Human skin penetration and local effects of topical nano zinc oxide after occlusion and barrier impairment

Public health concerns continue to exist over the safety of zinc oxide nanoparticles that are commonly used in sunscreen formulations. In this work, we assessed the effects of two conditions which may be encountered in everyday sunscreen use, occlusion and a compromised skin barrier, on the penetration and local toxicity of two topically applied zinc oxide nanoparticle products. Caprylic/capric triglyceride (CCT) suspensions of commercially used zinc oxide nanoparticles, either uncoated or with a silane coating, were applied to intact and barrier impaired skin of volunteers, without and with occlusion for a period of six hours. The exposure time was chosen to simulate normal in-use conditions. Multiphoton tomography with fluorescence lifetime imaging was used to noninvasively assess zinc oxide penetration and cellular metabolic changes that could be indicative of toxicity. We found that zinc oxide nanoparticles did not penetrate into the viable epidermis of intact or barrier impaired skin of volunteers, without or with occlusion. We also observed no apparent toxicity in the viable epidermis below the application sites. These findings were validated by ex vivo human skin studies in which zinc penetration was assessed by multiphoton tomography with fluorescence lifetime imaging as well as Zinpyr-1 staining and toxicity was assessed by MTS assays in zinc oxide treated skin cryosections. In conclusion, applications of zinc oxide nanoparticles under occlusive in-use conditions to volunteers are not associated with any measurable zinc oxide penetration into, or local toxicity in the viable epidermis below the application site

Cellular metabolism and pore lifetime of human skin following microprojection array mediation

Skin-targeting microscale medical devices are becoming popular for therapeutic delivery and diagnosis. We used cryo-SEM, fluorescence lifetime imaging microscopy (FLIM), autofluorescence imaging microscopy and inflammatory response to study the puncturing and recovery of human skin ex vivo and in vivo after discretised puncturing by a microneedle array (Nanopatch®). Pores induced by the microprojections were found to close by ~25% in diameter within the first 30 min, and almost completely close by ~6 h. FLIM images of ex vivo viable epidermis showed a stable fluorescence lifetime for unpatched areas of ~1000 ps up to 24 h. Only the cells in the immediate puncture zones (in direct contact with projections) showed a reduction in the observed fluorescence lifetimes to between ~518-583 ps. The ratio of free-bound NAD(P)H (α1/α2) in unaffected areas of the viable epidermis was ~2.5-3.0, whereas the ratio at puncture holes was almost double at ~4.2-4.6. An exploratory pilot in vivo study also suggested similar closure rate with histamine administration to the forearms of human volunteers after Nanopatch® treatment, although a prolonged inflammation was observed with Tissue Viability Imaging. Overall, this work shows that the pores created by the microneedle-type medical device, Nanopatch®, are transient, with the skin recovering rapidly within 1-2 days in the epidermis after application

Genome Reference and Sequence Variation in the Large Repetitive Central Exon of Human MUC5AC

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin

Repopulation Efficiencies of Adult Hepatocytes, Fetal Liver Progenitor Cells, and Embryonic Stem Cell-Derived Hepatic Cells in Albumin-Promoter-Enhancer Urokinase-Type Plasminogen Activator Mice

Fetal liver progenitor cell suspensions (FLPC) and hepatic precursor cells derived from embryonic stem cells (ES-HPC) represent a potential source for liver cell therapy. However, the relative capacity of these cell types to engraft and repopulate a recipient liver compared with adult hepatocytes (HC) has not been comprehensively assessed. We transplanted mouse and human HC, FLPC, and ES-HPC into a new immunodeficient mouse strain (Alb-uPAtg(+/−)Rag2(−/−)γc(−/−) mice) and estimated the percentages of HC after 3 months. Adult mouse HC repopulated approximately half of the liver mass (46.6 ± 8.0%, 1 × 106 transplanted cells), whereas mouse FLPC derived from day 13.5 and 11.5 post conception embryos generated only 12.1 ± 3.0% and 5.1 ± 1.1%, respectively, of the recipient liver and smaller cell clusters. Adult human HC and FLPC generated overall less liver tissue than mouse cells and repopulated 10.0 ± 3.9% and 2.7 ± 1.1% of the recipient livers, respectively. Mouse and human ES-HPC did not generate HC clusters in our animal model. We conclude that, in contrast to expectations, adult HC of human and mouse origin generate liver tissue more efficiently than cells derived from fetal tissue or embryonic stem cells in a highly immunodeficient Alb-uPA transgenic mouse model system. These results have important implications in the context of selecting the optimal strategy for human liver cell therapies