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FINAL MASTER PROJECT Master: Nanostructured Materials for Nanotechnological Applications THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages Carlos Piñol Domingo THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 2 FINAL MASTER PROJECT Master: Nanostructured Materials for Nanotechnological Applications THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages ■ STUDENT: ▪ Carlos Piñol Domingo ■ CERTIFY: ♦ DIRECTOR: ▪ Dr. Gerardo F. Goya Rossetti_Condensed Matter Physics department (INA), Spain. ♦ CO-DIRECTORS: ▪ Dr. María Carmen Visus Miguel_Patology department, (Hillman Cancer Center- UPCI), Pittsburgh, USA. ▪ Dr. Eric Grelet_Self-Organization of Liquid crystals department (CRPP-CNRS), Bordeaux, France. Science Faculty / University of Zaragoza 2012 THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 3 INDEX ► GENERAL ABSTRACT …………………………………………………………………………..… 5 ► RESUMEN GENERAL …………………….……………………………………………………….. 7 I. GENERAL INTRODUCTION ………………………………………………..………………… 9 1.- Nanobiotechnology ………………..…………………………..………………………………..….. 9 2.- Nanoparticles and nanofunctionalization ………………………..……………………………..… 10 • Bibliography ……………………………………………………………………………………………….. 10 II. EXAMPLE 1: Targeting Cancer Stem Cells with antibody-functionalized magnetic nanoparticles for magnetic hyperthermia …………………………………………………...… 12 1.- INTRODUCTION …………………………………………………………………………….. 12 1.1.- Cancer …………………………………………………………………………………… 12 1.2.- Cancer Stem Cells (CSCs): Concept, tumor cell lines and markers ………..…………… 12 1.3.- Magnetic nanoparticles (MNPs) as agents for hyperthermia ………………….………… 14 2.- AIMS ………………………………………………………………………………..………… 16 3.- EXPERIMENTAL SECTION ……………………………………………………….……….. 16 3.1. Material …………………………………………………………………………………… 16 3.2. Protocols ……………………………………………………………………………..…… 17 3.2.1.- MTT protocol for cytotoxicity studies …………………………………………………….. 17 3.2.2.- Protocol for studying the affinity between MNPs and tumor cell lines ……………………. 17 3.2.3.- Protocol for TEM/EDX sample preparation …………………………..…………………… 18 3.2.4.- Protocols for the studies both the interaction between MNPs:anti_CDs and the targeting of CSCs with these antibody-functionalized MNPs …………………………………...……… 18 3.2.4.1.- Protocol for spectrofluorometer sample preparation …………………….……….. 19 3.2.4.2.- Protocol for sample preparation for fluorescence microscopy …………………… 19 4.- RESULTS AND DISCUSSION ……………………………………………………..……….. 20 4.1.- Cytotoxicity studies of the MNPs on the tumor cell lines by MTT essay ………...…….. 20 4.2.- Study of the affinity between MNPs and tumor cell lines ………………………...…….. 21 4.3.- Study of the surface and intracellular distribution of the MNPs treated-tumor cells by TEM/ EDX techniques ……………………………..…………….……………………… 22 4.4.- Fluorescence microscopy studies of the interaction between MNPs:anti_CDs and the targeting of CSCs with these antibody-functionalized MNPs …………………………… 23 5.- CONCLUSIONS ……………………………………………………………………………… 28 • Bibliography ……………………………………………................……………………………………… 29 ▫ Acknowledgements ………………………………………………………………………………………... 30 III. EXAMPLE 2: Regioselective control of molecular nanoprobes on filamentous virus particles and self-assembly of these functionalized rod-like nanoparticles ……………………………. 31 1.- INTRODUCTION ………………………………………………….…………………………. 31 1.1.- M13 bacteriophage: wild type and mutant virus ………………………………………… 31 1.2.- Liquid crystalline organization ………………………………………………………….. 32 1.3.- Applications of the virus-based hybrid materials ………………………...…………….…… 33 THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 4 2.- AIMS ……………………………………………………………………….…………………. 34 3.- EXPERIMENTAL SECTION …………………………………………….………………….. 34 3.1.- Precautions …………………………………………………………….………………… 35 3.2. M13-C7C amplification process ………………………………………………………….. 35 3.3.- M13-C7C titration process ………………………………………….…………………… 35 3.4.- Functionalization process …………………………………………….………………….. 35 4.- RESULTS AND DISCUSSION ……………………………………………………………… 36 4.1.- Biology …………………………………………………………………...……………… 36 4.2.- Chemistry ……………………………………………………………….……………….. 38 4.3.- Physics …………………………………………………………………..……………….. 39 5.- CONCLUSIONS ……………………………………………………………………………… 40 • Bibliography ……………………………………………………………………………………………….. 41 ▫ Acknowledgements ……………………………………………………………………………..…………. 42 IV. GENERAL CONCLUSIONS …………………………………………………………..……… 43 ○ ABBREVIATIONS …………………………………………………………………………………….……… 45 ■ APPENDIXES_EXAMPLE 1 ……………………………………………………………………………….. 47 ▪ Protocols for working with tumor cells ………………………………………………………..…………………….. 47 ▪ Fluorescence images of the cytotoxicity essays ………………………………………………..……………………. 49 ▪ Fluorescence images of the SH-SY5Y tumor cell line ……………………………………………………………… 51 ■ APPENDIXES_EXAMPLE 2 …………………………………………………………….…………………. 52 ▪ Basic protocols for working with phages ……………………………………………………………………………. 52 ▪ Photos taken during the course of the project ………………………………………………………………………. 53 THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 5 ► GENERAL ABSTRACT This work, composed of two subprojects, has been developed within the framework of two independent research lines on the functionalization and application of nanoprobes to nanobiotechnology field, specifically in biomedicine or biotechnology. The multidisciplinary research proposal involves physicists, chemists, biochemists and medical doctors, and therefore a part of the student’s training work was performed at several centers abroad: The Hillman Cancer Center (UPCI), Pittsburgh, USA; the Centre de Recherche Paul Pascal (CRPP), Bordeaux and the Institut National de la Recherche Agronomique (BFP-IBVM), Villenave d’Ornon, France. Example 1. The Cancer Stem Cell (CSCs) concept says that only a small proportion of cells within the tumor have tumorigenic capacity. These cells are usually chemo and radio resistance that is why it is believed that are responsible for tumor relapse and recurrence of cancer patients. There is an urgent need to develop cancer therapies that are effective in killing these specific tumor cells. Magnetic nanoparticles (MNPs), functionalized with specific monoclonal antibodies, can be generated to recognize CSCs target molecules within the tumors. Hyperthermia is a new approach of using these MNPs positioned in an alternating magnetic field (AMF) to produce localized heat and kill CSCs. Magnetic nanoparticlesinduced hyperthermia is under investigation in different clinical trials on cancer patients. However, there is no information available on whether hyperthermia can eliminate CSCs. Therefore, the final goal of this project involve the application of hyperthermia to destroy CSCs and study the effect of this treatment in reducing tumor growth, increased cell death and improving the response to conventional therapies as chemo-radio therapy. More particularly, this work has focused on the first steps of this strategy related to the preparation of antibody-functionalized MNPs (MNPs:anti-CDs) and their application to target CSCs for several tumor cell lines by fluorescence microscopy. It has also been supplemented with cytotoxicity studies by MTT assays and surface and intracellular distribution studies by scanning electron microscopy (SEM) technique. Example 2. It is known that elongated viruses in concentrated solution self-organize into liquid-crystalline structures (mesophases) which exhibit different intermediate states between disordered liquids and perfectly organized crystals. Therefore, the introduction of specific functions by molecule grafting or nanoparticle bonding on the viral particles with a precise positional control, combined with the self-assembly properties of these functionalized THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 6 bioparticles, would give access to a wide range of applications in biosensing, memory devices, nanocircuits, light-harvesting systems, nanobatteries or MRI contrast agents. We used the mutant M13-C7C phage which presents a genetic mutation that allows the creation of unique binding sites on only one end of the filamentous virus through a pair of cysteine residues. The final goal of the project is to develop hybrid materials formed by the monitored assembly of these mutant M13-C7C phages with magnetic nanoparticles, with a 1:1 molar ratio, to induced highly sensitive nanorods by magnetic field, which are otherwise absent from the viral coat protein of the wild type phage. More specifically, this work has focused in the mass production, amplification and purification processes, of the mutant M13-C7C phage which is during its growth in competition with the wild type one. We have then specifically studied the functionalization of these mutant phages with maleimide fluorophores, followed by the self-organization properties in order to validate our scientific approach. • Keywords: Nanobiotechnology; Nanofunctionalization; Cancer stem cells (CSCs); Magnetic nanoparticles (MNPs); Monoclonal antibodies; Viral nanoparticles (VNPs); Liquid crystalline structures. THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 7 ► RESUMEN GENERAL Este trabajo, compuesto de dos subproyectos, se ha desarrollado en el marco de dos líneas de investigación independientes en la funcionalización y aplicación de nanosondas en el campo de la nanobiotecnología, específicamente en biomedicina o biotecnología. La propuesta de investigación multidisciplinar implica físicos, químicos, bioquímicos y médicos, y por lo tanto una parte del trabajo de formación del estudiante se realizó en varios centros en el extranjero: el Hillman Cancer Center (UPCI), Pittsburgh, USA; el Centre de Recherche Paul Pascal (CRPP), Bordeaux y el Institut National de la Recherche Agronomique (BFPIBVM), Villenave d’Ornon, Francia. Ejemplo 1. El concepto de célula Madre del Cáncer (CMC) dice que sólo una pequeña proporción de células dentro del tumor tienen capacidad tumorigénica. Estas células son normalmente quimio y radio resistentes, y es por lo que se cree que son responsables de la recaída y reaparición del tumor de pacientes con cáncer. Hay una necesidad urgente en desarrollar terapias contra el cáncer que sean efectivas en matar CMCs. Nanopartículas magnéticas (NPMs), funcionalizadas con anticuerpos monoclonales específicos, pueden ser generadas para reconocer moléculas diana de CMCs dentro de tumores. La Hipertermia Magnética (HTM) es un nuevo enfoque en utilizar las nanopartículas magnéticas (NPMs) posicionadas en un campo magnético alterno para producir calor localizado y matar las células tumorales. La hipertermia (HT) inducida mediante nanopartículas está bajo investigación en diferentes ensayos clínicos en pacientes con cáncer. Sin embargo no hay información disponible de sí la HT puede eliminar CMCs. Por lo tanto, el objetivo final de este proyecto implica la aplicación de la HT para destruir CMCs y estudiar el efecto de este tratamiento en reducir el crecimiento del tumor, aumentando la muerte celular y mejorando la respuesta a terapias convencionales como la quimio-radio terapia. En concreto, este trabajo se ha centrado en los primeros pasos de esta estrategia, relacionadas con la preparación de NPMs funcionalizadas con anticuerpos específicos (NPMs:anti-CDs) y su aplicación en el marcaje de CMCs para varias lineas celulares tumorales mediante microscopía de fluorescencia. También se ha complementado con estudios de citotoxicidad mediante ensayos de MTT y estudios de la distribución intracelular mediante la técnica de microscopía electrónica de barrido (MEB). Ejemplo 2. Es conocido que los virus elongados en soluciones concentradas se autoorganizan en estructuras de cristales líquidos las cuales exhiben diferentes estados THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 8 intermedios entre líquidos desordenados y cristales perfectamente organizados (mesofases). Por lo tanto, la introducción de funciones específicas mediante el injerto de moléculas o la adhesión de nanopartículas en las partículas virales con un control posicional preciso, combinadas con las propiedades de auto-ensamblado de estas biopartículas funcionalizadas, podría dar acceso a un amplio rango de aplicaciones en biosensores, dispositivos de memoria, nanocircuitos, sistemas acumuladores de luz, nanobaterías o agentes de contraste para MRI. Nosotros usamos el fago mutante M13-C7C el cual presenta una mutación genética que permite la creación de sitios de unión únicos exclusivamente en un extremo del fago, a través de un par de residuos de cisteína. El objetivo final del proyecto es desarrollar materiales híbridos formados por el ensamblado controlado de estos virus mutados M13-C7C con nanopartículas magnéticas (NPMs), con un relación molar 1:1, para inducir nanovarillas (nanorods) altamente sensibles mediante campos magnéticos, los cuales están por otra parte ausentes en la proteína de la cubierta viral de el fago de tipo silvestre. Más concretamente, este trabajo se ha centrado en los procesos de producción en masa, amplificación y purificación, del fago mutado M13-C7C el cual está durante su crecimiento en competición con el tipo salvaje. Después hemos estudiado específicamente la funcionalización de estos fagos mutantes con fluoróforos de maleimida, seguido de las propiedades de auto-organización para tratar de validar un enfoque científico. • Palabras clave: Nanobiotecnología; Nanofuncionalización; Células madre del cáncer (CMCs); Nanopartículas magnéticas (NPMs); Anticuerpos monoclonales; Nanopartículas virales (NPVs); Estructuras cristalinas líquidas. THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 9 I. GENERAL INTRODUCTION 1. Nanobiotechnology Nanobiotechnology (bionanotechnology or nanobiology), a mix between nanotechnology and biotechnology, is extensively interdisciplinary because it combines physical principles, chemical properties, and biological characteristics. The great promise of nanobiotechnology stems from our burgeoning ability to design and synthesize selfassembling biostructures with better functionality, sensitivity, and specificity than synthetic organic or inorganic molecules [1]. This intersection of research fields is based on the perception that nanotechnology can offer biology new tools with a depth of understanding, while biological principles guide nanotechnology to new forms of highly complex functional nanosystems. Biological systems can guide the physical world of electronics, materials science, computation, and manufacturing in the assembly of complex functional devices and systems operating at the molecular and even atomic level [see the FIG. 1] [2]. Nanomedicine is one of the fastest moving and most exciting research areas is the interface between nanotechnology, biology and medicine. This field presents many exciting possibilities for healthcare offering nanostructures as new therapeutic alternatives, which could revolutionize the areas of targeted/controlled drug delivery, disease detection, magnetic hyperthermia applied to cancer, biosensors or tissue engineering, and so on [3]. Another emerging and highly interdisciplinary field is Viral Nanotechnology, inhabiting the interface between virology, chemistry and materials science, in which viral nanoparticles (VNPs) are applied in diverse areas such as electronics, energy and next-generation medical devices [4,5]. Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 16 2. AIMS Several experiments designed to achieve the targeting of CSCs with MNPs, previously functionalized with anti-CDs, are reviewed in this work: - To determine the cytotoxicity of the Magnetic Nanoparticles (MNPs) on the tumor cell lines. Cytotoxicity studies will be carried out for each line by MTT essays, modifying the concentration of nanoparticles (NPs) and the times of incubation. - To study the surface and intracellular distribution of the NPs treated-tumor cells. It will carry out the incubation of the tumor cells lines with the MNPs, analyzing both the affinity between MNPs and tumor cells by Neubauer chamber counting and also the intracellular distribution of the cancer cells by Scanning Electron Microscopy (SEM). - To functionalize the MNPs with specific anti-CDs and CSCs targeting. The synthesis of the fully functional MNPs with highly specific markers, anti-CD44 and anti-CD133, and the characterization of the resulting complex (MNPs-anti_ABs) in terms of stability and functionality, with the targeting of CSCs, will be studied by fluorescence microscopies. 3. EXPERIMENTAL SECTION 3.1. MATERIAL [see the FIG. 3] ▪ Magnetic nanoparticles (MNPs). Monodisperse MNPs with a particle size of 40 nm, composed of a Fe 3 O 4 core and PEI shell, were used. ▪ Tumor cell lines. Four tumor cell lines have been used throughout the life of this project. On the one hand, SCCHN cell line PCI-13, breast cancer cell line MDA-MB-231 and pancreatic cancer cell line MIA PaCa-2 were used during the stay in the Hillman Cancer Center. These cell lines were either established at the University of Pittsburgh Cancer Institute. On the other hand, human neuroblastoma cell line SH-SY5Y, a cell line established at the Institute of Nanoscience of Aragón (INA), was used during the stay in the INA. ▪ Monoclonal antibodies (ABs or anti-CDs). It has worked with two monoclonal antibodies: ▫ CD44 (HI44a) – FITC Antibody, human. [Clone: HI44a (isotype: mouse IgG2a), purchased from MACS Miltenyi Biotec]. This CD44 antibody (anti-CD44), conjugated to Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 17 Fluorescein isothiocyanate (FITC), anti-CD44_FITC, recognizes an epitope of the CD44 antigen. ▫ CD133/1 (AC133) – PE antibody, human [Clone AC133 (isotype: mouse IgG1), purchased from MACS Miltenyi Biotec]. This CD133 antibody (anti-CD133), conjugated to R- phycoerythrin (PE), anti-CD133_PE, recognizes an epitope of the CD133 antigen. 3.2. PROTOCOLS ▫ Note: In the APPENDIX 1 are shown some protocols when working with tumor cells. 3.2.1. MTT protocol for cytotoxicity studies. DAY 1 – The tumor cells were seeded onto three 96-well plates (a plate for each essay time) at 10 6 cells mL -1 (10 5 cells per well). Each plate contained three-quarter parts of the wells with the three tumor lines, corresponding to a quarter for each one; and the last quarter of every plate without cells (only growth medium), used as a negative control. DAY 2 – 100 µL of MNPs medium at different concentrations were added to each one of the wells. Seven different concentrations, for each line of the plate, ranged from 1 to 200 µg MNPs mL -1 medium (1, 2, 5, 10, 50, 100 and 200 µg MNPs mL -1 medium), and the last line without MNPs used as a negative control (prepared in triplicate). These samples were cultured overnight at 37ºC. DAY 3, 4 and 5 – The following procedure was repeated with the pertinent plaque for each time. To perform the colorimetric test, 100 µL of medium of each well were removed gently, incubating the samples with 10 µL (5 mg mL -1 ) MTT at 37ºC for 3h. The resultant formazan crystals were dissolved in 100 µL of lysis buffer. The absorbance was measured by a microplate reader (ELISA) at 550 nm. An important aspect to take in account is the removal of any bubbles to avoid interferences in the measurements. 3.2.2. Protocol for studying the affinity between MNPs and tumor cell lines. DAY 1 – Three cell culture flasks for every tumor cell line containing 6.7 x 10 5 tumor cells each of them (1.34 x 10 5 cells mL -1 ) were prepared. Subsequently, 15 µL of MNPs (10.32 µg mL -1 ) were added into two of the three replicates. The last one was used like control. Then, all samples were incubated overnight at 37ºC. Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 18 DAY 2 – The harvesting and the counting of tumor cells lines were carried out, as are explained in the point protocols for working with tumor cells of the APPENDIX 1. Then, the samples were passed down a magnetic column (OctoMACS TM Separator, MACS Miltenyi Biotec), obtaining the respective flow thru of each sample. Subsequently, the magnetic columns were disengaged from the separator and several PBS volumes were passed down through to obtain the eluates. Finally, it was carried out the counting of the tumor cells, of both flow thru and elutes, in the same way than the last time. 3.2.3. Protocol for TEM/EDX sample preparation. DAY 1 – The SH-SY5Y cells were seeded onto a 24-well plate at 5 x 10 4 cells mL -1 (5 x 10 4 cells per well). A sterile cover slip had been added in each well used in the assay, previously. The tumor cells were cultured overnight at 37ºC. DAY 2 – Cells were washed twice with PBS and the MNPs media, at different concentration, were added into wells each. The first well was used as a negative control and the following to the well 6 contained 1.25, 2.5, 5, 25 and 25 µg MNPs mL -1 medium (two replicas). The tumor cells with and without MNPs were cultured overnight at 37ºC. DAY 3 – The growth medium of each well was removed, containing any cell that may have been peeled away and the MNPs that not have been incorporated or anchored into the tumoral cells. The samples were washed twice and afterwards fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer with a 3% sucrosa solution for 90 min at 37ºC. After the fixation, the samples were re-washed twice and the dehydratation process was conducted by incubating the cells at RT with increasing concentrations of methanol, through following steps in duplicate: Firstly, MeOH 30%, 50%, 70% for 5 min each one; secondly MeOH 100% for 10 min; and finally, MeOH 100% anhydride for 5 min. The prepared samples were preserved into anhydride methanol in the refrigerator at 4-8ºC, waiting for the next step. Finally, all cover slips were sputter-coated with gold, remaining as intact samples until they were analyzed by SEM (SEM INSPECT F50, FEI Company) and EDX. 3.2.4. Protocols for the studies both the interaction between MNPs:anti_CDs and the targeting of CSCs with these antibody-functionalized MNPs. Firstly, 10 µL of each anti-CD (or only one, depending of the sample) anti- CD44_FITC or/and anti-CD133_PE and different ranges of MNPs concentration were added into each eppendorf, containing 500 µL of TRIS buffer at pH:7.9. In addition, several Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 19 negative controls were also prepared, where the first one corresponded to the negative control with only buffer (without MNPs and anti-CDs), the second one only contained 10µL of each anti-CD and the last one contained exclusively MNPs. These samples were stirred by vortex and incubated for 1 hr at RT. Secondly, magnetic separation of the samples were carried out using a Magnetopure size big (Chemicell). To this end, each eppendorf was inserted into the magnetized wells and steeping for 30 min. Finally, the supernatants were isolated and analyzed by spectrofluorometry on the one hand, and the pellets by fluorescence microscopy on the other. 