Development and validation of the first point-of-care assay to objectively monitor adherence to HIV treatment and prevention in real-time in routine settings.

OBJECTIVE: HIV prevention and treatment studies demonstrate that pharmacologic adherence metrics are more accurate than self-report. Currently available metrics use liquid-chromatography/tandem-mass-spectrometry (LC-MS/MS), which is expensive and laboratory-based. We developed a specific and sensitive antibody against tenofovir, the backbone of treatment and prevention, but conversion to a lateral flow assay (LFA) - analogous to a urine pregnancy test - is required for point-of-care testing. We describe the development of the first LFA to measure antiretroviral adherence in real-time. METHODS: Previous work in a directly observed therapy study of providing tenofovir disoproxil fumarate (TDF) to HIV-noninfected volunteers at various simulated adherence patterns defined the appropriate cut-off for the LFA (1500 ng tenofovir/ml urine). We developed the LFA using a sample pad for urine; a conjugate pad coated with TFV-specific antibodies conjugated to colloidal gold nanoparticles; a nitrocellulose membrane striped with tenofovir-antigen (test line) and a control line; with an absorbent pad to draw urine across the reaction membrane. RESULTS: We tested 300 urine samples collected from the directly observed therapy study by this LFA and the gold-standard method of LC-MS/MS. The LFA demonstrated 97% specificity (95% CI 93-99%) and 99% sensitivity (94-100%) compared with LC-MS/MS. The LFA accurately classified 98% of patients who took a dose within 24 h as adherent. CONCLUSION: We describe the development and validation of the first point-of-care assay to measure short-term adherence to HIV prevention and treatment in routine settings. The assay is low-cost, easy-to-perform and measures the breakdown product (tenofovir) of both TDF and tenofovir alafenamide (TAF). This assay has the potential to improve HIV and PrEP outcomes worldwide by triggering differentiated service delivery with further study merited.

Targeted Gold Nanocluster-Enhanced Radiotherapy of Prostate Cancer.

For over a hundred years, X-rays have been a main component of the radiotherapeutic approaches to treat cancer. Yet, to date, no radiosensitizer has been developed to selectively target prostate cancer. Gold has excellent X-ray absorptivity and is used as a radiotherapy enhancing material. In this work, ultrasmall Au25 nanoclusters (NCs) are developed for selective prostate cancer targeting, radiotherapy enhancement, and rapid clearance from the body. Targeted-Au25 NCs are rapidly and selectively taken up by prostate cancer in vitro and in vivo and also have fast renal clearance. When combined with X-ray irradiation of the targeted cancer tissues, radiotherapy is significantly enhanced. The selective targeting and rapid clearance of the nanoclusters may allow reductions in radiation dose, decreasing exposure to healthy tissue and making them highly attractive for clinical translation.

Determination of Gold Ions in Human Urine Using Genetically Engineered Microorganisms on a Paper Device.

This paper presents a whole-cell biosensor that operates in conjunction with a smartphone-based fluorescence diagnostic system on a paper device to monitor the concentration of gold ions in human urine. The heavy metal-tolerant bacteria Cupriavidus metallidurans was genetically engineered for use as a chassis in a red fluorescent protein (RFP)-based microbial sensor. The biosensor is highly sensitive to gold ions, with a detection limit of 110 nM. The proposed smartphone-based analysis system provides a user-friendly approach to design tools of personal health monitoring for reporting the presence of gold ions in human urine.

Biokinetics of ultrafine gold nanoparticles (AuNPs) relating to redistribution and urinary excretion: a long-term in vivo study.

Gold nanoparticles (AuNPs) of ultrafine size have drawn attention for their use in drug delivery systems. Tissue toxicity may be an issue when AuNPs are used for such applications. We investigated the long-term biokinetics (90 d), redistribution, and urinary excretion of three different-sized (2 ± 0.5 nm, 5 ± 1 nm, and 10 ± 2 nm) AuNPs after a single intravenous (i.v.) administration of 1250 µg/kg dose in mice. ICP-AES analysis of lungs, liver, spleen, heart, kidney, brain, blood, and urine revealed highest accumulation of gold in spleen around 15 d after injection. A low concentration was detected in brain after 1 d without any residual AuNPs after 30 d. Ultrastructural study of brain tissue also showed few AuNPs in lysosome with no changes in cellular architecture. Renal retention of AuNPs was limited indicating low nephrotoxic potential. AuNPs were detectable in urine till 30 d after single injection indicating slow excretion from the body. No evidence of significant toxicity was observed in hemogram, serum biochemistry, and tissue histology. No mortality, changes in behavior, hair color, weight, and food intake was observed as compared to control mice. Therefore, we conclude that the ultrafine AuNPs are predominantly excreted in urine without any systemic toxicity following i.v. administration and are hence safe for use in drug delivery systems.

