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Bakteriofágy V Nanotechnologiích

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Bakteriofágy V Nanotechnologiích PŘÍRODOVĚDECKÁ FAKULTA Bakteriofágy v nanotechnologiích Bakalářská práce MARTIN STAROSTKA Vedoucí práce: Mgr. Ivana Mašlaňová, Ph.D. Ústav experimentální biologie obor Speciální biologie Brno 2020 BAKTERIOFÁGY V NANOTECHNOLOGIÍCH Bibliografický záznam Autor: Martin Starostka Přírodovědecká fakulta Masarykova univerzita Ústav experimentální biologie Název práce: Bakteriofágy v nanotechnologiích Studijní program: Experimentální biologie Studijní obor: Speciální biologie Vedoucí práce: Mgr. Ivana Mašlaňová, Ph.D. Rok: 2020 Počet stran: 82 Klíčová slova: nanotechnologie, nanomedicína, bakteriofágy, fágový displej, biosenzory, nanovlákna, fágové vakcíny, nanopóry BAKTERIOFÁGY V NANOTECHNOLOGIÍCH Bibliographic record Author: Martin Starostka Faculty of Science Masaryk University Department of Experimental Biology Title of Thesis: Bacteriophages in nanotechnology Degree Programme: Experimental Biology Field of Study: Special Biology Supervisor: Mgr. Ivana Mašlaňová, Ph.D. Year: 2020 Number of Pages: 82 Keywords: nanotechnology, nanomedicine, bacteriophage, phage display, biosensors, nanowires, phage vaccines, nanopores BAKTERIOFÁGY V NANOTECHNOLOGIÍCH Abstrakt Bakteriofágy jsou nejvíce abundantní entitou na této planetě. Široké spektrum jejich vlastností a morfologie spolu s jednoduchými možnostmi genetické modifikace poskytuje účinný nástroj pro celou řadu aplikací napříč průmyslem. Tato práce je zaměřena na využití fágů v relativně novém, rychle se rozvíjejícím vědním oboru, nanotechnologii. Selekční nástroj genového inženýrství, metoda fágového displeje, poskytuje jednoduchou možnost modifikace fágového povrchu. Identifikace a selekce peptidů s afinitou k organickým i anorganickým substrátům umožňuje vznik materiálů na bázi proteinů. Fágová schopnost samosestavení propůjčená těmto materiálům z nich činí ideální nástroj pro nanotechnologickou aplikaci, zejména pak v nanomedicíně. BAKTERIOFÁGY V NANOTECHNOLOGIÍCH Abstract Bacteriophages are the most abundant entity on this planet. The wide spectrum of their properties and morphology together with the ease of their genetic manipulation makes them an excellent tool for application throughout the industry. This thesis takes a closer look at the use of phages at the relatively new, rapidly developing field of nanotechnology. The selection instrument of genetic engineering, the phage display method, provides a simple way to modify the phage surface. Identification and selection of peptides with the affinity to organic and inorganic substrates allows the development of protein-based materials. The self-assembly property of phages makes these materials even more interesting for the application in nanotechnology, especially in nanomedicine. BAKTERIOFÁGY V NANOTECHNOLOGIÍCH Čestné prohlášení Prohlašuji, že jsem svoji bakalářskou práci vypracoval samostatně pod vedením vedoucího práce s využitím informačních zdrojů, které jsou v práci citovány. V Brně 13. března 2020 ....................................... Martin Starostka BAKTERIOFÁGY V NANOTECHNOLOGIÍCH Poděkování Na tomto místě bych rád poděkoval své školitelce Mgr. Ivaně Mašlaňové, Ph.D. a svému konzultantovi doc. Ing. Romanu Grögerovi, Ph.D. za odborné vedení, cenné rady, připomínky a trpělivost při vypracování této práce. Šablona DP 3.0.6-SCI (2019-12-05) © 2014, 2016, 2018, 2019 Masarykova univerzita OBSAH Obsah Seznam obrázků 15 Seznam pojmů a zkratek 16 1 Úvod 19 2 Nanotechnologie 21 2.1 Historie a vývoj .................................................................................................................. 21 2.2 Nanomedicína..................................................................................................................... 22 2.3 Nanotechnologie pro separaci mikročástic ............................................................ 27 3 Bakteriální viry 29 3.1 Charakteristika .................................................................................................................. 29 3.2 Morfologie ............................................................................................................................ 29 3.3 Životní cyklus...................................................................................................................... 34 3.4 Terapeutické využití bakteriofágů ............................................................................. 36 4 Fágová nanotechnologie 41 4.1 Biosenzory ........................................................................................................................... 42 4.2 Vláknité fágy jako základ nanovláken a jiných nanomateriálů ....................... 47 4.3 Fágy pro přenos genů a vakcín .................................................................................... 