Conformational change of hydroxyapatite/gelatin nanocomposite by glutaraldehyde

The hydroxyapatite (HAp)/gelatin (GEL) nanocomposite was prepared through the coprecipitation and then cross-linked by using glutaraldehyde (GA). From FT-IR measurement the spectral features for amide bands and phosphate bands were severely modified by the cross-linkage and the organic content increased with the degree of cross-linkage. From Transmission Electron Microscopy (TEM) and electron diffraction analyses we could confirm the preferentially directional growth of needle-like HAp particles, which were embedded in GEL by the mineralization. Also we could observe worm-like stria patterns of contrast, which were revealed by the mineralization on the individual GEL fibrils. Moiré images were observed in a highly cross-linked HAp/GEL nanocomposite sample and we think the cross-linkage induced the assembly of unordered individual fibrils along the preferential direction.     

Poly(hydroxyethyl methacrylate-co-methacrylated-beta-cyclodextrin) hydrogels: synthesis, cytocompatibility, mechanical properties and drug loading/release properties

Copolymerization of hydroxyethyl methacrylate (HEMA) with a methacrylated-derivative of β-cyclodextrin (β-CD) was evaluated as a way to obtain hydrogels with tunable mechanical and drug loading and release properties, particularly for preparing medicated soft contact lenses. A fully methacrylated β-CD monomer was synthesized and added to the HEMA and cross-linker solution at concentrations ranging from 0.042 to 0.333 g ml⁻¹ (i.e. 0.23–1.82 mol.%). Thermal polymerization led to transparent hydrogels with a degree of conversion above 74%, which showed a high cytocompatibility and did not induce macrophage response. The greater the content in methacrylated β-CD was, the higher the glass transition temperature, the lower the degree of swelling and free water proportion, and the greater the storage and loss moduli of the swollen disks. These findings are directly related to the increase in the degree of cross-linking caused by the methacrylated β-CD. Loading studies were carried out with hydrocortisone and acetazolamide, both able to form complexes with CDs in water and in lacrimal fluid. Hydrocortisone loading progressively decreased as the content in methacrylated β-CD rose due to a decrease in the volume of aqueous phase of the hydrogel. Acetazolamide loading showed a maximum for an intermediate content in β-CD (0.125–0.167 g ml⁻¹) owing to a balance between complexation with β-CD and hydrogel mesh size. The hydrogels sustained drug delivery for several days, the acetazolamide release rate being dependent on the β-CD content. An adequate selection of the content in β-CD enables pHEMA-co-β-CD hydrogels suitable for specific biomedical applications to be obtained.     

Electrochemical screening of self-assembling beta-sheet peptides using supported phospholipid monolayers

In the context of the medical applications of β-sheet self-assembling peptides, it is important to be able to predict their activity at the biological membrane level. A study of the interaction of four systematically varied 11-residue (P11-1, P11-2, P11-6 and P11-7) and one 13-residue (P13-1) designed β-sheet self-assembling peptides with DOPC monolayers on a mercury electrode is reported in this paper. Experiments were carried out in 0.1 mol dm⁻³ KCl electrolyte with added phosphate buffer (0.001 mol dm⁻³) at pH  7.6. The capacity–potential curves of the coated electrode in the presence and absence of the different peptides were measured using out-of-phase ac voltammetry. The frequency dependence of the complex impedance of the coated electrode surfaces in the presence and absence of the peptides was estimated between 65,000 and 0.1 Hz at −0.4 V versus Ag/AgCl 3.5 mol⁻³ dm⁻³ KCl. The monolayer permeabilising properties of the peptides were studied by following the reduction of Tl(I) to Tl(Hg) at the coated electrode. Of the five peptides studied, P11-2, P11-7 and P13-1 interact most strongly with the DOPC layer. P11-1 which has a polar primary structure shows no obvious interaction with the phospholipid but surprisingly, it permeabilises the phospholipid layer to Tl⁺.     

