Engineering M13 Bacteriophage Nanoplatforms for Diagnostic and Therapeutic Applications

M13 bacteriophage, a naturally monodisperse multifunctional nanostructure, consists of thousands of distinct protein subunits organized in a filamentous viral capsid; 900nm in length and 6nm in diameter. All M13 capsids are amenable to mutation and can be tuned for the binding and nucleation of inorganics and nanoparticles, and for the expression of ligands, functional moieties, and even enzymes. To harness these capabilities for medical imaging and therapy, the author has (i) tailored the assembly of M13 into ultra-short, ‘inho’, phage derived particles, (ii) developed a chlorotoxin (CTX) motif on the M13 p3 capsid to enable phage particle crossing of the blood-brain-barrier and homing to glioblastoma cancer cells, and (iii) built ‘inho’ phage derived transgene cassettes for phage gene delivery in mammalian cells. Tight control over the genetic sequence provided by ‘inho’ phagemids allow production of phage particles ranging in length from 25nm to over 2500nm, as dictated by the length of the packaged DNA. This length control over the phage filament is used to demonstrate the impact of the particle length on the morphology of phage templated metal nanofoams and on the in-vitro and in-vivo tissue trafficking of targeted phage nanocarrriers. An optimal length for enhancing ion transport and active material access in MnOx cathodes is described. Chlorotoxin-phages, conjugate with indocyanine green dye (ICG), are visualized in-vivo in the second window near infrared (SWIR) and home effectively to mouse brain tumor. Ultra-short, 50nm chlorotoxin-phage particles are shown to vastly improve this localization specificity. Additionally, the ‘inho’ phagemid system is engineered to produce ITR-flanked transgene cassettes. Such reporter genes packaged within targeted, cationically modified, ‘inho’ phages are able to transduce liver and brain cancer cells. The closed-ended, single-stranded ‘inho’ phage-derived cassettes have capacity up to 20 kilobases and can be delivered within phage particles as well as non-viral delivery vehicles. Ultimately, therapy or imaging agent carrying, miniaturized, chlorotoxin-targeted, M13 phage is considered here as a complete nanotheranostic platform that could augment the therapeutic efficacy of combination drugs shuttled to the site of glioma. The described multimodal, nanoplatform is re-designable for applications in nanomaterials, diagnostics, and across disease types.Ph.D

Engineering M13 bacteriophage platforms for cancer therapy applications

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2015.Cataloged from PDF version of thesis.Includes bibliographical references (pages 46-48).Two novel schemes for engineering M13 bacteriophage for application in the diagnosis, imaging and treatment of human tumors are proposed. Firstly, by exploiting the uniquely malleable biology of the M13 filamentous phage, we have engineered filamentous phages of shorter lengths by constructing our own set of small viral ssDNA that are packaged by M13 capsid proteins. These 'inho' phages can be sized to ~50nm and above in length. The small phage retains the M13 major and minor coat proteins which have previously been manipulated to serve as tethers to carry various therapy and imaging agents and target specific cancer sites. Now with the ability to control the aspect ratio of these rigid, rod-like phages we can further improve on M13 based cancer detection by optimizing for phage blood circulation and tumor extravasation. Secondly, we have added to our cancer targeting M13 platform collection by cloning for chlorotoxin display on the tail p3 capsid protein of M13. Chlorotoxin can induce passage across blood-brain barrier, targets for cancer cells, and specifically internalizes to glioma cells. Expression of chlorotoxin on M13 will allow us to capitalize on its strong affinity for tumors of neuroectodermal origin and expand the M13 therapy and imaging platform to tumor masses in the brain.by Uyanga Tsedev.S.M

The Origins and Development of the “Third Neighbor Policy” of Mongolia: A Prospective of the China Factor

TesisLima EsteEscuela Profesional de DerechoDerecho PenalEl presente trabajo de investigación se pretende determinar y explicar que el Estado debe de resarcir directamente a las personas que sufrieron prisión preventiva injusta, impuesta por los juzgados penales de Lima Metropolitana en los último diez años (2005-2015), iniciando la presente investigación con la realidad problemática en donde se describe que esta medida no solo afecta inculpado sino al entorno familiar, además que se restringen derechos fundamentales como la libertad individual y la presunción de inocencia causándoles daños y perjuicios a las personas, conforme a la teoría de Responsabilidad Patrimonial Extracontractual (Daños emergentes y Lucro cesante). El punto de trabajo de la presente investigación es de tipo descriptivo y en concordancia con el diseño de investigación establecido es de tipo científico. La presente investigación se realizó en la Fiscalía Provincial Penal de Lima, en el Juzgado Penal de Lima Metropolitana y un ex interno del Penal San Pedro de Lurigancho, durante el año 2016

Creating fluorescent quantum defects in carbon nanotubes using hypochlorite and light

Covalent doping of single-walled carbon nanotubes (SWCNTs) can modify their optical properties, enabling applications as single-photon emitters and bio-imaging agents. We report here a simple, quick, and controllable method for preparing oxygen-doped SWCNTs with desirable emission spectra. Aqueous nanotube dispersions are treated at room temperature with NaClO (bleach) and then UV-irradiated for less than one minute to achieve optimized O-doping. The doping efficiency is controlled by varying surfactant concentration and type, NaClO concentration, and irradiation dose. Photochemical action spectra indicate that doping involves reaction of SWCNT sidewalls with oxygen atoms formed by photolysis of ClO− ions. Variance spectroscopy of products reveals that most individual nanotubes in optimally treated samples show both pristine and doped emission. A continuous flow reactor is described that allows efficient preparation of milligram quantities of O-doped SWCNTs. Finally, we demonstrate a bio-imaging application that gives high contrast short-wavelength infrared fluorescence images of vasculature and lymphatic structures in mice injected with only ~100 ng of the doped nanotubes.Koch Institute Frontier Research Program. Marble Center for Cancer Nanomedicine (Cancer Center Support core Grant P30-CA14051)National Science Foundation (U.S.) (Grant CHE-1803066)Robert A. Welch Foundation (Grant C-0807

Genetic Control of Aerogel and Nanofoam Properties, Applied to Ni–MnO x Cathode Design

Aerogels are ultralight porous materials whose matrix structure can be formed by interlinking 880 nm long M13 phage particles. In theory, changing the phage properties would alter the aerogel matrix, but attempting this using the current production system leads to heterogeneous lengths. A phagemid system that yields a narrow length distribution that can be tuned in 0.3 nm increments from 50 to 2500 nm is designed and, independently, the persistence length varies from 14 to 68 nm by mutating the coat protein. A robotic workflow that automates each step from DNA construction to aerogel synthesis is used to build 1200 aerogels. This is applied to compare Ni–MnOx cathodes built using different matrixes, revealing a pareto-optimal relationship between performance metrics. This work demonstrates the application of genetic engineering to create “tuning knobs” to sweep through material parameter space; in this case, toward creating a physically strong and high-capacity battery