3.2.4.1. Protocol for spectrofluorometer sample preparation. The supernatants obtained were analyzed by spectrofluorometry, using a LS5S luminescence spectrometer (Perkin Elmer Life Sciences). The intrinsic fluorescence spectrum was monitored from 496–600 nm upon excitation at 496 nm in 0.5 cm path length plastic disposable cuvettes. In advance, optimization process was carried out with the best slit width and preparation of the standard curves for both anti-CDs and MNPs, or without MNPs. 3.2.4.2. Protocol for sample preparation for fluorescence microscopy. DAY 1 – SH-SY5Y cells were seeded onto a 24-well plate at 10 5 cells mL -1 (10 5 cells per well). A sterile cover slip had been added in each well used in the assay, previously. The tumor cells were cultured overnight at 37ºC. DAY 2 – Cells were washed twice with PBS and the culture mediums containing the pellets related to the essay in basic media (TRIS buffer at pH=7.9) were added in each well. The first two wells were used as a negative control, where the first one corresponded to the negative control without MNPs or anti-CDs, and the second one was the negative control only with 10 µL of each anti-CD. These samples were cultured overnight at 37ºC. DAY 3 – The growth medium of each well was removed, containing any cell that may have been peeled away and the conjugates that not have been incorporated or anchored into the tumoral cells. The samples were washed twice and afterwards fixed with 4 % PFA for 15 min at RT. After the fixation, by helping with tweezers and outside the laminar-flow, the cover slips were washed three times sequentially submerging into different beakers with the following sequence of fluids: PBS, PBS, Milli-Q. Then, the treated cover slips were fixed on slides. To that end, 5 µL of Mowiol-DAPI solution was added on a slide for Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 20 every sample and dropped carefully the cover onto the solution, trying to avoid formation of air bubbles between slide and cover. DAY 4 – Finally, each cover was fixed stronger on the cover slip with nail polish. The fluorescent samples must be kept in the dark to avoid any degradation. 4. RESULTS AND DISCUSSION 4.1. Cytotoxicity studies of the MNPs on the tumor cell lines by MTT essay The cytotoxicity produced by MNPs is an important obstacle that needs to be overcome for biomedical applications. Therefore, it is fundamental to determine how the MNPs are affecting to the viability of any type of cell inside the bloodstream. In this project was delimited the study for several tumor cell lines. For that, MTT cell viability assays were carried out to analyze the toxicity caused by the MNPs in several tumor lines (Mia PaCa-2, PCI-13 and MDA-MB-231) at different times; in this case, every 24 hours for three days (see the protocol in previous experimental section 3.2.1.). The results showed a decreasing trend for each cell viability bar, pertaining to each tumor cell line, when the MNPs concentration is increased from 10 µg mL -1 to 200 µg mL -1 [see the FIG. 4]. More specifically, there was not observed a loss of viability to 5 µg mL -1 , and from here there were a proportionately reduction to the higher concentration studied, with a Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 21 net loss of cell viability around the 20 percent. There seems not to exist an obvious loss of the viability of tumor cell lines over time, from day 1 to 3. Furthermore, these cancer cells seem to attach (or internalize, it is not clear) with the MNPs, as there seems shown in the fluorescence images taken during this cytotoxicity essay (see the photos in the Appendix 1). It was also observed the agglomeration of the MNPs, at high concentration, which is a problem to take into account. It should also be noted that if the results would have been positive, showing a toxic effect these MNPs for the tumour cell lines studied, the application of hyperthermia would not have been needed. Actually, it is fundamental to work with non-toxic MNPs because if they were capable to destroy very resistant cells, such as tumor cells, to kill the normal cells would be more very simple. 4.2. Study of the affinity between MNPs and tumor cell lines In order to get an idea regarding the affinity between the MNPs and the tumor cell lines, whether or not take place their uptake, a simple essay of cell counting was carried out by Neubauer chamber counting (explained in the experimental section 3.2.2.) [see the FIG. 5]. The results for every tumor cell line could be three replicas of the same essay because of the similarity. The flow-thru, which corresponds to the cells not attached on the magnetic column, only show positive results for the cells without MNPs. On the contrary, Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 22 exclusively the population of the cells with the MNPs has been obtained in the eluates, which correspond to the cells attached on the magnetic column. Thanks to this experiment, it has been proved that practically all cells attach or internalize the MNPs for each tumor line studied (PCI-13, MIA PaCa-2 and MDA-MB-231). 4.3. Study of the surface and intracellular distribution of the MNPs treated-tumor cells by TEM/EDX techniques We have already demonstrated that tumor cells interact with the MNPs, but it doesn’t know if there exists the internalization of them. Subsequently, a much more comprehensive study was carried out using two more powerful techniques such as Scanning Electron Microscopy (SEM), which provides high-resolution and long-depth-of-field images of the sample surface, and Energy Dispersive X-ray Spectroscopy (EDX), offering elemental identification of almost any element of the periodic table (explained in the experimental section 3.2.3.). Clear differences in the tumor cell surface were observed between the cultured tumor cells with and without MNPs [see the FIG. 7]. The blank sample, belonging to the control without MNPs, showed the normal morphology of these kind of cells, which is a large extension of the axon (that conducts electrical impulses away from the neuron's cell body), and the long and branched projections of the dendrites (that act to conduct the electrochemical stimulation received from other neural cells to the cell body). In contrast, in the case of the tumor cells cultured with MNPs, it was observed a reduced cell extension with much more Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 23 delimited cell morphology due to the presence of short dendrites even further branched. Furthermore, unlike the control, these samples exhibited a cell membrane filled with roughness. These membrane deformations seem to show that the tumor cells contain material inside, therefore, it is likely that the MNPs were also internalized apart from carried out the MNPs adhesion on the surface. These observations present the same trend with increasing concentration of MNPs from 5 to 25 µg mL -1 . Thanks to the second technique used, EDX, it was definitely corroborated that we have effectively iron material in the samples, corresponding to the MNPs. Clearly, it is observed an expanding red spot on the tumor cells with increasing concentration of MNPs [see the FIG. 7]. These figures give us also information about the distribution of the carbon (integral element of living systems) or the silice (the SiO2 is the major component of cover slip) in each sample. These samples were also analyzed by fluorescence microscopy obtaining good photos (see the fluorescence images of the SH-SY5Y tumor cell line in the APPENDIX 2). 