Bacteriophage bionanowire as a carrier for both cancer-targeting peptides and photosensitizers and its use in selective cancer cell killing by photodynamic therapy.

A photosensitizer, pyropheophorbid-a (PPa), is conjugated to SKBR-3 breast cancer cell-specific biological nanowire phage, to form a novel PPa-phage complex, which is further successfully used in selectively killing SKBR-3 breast cancer cells by the mechanism of photodynamic therapy (PDT).

Failure Mode Identification of Insulin Drug Products - Impact of Relevant Stress Conditions on the Quality of the Drug.

Fast-acting insulin drug products (DPs) are carried and administered by diabetic patients to maintain their blood glucose level throughout the day, exposing the DPs to stress conditions. Apidra, Novolog, and Humalog insulin DPs were tested under various stress conditions. Dynamic light scattering (DLS), and size exclusion chromatography (SEC) were used to monitor the stability and aggregation. Thermal stress alone did not influence the stability. However, 24 hr exposure to vigorous mechanical stress shifted the DLS size peaks of Novolog and Humalog from 5 ± 1 nm to > 50.9 ± 25.6 nm, and the SEC native protein peak areas decreased 52% for Novolog and 18.4% for Humalog. Combined stress accelerated protein aggregation more drastically. Novolog and Humalog size shifted (>75 nm) after 3 hr and the peak area decreased > 97.9% after 6 hr exposure, indicating that high temperature accelerated the aggregation triggered by agitation. Soluble aggregates were captured by DLS early on compared to SEC. Apidra was comparably stable indicating DP formulation plays a critical role in stability. Our study provides a greater understanding of potential failure modes patients and care givers may encounter while handling insulin DPs.

Upconversion nanoparticles: synthesis, surface modification and biological applications.

New generation fluorophores, also termed upconversion nanoparticles (UCNPs), have the ability to convert near infrared radiations with lower energy into visible radiations with higher energy via a nonlinear optical process. Recently, these UCNPs have evolved as alternative fluorescent labels to traditional fluorophores, showing great potential for imaging and biodetection assays in both in vitro and in vivo applications. UCNPs exhibit unique luminescent properties, including high penetration depth into tissues, low background signals, large Stokes shifts, sharp emission bands, and high resistance to photobleaching, making UCNPs an attractive alternative source for overcoming current limitations in traditional fluorescent probes. In this article, we discuss the recent progress in the synthesis and surface modification of rare-earth doped UCNPs with a specific focus on their biological applications. FROM THE CLINICAL EDITOR: Upconversion nanoparticles - a new generation of fluorophores - convert near infrared radiations into visible radiations via a nonlinear optical process. These UCNPs have evolved as alternative fluorescent labels with great potential for imaging and biodetection assays in both in vitro and in vivo applications.

Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization.

Filamentous phage as a bacteria-specific virus can be conjugated with an anticancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and rearrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells.

Sirt2 Regulates Radiation-Induced Injury.

Sirtuin 2 (SIRT2) plays a major role in aging, carcinogenesis and neurodegeneration. While it has been shown that SIRT2 is a mediator of stress-induced cell death, the mechanism remains unclear. In this study, we report the role of SIRT2 in mediating radiation-induced cell death and DNA damage using mouse embryonic fibroblasts (MEFs), progenitor cells and tissues from Sirt2 wild-type and genomic knockout mice, and human tumor and primary cell lines as models. The presence of Sirt2 in cells and tissues significantly enhanced the cell's sensitivity to radiation-induced cytotoxicity by delaying the dispersion of radiation-induced γ-H2AX and 53BP1 foci. This enhanced cellular radiosensitivity correlated with reduced expression of pro-survival and DNA repair proteins, and decreased DNA repair capacities involving both homologous repair and non-homologous end joining DNA repair mechanisms compared to those in Sirt2 knockout (KO) and knockdown (KD) phenotypes. Together, these data suggest SIRT2 plays a critical role in mediating the radiation-induced DNA damage response, thus regulating radiation-induced cell death and survival.

Virus-based photo-responsive nanowires formed by linking site-directed mutagenesis and chemical reaction.

Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators.

Development of an optimized protocol for studying the interaction of filamentous bacteriophage with mammalian cells by fluorescence microscopy.

Filamentous bacteriophage has been proposed as a vehicle that can carry and deliver therapeutics into mammalian cells for disease treatment, thus a protocol for imaging phage-cell interaction is essential. Because high signal intensity is necessary to study the mechanism of interaction between filamentous bacteriophage and mammalian cells, it is important to optimize the procedure for fluorescence labeling of phage in order to understand such interaction. Here, we describe a procedure that gives intense fluorescence labeling and can show interactions between fd-tet bacteriophage selected from phage libraries and mammalian cells (SKBR-3 and MCF-10A). The indirect labeling of phage with dye-conjugated antibody and cytoskeleton associated proteins was significantly enhanced in the presence of a cross-linking reagent called dithiobissuccinimidylpropionate (DSP) as shown by qualitative and quantitative fluorescence microscopy. The use of DSP resulted in high signal intensity in fluorescence imaging of phage-cell complex. The DSP cross-linker is believed to preserve soluble unbound proteins for fluorescence imaging. The interaction between the phage and mammalian cells was further confirmed by scanning electron microscopy.

Architectonics of phage-liposome nanowebs as optimized photosensitizer vehicles for photodynamic cancer therapy.

Filamentous M13 phage can be engineered to display cancer cell-targeting or tumor-homing peptides through phage display. It would be highly desirable if the tumor-targeting phage can also carry anticancer drugs to deliver them to the cancer cells. We studied the evolution of structures of the complexes between anionic filamentous M13 phage and cationic serum-stable liposomes that encapsulate the monomeric photosensitizer zinc naphthalocyanine. At specific phage-liposome ratios, multiple phage nanofibers and liposomes are interwoven into a "nanoweb." The chemical and biological properties of the phage-liposome nanoweb were evaluated for possible application in drug delivery. This study highlights the ability of phage-liposome nanowebs to serve as efficient carriers in the transport of photosensitizers to cancer cells.