Visual and colorimetric detection of p-aminophenol in environmental water and human urine samples based on anisotropic growth of Ag nanoshells on Au nanorods.

A simple, sensitive, selective and high-resolution colorimetric method has been developed for the detection of p-aminophenol in environmental water and human urine samples. In the presence of p-aminophenol, silver ions are reduced to silver atoms and subsequently Ag nanoshells anisotropically grow on the surface of Au nanorods to generate orange slice-like Au@Ag core-shell nanocrystals, thereby resulting in the blue-shift of longitudinal surface plasmon resonance band of Au nanorods accompanying a sharp-contrast multicolor change. Using Au@Ag core-shell nanocrystals as the transducer, sub-micromolar p-aminophenol can be detected by the colorimetric method and 10 µmol L(-1) p-aminophenol can be visual readout by the naked eyes. Furthermore, a simple, cheap, portable test kit is constructed for the visual assay of urinary p-aminophenol without complicated sample pretreatment and sophisticated instruments. The proposed colorimetric method has the potential for the rapid and on-site analyses of p-aminophenol in environmental water and human urine samples.

Gold nanoparticles as physiological markers of urine internalization into urothelial cells in vivo.

BACKGROUND: Urothelial bladder is the reservoir of urine and the urothelium minimizes the exchange of urine constituents with this tissue. Our aim was to test 1.9 nm biocompatible gold nanoparticles as a novel marker of internalization into the urothelial cells under physiological conditions in vivo. METHODS: We compared normal and neoplastic mice urothelium. Neoplastic lesions were induced by 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water for 10 weeks. Nanoparticles, intravenously injected into normal and BBN-treated mice, were filtered through the kidneys and became constituents of the urine within 90 minutes after injection. RESULTS: Gold nanoparticles were densely accumulated in the urine, while their internalization into urothelial cells depended on the cell differentiation stage. In the terminally differentiated superficial urothelial cells of normal animals, nanoparticles were occasionally found in the endosomes, but not in the fusiform vesicles. Regions of exfoliated cells were occasionally found in the normal urothelium. Superficial urothelial cells located next to exfoliated regions contained gold nanoparticles in the endosomes and in the cytosol beneath the apical plasma membrane. The urothelium of BBN-treated animals developed fat hyperplasia with moderate dysplasia. The superficial cells of BBN-treated animals were partially differentiated as demonstrated by the lack of fusiform vesicles. These cells contained the gold nanoparticles distributed in the endosomes and throughout their cytosol. CONCLUSION: Gold nanoparticles are a valuable marker to study urine internalization into urothelial cells in vivo. Moreover, they can be used as a sensitive marker of differentiation and functionality of urothelial cells.

Biodistribution and acute toxicity of naked gold nanoparticles in a rabbit hepatic tumor model.

There is a paucity of data regarding the safety of administering solid gold nanoparticles (AuNPs) in large animal tumor models. We assessed the acute toxicity and biodistribution of 5 nm and 25 nm solid AuNPs in New Zealand White rabbits (n = 6 in each) with implanted liver Vx2 tumors 24 h after intravenous injection. Gold concentration was determined by inductively coupled plasma atomic emission spectrometry (ICP) and imaged with transmission electron microscopy (TEM). There was no clinico-pathologic evidence of renal, hepatic, pulmonary, or other organ dysfunction. After 25 nm AuNP administration, the concentration of white blood cells increased after treatment (p = 0.001). Most other blood studies were unchanged. AuNPs were distributed to the spleen, liver, and Vx2 tumors, but not to other tissues. The urinary excretion of AuNPs was bimodal as measured by ICP. 25 nm AuNPs were more evenly distributed throughout tissues and may be better tools for medical therapy.

Unexpected toxicity of monolayer protected gold clusters eliminated by PEG-thiol place exchange reactions.

