Proteomska karakterizacija nanodelcev za ovrednotenje njihove citotoksičnosti in imunotoksičnosti ter razvoj novih vrst nanodelcev

Strojan, Klemen (avtor), Pavlin, Mojca (mentor), Bregar, Vladimir B. (komentor)

Nanodelci so delci zelo majhnih, nanometrskih dimenzij. Človek je lahko izpostavljen nanodelcem preko hrane, kozmetike in tekstila. Možna je tudi izpostavitev ob zdravljenju nekaterih specifičnih bolezni. Mehanizmi delovanja nanodelcev v organizmu še niso povsem jasni. Preden nanodelci pridejo v stik s celicami oziroma tkivi organizma so izpostavljeni medijem, ki vsebujejo makromolekule (proteine, lipide, nukleinske kisline). Odvisno od lastnosti makromolekul, nanodelcev in medija, med makromolekulami in površino nanodelcev potečejo interakcije, ki vodijo do nastanka t.i. biokorone. Biokorona je plast makromolekul, ki tvorijo interakcije s površino nanodelca, kadar pa govorimo o interakcijah med proteini in površino nanodelca, govorimo o proteinski koroni. Količine in tipe proteinov, ki so vezani na površino nanodelca, določajo fizikalno-kemijske lastnosti nanodelcev, lastnosti proteinov in lastnosti okoliškega medija. Osredotočili smo se na dva tipa magnetnih nanodelcev, relevantnih za biomedicinske aplikacije, ter na industrijsko proizvedene nanodelce, ki so prisotni v produktih za vsakdanjo rabo. V prvem delu disertacije smo pokazali, da je za interakcije med proteini in nanodelci bistvena predpriprava nanodelcev. Silicijeve nanodelce in kobalt feritne nanodelce, oplaščene s poliakrilno kislino, smo pred izpostavitvijo proteinom redčili v štirih različnih medijih ter dokazali, da izbira medija za predpripravo nanodelcev pomembno določa kvalitativno in kvantitativno sestavo proteinske korone. Nadalje smo preverili, če je spremenjena proteinska korona povezana z nastankom citokinov IL-6 in TNF-α v celični liniji THP-1. Povezave med spremenjeno proteinsko korono in izražanjem citokinov nismo dokazali, vendar pa lahko nanodelci vplivajo tudi na druge dele imunskega sistema. Citotoksičnost kobalt feritnih nanodelcev oplaščenih s polietileniminom smo želeli zmanjšati z dodatnim oplaščenjem z glutationom. Na celični liniji CHO smo pokazali, da lahko z glutationskim oplaščenjem dosežemo najmanj 30% višjo viabilnost v primerjavi z neoplaščeno formulacijo in zmanjšanje nastanka reaktivnih kisikovih zvrsti. Ključen korak je bil dodatek glutationa ob sintezi, saj z dodatkom prostega glutationa v celični medij ob hkratni izpostavitvi nanodelcem nismo dosegli želenega učinka. Primerjali smo tudi pot internalizacije neoplaščene in oplaščene formulacije, kjer nismo opazili razlik. Nanodelce smo našli izključno v znotrajceličnih veziklih, kar je v nasprotju s hipotezo lizosomalnega pobega. Dokazali smo, da so nanodelci kljub dodatnemu oplaščenju še vedno uporabni za transfekcijo, ki je glavna aplikacija omenjenih nanodelcev. Na celičnih modelih zdravega in rakastega urotelija smo dokazali selektivno endocitozo kobalt feritnih nanodelcev, oplaščenih s poliakrilno kislino, v rakaste celice urotelija. Na podlagi predpostavke o selektivni endocitozi in razumevanja citotoksičnosti kobalt feritnih nanodelcev oplaščenih s polietileniminom smo preverili potencialno obliko zdravljenja urotelijske papilarne neoplazme. Z dodatnim oplaščenjem citotoksičnih nanodelcev z govejim serumskim albuminom smo želeli zmanjšati poškodbe celične membrane, a hkrati ohraniti toksičnost, ki se izrazi po internalizaciji. Na tak način bi, ob predpostavki selektivne endocitoze, dosegli selektivno citotoksičnost proti rakastim celicam. Kratkotrajni učinek (merjenje viabilnosti po treh urah) je delno potrdil našo hipotezo, medtem ko dolgotrajni učinek (merjenje viabilnosti po štiriindvajsetih urah) ni bil v skladu s pričakovanimi rezultati. V predzadnjem delu disertacije je predstavljena kvantitativna analiza objavljene literature, ki združuje podatke o fizikalno-kemijskih lastnostih nanodelcev, sestavi proteinske korone in učinke na imunski sistem in vitro. Prva ugotovitev analize je bila, da je študij, ki združujejo vse tri kategorije podatkov, izredno malo, razlikovali so se tudi pogoji pod katerimi so bili posamezni eksperimenti izvedeni. Na podlagi 21 študij, ki so ustrezale našim kriterijem, smo naredili kvantitativno analizo. Primerjali smo dve skupini nanodelcev in sicer tiste, ki so sprožili povečan nastanek citokinov, proti ostalim. Pomembne razlike so bile med tipi nanodelcev v obeh skupinah, medtem ko razlik v hidrodinamskem premeru in zeta potencialu med skupinama nismo odkrili. Na podlagi našega vzorca smo sklenili, da sta sestava proteinske korone in sproščanje citokinov neodvisni spremenljivki. Ob koncu je predstavljena študija, s katero smo povezanost med lastnostmi nanodelcev, sestavo proteinske korone in sproščanjem citokinov preverili z lastnimi eksperimenti. Kljub temu, da smo lahko bistveno bolje kontrolirali pogoje posameznih meritev, nismo mogli podati trdnih zaključkov.