4.4. Fluorescence microscopy studies of the interaction between MNPs:anti_CDs and the targeting of CSCs with these antibody-functionalized MNPs The goal of these essays was to optimize the conditions for achieving the maximum interaction between NPMs and anti-CDs, and thus reaching together the target (the CSCs) with the maximum guarantees. As already discussed, the labeling of MNPs with anti-CDs is a Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 24 crucial step for the selective and specific labeling of the CSCs within the tumor. Therefore, an experiment to study the interaction between MNPs and anti-CDs in different media, mimicking the behavior of these conjugates in hypothetical living conditions which would be found inside of blood stream, was designed. After doing several initial essays without satisfactory results, possibly because this functionalization method chosen, by electrostatic interactions, does not seem sufficiently consistent to take account in carrying out more complex essays such as when it works in PBS or cRPMI it was decided to simplify the experiment. In doing so, several essays working in basic conditions (TRIS buffer pH:7.9) were evaluated to study the affinity between MNPs:anti-CDs (explained in the experimental section 3.2.4.) [see the FIG. 8]. The functionalization process was carried out by electrostatic interactions [see the FIG. 3], where the carboxyl groups of the FcR of the anti-CDs were made interact with the free amine groups on the surface of the MNPs. Both anti-CDs, anti-CD44 and anti-CD133, have an isoelectric point of 7.3, therefore, the carboxyl groups present negative charge for this pH and for more basic values. For the case of the MNPs the isoelectric point (Zeta potential) is 9.2, so the closer the pH value are to 9.2, the more positive charges the MNPs have on the surface (reason because the TRIS buffer pH:7.9 was chosen). Moreover, thanks to the interaction between the anti-CDs and the MNP is realized in the FcR, the Fab region is free to interact with the CSCs markers (fundamental step). In principle, these electrostatic interactions achieved will remain at the physiologic pH, 7.3 (blood conditions), disappearing for lower values because of the protonation of the carboxyl groups (loss of the negative charges). Example 1: TARGETING CANCER STEM CELLS WITH ANTIBODY-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CARLOS PIÑOL DOMINGO Page 25 Once the magnetic separation was carried out, after the incubation process between the anti-CDs and the MNPs, two types of samples were collected: Supernatants and pellets. The observation of a loss of anti-CDs in the supernatants with the resultant presence of them in the pellets will corroborate the affinity between the MNPs and the anti-CDs. Besides, the incubation of these MNPs:anti-ABs_FLU with the tumor cells will allow to locate the CSCs inside the tumor sample. On the one hand, the supernatant samples, containing the anti-CDs_FLU unreacted free from the MNPs or MNPs:anti-ABs_FLU, were obtained. According to our estimates, the peak intensity for each of these sample (measured by the spectrofluorometer) should be lower gradually, meaning a less amount of anti-CDs in the supernatant since they will have reacted with the MNPs, for higher values of the MNPs concentration (added during the incubation). This is because a higher amount of MNPs permits an increased interaction surface for the anti- CDs. The hypothesis seems to be fulfilled in outcomes obtained in the graphic [represented in the FIG. 9]. Specifically, this is true for all the samples except for the sample which had been incubate with the highest MNP concentration. An adequate MNP concentration there might be, in this case around 50 µg mL -1 , producing the MNPs agglomeration for higher concentrations and avoiding the interaction between MNPs and anti-CDs_FLU. Besides, the affinity between MNPs and anti-CDs seems to change slightly for anti-CD44 or anti-CD133, because the agglomeration effect previously described is lower in the case of the first anti-CD. Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 32 These rod-like colloids can be handled easily in the laboratory and have long been used in molecular biology studies because they are amenable to genetic modification of virus capsid proteins. Besides, a very interesting characteristic of these VNPs is that non-reactive cysteine residues are exposed to exterior surface because evolution had disfavored interparticle disulfide bonds. Therefore, this presents a unique opportunity to genetically monitor the position of the cysteine residue on strategic locations of the virus and protein shells, after which the thiol group can be selectively targeted with thiol-selective probes [7]. The mutant M13-C7C bacteriophage [see the FIG. 2] is based on a simple M13 phage vector modified, in the end of the pIII protein, by pentavalent display of a loopconstrained heptapeptide. Each loop is flanked by a pair of cysteine residues, which could be oxidized during the phage functionalization to a disulfide linkage. In this case, the infectivity function of PIII is not affected because the displayed peptide is sufficiently short (<50 residues). Furthermore, the LacZ gene has been genetically inserted into the mutant phage vector, which is used as a characterization method to distinguish between the mutant M13- C7C and the M13 (wild-type) phages thanks to the mutant phage plaques appear blue when plate on media containing XGal and IPTG. An important concept in biology is the plaqueforming unit (PFU) which is a measure of the number of particles capable of forming plaques (colonies) per unit volume, such as virus particles. Unfortunately, there exists a counterproductive part resulting in a longer replication cycle of the mutant phage compared with the wild type. As a result, there is the possibility of in vitro selection for any contaminating wild-type phage during the amplification steps, even vanishingly small levels of contamination can result in a majority of the phage pool [8]. 1.2. Liquid crystalline organization ‘Soft’ matter, including polymers, colloids, amphiphiles, liquid crystals and a lot of biological systems, presents two fundamental features. The first one, the ordering is generally intermediate between that of a crystalline solid and that of a liquid, and the second one is the range of organization shown in the structures formed, typically in the ‘nanoscale’ ordering (1- 1000 nm). An important distinction between different types of soft material is the difference between direct and indirect ordering of molecules via supramolecular aggregates [9]. Liquid crystals are materials presenting at least one intermediate phase between isotropic liquid and crystalline solid phases. The calamitic liquid crystals are the most Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 33 common type of self-assembly for these structures, composed exclusively of anisotropic particles exhibiting a rod-shape. Liquid crystals composed of rod-like colloids exhibit different phase sequences with increasing concentration, behaving as lyotropic liquid crystals. The simplest liquid crystalline phase is the so called nematic (N), in which the molecules are positionally totally disordered, but arrange in a same preferential direction. The Smectic (Sm) phase shows a positional order since the centres of mass are arranged in layers (Sm layers) [see the FIG. 3] [10-12]. 1.3. Applications of the virus-based hybrid materials There exists an ample utilization of the phage display technology for material science applications. Liquid crystalline self-ordering systems with nanosized features of engineered viruses may provide new pathways to organize electronic, optical, and magnetic materials that would not be feasible via the traditional top-down fabrication techniques [3, 13]. For example, the wire-structures of viruses have inspired studies of their applications as conductive nanowires for electrical devices such as nanocircuits or nanobatteries, by mineralizing continuous metal layers on the virus surface, to obtain nanowires that exhibit crystallographic ordering [4]. Also, studies of the use of rod-like viruses as biological scaffolds for the construction of light-harvesting systems or magnetic systems are noteworthy both for their potential uses in solar cells, photocatalysts, and optical sensors or memory devices, respectively [see the FIG. 4 (left)] [5]. Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 34 More recently, nanometer-sized constructs, such as dendrimers, liposomes, nanoshells, nanotubes, nanoemulsions, quantum dots, and even viruses are being developed to increase detection sensitivity and efficacy for therapeutic applications as bioimaging agents intended as noninvasive probes (near infrared fluorescent dyes, magnetic contrast agents) or to target biomarkers of disease [7, 14]. For example, the crystalline phase of the VNPs could be adjusted to create enhanced suspension stability for magnetic resonance imaging (MRI) contrast agents [5]. Also, the VNPs could be an invaluable tool to improve detection, early and accurate biosensing, of small lumps of cancer cells. These “smart” nanoparticles were equipped with bright fluorescent dyes for imaging and a chemical tags to help it find and latch onto cancer cells to increase imaging sensitivity [see the FIG. 4 (right)] [15]. 2. AIMS The validation of a method to carry out the mass production of the mutant M13-C7C phages and the optimization process to functionalize with any maleimide probe are reviewed in this work: - Amplification, purification and characterization of the M13-C7C phages. The growth of viruses by M13-C7C-infected bacteria solution and analysis of the phages obtained, by titration process and transmission electron microscopy (TEM), will be carried out. - To functionalize phages with nanoprobes. The functionalization of these rod-like particles, with a previously disulfide reduction process, and labeling with maleimidefunctionalized fluorophores of the M13-C7C phage will performed. - Self-organization studies. Liquid crystal line phases will be studied by fluorescence and differential interference contrast microscopy (DIC), in order to validate our scientific approach, i.e. the regioselective functionalization of viral nanoparticles (VNPs). 3. EXPERIMENTAL SECTION ▫ Note: In the APPENDIX 2 are shown some protocols for working with phages. Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 35 3.1. Precautions. It is important to note the special precautionary measures that were taken into account in every step before and during the amplification process always to prevent the contamination of the mutant viruses with the undesired wild-type ones. Basically, the multiplication of the wild type is many times greater than the mutant viruses and consequently the slightest contamination would lead a guaranteed failure. Therefore, the first step was to disassemble and thoroughly clean the micropipettes with 70% EtOH, which were used exclusively in the inoculation processes. Besides, some considerations such as the thorough cleaning of the laminar hood with 70% EtOH every time was used, the use of virgin bacterial culture Petri dish without a previous bacteria extraction or the opening of a new box of aerosol-resistance tips for each new amplification, were strictly undertaken [8]. 3.2. M13-C7C amplification process. A volume of LB was inoculated with the mutant M13- C7C solution at a concentration of 10 11 pfu mL -1 (1mL/L for the infecting solution and 0.5 mL/L for the mass production). At the same time, a volume of E. coli ER2738 culture (10mL/L for both cases), previously grown overnight, were also added. The mix was shaken at 37ºC, 230 rpm for different incubation times (5 hrs for the infecting solution and 8 hrs. for the mass production) [8, 16]. 3.3. M13-C7C titration process. Firstly, 200 µL of serial dilutions of M13-C7C phage in LB were prepared into sterile tubes. After, 200 µL of E. coli ER2738 (OD 599 =0.5) were put into each one of the M13-C7C serial dilutions, vortex and incubate for 5 min at room temperature. Then, 3 mL of top agar at 56ºC were also added, vortex and the mix were poured on the top of the IPTG/XGal_LB-agar plates prepared previously. Finally, let cool down, cover with parafilm and grow overnight at 37ºC [8, 16]. 3.4. Functionalization process. 50 mL of the M13-C7C mass production (1 mg mL -1 ) in 20 mM Na 2 HPO 4 buffer pH 7.0 was reduced with 1.22 µM TCEP·HCl about 30 min at room temperature. Then, the solution was mixed with 3.05 µM of DyLight 550 Maleimide (1 mL of DyLight 550 Sulfhydryl-Reactive Dye purchased from the Thermo Scientific Company) dissolved in DMSO, 1:10 4 dilution to prevent virus damage, stirring gently it at 4ºC for 2 hrs. It must be taken into account that is needed 10 times more of reduction agent and dye per virus molecules, because there are 5 tails per M13-C7C molecule with 1 disulfide group in each tail. Besides, it has been used about 2 and 8 molar excess of reduction agent and dye, respectively [17-19]. Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 36 4. RESULTS AND DISCUSSION ▫ Note: In the APPENDIX 2 are shown some photos taken during the course of the project. 4.1. BIOLOGY Before starting the experiments, a 1:100 dilution of the M13-C7C bacteriophage (100 mL of Ph. D.-C7C [phage display Library], ~10 13 pfu mL -1 , purchased from the New England Biolabs Company) was prepared, because a 10 11 pfu mL -1 value is required for the amplification process. The first step was to prepare 50 mL of the infecting solution, corresponding to a first amplification, needed to carry out subsequently the M13-C7C mass production. It was mandatory to check the complete absence of wild type viruses (M13 phages) in the infecting solution before to proceed with the following steps. In the FIG. 5 are shown four filamentous phages with an estimated length of 1006 nm (accomplished by Alexis de la Cotte of the CRPP). Many impurities are observed in the picture due to the LB medium drying. An optimization process was now realized to optimize the steps of multiplication and purification needed for the mass production. Specifically, Optical Density (O.D.) measurements at different incubation times were taken to study the bacteria growth curve [see the FIG. 6]. Viruses need to infect bacteria to multiply. Therefore, virus amplification also occurred during bacterial amplification, corresponding to the exponential phase. The results indicate that the exponential phase before it reaches the plateau (stationary phase) is between 4 and 10 hrs. It should be noted that the probability of infecting the amplification solution Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 37 with the wild type viruses increases with incubation time, due to a higher number of mutations. After several amplifications essays, it was confirmed that a suitable amplification time is 8 hrs. Besides, the purification process was also maximized, adapting the centrifugation steps (times and speeds) [see the FIG. 7]. Once the process was optimized, the M13-C7C mass production was carried out to produce the M13-C7C in huge amounts. For do that, 10 independent experiments of amplification with a final volume of 2 L were made to obtain about 284 mg of the mutant M13-C7C phage, free from wild type (see the experimental section 3.2.). A purification process of the M13-C7C phages is needed to carry out after each amplification process. This process is composed of a depletion step and several centrifugation steps [see the FIG. 7], which aims is obtain the M13-C7C in high concentration diluted in dH 2 O and free of bacteria, LB and other