A three-dimensional human skin model to evaluate the inhibition of Staphylococcus aureus by antimicrobial peptide-functionalized silver carbon nanotubes.

OBJECTIVE: To investigate the toxicity and antibacterial application of antimicrobial peptide-functionalized silver-coated carbon nanotubes against Staphylococcus infection using a full thickness human three-dimensional skin model. MATERIALS AND METHODS: The three-dimensional skin formation on the scaffolds was characterized by electron microscopy and investigation of several skin cell markers by real time-reverse transcriptase polymerase chain reaction. Functionalized silver-coated carbon nanotubes were prepared using carboxylated silver-coated carbon nanotubes with antimicrobial peptides such as TP359, TP226 and TP557. Following the characterization and toxicity evaluation, the antibacterial activity of functionalized silver-coated carbon nanotubes against Staphylococcus aureus was investigated using a bacterial enumeration assay and scanning electron microscopy. For this purpose, a scar on the human three-dimensional skin grown on Alvetex scaffold using keratinocytes and fibroblasts cells was created by taking precaution not to break the scaffold beneath, followed by incubation with 5 µg/mL of functionalized silver-coated carbon nanotubes re-suspended in minimum essential medium for 2 h. Post 2-h incubation, 200 µL of minimum essential medium containing 1 × 104 colony forming units of Staphylococcus aureus were incubated for 2 h. After incubation with bacteria, the colony forming unit/gram (cfu/g) of skin tissue were counted using the plate count assay and the samples were processed for scanning electron microscopy analysis. RESULTS: MTT assay revealed no toxicity of functionalized silver-coated carbon nanotubes to the skin cells such as keratinocytes and fibroblasts at 5 µg/mL with 98% cell viability. The bacterial count increased from 104 to 108 cfu/g in the non-treated skin model, whereas skin treated with functionalized silver-coated carbon nanotubes showed only a small increase from 104 to 105 cfu/g (1000-fold viable cfu difference). Scanning electron microscopy analysis showed the presence of Staphylococcus aureus on the non-treated skin as opposed to the treated skin. CONCLUSION: Thus, our results showed that functionalized silver-coated carbon nanotubes are not only non-toxic, but also help reduce the infection due to their antibacterial activity. These findings will aid in the development of novel antibacterial skin substitutes.

Neuroligin 4X overexpression in human breast cancer is associated with poor relapse-free survival.

The molecular mechanisms involved in breast cancer progression and metastasis still remain unclear to date. It is a heterogeneous disease featuring several different phenotypes with consistently different biological characteristics. Neuroligins are neural cell adhesion molecules that have been implicated in heterotopic cell adhesion. In humans, alterations in neuroligin genes are implicated in autism and other cognitive diseases. Until recently, neuroligins have been shown to be abundantly expressed in blood vessels and also play a role implicated in the growth of glioma cells. Here we report increased expression of neuroligin 4X (NLGN4X) in breast cancer. We found NLGN4X was abundantly expressed in breast cancer tissues. NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) was analyzed. Correlation between NLGN4X levels and clinicopathologic parameters were analyzed within Oncomine datasets. Evaluation of these bioinfomatic datasets results revealed that NLGN4X expression was higher in triple negative breast cancer cells, particularly the basal subtype and tissues versus non-triple-negative sets. Its level was also observed to be higher in metastatic tissues. RT-PCR, flow cytometry and immunofluorescence study of MDA-MB-231 and MCF-7 breast cancer cells validated that NLGN4X was increased in MDA-MB-231. Knockdown of NLGN4X expression by siRNA decreased cell proliferation and migration significantly in MDA-MB-231 breast cancer cells. NLGN4X knockdown in MDA-MB-231 cells resulted in induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry. High NLGN4X expression highly correlated with decrease in relapse free-survival in TNBC. NLGN4X might represent novel biomarkers and therapeutic targets for breast cancer. Inhibition of NLGN4X may be a new target for the prevention and treatment of breast cancer.

Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin.

The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolerance induction and outlines in detail current available strategies and alternatives that may allow achieving full-thickness skin replacement and repair.

Silver nanoparticles protect human keratinocytes against UVB radiation-induced DNA damage and apoptosis: potential for prevention of skin carcinogenesis.