Slow isomerization step in the interaction between mouse dopamine transporter and dopamine re-uptake inhibitor N-(3-iodoprop-2E-enyl)-2beta-carbo-[3H]methoxy-3beta-(4'-methylphenyl)nortropane

The kinetics of the association and dissociation of the tritium-labeled selective and potent dopamine transporter inhibitor N-(3-iodoprop-2E-enyl)-2β-carbo-[³H]methoxy-3β-(4′-methylphenyl)nortropane ([³H]PE2I) with the transporter of mouse striatal membranes was studied. The analysis revealed that the specific binding of [³H]PE2I occurs within a homogeneous population of binding sites in these membranes. The relatively slow binding process was characterized by the pseudo-first-order rate constant k_obs. The plot of these rate constants versus free radioligand concentration was hyperbolic, demonstrating that at least two kinetically distinguishable steps can be identified in the interaction of dopamine transporter with this inhibitor. The fast and reversible binding step, characterized by dissociation constant K_A = 51 ± 23 nM, is followed by a slow but also reversible isomerization step of the complex, characterized by the isomerization rate constant k_i = (7 ± 2)10⁻² s⁻¹ and by the rate constant k_−i = (3.9 ± 0.5)10⁻³ s⁻¹ for the reverse process. This isomerization step increases the apparent affinity of the ligand and probably consists of a conformational transition of the transporter protein, induced by the inhibitor molecule.     

2-Cys peroxiredoxin PfTrx-Px1 is involved in the antioxidant defence of Plasmodium falciparum

Peroxiredoxins (Trx-Px) are ubiquitous antioxidant enzymes that catalyse the thioredoxin-dependent reduction of hydroperoxides. The number of characteristic active site (VCP/T) motifs defines these proteins as 1-Cys and 2-Cys Trx-Px. Steady-state kinetic parameters of Plasmodium falciparum 2-Cys Trx-Px (PfTrx-Px1) were determined using stopped flow rapid kinetics. The bi-substrate reaction displays ping-pong kinetics and the K_m values for H₂O₂ and thioredoxin were determined to be 0.78±0.14 μM and 18.94±3.01 μM, respectively. The V_max^app and k_cat^app for H₂O₂ were found to be 4±0.6 U mg⁻¹ and 1.67±0.25 s⁻¹, respectively and those for thioredoxin are 23.0±0.2 U mg⁻¹ and 9.65±0.1 s⁻¹, emphasising the specificity of the enzyme for the substrate H₂O₂. After subjection to exogenous and endogenous oxidative stress, P. falciparum blood stage forms showed a marked elevation of PfTrx-Px1 mRNA and protein levels consistent with the hypothesis that it is an important component of the parasite’s antioxidant machinery. Gel filtration, cross-linking and electron microscopy (EM) revealed that the protein forms decamers consisting of pentamers of homodimers that have a doughnut-like shape consistent with the structures of related proteins. No dimeric forms of the protein were detectable after gel filtration suggesting that PfTrx-Px1 predominantly exists as an oligomer.     

Linkage of low and high affinity anti-fluorescein idiotype families

Data presented in this study describes the isolation and characterization of two anti-fluorescein (Fl) hybridoma proteins 3–24 and 12–40, both IgG₁, with a K_a = 2.8 and 3.4 × 10⁶ M⁻¹, respectively, at 37°C. These clones inhibited (6.8 ± 2.8 − 20.8 ± 0.6% at l μg/well) the idiotype-anti-idiotype interactions (IAII) of anti-Fl clones 3–13 and 3–17, which define a previously described low affinity idiotype family. Antibodies 3–24 and 12–40 also inhibited (45.0 ± 3.0 and 61.3 ± 5.6%, respectively, at 1 μg/well) an IAII denfied by a high affinity (K_a = 5.2 ± 1.5 × 10⁹ M⁻¹ at 37°C) anti-Fl clone, 4-4. Hybridoma proteins 3–13 and 3–17 possess similar affinities for Fl (K_a = 3.8 ± 5.1 and 5.9 ± 4.0 × 10⁴ M⁻¹) and are known to be idiotypically unrelated to clone 4-4. While 3–24 and 12–40 appeared very similar, non-identity of their active sites was established by heterologous idiotypic inhibitions, fine specificity of binding and spectral measurements (Q_max and λ_max) of bound Fl. All IAII (3–13, 3–17, 9–40 and 4-4) were inhibited>80% by the presence of 10⁻⁴M F1 or F1-BSA, In addition, four intermediate affinity (6.0 × 10⁶ K_a 5.3 × 10⁸ M⁻¹) anti-FI clones, comprising a second previously described idiotype family (designated the 9–40 family) were further analyzed. Inhibition of the 9–40 IAII by all heterologous proteins in the 9–40 family (except clone 5–27), and clones 3–24, 12–40 and 4-4 ranged from 87.7 ± 1.3 to 95.4 ± 1.0% at 1μg/well. Titration of the 9–40 IAII inhibition by antibodies 9–40, 3–24, 12–40 or 4-4 generated essentially superimposable profiles. In reciprocal inhibition experiments, using the 4-4 IAII, clones 3–24, 12–40, 9–40 and 4-4 gave distinct idiotypic titration patterns. Thus, members of the 9–40 family, 3–24 and 12–40 were more closely related to intermediate affinity clone 9–40 than high affinity clone 4-4. Finally all members of the 9–40 family also significantly inhibited both the 3–13 and 3–17 IAII (11.8 ± 3.1 − 32.9 ± 6.1 at 1 μg/well) and gave distinct idiotypic inhibition profiles. Clones 3–24 and 12–40, characterized in this report, and the 9–40 family provide linkage between idiotypically distinct anti-Fl hybridoma proteins differing in affinity by> 20,000-fold. This linkage provides a greater span in affinity, than in all previously reported idiotypic families, within restricted or unrestricted systems.     

Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system

Isolated hepatocytes in spheroid configuration exhibit a high degree of cell–cell contacts, which are important in the maintenance of viability and liver specific functions. In the absence of a vascular network, the cells in a large spheroid size experience mass transfer limitations of metabolites and oxygen in the core of aggregates. In this paper transport phenomena related to the diffusion and reaction of oxygen, glucose and lactate are mathematically described and experimentally verified for hepatocyte spheroids cultured in a rotating-wall polystyrene system (RWPS) not permeable for gases and in a rotating-wall membrane system (RWMS) with oxygen-permeable membrane. The concentration profiles of glucose, oxygen and lactate in the hepatocyte spheroids were estimated for different diameters of aggregates by solving the mass transfer equations for simultaneous diffusion and reaction, by finite element method. Simulation results evidenced that, for aggregates with size lower than 300 μm cultured in both RWPS and RWMS systems, the concentration profiles of glucose and lactate towards the core of spheroids (effective diffusion coefficients in the order of 10⁻¹¹ m²/s) are not significantly affected by the metabolic rate (c.a 10⁻⁶ μg/mm³/s for glucose and about one order of magnitude less for lactate). On the contrary, the transport of oxygen (diffusion coefficient: 3.4×10⁻¹⁰ m²/s, reaction rate: 1.5×10⁻⁵ μg/mm³/s) is critically affected by the size of the multicellular spheroids and significant gradients in oxygen concentration may develop in spheroids. Aggregates with a size greater than 200 μm suffer severe oxygen limitation in the most part of its size attaining the lowest partial pressure in the centre. The improved viability predicted by the model culturing hepatocyte spheroids in the RWMS, characterized by a higher O₂ permeability with respect to RWPS, was experimentally confirmed. The results demonstrated that the mathematical model used in this study represents a useful support to experimental procedures in order to obtain hepatocyte spheroids with optimal size.     

SLOS carrier frequency in Poland as determined by screening for Trp151X and Val326Leu DHCR7 mutations

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations in the DHCR7 gene. Previous studies estimated the prevalence of SLOS between 1 in 10,000 to 1 in 70,358 based on case frequency surveys. Although panethnic, SLOS appears to be most frequent in Central European populations (Czech Republic 1 in 10,000, Slovakia 1 in 15,000 – 1 in 20,000). In Polish individuals with SLOS two DHCR7 mutations, c.452G > A (p.Trp151X) and c.976G > T (p.Val326Leu), account for 65.2% of all observed DHCR7 mutations. We analyzed 2169 samples for the p.Trp151X mutation and 2087 for the p.Val326Leu mutation. The combined carrier frequency of these two mutations of was 2.40 ± 0.32%, yielding a calculated incidence of SLOS in Poland of 2.5 4 × 10⁻⁴–4.3 5 × 10⁻⁴ (1 in 2,300 to 1 in 3,937) placing SLOS among the most common recessive genetic disorders in Poland.     