impurities. These last two processes, the amplification and purification, are the most important steps in the work, consequently where more time has been invested, because a large-scale production of pure mutant M13-C7C viruses is necessary to form smectic phases of liquid crystals. Finally, a checking process by titration was carried out for each sample amplified (explained in the experimental section 3.3.) to corroborate that the amplification had been successful [see the FIG. 8]. Specifically, this characterization process allows to know both Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 38 whether or not the amplified culture is infected, by the wild type M13, and the PFU mL -1 value. The results achieved were satisfactory, even exceeding our expectations, obtaining considerable amounts of M13-C7C with only blue colonies in all plates. To confirm of absolutely way that the viruses correspond exclusively to mutant M13 an amplification of DNA is suggested [8]. But this process is only suitable when it works with analytical quantities. Therefore, the functionalization of the mutant M13-C7C bacteriophages with fluorescent probes and the study the liquid crystalline phases obtained will be done to confirm in a definitive way these biological results. 4.2. CHEMISTRY The second part, the chemistry, a functionalization method was proposed both to validate the amplification process, to verify this once and for all and also to validate this functionalization method. After the functionalization process [see the FIG. 9], composed of a first reduction step of thiol groups and another corresponding to the labeling with the 550 maleimide dye (explained in the experimental section 3.4.), two dialysis processes were carried out. Firstly, with dH 2 O to remove the excess of dyes and the second with 20 mM TRIS, pH 8.1, 20 mM NaCl, to change the pH for suitable working conditions with virus liquid crystals. It is noted that in this purification step, corresponding to dialysis step, did not work to remove the excess of dyes for both dH 2 O and TRIS buffer. This is because a pink solution, Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 39 corresponding to the dye color in solution, was not obtained after the first dialysis step. Despite that, the dialysis process is needed to remove protein remains and other impurities that are impossible to remove by centrifugation. Therefore, to overcome this obstacle and to remove the excess of dye, several ultracentrifugation steps were carried out. 4.3. PHYSICS An ultracentrifugation process was fundamental, in any case, to concentrate the viruses for the sample preparation. Working with liquid crystals, and study the smectic (Sm) phases, must be achieve concentrations around 90-140 mg mL -1 of virus. To do this, the sample preparation is a delicate and laborious process, because we need homogeneous sample which is highly concentrated. The iridescence phenomena, due to visible light diffraction of the smectic layers (having a periodicity of about 1 µM), were observed within the sample once a homogenous smectic phase is achieved [see Appendix, the Image 12]. Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 40 Self-organization studies. Three images of the smectic phases, using different observation conditions, in two different zones were taken by confocal microscopy [see the FIG. 10]. Clear lines are observed by differential interference contrast (DIC) microscopy. Each line corresponds to a layer of virus with a width of the size of the M13 virus (1.006 µm). Two different results are obtained when the fluorescence is measured. On the one hand, exciting at 550 nm our 550 DyLight emits red light, showing the functionalized tails colored. On the other hand, it is not observed the pattern of lines in the sample when the sample is excited at 488 nm. This confirms that the goal has been achieved, obtaining phages functionalized with fluorophores with a regioselective control. Besides, thanks to this functionalization step it could verify that the amplification process was a success. An important consideration, constituting the most direct application of our results, is that an optical technique more easily available in any lab, such as fluorescence microscopy, can be now used to study these self-organization structures. Therefore, it is not already necessary to use an advanced technique, such as DIC, to observe the Sm or lamellar phases whenever working with these fluorescence mutant M13-C7C viruses. 5. CONCLUSIONS A simple approach for the mass production of mutant M13-C7C viruses by amplification process, free of the wild type, is described. The main advantage of this M13- C7C amplification process is the large-scale production of the mutant phages exhibiting cysteine residues at one of its extremities. However, the special precautionary measures to be taken to avoid any contamination with the wild type viruses, before and during the amplification process, are strong. Herein lies the importance of this method validated, namely the neatness worked with over the life of this project. With regard to the biology part, the first challenge was to obtain the infecting solution, without the M13 viruses, and perform the optimization process. Secondly, the multiplication processes, with the corresponding purification steps, were carried out many times until a sufficient quantity of mutant viruses were reached. The results achieved were more than satisfactory obtaining considerable amounts of mutant M13-C7C viruses. To do Example 2: REGIOSELECTIVE CONTROL OF MOLECULAR NANOP ROBES ON FILAMENTOUS VIRUS PARTICLES AND SELF-ASSEMBLY OF THESE FUNCTIONALIZED ROD-LIKE NANOPARTICLES CARLOS PIÑOL DOMINGO page 41 this, a checking process by titration was carried out, obtaining exclusively blue colonies for each phage dilution on every plate. The chemistry section consisting in a method of functionalization was then realized, achieving the validation of this method as well as definitively the amplification process also. To do this, a first reduction step of thiol groups and another corresponding to the labeling with a maleimide dye, with the corresponding purification steps, were realized. Finally, related to the physics part, the self organization studies of these functionalized rod-like nanoparticles were carried out. To that end, the sample was prepared through a delicate process. Three images of the smectic phases, using different conditions, were taken by confocal microscopy. Bright lines, each corresponding to a layer of virus were observed by differential interference contrast (DIC) microscopy. Two different results, exciting at 550 nm or 488 nm, were obtained when the fluorescence was measured, showing the functionalized tails colored due to a pattern in the sample or non-pattern observed, respectively. This confirms that the aim was achieved, obtaining phages functionalized with fluorophores with a regioselectivity control. Over the years noticeable advances are being made in the fabrication of nanostructured novel materials from VNPs due to the unique capabilities of the viruses, such as high monodispersity, chemical specificity and the anisotropic character. We anticipate that our approach, using a liquid crystalline self-ordering system of engineered mutant viruses, may provide new pathways to organize electronic, optical, and magnetic materials among other potential applications. Therefore, our next challenge could be to develop hybrid materials formed by the monitored assembly of these mutant M13-C7C phages with magnetic nanoparticles, with a 1:1 molar ratio, to induced highly sensitive nanorods by magnetic field. Bibliography [1] J. Pokorski et al., Mol. Pharmaceutics. 