55 4.4 Nanopóry .............................................................................................................................. 62 5 Závěr 65 Použité zdroje 66 13 SEZNAM OBRÁZKŮ Seznam obrázků Obr. 1: Schématické zobrazení kvantových teček .............................................. 23 Obr. 2: Grafické znázornění modelového bakteriofága T4 .............................. 30 Obr. 3: Rekonstrukce snímku z kryoelektronové mikroskopie ................ 31 Obr. 4: Životní cyklus bakteriofága ........................................................................ 36 Obr. 5: Schématické zobrazení metody fágového displeje .............................. 41 Obr. 6: Upevnění bakteriofága T4 ke zlatému povrchu .................................... 45 Obr. 7: Různé typy nanostruktur vzniklých po fágové modifikaci ................ 49 Obr. 8: Fágem řízená syntéza nanovlákna ............................................................ 50 Obr. 9: Schématické zobrazení modifikovaného fága cíleného na denaturovaný kolagen ................................................................................................. 60 15 SEZNAM POJMŮ A ZKRATEK Seznam pojmů a zkratek AgNPs – Silver nanoparticles; stříbrné nanočástice ASCs – Adult stem cells; kmenové buňky ATP – Adenosine triphosphage; adenosintrifosfát AuNPs – Golden nanoparticles; zlaté nanočástice BIND – Bactrial ice nucleation diagnostic; bakteriální diagnostika ledovou nukleací BREX – Bacteriophage exclusion; bakteriofágové vyloučení Cas – CRISPR-associated protein; proteiny asociované s CRISPR systémem CRISPR – Clustered regularly interspaced short palindromic repeats; krátké palindromové repetice přerušované mezerníky DISARM – Defence island system associated with restriction-modification; obranný systém asociovaný s restrikčně-modifikačním systémem EGFR – Epidermal growth factor receptor; receptor epidermálního růstového faktoru ELISA – Enzyme-linked imunosorbent assay G- – Gram-negative bacteria; gram-negativní bakterie G+ – Gram-positive bacteria; gram-pozitivní bakterie GC IONPs – Iron oxide nanoparticles coated with glycol-chitosan; nanočástice oxidu železa kryté glykol-chitosanem GC – Glycol-chitosan; glykol-chitosan Hoc – Higly immunogenic outer capsid protein; vysoce imunogenní vnější kapsidový protein IONPs – Iron oxide nanoparticles; nanočástice oxidu železa Li-ion – Lithium-ion battery; lithium-iontová baterie 16 SEZNAM POJMŮ A ZKRATEK MALDI TOF MS – Matrix assisted laser desorption/ionization time of flight mass spektrometry; hmotnostní spektrometrie za použití ionizace laserem v přítomnosti matrice s detektorem doby letu PA – protective antigen; protektivní antigen PCR – Polymerase chain reaction; polymerázová řetězová reakce PEDOT – poly (3,4-ethylendioxythiofen) QCM – Quartz crystal microbalance; křemen-krystalová mikrobalance QD – Quantum dots; kvantové tečky SEPTIC – Sensing of phage-triggered ion sensing; snímání fágem indukované iontové kaskády Soc – Small outer capsid protein; malý vnější kapsidový protein SPR – Surface plasmon resonance; povrchová plasmonová rezonance VLP – Virus-like particle; viru podobná částice 17 ÚVOD 1 Úvod Nanotechnologie je technický obor studující vlastnosti hmoty na úrovni nanometrů. Tato dimenze poskytuje materiálům naprosto odlišné vlastnosti od těch, které denně pozorujeme v makrosvětě. Vlastnosti nanomateriálů jsou ovlivňovány převážně kvantově-mechanickými jevy. Cílem odvětví je aplikace těchto poznatků pro vývoj nástrojů schopných posunout hranice našich technologických možností stejně jako usnadnit náš každodenní život. K velkému rozvoji nanotechnologie pomohl Richard Faynmen, který v roce 1959 přednesl svou přednášku na téma: „There is plenty of room at the bottom: an invitation to enter a new field of physics“ na sjezdu Americké fyzikální společnosti. Zlatá doba nastala v 80. letech minulého století s objevem fullerenů a uhlíkových nanotrubek. Od té doby hrají nanotechnologie důležitou roli ve vědě každého státu, např. v USA roku 2003 byl pokrok v nanotechnologiích uzákoněn jakožto národní priorita. V současné době jsou nanotechnologie využívány nejvíce v elektronice, strojírenství a chemickém průmyslu. Pro tuto práci je však klíčovou oblastí nanomedicína. Nanomedicína je rychle se rozvíjející obor medicíny, který se snaží aplikovat poznatky a technologické postupy nanotechnologií k léčbě a diagnostice nemocí, se kterými si současné postupy neumí poradit. Tato práce je zaměřena na teoretické využití bakteriofágů, virů infikujících bakterie, ve spojení s postupy nanotechnologického odvětví. Fágy lze aplikovat nejen samostatně, ale také v kombinaci s organickými a anorganickými materiály, hlavně pak s nanomateriály. Možné uplatnění fágové nanotechnologie lze nalézt právě v nanomedicíně. Již od dob svého prvního objevu hrály bakteriofágy
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