However, not all preconditioning solutions are equivalent. A preconditioning strategy may be useful in accelerating the short-term cytotoxicity test toward a longer-term result.

The effect of dental alloys on mouse lymphocyte subpopulations.

The aim of the present study was to determine the effect of nickel-containing alloys on lymphocyte subsets in an experimental setting. Plates of alloys containing nickel (Ceramalloy, Talladium, Cerillium, Rexillium) or gold (Orion) were implanted subcutaneously into mice. The levels of CD4+ and CD8+ T-lymphocyte subpopulations and of Smig+ B lymphocytes were determined at various intervals following implantation, using monoclonal antibodies and flow cytometry. No changes were detected in the proportion of the lymphocyte subsets tested. One month after implantation, the mean fluorescence intensity of CD4, CD8 or Smig, in the peripheral blood lymphocytes (PBL) of the nickel alloy-implanted animals, was significantly higher than that prior to this procedure. Only a mild increase in CD4 and CD8 was noted after implantation of the gold alloy. The observed effects are most likely attributable to the surgical trauma, and do not indicate that nickel-containing dental alloys influence T cell subsets in this murine model.

The influence of dental metal alloys on cell proliferation and fibronectin arrangement in human fibroblast cultures.

The biocompatibility of six single-phase dental metal alloys was studied by determining cell proliferation rates correlated to the arrangement of fibronectin (FN) in fibroblast cultures. Immunocytochemical methods were used to detect cell proliferation by 5-bromodeoxyuridine (BrdU) incorporation, and FN organization [i.e. diffuse in the extracellular matrix and organized in fibrils or in focal adhesions (FA)] in human fibroblast cultures. Cell proliferation rates were related to FN arrangement and in particular a higher percentage of cells in the S-phase was related to a predominance of FA. The greatest difference in behaviour compared to that of the controls was detected after 120 and 168 hr: at these times, as well as at previous ones, the alloy with the highest Au content seemed the most biocompatible among those tested, as it behaved in a very similar way to the controls. In contrast, fibroblasts exposed to the other five alloys showed different behaviours from the controls. It is assumed that a correlation exists between FN organization and the percentage of BrdU-positive cells, and that these features vary in the presence of different alloys. The observation of FN arrangement together with cell proliferation rates could be another useful tool in determining the biocompatibility of dental metal alloys.

A comparison of two systems for bonding type II gold alloy to dentine: an in vitro study.

This in vitro investigation compares the shear bond strength of two systems that can be used to bond gold alloys to dentine. In the first group, sandblasted gold alloy surfaces were tin-plated and bonded to dentine using C&B-Metabond, an adhesive resin cement. In the second group sandblasted gold alloy surfaces were silicoated (Kulzer) and bonded to dentine using Denthesive II, Adhesive Bond II and Twinlook cement. Forty eight hours after bonding the bonds were stressed to failure in the sheer mode in an Instron testing machine. All data were statistically analysed (Student-t-test). The results in MPa were: Tin-plated gold to dentine 18.23; silicoated gold to dentine: 10.35. C&B-Metabond demonstrated a statistically significantly higher bond strength (p < 0.01). It can be concluded that tin-plating in conjunction with C&B-Metabond produces a stronger bond than silicoating in conjunction with Denthesive II and Twinlook cement when used to bond gold alloys to dentine.