8 (1), pp 29–43 (2011). [2] K. Koudelka et al., Current Opinion in Chemical Biology. 14, 810–817 (2011). [3] S-W. Lee, A. M. Belcher et al., Science. Vol 296 (2002). [4] C.M. Soto et al., Current Opinion in Biotechnology. 21, 426–438 (2010). THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 48 • Harvesting of tumor cell lines (standard protocol for adherent cells) Tumor cells, a type of adherent cells, are harvested when have reached a too much population density or it is interested in carrying out an essay. Firstly, it was removed and discarded the spent cell culture medium from each culture flask and proceeding with the wash step, without wasting any time to prevent cell drying. To this end, PBS (wash solution) was gently added on the opposite side of the attached cell layer of the vessel to avoid disturbing it, and rock the vessel back and forth several times (repeat twice in total). This wash step removes any traces of serum, calcium, and magnesium, apart of dead cells or other impurities, that they would inhibit the action of the dissociation reagent. Secondly, it was removed and discarded the wash solution from each culture vessel and was added the pre-warmed accutase (trypsin) into the tumor cells. This is a dissociation reagent to disengage the cells from the vessel. It was used enough reagent to cover the cell layer, gently rocking the container to get complete coverage of the cell layer. It now was incubate the culture vessel at 37º for 3-7 min (the actual incubation time varies with the cell line used) to facilitate cell separation. In difficult cases, it can tap the vessel to expedite cell detachment. Thirdly, once cells were been detached, it was added the equivalent of twice the volume used for the dissociation reagent of pre-warmed complete growth medium to neutralize the trypsin effect. It was dispersed the medium by pipetting over the cell layer surface several times. Then, tumor cells were transferred to a conical tube and were centrifuged, at 200 × g during a few minutes (about 7 min), for removing pollutants. Finally, the cell pellet was resuspended in a minimal volume, depending on the estimated cell density, of pre-warmed cRPMI for cell counting. • Counting of tumor cells It is essential to know the tumor cell concentration for each sample before carrying out an experiment, to work with the appropriate conditions and obtaining reproducible essay. Therefore, 100 µL of tumor cell sample are removed and mixed 1:1 with 4% trypan blue solution, a diazo dye, which distinguishes between dead and living cells. Viable cells, with intact cell membranes, exclude trypan blue while dead cells stain blue due to trypan blue uptake. Then, the total number of cells is calculated using a hemacytometer. This is a counting chamber which determines the cell concentration in a liquid sample. THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 49 THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 50 FLUORESCENCE IMAGES OF THE CYTOTOXICITY ESSAYS. 1) Blank (only MNPs): 1.a) 0 µg mL -1 , 1.b) 100 µg mL -1 . 2) MNPs_MDA-MB-213_12h: 2.a) 0 µg mL -1 , 2.b) 5 µg mL -1 , 2.c) 5 µg mL -1 , 2.d) 100 µg mL -1 . 3) MNPs_MIA PaCa-2_36h: 3.a) 0 µg mL -1 , 3.b) 50 µg mL -1 , 3.c) 100 µg mL -1 , 3.d) 200 µg mL -1 . 4) MNPs_PCI-13_60h: 4.a) 0 µg mL -1 , 4.b) 50 µg mL -1 , 4.c) 100 µg mL -1 , 4.d) 200 µg mL -1 . Images taken by the inverted fluorescence microscope AXIOVERT 40 CEL in the image service of the Hillman Cancer Center (Pittsburgh, USA). THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 51 ▪ FLUORESCENCE IMAGES OF THE SH-SY5Y TUMOR CELL LINE. A) Study of the interaction between MNPs:anti_CDs in TRIS buffer, pH:7.9. SUPERNATANTS: cells + 5 µL of each anti CDs + MNPs. A.1) 5 µg mL -1 MNPs: A.1.a) Without filter, A.1.b) Green filter, A.1.c) Red filter. A.2) 25 µg mL -1 MNPs: A.2.a) Without filter, A.2.b) Green filter, A.2.c) Red filter. A.3) 100 µg mL -1 MNPs: A.3.a) Without filter, A.3.b) Green filter, A.3.c) Red filter. B) Preparation samples for SEM. B.1) BLANK1 (only cells): B.1.a) cells, 20X, B.1.b) cells, 40X. B.2) BLANK2 (cells + MNPs): B.2.a) 1 µg mL -1 MNPs, B.2.b) 5 µg mL -1 MNPs, B.2.c) 25 µg mL -1 MNPs, B.2.d) 100 µg mL -1 MNPs. Images taken by the inverted fluorescence microscope NIKON ECLIPSE TE2000-S in the image service of the INA. THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 52 ■ APPENDIX 2 ▪ BASIC PROTOCOLS FOR WORKING WITH PHAGES • Culture media - LB-Agar plates: To prepare 5 plates: 100 mL of pre-warmed LB-Agar (5 plates per 100 mL), warming up in the microwave, is poured in every plate, filling 1/3 of the plate, and wait to solidification. Store the plates upside down wrapped with parafilm, to prevent them from drying out, and store at 4ºC. - IPTG/XGal_LB-agar plates. To prepare 25 plates: 0.1 mM of IPTG and XGal (corresponds to 100 µL/200 µL of 0.1 M IPTG/0.05 M XGal stock solution, respectively) were added into 100 mL of LB-Agar (8 plates per 100 mL), previously dissolved and cooled to 65ºC or less. As quickly as possible after their preparation, the solution is poured into the plates, until cover the surface completely, and wait to solidification. Store the plates upside down inside the bag at 4ºC. • Culture bacteria - Preparation of plating bacteria: To prepare 3 E.coli_LB-Agar plates: Streak out the master stock culture of E. coli K12 ER2738 onto each LB-Agar plate, helped by a sterile stick. Then, the plates were inverted and incubated at 37ºC overnight. Store for a maximum of 2 weeks, wrapped with parafilm at 4ºC in the dark. - Preparation of bacteria solution: To prepare 100 mL of E.coli_LB solution: Firstly, 100 mL of LB were inoculated, in a 100 mL sterile erlenmeyer, with a single well-isolated bacterial colony, picked from the minimal agar plate previously prepared. Then, 133 µL of Tetracycline (7.5 mg mL -1 ) were added to the solution. Tetracycline is a broad-spectrum polyketide antibiotic (explain). Finally, the liquid culture was agitated, in a rotatory shaker, overnight at 37ºC and 180 rpm. ▫ Note: It must work in the amplification process with values of OD around 0.5 for the bacteria solution, because this corresponds at the beginning of the exponential phase. THE USE OF FUNCTIONALIZATION APPLIED TO NANOBIOTECHNOLOGY: Targeting cancer stem cells and regioselective functionalization of phages CARLOS PIÑOL DOMINGO Page 53 ▪ PHOTOS TAKEN DURING THE COURSE OF THE PROJECT . 1) PREPARATION OF PLATING BACTERIA: E.coli K12 ER 2738 grown in LB medium. 2) PREPARATION OF SOLUTION BACTERIA: Turbid solution after overnight incubation. 3) AMPLIFICATION PROCESS: Incubation process. 4) PURIFICATION PROCESS: Step 1_Elimination bacteria-Plastic bottles with the bacteria pellets after the centrifugation process. 5) PURIFICATION PROCESS: Steps 2-3_Comparation between both samples: after the depletion process (turbid solution) and after the step3-erlemeyer containing the supernatant removed (clear solution). 6) TITRATION PROCESS: Preparing the phage dilutions. 7) TITRATION PROCESS: Let cool down the IPTG/XGal-LB agar plates with the top agar-dilutions samples. 8) SAMPLE PREPARATION FOR SEM: M13-C7C sample and washing solutions. 9) FLUORESCENCE LABELLING PROCESS: Removing the excess of dye by centrifugation process. 10) SAMPLE PREPARATION FOR LIQUID CRYSTALS STUDIES: Melting the ends of capillary to seal hermetically. 11) SAMPLE PREPARATION FOR LIQUID CRYSTALS STUDIES: Sealing the sides of sample with UV polymer glue . 12) SAMPLE PREPARATION FOR LIQUID CRYSTALS STUDIES: Iridescence phenomena observed within the sample with Sm phase organization. All Photos were taken at the Centre de Recherche Paul Pascal (CRPP-CNRS), Bordeaux, France.