Cellular internalization of quantum dots.

Cell-penetrating peptides (CPPs) can facilitate uptake of quantum dots (QDs) for a variety of basic and applied sciences. Here we describe a method that utilizes simple noncovalent interactions to complex QDs and CPPs. We further describe methods to study uptake mechanisms of the QD/CPP complex. The inhibitor study coupled with the RNA interference (RNAi) technique provides a comprehensive approach to elucidate cellular entry of the QD/CPP complex.

Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence.

Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell-penetrating peptides (CPPs) and their cargoes. However, a major limitation of endocytic pathway is entrapment of the CPP-cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity. Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin-2 (HA2) remarkably enhances the cytosolic delivery of proteins in human A549 cells. To determine the endosome-disruptive effects, recombinant DNA plasmids containing coding sequences of HA2, CPPs and red fluorescent proteins (RFPs) were constructed. The fusion proteins were purified from plasmid-transformed Escherichia coli, and their effects on protein transduction were examined using live cell imaging and flow cytometry. Our data indicate that endocytosis is the major route for cellular internalization of CPP-HA2-tagged RFP. Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP-mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm. Furthermore, incorporating the HA2 fusion peptide of the CPP-HA2 fusion protein improved cytosolic uptake without causing cytotoxicity. These findings strongly suggest that the CPP-HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs.

Arginine-rich cell-penetrating peptides deliver gene into living human cells.

Transgenesis is a process that introduces exogenous nucleic acids into the genome of an organism to produce desired traits or evaluate function. Improvements of transgenic technologies are always important pursuit in the last decades. Recently, cell-penetrating peptides (CPPs) were studied as shuttles that can internalize into cells directly and serve as carriers to deliver different cargoes into cells. In the present study, we evaluate whether arginine-rich CPPs can be used for gene delivery into human cells in a noncovalent fashion. We demonstrate that three arginine-rich CPPs (SR9, HR9, and PR9) are able to transport plasmid DNA into human A549 cells. For the functional gene assay, the CPP-delivered plasmid DNA containing the enhanced green fluorescent protein (EGFP) coding sequence could be actively expressed in cells. The treatment of calcium chloride did not facilitate the CPP-mediated transfection efficiency, but enhance the gene expression intensity. Mechanistic studies further revealed that HR9/DNA complexes mediate the direct membrane translocation pathway for gene delivery. Our results suggest that arginine-rich CPPs, especially HR9, appear to be a high efficient and promising tool for gene transfer.

A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides.

Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84-93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis.

Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium.

Owing to the cell membrane barriers, most macromolecules and hydrophilic molecules could not freely enter into living cells. However, cell-penetrating peptides (CPPs) have been discovered that can translocate themselves and associate cargoes into the cytoplasm. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) can form stable complexes with plasmid DNA at the optimized nitrogen/phosphate ratio of 3 and deliver plasmid DNA into Paramecium caudatum in a noncovalent manner. Accordingly, the transported plasmid encoding the green fluorescent protein (GFP) gene could be expressed in cells functionally assayed at both the protein and DNA levels. The efficiency of gene delivery varied among these CPPs in the order of HR9>PR9>SR9. In addition, these CPPs and CPP/DNA complexes were not cytotoxic in Paramecium detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diohenyltetrazolium bromide (MTT) assay. Thus, these results suggest that the functionality of arginine-rich CPPs offers an efficient and safe tool for transgenesis in eukaryotic protozoans.

Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism.

Functional peptides that transfer biomaterials, such as semiconductor quantum dots (QDs), into cells in biomaterial research have been developed in recent years. Delivery of QDs conjugated with cell-penetrating peptides (CPPs) into cells by the endocytic pathway was problematic in biomedical applications because of lysosomal trapping. Here, we demonstrate that histidine- and arginine-rich CPPs (HR9 peptides) stably and noncovalently combined with QDs are able to enter into cells in an extremely short period (4 min). Interrupting both F-actin polymerization and active transport did not inhibit the entry of HR9/QD complexes into cells, indicating that HR9 penetrates cell membrane directly. Subcellular colocalization studies indicated that QDs delivered by HR9 stay in cytosol without any organelle capture. Dimethyl sulphoxide, ethanol and oleic acid, but not pyrenebutyrate, enhanced HR9-mediated intracellular delivery of QDs by promoting the direct membrane translocation pathway. HR9 and HR9/QDs were not cytotoxic. These findings suggest that HR9 could be an efficient carrier to deliver drugs without interfering with their therapeutic activity.

Quantitative analysis of cholesterol nucleation with time in supersaturated model bile.

The amount of cholesterol (Ch) crystals formed in supersaturated taurochenodeoxycholate (TCDC) - lecithin (L) solutions of the same Ch saturation index (CSI) but at different Ch thermodynamic activities (Ch A(T)) was quantified at different time intervals. The initial Ch nucleation rate (i.e., amount of Ch crystals formed with respect to time) in a Ch A(T) = 1.73 and TCDC to L molar ratio (TCDC:L) = 5.1 system was faster than that in a Ch A(T) = 1.42 and TCDC:L = 3.4 system. Shaking could enhance the early appearance of Ch crystals and cause the fast initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. The final Ch nucleation rates were faster than the initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. According to a light scattering analysis of vesicle concentration in supersaturated TCDC-L solutions, vesicles provide nucleation sites only in the Ch nucleation process and the vesicle concentration may not be an important factor for the Ch nucleation rate. A model of a mixed TCDC-L micelle releasing Ch molecules together with the surface area of Ch crystals formed was used in the interpretation of the Ch nucleation.