Modification of Hydroxyapatite/Gelatin Nanocomposite Using Polyacrylamide

A hydroxyapatite (HAp)/gelatin (GEL) nanocomposite was mixed with mineralized polyacrylamide (PAM) to produce a macrocomposite. The mineralization of PAM was carried out by solution-precipitation using Ca(OH)₂ and H₃PO₄. The crystal growth of HAp in PAM was moderately changed from amorphous-like nanocrystalline to crystalline with the increase of PAM. The dry body of HAp/PAM nanocomposite cracked after the immersion test in water, but the cross-linked sample using glutaraldehyde did not crack. The macrocomposite of HAp/GEL nanocomposite and HAp/PAM nanocomposite showed good toughness, but cracked after the immersion test in water. The cross-linked macrocomposite sample did not crack after the immersion test in water.     

Modafinil more effectively induces wakefulness in orexin-null mice than in wild-type littermates

Narcolepsy—cataplexy, a disorder of excessive sleepiness and abnormalities of rapid eye movement (REM) sleep, results from deficiency of the hypothalamic orexin (hypocretin) neuropeptides. Modafinil, an atypical wakefulness-promoting agent with an unknown mechanism of action, is used to treat hypersomnolence in these patients. Fos protein immunohistochemistry has previously demonstrated that orexin neurons are activated after modafinil administration, and it has been hypothesized that the wakefulness-promoting properties of modafinil might therefore be mediated by the neuropeptide. Here we tested this hypothesis by immunohistochemical, electroencephalographic, and behavioral methods using modafinil at doses of 0, 10, 30 and 100 mg/kg i.p. in orexin^−/− mice and their wild-type littermates. We found that modafinil produced similar patterns of neuronal activation, as indicated by Fos immunohistochemistry, in both genotypes. Surprisingly, modafinil more effectively increased wakefulness time in orexin^−/− mice than in the wild-type mice. This may reflect compensatory facilitation of components of central arousal in the absence of orexin in the null mice. In contrast, the compound did not suppress direct transitions from wakefulness to REM sleep, a sign of narcolepsy–cataplexy in mice. Spectral analysis of the electroencephalogram in awake orexin^−/− mice under baseline conditions revealed reduced power in the θ band frequencies (8–9 Hz), an index of alertness or attention during wakefulness in the rodent. Modafinil administration only partly compensated for this attention deficit in the orexin null mice. We conclude that the presence of orexin is not required for the wakefulness-prolonging action of modafinil, but orexin may mediate some of the alerting effects of the compound.     

Preliminary characterization of an Na,K,Cl co-transport activity in cultured human astrocytes

We have studied the potassium uptake using ⁸⁶Rb⁺ into monolayers of secondary cultures of human astrocytes prepared from cerebral hemispheres of a 4-month-old fetus. With the use of inhibitors we could attribute 30–40% of the ⁸⁶Rb⁺ uptake to an Na⁺,K⁺-ATPase, 50–60% to an anion-cation co-transporter and 10% to potassium leak channels. The anion-cation co-transporter was dependent on the simultaneous presence of both sodium and chloride in the incubation medium and is therefore most likely an Na⁺,K⁺,Cl⁻ co-transporter. This is the first evidence of such an Na⁺,K⁺,Cl⁻ co-transport in human astrocytes.     

Fourier-transform infrared spectroscopy as a tool for detecting early lymphocyte activation: a new approach to histocompatibility matching

Fourier transform infrared (FT-IR) spectroscopy due to its speed and sensitivity is becoming an increasingly powerful tool in the study of cell composition. We outline the potential of FT-IR microspectroscopy in monitoring mitogenic and cell mediated lymphocyte activation. We demonstrate the potential of FT-IR spectroscopy in histocompatibility testing by showing that significant spectral differences (p < 0.001, for the mean integrated intensity of phosphodiester band located in the 1142-996 cm⁻¹ region) exist between lymphocyte cocultures from pairs of HLA matched and mismatched volunteers after only 90 min of incubation. The preliminary results indicate that early spectral changes measured are due to HLA differences between individuals, although the relative contribution of class I and class II differences has yet to be determined. FT-IR spectroscopy represents a novel approach to histocompatibility matching and the rapidity and sensitivity of the technique indicates a potential role in matching protocols for clinical use, particularly in allogeneic bone marrow transplantation.     