Ultraviolet (UV)-B radiation from the sun is an established etiological cause of skin cancer, which afflicts more than a million lives each year in the United States alone. Here, we tested the chemopreventive efficacy of silver-nanoparticles (AgNPs) against UVB-irradiation-induced DNA damage and apoptosis in human immortalized keratinocytes (HaCaT). AgNPs were synthesized by reduction-chemistry and characterized for their physicochemical properties. AgNPs were well tolerated by HaCaT cells and their pretreatment protected them from UVB-irradiation-induced apoptosis along with significant reduction in cyclobutane-pyrimidine-dimer formation. Moreover, AgNPs pre-treatment led to G1-phase cell-cycle arrest in UVB-irradiated HaCaT cells. AgNPs were efficiently internalized in UVB-irradiated cells and localized into cytoplasmic and nuclear compartments. Furthermore, we observed an altered expression of various genes involved in cell-cycle, apoptosis and nucleotide-excision repair in HaCaT cells treated with AgNPs prior to UVB-irradiation. Together, these findings provide support for potential utility of AgNPs as novel chemopreventive agents against UVB-irradiation-induced skin carcinogenesis. FROM THE CLINICAL EDITOR: Excessive exposure to the sun is known to increase the risk of skin cancer due to DNA damage. In this work, the authors tested the use of silver nanoparticles as protective agents against ultraviolet radiation. The positive results may open a door for the use of silver nanoparticle as novel agents in the future.

Enhanced intracellular translocation and biodistribution of gold nanoparticles functionalized with a cell-penetrating peptide (VG-21) from vesicular stomatitis virus.

Reduced toxicity and ease of modification make gold nanoparticles (GNPs) suitable for targeted delivery, bioimaging and theranostics by conjugating cell-penetrating peptides (CPPs). This study presents the biodistribution and enhanced intracellular uptake of GNPs functionalized with VG-21, a CPP derived from vesicular stomatitis virus glycoprotein (G). Cell penetrating efficiency of VG-21 was demonstrated using CellPPD web server, conjugated to GNPs and were characterized using, UV-visible and FTIR spectroscopy, transmission electron microscopy, dynamic light scattering and zeta potential. Uptake of VG-21 functionalized GNPs (fGNPs) was tested in eukaryotic cell lines, HEp-2, HeLa, Vero and Cos-7, using flow cytometry, fluorescence and transmission electron microscopy (TEM), and inductively coupled plasmon optical emission spectroscopy (ICP-OES). The effects of nanoparticles on stress and toxicity related genes were studied in HEp-2 cells. Cytokine response to fGNPs was studied in vitro and in vivo. Biodistribution of nanoparticles was studied in BALB/c mice using TEM and ICP-OES. VG-21, GNPs and fGNPs had little to no effect on cell viability. Upon exposure to fGNPs, HEp-2 cells revealed minimal down regulation of stress response genes. fGNPs displayed higher uptake than GNPs in all cell lines with highest internalization by HEp-2, HeLa and Cos-7 cells, in endocytotic vesicles and nuclei. Cytokine ELISA showed that mouse J774 cells exposed to fGNPs produced less IL-6 than did GNP-treated macrophage cells, whereas TNF-α levels were low in both treatment groups. Biodistribution studies in BALB/c mice revealed higher accumulation of fGNPs than GNPs in the liver and spleen. Histopathological analyses showed that fGNP-treated mice accumulated 35 ng/mg tissue and 20 ng/mg tissue gold in spleen and liver respectively, without any adverse effects. Likewise, serum cytokines were low in both GNP- and fGNP-treated mice. Thus, VG-21-conjugated GNPs have enhanced cellular internalization and are suitable for various biomedical applications as nano-conjugates.

A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system.

The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs), which consisted of poly(2-hydroxyethyl methacrylate) nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV)-F gene construct (a model for a DNA vaccine). The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM), fluorescence activated cell sorting (FACS), and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR), we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo.

Secondary RNA structure and its role in RNA interference to silence the respiratory syncytial virus fusion protein gene.

RNA interference (RNAi) is a post-transcriptional, gene silencing mechanism which uses small interfering RNA molecules (siRNA) for gene silencing. Respiratory Syncytial Virus (RSV) is an important respiratory pathogen of medical significance that causes high mortality in infants. The fusion (F) protein of RSV is a good target for therapeutic purposes as it is primarily responsible for penetration of the virus into host cells and subsequent syncytium formation during infection. In the present study, four siRNAs were designed and used individually as well as a mixture, to silence the RSV F gene. The relationship between siRNA design, target RNA structure, and their thermodynamics was also investigated. Silencing of F gene was observed using indirect immunofluorescence, western blot, reverse transcription PCR, and progeny viral titers. Our results show F gene silencing by all the four siRNAs individually and collectively. RT-PCR analysis revealed a decrease in mRNA level which corresponded to decreased F protein expression. siRNAs also inhibited RSV progeny as shown by viral titer estimation on infected HEp-2 cells. The present study demonstrates the silencing of the F gene using siRNA. Thermodynamic characteristics of the target RSV mRNA and siRNA seem to play an important role in siRNA gene silencing efficiency.