Gopal Abbineni

Upconversion nanoparticles: synthesis, surface modification and biological applications

UNLABELLED 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.

Nanofibrous bio-inorganic hybrid structures formed through self-assembly and oriented mineralization of genetically engineered phage nanofibers.

Nanofibrous organic–inorganic hybrid structures, where inorganic nanocomponents are formed or assembled within the aligned organic nanofibrous matrix, are important materials that can find applications in electronics, photonics, catalysis, and tissue engineering.[1–11] They not only provide a means for supporting and ordering the functional inorganic materials such as nanoparticles,[4–6,8] but also can serve as building blocks to further self-assemble into higher-order structures.[5,11,12] The common approaches to the synthesis of the nanofibrous organic-inorganic hybrid structures include electrospinning,[2,4,10] polymer-templating,[3] biotemplating,[5–7,11] and directional freezing.[1] As a matter of fact, the nanofibrous organic-inorganic hybrid structures are important building blocks in natural mineralized biomaterials. One of the best examples is the mineralized collagen fibrils constituting the extracellular matrix (ECM) of bone.[13] Bone is made of cells embedded in ECM, which is hierarchically organized from proteins, including type I collagen and non-collagenous proteins (NCPs) such as bone sialoprotein (BSP), and calcium hydroxylapatite (HAP, Ca10(PO4)6(OH)2). The collagen molecules (~1.5 nm wide and 300 nm long) are self-assembled into wider (up to 200 nm wide) and longer (several µm long) fibrils in a side-to-side and head-to-tail format, which are further hierarchically self-assembled to form much wider and longer collagen fibers (up to several tens µm wide and long).[14,15] HAP is found within the gaps and grooves of the collagen fibers with its c-axis preferentially along the collagen fibers.

Self-Assembly of Drug-Loaded Liposomes on Genetically Engineered Target-Recognizing M13 Phage: A Novel Nanocarrier for Targeted Drug Delivery**

Liposomes have been a center of research for many years due to their numerous applications in chemistry, biology, medicine, and nanotechnology. They can be used to entrap materials such as drugs either within the central aqueous compartment if they are water soluble, or within the hydrophobic domain of the lipid bilayer if they are oil soluble. In addition, their surface can bemodified to realize targeted delivery. For example, in biomedicine, liposomes are used as vehicles to deliver drugs and genes to specific parts of the body. When used in the delivery of certain anticancer drugs, liposomes help to shield healthy cells from the drug toxicity and prevent concentration in vulnerable tissues. In addition, the liposomes allowmuch smaller doses of drug to be used, thus reducing the side effects of that drug. On the other hand, zinc phthalocyanine (ZnPc) is a potential drug that is being tested as a photosensitizer for photodynamic therapy (PDT). PDT involves the systemic administration of a photosensitizer followed by illumination with light of an appropriate wavelength, which results in the formation of singlet oxygen (O2) for destroying cancer cells. [6] ZnPc has a high absorption coefficient at 650–700 nm with optimal tissue penetration. It has a long lifetime in the triplet excited state, thus resulting in the highly efficient production of O2 which is the main cytotoxic species in PDT. However, it is insoluble in water and must be incorporated into unilamellar liposomes (Figure 1a). The main drawback of liposomes is their instability in biological media, as well as their sensitivity to many external parameters, such as temperature or osmotic pressure. This instability problem affects the application of liposomes in drug/gene delivery and PDT. Attempts have been made to stabilize liposomes by adsorbing some

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.

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).

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.

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. Mol Cancer Ther; 9(9); 2524–35. ©2010 AACR.

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. Microsc. Res. Tech., 2010.

Sensing humidity using virus-nanoparticle assembly

A virus-gold nanoparticle (AuNP) composite film was prepared through the layer-by-layer assembly of cationic filamentous virus (M13 phage) particles (displaying a positively charged peptide on the major coat) and anionic spherical AuNPs. The surface plasmon resonance (SPR) for AuNPs in the virus-nanoparticle nanocomposite films was found to be sensitive to the environmental humidity. In air, both λmax and peak absorbance of the SPR spectra of the nanocomposite films are linearly dependent on the environmental humidity. We believe that when the environmental humidity is reduced, the height of the phage particles in the nanocomposite films is reduced, leading to the shortening of the spacing between neighboring gold nanoparticles in the films. Therefore, the SPR band systematically redshifts with the decrease in humidity. Furthermore, the response of the virus-nanoparticle film to the humidity is rapid and reversible, which favors in it for humidity sensing application.