Selected Biomarkers Revealed Potential Skin Toxicity Caused by Certain Copper Compounds.

Copper is an essential mineral and plays important roles in skin growth and activity. Copper delivery through skin can provide beneficial effects but its potential to induce skin irritation reactions is often overlooked. Data on dermal toxicity caused by copper compounds is scant. Some recognized in vitro skin toxicity methods are unsuitable for all metal compounds. Here, we employ a keratinocyte-based model and evaluated the skin irritation potential of copper compounds at cellular, genomic and proteomic levels. We determined cell viability and cytotoxicity by using tetrazolium reduction assay and Lactate Dehydrogenase (LDH) assay, performed real-time PCR and protein quantification to assess the expression of biomarkers after treating cells with copper peptide (GHK-Cu), copper chloride (CuCl2) and copper acetate (Cu(OAc)2). These copper compounds exhibited different irritancy potentials at the same treatment concentrations. GHK-Cu was not cytotoxic and did not induce any significant change in the expression levels of various skin irritation-related biomarkers. IL-1α and IL-8, HSPA1A and FOSL1 were significantly upregulated following 24-h treatment with CuCl2 and Cu(OAc)2 at 58 and 580 µM without concomitant inhibition in cell viability. GHK-Cu has a low potential of inducing skin irritation and therefore provides a safer alternative for the delivery of copper through skin.

High durability and low toxicity antimicrobial coatings fabricated by quaternary ammonium silane copolymers.

Adhesion and subsequent growth of microorganisms on material surfaces are a major concern in many biomedical applications. Currently, various polymers are immobilized on material surfaces to prevent microbial colonization. However, there are several challenges with regard to the coating materials, including their inability to kill microorganisms, complexity of surface grafting, limited durability and toxicity towards humans. To address these challenges, we synthesize a novel quaternary ammonium silane (QAS) antimicrobial copolymer to confer the antimicrobial effect via a simple thermal-curing coating process. The QAS copolymers were less toxic to 3 human cell lines than a commercial antimicrobial QAS monomeric agent, namely, dimethyloctadecyl[3-(trimethoxysilyl) propyl]ammonium chloride (DTPAC). Moreover, the QAS coatings demonstrated superior antimicrobial efficacy and durability than those of the DTPAC coatings. In conclusion, the novel QAS copolymers are useful to prevent substrates from microbial infections, yet with low toxicity to humans and long durability. In addition, the synthetic process is potentially scalable for industrial applications.

Microneedle-Mediated Delivery of Copper Peptide Through Skin.

PURPOSE: Copper peptide (GHK-Cu) plays an important role in skin regeneration and wound healing. However, its skin absorption remains challenging due to its hydrophilicity. Here we use polymeric microneedle array to pre-treat skin to enhance GHK-Cu skin penetration. METHODS: Two in vitro skin models were used to assess the capability of microneedles in facilitating skin delivery of GHK-Cu. Histological assay and confocal laser scanning microscopy were performed to characterize and quantify the microconduits created by the microneedles inside skin. Cellular and porcine models were used to evaluate the safety of microneedle-assisted copper peptide delivery. RESULTS: The depth and percentage of microneedle penetration were correlated with application forces, which in turn influenced the extent of enhancement in the skin permeability of GHK-Cu. In 9 h, 134 ± 12 nanomoles of peptide and 705 ± 84 nanomoles of copper permeated though the microneedle treated human skin, while almost no peptide or copper permeated through intact human skin. No obvious signs of skin irritation were observed with the use of GHK-Cu after microneedle pretreatment. CONCLUSIONS: It is effective and safe to enhance the skin permeation of GHK-Cu by using microneedles. This approach may be useful to deliver similar peptides or minerals through skin.

Surface plasmon enhanced fluorescence of cationic conjugated polymer on periodic nanoarrays.

