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Kemp et al. Page 4 carried out in a PHI 5400 instrument with a 200 W Mg Kα probe beam. The spectrometer was configured to operate at high resolution with pass energy of 20 eV. TEM was used to determine the size distribution of the particles on a Philips CM12 TEM. Anticoagulant Efficacy a) Activated Partial Thromboplastin Time (aPTT):29—An appropriate amount of DAPHP capped Au and Ag nanoparticles or Au and Ag nanoparticles reduced with glucose were added with 0.1 ml of automated aPTT reagent to cups and warmed for 1 min at 37 °C. Citrated plasma (0.1 ml) was added and the mixture was incubated at 37 °C for 5 min. CaCl2 (0.1 ml of 0.025 M) was added to recalcify the citrated plasma, the fibrometer was started and the clotting time was measured. Each determination was performed in triplicate and experiment repeated six times. b) Clot dynamics in human whole blood using Thrombelastography (TEG)— Human healthy male and female volunteers (age ranges from 25 – 45 years old) were enrolled in the study. Siliconized Vacutainer tubes (Becton Dickinson, Rutherford, NJ) were used to collect whole blood. To maintain a ratio of citrate to whole blood of 1:9 (v/v), the tubes contained 3.2% trisodium citrate. Blood samples were placed on a slow speed rocker until thrombelastography (TEG) analysis. Thrombelastography Whole Blood Coagulation Analyzer, Model 5000 Thrombelastograph, Haemoscope Corporation, Skokie, Illinois, was used. TEG is based on the measurement of the physical viscoelastic characteristics of blood clots. An oscillating plastic cylindrical cuvette (“cup”) and a coaxially suspended stationary piston (“pin”) with a 1-mm clearance between the surfaces are used to monitor clot formation at 37 °C. Every 4.5 seconds, with a 1-second mid cycle stationary period, the cup oscillates in either direction, resulting in a frequency of 0.1 Hz. A torsion wire that acts as a torque transducer suspends the pin. Fibrin fibrils link the cup to the pin during clot formation, and the rotation of the cup is transmitted to the pin via the viscoelasticity of the clot. Customized software (Haemoscope Corporation, Skokie, IL) and an IBM-compatible personal computer display the rotation. The pin’s torque is plotted as a function of time, as shown by the different TEG clot parameters.30–34 The effect of Au-DAPHP or Ag-DAPHP versus heparin and all respective controls were tested for their efficacy on clot dynamics in human blood using TEG. Evaluation of Anti-inflammatory Test Compounds in a Rodent Model of Carrageenan- Induced Paw Edema:35 Animals were randomized by weight and group rats into 10 different groups, with 5 male rats (180–200 g) per group. The protocol is as previously described35 where briefly paw volumes were initially weighed and recorded by placing a beaker of water on balance and tare to zero and record readout on scale. This was followed by placing right hind limb in the beaker of water on the scale with the top of the hock centered in the meniscus of the water. The foot was dried after each measurement as wet feet will change the readout. Scale was re-tared between measurements to account for decrease in water volume. After foot measurements were recorded, the rats (5 rats/treatment group) were pretreated with either: a) Indomethacin, s.c. at 5 mg/kg, 30 min prior to injection with carrageenan; b) Test compounds or vehicle, s.c., 30 min prior to injection with carrageenan in sub-plantar region of right hind foot. Test compounds include the followings: gold-glucose, silver-glucose, gold-HA, silver-HA, gold-heparin, silver- heparin, heparin, and vehicle. At the end of the pretreatment period, the rats were anesthetized and injected with 0.1 ml of 0.3% carrageenan solution in the sub-plantar region of the right hind foot, using a 1⁄2 inch 26 gauge needle attached to a 1 cc syringe, and returned to their cage. Biomacromolecules. Author manuscript; available in PMC 2010 March 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptPDF Image | Gold and Silver Nanoparticles Stabilized Glycosaminoglycans
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