Human and chimpanzee monoclonal antibodies

Monoclonal antibody-secreting cell lines were isolated after transformation of peripheral blood leukocytes with Epstein-Barr virus. Blood samples were obtained from human donors having circulating antibodies against hepatitis viruses (HAB, HBV), rubella, or rabies virus and from a chimpanzee infected with HAV. Dextran-isolated leukocytes were submitted to Epstein-Barr virus infection at low cell concentrations (1 × 10⁴ cells · mml⁻¹. Proliferating clones could be observed in 50–100% of the cultures within 4–6 weeks. Out of 1 ml blood (1 × 10⁶ leukocytes) 1–10 stable clones were isolated, secreting specific anti-viral antibodies. These clones were fused with an aminopterin-sensitive, ouabain-resistant, non-immunoglobulin producing mouse-human hybridoma (Org MHH.1). From such fusions 10–90% of the cultures yielded viable hybridomas of which 45% produced antibodies with the same specificity as of the parental EBV transformant. Immunoglobulin production of both EBV transformants and hybridomas was shown to be stable for more than 6 months and at a concentration up to 100 μg · ml⁻¹ · 48 h⁻¹.

Chimpanzee EBV-transformed lymphocytes proliferated excellently in vitro. Mouse-human hybridomas, however, could be more easily cultivated, cloned and scaled up than the parental EBV-transformed lymphocytes.

In conclusion, stable, monoclonal antibody-secreting cell lines of either human or chimpanzee origin could be isolated with an efficiency that exceeds by 10–100-fold standard murine hybridoma technology.     

Cristallographic behaviour of fluoridated hydroxyapatites containing Mg and CO3 ions

Fluoridated hydroxyapatites containing small amounts of magnesium and carbonate ions were synthesized at 80 and 60° C to examine their inhibiting properties regarding apatite crystal growth, in contrast to the promoting action of fluoride. The shortening of a-axis and c-axis dimensions of the apatite crystals, as revealed by X-ray diffraction analysis, suggested that both magnesium and carbonate ions were substituted into the apatite crystals. The a-axis dimensions also decreased with the degree of fluoridation. The infrared spectra due to CO²⁻₃ ions at 875 cm⁻¹ were shifted with increasing fluoride content. The overall crystallinity was inhibited in comparison with that of Mg and CO₃-free fluoridated hydroxyapatites, but recovered considerably with increased fluoride content. The apparent solubility of the apatites at pH 4.0 and 37°C was higher than that of Mg and CO₃-free fluoridated hydroxyapatites at lower fluoride contents, but gradually approached the latter at higher fluoride content. After 1 month's incubation, dicalcium phosphate dihydrate was formed from fluoride-free Mg-CO₃ apatite synthesized at 60°C.     

Measurements of total body water with a foot-to-foot impedancemeter

The objective was to set-up a method for measuring total body water volumes (TBW) using a Tefal foot-to-foot impedancemeter (FFI) by comparison with a multifrequency medical impedancemeter and to validate this method against deuterium dilution data. The investigation was carried out in 57 Caucasian adult subjects. Impedancemeters were a Tefal Bodymaster Vision^® (foot-to-foot) featuring a square wave signal and a Xitron Hydra 4200^® (5–1000 kHz) using BIS method. TBW was measured by the Xitron using a new method that we have developed which applies the BIS method directly to extra and intracellular fluids combined. Although the high frequency impedance of the FFI (R_hf) was higher than the Xitron infinite frequency resistance and corresponded to a frequency around 100 kHz, TBW differences between the FFI and Xitron were not significant, 0.17 ± 2.17 L for men (P = 0.694) and 0.04 ± 1.88 L for women (P = 0.902). Then, our method was tested on another Caucasian population in which R_hf had been measured with the same FFI, together with TBW measurements by deuterium dilution. TBW differences between the FFI and dilution were −0.38 ± 2.27 L for men (P = 0.237) and −0.72 ± 2.37 L for women (P = 0.06).

Our method permits, at least in a Caucasian healthy population, to measure TBW using this FFI with the same accuracy as a whole body multifrequency medical impedancemeter, and the measurement, made in upright position, is much quicker.     