• Jezik: Slovenski jezik
• Ključne besede: nanodelci, proteinska korona, imunotoksičnost
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: FE - Fakulteta za elektrotehniko
• Leto izida: 2018
• PID: 20.500.12556/RUL-99599
• COBISS.SI-ID: 11948372
• Datum objave v RUL: 07.02.2018

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Proteomic characterization of nanoparticles for evaluation of their cytotoxicity, immunotoxicity and development of new types of nanoparticles

Izvleček

Nanoparticles are particles of small, nano metric dimensions. Humans are exposed to nanoparticles through food, cosmetics and textile. Moreover, nanoparticles are also increasingly used in healthcare, so there is a possibility of exposure when treating specific diseases. Still, the mechanisms of their action in humans are not clear. Once in the organism, nanoparticles are immediately exposed to physiological fluids containing macromolecules. Based on nanoparticle, macromolecule and medium properties interactions between nanoparticle surface and macromolecules lead to the formation of biocorona. When describing only interactions between nanoparticle surface and proteins this layer of macromolecules, i.e. proteins, is called protein corona. Physicochemical properties of nanoparticles, properties of proteins and properties of surrounding media determine types and quantities of protein bound to nanoparticle surface. We have focused on two types of magnetic nanoparticles relevant for biomedical applications and different industrially engineered nanoparticles present in consumer products. In the first part of this dissertation we have shown that the selection of media for nanoparticle dilution is a crucial parameter of protein corona composition. Silica nanoparticles and polyacrylic acid coated cobalt ferrite nanoparticles were diluted in four different media prior to corona formation. We have shown that selection of media determined qualitative and quantitative composition of protein corona. Additionally, we tested if differences in corona composition influenced secretion of cytokines IL-6 and TNF-α in the cell line THP-1. There was no connection between differences in protein corona composition and cytokine secretion, but nanoparticles could also affect other parts of immune system. Next, we tried to reduce cytotoxicity of cobalt ferrite nanoparticles coated with polyethyleneimine by additional coating with glutathione. Using CHO cell line we have shown that additional glutathione coating results in 30% higher viability compared to non-coated formulation and reduction of reactive oxygen species formation. The key was to add glutathione during nanoparticle synthesis; when added directly to the cell culture media together with nanoparticles, it did not induce the same effect. There were no differences in internalization pathways between nanoparticles. Furthermore, nanoparticles were found exclusively in intracellular vesicles, which is not in accordance with lysosomal escape hypothesis. Moreover, the additional layer of glutathione did not reduce applicability of nanoparticles as a transfection vector. Cell models of healthy and cancerous urothelium were used to prove selective endocytosis of cobalt ferrite nanoparticles coated with polyacrylic acid into cancerous urothelial cells. Based on this finding and our understanding of cytotoxicity of polyethyleneimine coated nanoparticles, we tested potential bladder papillary neoplasm treatment. With additional layer of bovine serum albumin we tried to reduce direct membrane damage while retaining intracellular toxicity. In this way we would have achieved, based on selective endocytosis, selective cytotoxicity towards cancerous cells. We proved the short-term effect (viability after three hours) but were unable to demonstrate long-term effect (viability after twenty-four hours). In the next part of this dissertation we are presenting quantitative analysis of published literature, which combines data on physicochemical properties of nanoparticles, composition of protein corona and in vitro immune system effects. Our first conclusion was that the studies combining all three topics are relatively scarce; there were also differences in experimental protocols between the studies. Nonetheless, we performed a quantitative analysis of results collected from the 21 studies that matched our selection criteria. We compared nanoparticles that triggered cytokine secretion with those that did not. We found no differences between hydrodynamic diameter and zeta potential of the two groups, but showed connection between nanoparticle type and cytokine secretion. Based on our analysis we concluded that cytokine secretion and corona composition were independent variables. In the last part of dissertation we are showing results of our own study, which was designed to analyse connections between nanoparticle characteristics, protein corona composition and cytokine secretion. Despite the fact that we had exactly the same experimental conditions for all experiments, we have not come to a firm conclusion.

• Ključne besede: nanoparticles, protein corona, immunotoxicity