Shan-Yu Fung

Claudin 1 Mediates TNFα-Induced Gene Expression and Cell Migration in Human Lung Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies.

Self-assembling peptide-based nanoparticles enhance cellular delivery of the hydrophobic anticancer drug ellipticine through caveolae-dependent endocytosis

UNLABELLED A special class of self-assembling peptide (EAK16-II) has been found to stabilize the hydrophobic anticancer agent ellipticine (EPT) in aqueous solution. In this study, the mechanism of such peptide-EPT complexes to enhance cellular delivery and anticancer activity was evaluated. Results revealed that EAK16-II can form nanoparticles with EPT, having an average size of ∼100 nm. This nanoformulation had cytotoxicity to human lung carcinoma A549 cells that was comparable to EPT dissolved in dimethyl sulfoxide. It enhanced EPT uptake drastically when compared to the microformulation. Such enhanced uptake was significantly reduced by inhibitors specifically for the caveolae-dependent pathway. We also found both protonated and neutral forms of EPT present in the cells. Interestingly, both were found in the cytoplasm, co-localized with LysoTracker, whereas only protonated EPT was seen in the nucleus. The promising therapeutic efficacy, specific delivery pathway, and intracellular distribution pattern discovered in this work may help further develop EPT as a nanoformulation for clinical applications. FROM THE CLINICAL EDITOR A special class of self-assembling peptide (EAK16-II) has been found to stabilize ellipticine in aqueous solution. The authors demonstrate therapeutic efficacy, describe specific delivery pathways, and effective intracellular distribution pattern, which will aid the development of this technology for future clinical applications.

Programming the Cellular Uptake of Physiologically Stable Peptide–Gold Nanoparticle Hybrids with Single Amino Acids

The fast-developing field of nanotechnology has produced an enormous variety of nanoparticles (for example quantum dots, and metallic, magnetic, and polymeric nanoparticles) with different sizes and shapes for diverse biomedical applications in drug delivery, disease diagnostics, and medical imaging. These nanoparticles often require surface modification to ensure their biocompatibility and/or enhance the bioavailability. In many cases, functional biomolecules, such as antibodies, peptides, and nucleic acid ligands, have been extensively introduced onto nanoparticles to enable cellspecific recognition, assist cellular uptake, and alter intracellular localization. Thus, there is a tremendous need to fundamentally understand the interactions between these functionalized nanoparticles and biological systems, and how the nanoparticle surface chemistry affects the biological responses. As part of this large scope, we developed a novel peptide– gold nanoparticle hybrid system to investigate the surface chemistry of the nanoparticles in determining their cellular responses. We selected the gold nanoparticle (GNP) as the core of the system for 1) its ease of surface modification by gold–thiol or gold–amine linkages; 2) the intrinsic fluorescence of GNP aggregates/clusters on surfaces for the direct observation of their cellular uptake by confocal or fluorescence microscopy; and 3) clinical trials of GNPs in cancer therapy, thus enabling our future development of the hybrid system for potential clinical uses. The critical part of the hybrid system is the modulation of GNP surface properties by rationally designed peptide ligands (Supporting Information, Table S1). These peptide ligands contain three functional regions (Figure 1a): the gold binding motif at the N terminus, the hydrophobic spacing region in the middle, and the functional end group at the

The potential of nanoscale combinations of self-assembling peptides and amino acids of the Src tyrosine kinase inhibitor in acute lung injury therapy

Many newly discovered therapeutic agents require a delivery platform in order to translate them into clinical applications. For this purpose, a nanoscale formulation strategy was developed for the Src tyrosine kinase inhibitor PP2. The formulation utilizes the combination of the self-assembling peptides (EAK16-II) and amino acids to minimize the use of the toxic organic solvent DMSO; hence, the biocompatibility of the PP2 nanoformulations was significantly improved. They were found to be non-hemolytic and safe for intravenous and intratracheal administration; the formulations did not alter PP2 activity in Src inhibition on cultured cells. The PP2 nanoformulation was further evaluated on a lipopolysaccharide (LPS)-induced acute lung injury mouse model. Results revealed that the pretreatment of PP2 nanoformulation could decrease the inflammatory cell infiltration and the pro-inflammatory cytokine TNF-α production in the bronchoalveolar lavage fluid after LPS stimulation. The promising therapeutic efficacy and the formulation strategy developed in this work may help further translate PP2 and other hydrophobic therapeutic agents into clinical applications.

Formulation of hydrophobic therapeutics with self-assembling peptide and amino acid: A new platform for intravenous drug delivery

Clinical application of hydrophobic therapeutics is restricted by lack of an efficient vehicle which permits their solubility in aqueous environments. We have previously developed a novel formulation strategy to deliver a hydrophobic Src inhibitor, PP2, involving combinations of one self-assembling peptide (SAP) and one of 4 selected amino acids (AAs). The present study aims to develop a generalized drug delivery platform for intravenous application of hydrophobic drugs by combining self-assembling peptide, amino acid and low concentration of co-solvent. A multi-step screening pipeline is established which includes assessment of drug solubility and physicochemical characteristics, as well as functional efficacy and safety in vitro and in vivo. Using PP2 as an exemplary hydrophobic compound, 480 different combinations of 6 SAPs, 20 naturally existing AAs at 2 concentrations, and 2 co-solvents were evaluated. Among the combinations, 60 formulae dissolved PP2; 10 of which significantly reduced thrombin-induced IL-8 production, a sign of inflammatory response, in normal human lung epithelial BEAS2B cells. These formulations did not show cytotoxicity alone, but 2 reduced cell viability with presence of thrombin. We then performed a double-blinded test in a rat model of pulmonary ischemia-reperfusion. PP2 formulated with EAK16-I peptide plus methionine and 2% ethanol were administrated intravenously, significantly reducing severity of lung injury. The SAP-AA formulation strategy was also successfully applied to other hydrophobic compounds, suggesting this strategy could be applicable to other hydrophobics for a variety of clinical applications.

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds - PP2, rottlerin, and curcumin - in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.