Monolayer protected clusters (MPCs) are small, metal nanoparticles capped with thiolate ligands that have been widely studied for their size-dependent properties and for their ability to be functionalized for biological applications. Common water-soluble MPCs, functionalized by N-(2-Mercaptopropionyl)-glycine (tiopronin) or glutathione, have been used previously to interface with biological systems. These MPCs are ideal for biological applications not only due to their water-solubility but also their small size (<5 nm). These characteristics are expected to enable easy biodistribution and clearance. In this article, we show an unexpected toxicity is associated with the tiopronin monolayer protected cluster (TMPC), making it incompatible for potential in vivo applications. This toxicity is linked to significant histological damage to the renal tubules, causing mortality at concentrations above 20 µM. We further show how the incorporation of poly ethylene glycol (PEG) by a simple place-exchange reaction eliminates this toxicity. We analyzed gold content within blood and urine and found an increased lifetime of the particle within the bloodstream due to the creation of the mixed monolayer. Also shown was the elimination of kidney damage with the use of the mixed-monolayer particle via Multistix analysis, MALDI-TOF MS analysis, and histological examination. Final immunological analysis showed no effect on white blood cell (WBC) count for the unmodified particle and a surprising increase in WBC count with the injection of mixed monolayer particles at concentrations higher than 30 µM, suggesting that there may be an immune response to these mixed monolayer nanoparticles at high concentrations; therefore, special attention should be focused on selecting the best capping ligands for use in vivo. These findings make the mixed monolayer an excellent candidate for further biological applications using water-soluble nanoparticles.

Size-dependent tissue kinetics of PEG-coated gold nanoparticles.

Gold nanoparticles (AuNPs) can be used in various biomedical applications, however, very little is known about their size-dependent in vivo kinetics. Here, we performed a kinetic study in mice with different sizes of PEG-coated AuNPs. Small AuNPs (4 or 13nm) showed high levels in blood for 24h and were cleared by 7days, whereas large (100nm) AuNPs were completely cleared by 24h. All AuNPs in blood re-increased at 3months, which correlated with organ levels. Levels of small AuNPs were peaked at 7days in the liver and spleen and at 1month in the mesenteric lymph node, and remained high until 6months, with slow elimination. In contrast, large AuNPs were taken up rapidly ( approximately 30min) into the liver, spleen, and mesenteric lymph nodes with less elimination phase. TEM showed that AuNPs were entrapped in cytoplasmic vesicles and lysosomes of Kupffer cells and macrophages of spleen and mesenteric lymph node. Small AuNPs transiently activated CYP1A1 and 2B, phase I metabolic enzymes, in liver tissues from 24h to 7days, which mirrored with elevated gold levels in the liver. Large AuNPs did not affect the metabolic enzymes. Thus, propensity to accumulate in the reticuloendothelial organs and activation of phase I metabolic enzymes, suggest that extensive further studies are needed for practical in vivo applications.

Simultaneous determination of palladium, platinum, rhodium and gold by on-line solid phase extraction and high performance liquid chromatography with 5-(2-hydroxy-5-nitrophenylazo)thiorhodanine as pre-column derivatization regents.

In this paper, 5-(2-hydroxy-5-nitrophenylazo)thiorhodanine (HNATR) was synthesized. A new method for the simultaneous determination of palladium, platinum, rhodium and gold ions as metal-HNATR chelates was developed using a rapid analysis column high performance liquid chromatography equipped with on-line solid phase extraction technique. The samples (Water, human urine, geological samples and soil) were digested by microwave acid-digestion. The palladium, platinum, rhodium and gold ions in the digested samples were pre-column derivatized with HNATR to form colored chelates. The Pd-HNATR, Pt-HNATR, Rh-HNATR and Au-HNATR chelates can be absorbed onto the front of the enrichment column when they were injected into the injector and sent to the enrichment column [Zorbax Stable Bound, 10 mm x 4.6 mm, 1.8 microm] with a buffer solution of 0.05 mol L(-1) phosphoric acid as mobile phase. After the enrichment had finished, by switching the six ports switching valve, the retained chelates were back-flushed by mobile phase and travelling towards the analytical column. These chelates separation on the analytical column [Zorbax Stable Bound, 10 mm x 4.6 mm, 1.8 microm] was satisfactory with 72% acetonitrile (containing 0.05 mol L(-1) of phosphoric acid and 0.1% of Triton X-100) as mobile phase. The palladium, platinum, rhodium and gold chelates were separated completely within 2.5 min. Compared to the routine chromatographic method, more then 80% of separation time was shortened. By on-line solid phase extraction system, a large volume of sample (10 mL) can be injected, and the sensitivity of the method was greatly improved. The detection limits (S/N=3, the sample injection volume is 10 mL) of palladium, platinum, rhodium and gold in the original samples reaches 1.4, 1.8, 2.0 and 1.2 ng L(-1), respectively. The relative standard deviations for five replicate samples were 2.4-3.6%. The standard recoveries were 88-95%. This method was applied to the determination of palladium, platinum, rhodium and gold in human urine, water and geological samples with good results.