The fluorescence from conjugated polymer assembled onto lithographically fabricated gold nanoarrays using genetically engineered peptides as molecular linkers is studied. A 16-fold increase in the photoluminescence of the conjugated polymer is observed when assembled on the optimized nanostructures due to surface plasmon enhanced fluorescence. This is achieved using a water-soluble cationic conjugated polymer, poly[(9,9-bis(6'-((N,N,N-trimethylammonium)hexyl)-2,7-fluorene)-co-4,7-di-2-thienyl-2,1,3-benzothiadiazole] dibromide (PFDBT-N(+)), systematically tuning the vertical distance of PFDBT-N(+) from the gold nanopillar surface using solid-specific peptide linkers and horizontally optimizing the localized surface plasmon resonance by varying the geometric arrangements of the patterned metal nanoarrays. The diameter and tip-to-tip spacing of the nanopillars along with vertically tuning the distance of PFDBT-N(+) from the nanopillar affected the observed fluorescence enhancements. The collective optical properties of conjugated polymers combined with the photonic properties of nanoparticles provide a new means in the development of metal enhanced hybrid nanomaterials for biotechnology.

Surface-plasmon-enhanced fluorescence from periodic quantum dot arrays through distance control using biomolecular linkers.

We have developed a protein-enabled strategy to fabricate quantum dot (QD) nanoarrays where up to a 15-fold increase in surface-plasmon-enhanced fluorescence has been achieved. This approach permits a comprehensive control both laterally (via lithographically defined gold nanoarrays) and vertically (via the QD-metal distance) of the collectively behaving assemblies of QDs and gold nanoarrays by way of biomolecular recognition. Specifically, we demonstrated the spectral tuning of plasmon resonant metal nanoarrays and self-assembly of protein-functionalized QDs in a stepwise fashion with a concomitant incremental increase in separation from the metal surface through biotin-streptavidin spacer units.

Directed self-immobilization of alkaline phosphatase on micro-patterned substrates via genetically fused metal-binding peptide.

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self-assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site-specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N-terminus of AP (nGBP1-AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi-functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple-repeat constructs, 5GBP1-AP displayed the best bi-functional activity and, therefore, was chosen for self-immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1-AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self-immobilization of the bi-functional enzyme on micro-patterned substrates where genetically linked 5GBP1-AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site-specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic-binding peptides has a potential utility in a wide range of biosensing and bioconversion processes.

Assembly of nanomaterials through highly ordered self-assembled monolayers and peptide-organic hybrid conjugates as templates.

Synthesis and processing techniques have now been established for obtaining high quality monodisperse nanocrystals of various metallic and semiconducting materials, fullerenes of distinct properties, single- and multi-wall carbon nanotubes, polymeric dendrimers with tailored functionalities, as well as other nanophase constructs. The next key step towards novel applications of nanostructured materials concerns their positioning, arrangement, and connection into functional networks without mutual aggregation. In this review, we highlight the recent progress of using anthracene- and pyrene-based self-assembling molecules with tunable energetic (pi-pi interactions, hydrogen bonding, dipole-dipole interactions) and variable geometries to create stable, highly ordered, and rigid self-assembled monolayer (SAM) templates with adjustable superlattices on crystalline substrates. Based on aromatic SAM templates, stable and highly ordered self-assembled structures of optoelectronically active C60 have been obtained and shown to exhibit desirable electrical and optoelectronic properties, such as nonlinear transporting effect for molecular electronics and efficient photocurrent generation for mimicking photosynthesis in nature. By using genetically engineered polypeptides with surface recognition for specific inorganics, selective integration of nanoparticles onto aromatic SAM templates have also been realized. Through a combination of spatially confined surface chemical reaction and microcontact printing, sub-micron arrays of peptide-organic hybrid conjugates were successfully generated to serve as templates to achieve the patterned assembly of nanoparticles.

Arrays of covalently bonded single gold nanoparticles on thiolated molecular assemblies.

A simple approach to form arrays of covalently bonded single gold nanoparticles (AuNPs) is demonstrated. Asymmetric molecular assemblies composed of two layers of rigid aromatic molecules with different structures, arranged in hexagonal arrays on a template produced by edge-spreading lithography, are used to guide the assembly of AuNPs. Arrays of single AuNPs are achieved by taking advantage of the interplay of electrostatic interactions and covalent bonding in conjunction with the positional constraint on the template. Schiff base chemistry is highlighted in the surface chemical reaction to selectively modify nanoscale surface features with high yield.