Modulation by nitric oxide of rat brain GABAA receptors

The effect of nitric oxide (NO) on the function of GABA_A receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABA_A receptors were studied by preparing microsacs and evaluating ³⁶Cl⁻ accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor arginine there was a 15–25% reduction in the V_max for GABA-stimulated ³⁶Cl⁻ accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor . GABA_A receptor mediated Cl⁻ currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and -arginine reduced the Cl⁻ current elicited by 10 μM GABA. The -arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABA_A receptor function in cerebellar granule cells.     

Engineering embryonic stem cell derived glia for adenosine delivery

Based on the anticonvulsant and neuroprotective properties of adenosine, and based on the long-term survival potential of stem cell derived brain implants, adenosine releasing stem cells may constitute a novel tool for the treatment of epilepsy. Pluripotency and unlimited self-renewal make embryonic stem (ES) cells a particularly versatile donor source for cell transplantation. With the aim to test the feasibility of a stem cell-based delivery system for adenosine, both alleles of adenosine kinase (ADK), the major adenosine-metabolizing enzyme, were disrupted by homologous recombination in ES cells. Adk^−/− ES cells were subjected to a glial differentiation protocol and, as a result, gave rise to proliferating glial precursors, which could be further differentiated into mature astrocytes and oligodendrocytes. Thus, a lack of ADK does not compromise the glial differentiation potential of ES cells. The Adk^−/− ES cells yielded glial populations with an adenosine release of up to 40.1 ± 6.0 ng per 10⁵ cells per hour, an amount considered to be sufficient for seizure suppression. Our findings indicate that Adk^−/− ES cells constitute a potential source for therapeutic adenosine releasing grafts.     

gamma-Glutamyl transpeptidase activity in Ascaris suum

Using histochemical techniques γ-glutamyl transpeptidase activity has been localized mainly in the cuticle-hypodermis in Ascaris suum. The specific activity of γ-glutamyl transpeptidase was 3.87 ± 0.49 μmol h⁻¹ (g tissue)⁻¹ in cuticle-hypodermis as compared to gastrointestinal tract, where it was 0.07 ± 0.02 μmol h⁻¹ (g tissue)⁻¹, and muscle and reproductive tract where it was <0.001 μmol h⁻¹ (g tissue)⁻¹. When cuticle sections were incubated with labelled glutamine a large portion of the glutamine nitrogen was recovered as ammonia which indicated glutamine uptake and utilization by the cuticle-hypodermis. Collectively these histochemical and biochemical data support the fact that γ-glutamyl transpeptidase activity is found in the cuticle-hypodermis of A. suum and this may be an indication that amino acid absorption could occur through the cuticle via the γ-glutamyl cycle.     

Morphology and electrochemical behavior of Ag-Cu nanoparticle-doped amalgams

The aim of this study was to introduce Ag–Cu phase nanopowder as an additive to improve the corrosion behavior of dental amalgams. A novel Ag–Cu nanopowder was synthesized by the precipitation method. An amalgam alloy powder (World-Cap^®) was added and mixed with 5 wt.% and 10 wt.% of Ag–Cu nanopowders, respectively, to form experimental amalgam alloy powders. The original alloy powder was used as a control. Alloy powders were examined using X-ray diffraction, transmission electron microscopy (TEM), scanning electron microscopy and electron probe microanalysis. Amalgam disk specimens of metallurgically prepared were tested in 0.9% NaCl solution using electrochemical methods. The changes in the corrosion potential and anodic polarization characteristics were determined. Corrosion potential data were analyzed statistically (n = 3, analysis of variance, Tukey’s test, p < 0.05). The diameters of lamellar structure Ag–Cu nanoparticles were measured to be approximately 30 nm. The composition of the Ag–Cu nanoparticles determined by TEM–energy-dispersive spectroscopy was 56.28 at.% Ag–43.72 at.% Cu. A light-shaded phase was found mixing with dark Cu–Sn reaction particles in the reaction zones of Ag–Cu nanoparticle-doped amalgams. The Ag–Cu nanoparticle-doped amalgams exhibited zero current potentials more positive than the control (p < 0.05) and no current peak was observed at −325 mV that related to Ag–Hg phase and Cu₆Sn₅ phase in anodic polarization curves. The results indicated that the corrosion resistance of high-copper single-composition amalgam could be improved by Ag–Cu nanoparticle-doping.