Cholestatic hepatitis caused by acute gold potassium cyanide poisoning.

INTRODUCTION: Poisoning after oral ingestion of gold potassium cyanide is rarely reported. A case of suicidal ingestion of gold potassium cyanide (potassium dicyanoaurate; CAS# 13967-50-5) is described. CASE REPORT: A 27-year-old man attempted suicide by ingesting 5 mL gold potassium cyanide solution. He developed vomiting, hyperamylasemia, and hepatic dysfunction. Cyanide poisoning was not detected but acute gold toxicity was noted. Pathologic findings of the liver showed centrilobular cholestasis with eosinophilic degeneration. The whole blood and serum gold were 4361 and 6011 microg/L, respectively, and the 24-hour urine gold was 429 microg/d in samples obtained on day 4. CONCLUSION: Gold-induced hepatotoxicity has been seen infrequently in patients receiving gold therapy. Reported agents include sodium aurothiomalate, sodium aurothiopropranol sulfonate, aurothioglucose, aurothiopolypeptide (Auro-detoxin), auric sulfide, and gold thiosulfate, our report adds gold potassium cyanide.

Gold and palladium burden from dental restoration materials.

From 81 volunteers (16 without dental restorations, 65 with gold crowns or inlays) samples of saliva before and after chewing gum, blood, serum, urine and faeces were taken and analysed for gold (Au) and palladium (Pd). The Au concentration in all analysed biomonitors correlates significantly to the number of teeth with gold restorations. For Pd the correlations were still significant, but weaker than for Au. Persons with gold restorations show maximal Au and Pd concentrations, 10(2)-10(3) higher than the background burden. The calculated maximal daily Au load in saliva (1.38 mg Au per day) reaches the range of an oral Au therapy for rheumatoid arthritis with 6 mg Auranofin (= 1.74 mg Au per day). During this therapy severe and frequent side effects are reported. In contrast, the Au concentration in serum maximally reached from Au restorations, amounts to only approximately 1/20 of the Au level during arthritis therapy. But even under subtherapeutic doses of 1 mg Auranofin/day severe side effects have been reported (4 out of 56 cases). The mean Au blood concentration from 1 mg Auranofin daily was only 3 times higher than our maximum value. A toxicological classification of the Pd values is difficult, because no toxicological threshold limit has been established, especially for the low-level long-term burden with Pd.

Separation and enrichment of palladium and gold in biological and environmental samples, adapted to the determination by total reflection X-ray fluorescence.

The reductive co-precipitation of trace and ultra-trace elements together with mercury followed by complete evaporation of the mercury makes it possible to determine palladium and gold by total reflection X-ray fluorescence. Both elements can be detected without interferences at optimal sensitivity in the pg range. Thus, detection limits of, e.g., 2.5 ng L-1 for palladium and 2.0 ng L-1 for gold, in urine, were obtained. The precision was determined to 0.04 at a palladium concentration of about 200 ng L-1 urine and to 0.19 at a gold concentration of only 18 ng L-1. The recovery for a urine sample spiked with known amounts of palladium and gold amounted to > 95%. Results of the combined procedure are given for the determination of palladium and gold in the urine of non-exposed and occupationally exposed persons and in some other environmentally relevant samples.

Internal platinum, palladium, and gold exposure in environmentally and occupationally exposed persons.

In a pilot study the urinary platinum (Pt), palladium (Pd), and gold (Au) excretion was analyzed in 27 dental technicians, 17 road construction workers and 17 school-leavers using sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Detection limits in urine were 0.24 ng/l for Pt and Au and 0.17 ng/l regarding Pd. A standardized questionnaire was used to assess information about kind and degree of contact to these metals, the physical condition of the volunteers and confounding factors. Significant differences between the three study groups were found. The mean Pt, Pd, and Au excretions of the dental technicians were significantly higher than those of the road construction workers and school-leavers. This indicates that the occupational treatment of dental alloys leads to an internal exposure to these metals which is distinctly higher than that from automobile exhaust exposure. Significant differences between Monday morning (pre-shift) and Thursday afternoon (post-shift) urine samples of the dental technicians were not found. The Pt excretion of road construction workers working near a much traveled highway was comparable with that of school-leavers being less (only environmentally) exposed to automobile exhaust. Regarding Pd and Au the road construction workers showed a tendency to higher levels in urine when compared with the school-leavers, but statistically significant differences were not found. The tendency to higher urinary Pd and Au levels in the road-construction workers may be explained by their slightly greater number of noble metal containing artificial dentures, which may cause an additional exposure. A statistically significant effect of age on the urinary noble metal excretion was not detectable.

