Detection of the Presence of Gold Nanoparticles in Organs by Transmission Electron Microscopy.

Gold nanoparticles of 10 nm and 250 nm were intravenously injected in rats. At 24 h after administration, tissues were collected and prepared for transmission electron microscopy (TEM). In the liver and spleen of animals treated with 10 nm gold nanoparticles, groups of nanoparticles were observed that could be positively identified by Energy Dispersive X-ray (EDX) analysis to contain gold, while nanoparticles could not be detected in the heart, kidney and brain. The 10 nm gold nanoparticles were present in the phagocytic cells of the reticulo-endothelial system (RES). The 250 nm gold nanoparticles could not be detected in any of the organs investigated. Considering the number of 250 nm gold nanoparticles administered, calculations showed that it would indeed be almost impossible to detect the 250 nm gold nanoparticles in TEM preparations in view of the very low number of particles that would be theoretically present in one TEM tissue section. This shows that relatively high numbers of nanoparticles need to be administered to enable the detection of nanoparticles in organs by TEM. In a number of samples, several globular structures of approximately the expected size were found in liver cells and the endothelium of blood vessels in the brain. However, elemental analysis with EDX detection showed that these structures did not contain gold. Our studies thus indicate that the in vivo identification of nanoparticles cannot only depend on the detection of nanosized structures in cells. An additional identification of the composing elements of the nanomaterial is necessary for a positive identification of the nanomaterial.

Particle size-dependent organ distribution of gold nanoparticles after intravenous administration.

A kinetic study was performed to determine the influence of particle size on the in vivo tissue distribution of spherical-shaped gold nanoparticles in the rat. Gold nanoparticles were chosen as model substances as they are used in several medical applications. In addition, the detection of the presence of gold is feasible with no background levels in the body in the normal situation. Rats were intravenously injected in the tail vein with gold nanoparticles with a diameter of 10, 50, 100 and 250 nm, respectively. After 24 h, the rats were sacrificed and blood and various organs were collected for gold determination. The presence of gold was measured quantitatively with inductively coupled plasma mass spectrometry (ICP-MS). For all gold nanoparticle sizes the majority of the gold was demonstrated to be present in liver and spleen. A clear difference was observed between the distribution of the 10 nm particles and the larger particles. The 10 nm particles were present in various organ systems including blood, liver, spleen, kidney, testis, thymus, heart, lung and brain, whereas the larger particles were only detected in blood, liver and spleen. The results demonstrate that tissue distribution of gold nanoparticles is size-dependent with the smallest 10nm nanoparticles showing the most widespread organ distribution.

Two variants among Haemophilus influenzae serotype b strains with distinct bcs4, hcsA and hcsB genes display differences in expression of the polysaccharide capsule.

BACKGROUND: Despite nearly complete vaccine coverage, a small number of fully vaccinated children in the Netherlands have experienced invasive disease caused by Haemophilus influenzae serotype b (Hib). This increase started in 2002, nine years after the introduction of nationwide vaccination in the Netherlands. The capsular polysaccharide of Hib is used as a conjugate vaccine to protect against Hib disease. To evaluate the possible rise of escape variants, explaining the increased number of vaccine failures we analyzed the composition of the capsular genes and the expressed polysaccharide of Dutch Hib strains collected before and after the introduction of Hib vaccination. RESULTS: The DNA sequences of the complete capsular gene clusters of 9 Dutch Hib strains were assessed and two variants, designated type I and type II were found. The two variants displayed considerable sequence divergence in the hcsA and hcsB genes, involved in transport of capsular polysaccharide to the cell surface. Application of hcsA type specific PCRs on 670 Hib strains collected from Dutch patients with invasive Hib disease showed that 5% of the strains collected before 1996 were type II. No endogenous type II Hib strains were isolated after 1995 and all type II strains were isolated from 0-4 year old, non-vaccinated children only. Analysis of a worldwide collection of Hib strains from the pre-vaccination era revealed considerable geographic differences in the distribution of the type I and type II strains with up to 73% of type II strains in the USA. NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences. However, type I strains were shown to produce twice as much surface bound capsular polysaccharide. CONCLUSION: Type II strains were only isolated during the pre-vaccination era from young, non-vaccinated individuals and displayed a lower expression of capsular polysaccharide than type I strains. The higher polysaccharide expression may have provided a selective advantage for type I strains resulting in the rapid elimination of type II from the Dutch Hib population after introduction of nationwide Hib vaccination. However, this phenomenon does not explain the increase in the number of Hib vaccine failures in the Netherlands.