Cell-penetrating peptide-functionalized quantum dots for intracellular delivery.

Quantum dots (QDs) are luminescent semiconductor nanocrystals that are widely used as fluorescent probes in biomedical applications, including cellular imaging and tumor tracking. Cell-penetrating peptides (CPPs), also called protein transduction domains (PTDs), are short basic peptides that permeate cell membranes and are able to deliver a variety of macromolecule cargoes, such as DNAs, RNAs, proteins, and nanomaterials. Here we review strategies to couple QDs to CPPs, by either covalent linkages or noncovalent interactions, to provide a tool to study intracellular delivery. This facilitated transport of QDs by CPPs into cells is both simple and efficient. Accordingly, CPP-QD nanoparticles are likely to be of broad utility in biological research and advance the development of medical and pharmaceutical therapeutics.

Nona-arginine facilitates delivery of quantum dots into cells via multiple pathways.

Semiconductor quantum dots (QDs) have recently been used to deliver and monitor biomolecules, such as drugs and proteins. However, QDs alone have a low efficiency of transport across the plasma membrane. In order to increase the efficiency, we used synthetic nona-arginine (SR9), a cell-penetrating peptide, to facilitate uptake. We found that SR9 increased the cellular uptake of QDs in a noncovalent binding manner between QDs and SR9. Further, we investigated mechanisms of QD/SR9 cellular internalization. Low temperature and metabolic inhibitors markedly inhibited the uptake of QD/SR9, indicating that internalization is an energy-dependent process. Results from both the pathway inhibitors and the RNA interference (RNAi) technique suggest that cellular uptake of QD/SR9 is predominantly a lipid raft-dependent process mediated by macropinocytosis. However, involvement of clathrin and caveolin-1 proteins in transducing QD/SR9 across the membrane cannot be completely ruled out.

Estimation of cholesterol solubilization by a mixed micelle binding model in aqueous tauroursodeoxycholate:lecithin:cholesterol solutions.

In order to interpret the clinical efficacy of conjugated ursodeoxycholate (UDC) in cholesterol (Ch) gallstone patients, the Ch solubilization in mixed micelles in 40:40:32 mM tauroursodeoxycholate (TUDC):taurochenodeoxycholate (TCDC):lecithin (L) and 80:32 mM TUDC:L systems was estimated by using a model of Ch binding to mixed micelles. The Ch solubilization limit in mixed TUDC:L micelles was found to be higher than that in mixed TUDC:TCDC:L micelles. In the 80:32 mM TUDC:L system, the dissolution of the Ch pellet decreased after vesicles (liposomes) formed on the surface of the Ch pellet whereas the dissolution of microcrystalline Ch was rapid before and after vesicle formation in the solution, indicating that the total surface area of solid Ch exposed to the solution may be another important factor in inducing the dissolution of Ch gallstones. These phenomena suggest that although vesicles, occasionally formed in the bile of patients under the therapy of conjugated UDC, make a contribution to the solubilization of Ch gallstones, the model of Ch binding to mixed TUDC:L micelles can be used to estimate Ch solubility in TUDC:L system.

Epitope mapping of Mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes.

Phage display techniques have been widely employed to map the epitope structures which served as the basis for developing molecular vaccines. In the present study, we applied this technique to map the epitopes of Mycoplasma hyopneumoniae, the etiologic agent causing swine enzootic pneumonia, and evaluated directly the immune responses in mice of the selected phage-displayed epitopes (phagotopes). Two phage-displayed random peptide libraries were biopanned with the protein A-purified IgG of the rabbit anti-M. hyopneumoniae hyperimmune serum and the selected phage clones were sequenced and analyzed. Some of the inserts of the selected phagotopes showed a good match with the known proteins of M. hyopneumoniae. Others, which did not match with any known proteins, but shared extensive homology with each other, were clustered and classified as the conformational epitopes of M. hyopneumoniae. To evaluate the potential of using these phagotopes as effective vaccines, several phage clones were chosen to immunize mice. IgA coproantibody, IgA in bronchoalveolar lavage fluid and serum IgG responses were assayed. The serum raised by the phage clones clearly recognized several major mycoplasmal proteins indicating that the phagotope-induced immune responses were antigen-specific. The stronger IgG1 response revealed that the immune responses of the epitope-displaying phage were mainly through Th2 activation. The growth inhibition assay showed that the selected phage clones CS4 and varphi58 are potential vaccine candidates and suggested that the mycoplasmal 97 kDa, 56 kDa, 30 kDa and 23 kDa proteins may play important roles in the immune responses. The present work demonstrates that the whole epitope profile of a microorganism can be obtained through screening the phage displayed peptide libraries with the hyperimmune serum and reveals the potential of using epitope-displaying phages as peptide vaccines.