Determination of auranofin, a chrysotherapy agent, in urine by HPLC with a postcolumn reaction and visible detection.

Auranofin [(2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S) (triethylphosphine)gold(I): AF] is a unique orally active chrysotherapy agent. A HPLC method has been developed for determining AF in urine. The proposed method comprises initial chromatographic separation of AF followed by on-line decomposition by potassium iodide with a released mercapto group undergoing a color-developing reaction with 5,5'-dithiobis(2-nitrobenzoic acid). An aliquot (100 microliters) of a urine sample was chromatographed on a YMC AM-302 octadecylsilica column (4.6 mm i.d. x 15 cm, ambient) with a water-methanol (35:65) eluent delivered at a flow rate of 1 ml/min. A reagent solution for a postcolumn reaction comprised of 50 microM 5,5'-dithiobis(2-nitrobenzoic acid), 0.3 M potassium iodide and a 50 mM phosphate buffer (pH 7.4), was delivered at a flow rate of 0.5 ml/min. The postcolumn reactor consisted of a poly(tetrafluoroethylene) tube (0.5 mm i.d. x 5 m) at 60 degrees C. Detection wavelength was 412 nm. The identity of the AF peak was confirmed by a 3-dimensional chromatogram as well as by atomic absorption spectrophotometric analysis of gold in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and AF concentration in the range 0.1 to 10 microM, with a correlation coefficient of 0.999. The detection limit was 50 nM (S/N = 3 at 0.005 AUFS) and the reproducibility was within 4% for 5 determinations. The AF concentrations in the urine of a rabbit given AF intraperitoneally were determined.

Dicyanogold (I) is a common human metabolite of different gold drugs.

Gold based drugs and their metabolites have been characterized using reversed phase, ion pairing chromatography with an inductively coupled plasma mass spectrometer as an element specific detector. For a patient receiving gold sodium thiomalate the principal gold species in the urine is [Au(CN)2]-, which is also seen in a low molecular weight infiltrate of the blood. The same compound is also identified in the urine and blood of a patient taking auranofin and in patients taking solganol. This represents the first identification of a specific gold metabolite in biological fluids taken from patients undergoing gold therapy and the first evidence that different gold drugs have common metabolites.

Characterization of gold in urine and bile following administration of gold sodium thiomalate with chelating agents to rats.

Gold was characterized in the urine and bile of rats treated with D-penicillamine (D-PEN), 2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropane sulphonate (DMPS), or N-(2-mercapto-2-methylpropanoyl)-L-cysteine (bucillamine) immediately after gold sodium thiomalate (AuTM) injection by both gel chromatographic and electrophoretic methods. It is suggested that the gold in the urine and bile after AuTM administration was predominantly bound to high molecular weight compounds. The characterization of gold in the urine after administration of AuTM with D-PEN, DMSA, or DMPS showed that most of the gold was bound to the chelating agents. In the treatment with the chelating agents such as D-PEN and DMPS, the gold was mainly excreted as a gold-chelating agent compound in the bile and a minor portion of the gold was present in the form of a gold-L-cysteine compound and high molecular weight compounds. DMSA treatment showed that a major portion of the gold was bound to high molecular weight compounds in the bile and a minor portion of the gold was present in the forms of gold-DMSA and gold-L-cysteine compounds. The administration of AuTM and bucillamine indicated that the gold was mainly present as a gold-Me-bucillamine compound in the urine and a gold-bucillamine compound in the bile.

Whole body elimination routes of gold in humans after a single-dose application of the antirheumatic auranofin.

After removal of the gallbladder (cholecystectomy) 7 patients were administered the antirheumatic agent Auranofin in its commercially available tablet form (Ridaura) 3 d postoperative. The single dose consisted of 5 tablets (4.35 mg of gold). Gold was determined in samples of blood, plasma, urine, bile, and feces (1-2 mL and 2.5 g, respectively). The specimens were drawn 10 min to 8 d p.a. and on d 14 p.a. The determinations were performed by instrumental neutron activation analysis (INAA). The limit of detection was less than 1 ng of gold. The courses of the mean gold concentrations show maxima after 1.9 h in blood and plasma, and after 16 h in urine and bile. The mean half-lives (terminal phases) are 7.6 d (blood), 23 d (plasma), and 6.5 d (bile).