Uloga dva nanokompozita fulerenol/doksorubicin i fulerenol/gvožđe kod toksičnih efekata izazvanih doksorubicinom

Abstract

Tumori predstavljaju drugi vodeći uzrok smrti u svetu (posle kardiovaskularnih bolesti) i procena je da će učestalost pojave tumora u budućnosti porasti. Hemioterapija je jedna od najčešće korišćenih metoda lečenja tumora koju često prate sporedni sistemski štetni efekti. S obzirom da lečenje tumora jednim terapeutskim agensom često rezultuje ograničenim kliničkim ishodom, kombinovane terapije mogu sinergistički podići nivo antitumorske aktivnosti, dok sniženjem doze svakog pojedinačnog agensa se mogu umanjiti štetni efekti. Istovremena aplikacija različitih terapeutskih agenasa zahteva dostavnu platformu kako bi se omogućila normalizacija farmakokinetike i farmakodinamike agenasa. Fulerenol C60(OH)24 predstavljaju pogodnu platformu za dostavu lekova zbog svojih fizičko-hemijskih i bioloških karakteristika. U vodenom rastvoru fulerenol se nalazi u obliku negativno naelektrisanih polianjonskih nanočestica koje poseduju veliku površinu i imaju mogućnost vezivanja pozitivno naelektrisanih hemioterapeutika kao što je doksorubicin (DOX), kao i pozitivnih jona metala, u ovom slučaju Fe2+. Osnovna biološka karakteristika fulerenola je da ima ulogu antioksidativnog agensa. U ovom radu iskorišćene su fizičkohemijske osobine fulerenolskih nanočestica (FNP), DOX-a i Fe2+ u cilju formiranja stabilnih nanokompozita FNP/DOX i FNP/Fe2+. Nanokompoziti su okarakterisani različitim fizičkohemijskim metodama, pri čemu je ustanovljeno da su čestice nanokompozita FNP/DOX veličine između 20 i 58 nm, naelektrisanja od -6 mV, čestice nanokompozita FNP/Fe2+ veličine između 11 i 60 nm, naelektrisanja od -30.8 mV, i diskoidalne morfologije. Oba nanokompozita imaju dobru stabilnost u vodenom rastvoru, što ih čini biokompatibilnim i pogodnim za primenu u biomedicini. FNP dekorisan DOX-om, u obliku nanokompozita FNP/DOX, omogućava značajno obimniji unos DOXa u ćelije i smanjuje procenat ćelija u G2M fazi ćelijskog ciklusa, u odnosu na samostalnu primenu DOX-a, kod malignih ćelijskih linija dojke. DOX se u terapiji tumora koristi poslednjih četrdeset godina i još uvek predstavlja nezaobilazan lek u onkologiji. Njegovo dugotrajno korišćenje izaziva oksidativni stres, što za posledicu ima pojavu kardiomiopatije i hepatopatije. Kardio i hepatotoksičnost nanokompozita FNP/DOX u odnosu na komercijalni DOX ispitana je na odraslim muţacima pacova soja Wistar. Ultrastrukturna analiza tkiva srca i jetre pokazala je da tretman DOX dovodi do oštećnja pojedinih ćelijskih kompartmana, kao što su mitohondrije, miofibrili, kompromituje integritet sarkoleme i dovodi do hiperkontrakcije sarkomera, unutarćeliskog edema, vakuolizacije citoplazme, pojave mijelinskih figura, autofagozoma i mikrovezikularnih masnih promena. Nanokompozit FNP/DOX izaziva manje štetnih promena na srcu i jetri zdravih pacova soja Wistar, u poreĊenju sa komercijalnim DOX-om, pri ĉemu je doza od 4 mg/kg postigla bolji efekat nego doza od 2 mg/kg. Na molekularnom nivou, kvantifikovana je ekspresija gena: Bax i Bcl-2 koji uĉestvuju u apoptozi, kao i katalaza i mangan superoksid dismutaza(MnSOD), koji uĉestvuju u odgovoru ćelije na oksidativni stres. Nanokompozit FNP/DOX menja gensku ekspresiju antioksidativnih enzima, ukazujući na prisustvo manjeg oksidativnog stresa u tkivu srca i jetre, u poreĊenju sa dejstvom komercijalnog DOX. Nanokompozit FNP/DOX u tkivu srca, menja i gensku ekspresiju ključnih enzima apoptoze, Bax i Bcl-2, pri čemu inicira povećanu transkripciju antiapoptotskog Bcl-2 gena, omogućavajući ćellijama da se odupru programiranoj ćelijskoj smrti. Promene ekspresije iRNK za dva antioksdataivna gena kao i za dva gena koja učestvuju u apoptozi, u srcu i jetri, ne mogu da ukažu na jasan mehanizam preko koga FNP/DOX izaziva manje štetnih promena na srcu i jetri zdravih pacova soja Wistar, u poređenju sa komercijalnim DOX-om. Kardiotoksičnost doksorubicina se pripisuje slobodnoradikalskom oksidativnom stresu zasnovanom na gvožđu. Hidroksil radikal, direktno je odgovoran za oštećenja na DNK i nastaje u reakciji H2O2 i O2•− koja je katalizovana jonima gvožđa (Fe2+). Dodatno, kada se DOX veže za iRNK koja kodira feritin dolazi do modifikacije ekspresije feritina što utiče na metabolizam i homeostazu gvožđa. Stoga, helatori gvožđa mogu interferirati sa DOX na mnogo složeniji način nego što je Fentonova reakcija. U ovoj tezi iskorišćene su helatorske sposobnosti FNP da za sebe veže jone gvožđa u formi nanokompozita FNP/Fe2+ i ispitani su njegovi biološki efekti in vivo na pacovima soja Wistar. Nanokompozit FNP/Fe2+ u predtretmanu 1h pre primene DOX-a, je pokazao da izaziva manje štetnih promena na srcu i jetri zdravih pacova soja Wistar, u poređenju sa komercijalnim DOX-om. Rezultati na nivou genske ekspresije, prilikom primene nanokompozita FNP/Fe2+ u predtretmanu 1h pre doksorubicina, za dva antioksdataivna gena u srcu i jetri, ne mogu da ukaţu najasan mehanizam preko koga nanokompozit FNP/Fe2+ izaziva manje štetnih promena na srcu i jetri zdravih pacova soja Wistar, u poređenju sa komercijalnim DOX-om. Sveukupno gledano ovo istraživanje je omogućilo detaljniji uvid u kompleksnu interakciju između fulerenolskih nanočestica, doksorubicina, Fe2+ i ćelija, kako malignih tako i zdravih. Pokazan je protektivni potencijal fulerenolskih nanočestica kada se kombinuju sa citostatikom doksorubicinom i sa Fe2+, a što može imati pozitivne implikacije prilikom dizajniranja antitumorskih lekova.

Tumors are the second leading cause of death in the world (following cardiovascular diseases) and it is estimated that the incidence of tumors will increase in the future. Chemotherapy is one of the most common methods of tumor treatment which is often accompanied by systemic side effects. Treatment of tumors with one therapeutic agent often results in limited clinical outcome. Combination therapy, using a variety of agents and modalities of action, can synergistically increase thelevel of antitumor activity, lowering the dose of each single agent and thus reduce adverse side effects. The simultaneous application of various therapeutic agents requires a delivery platform to enable normalization of agents’ pharmacokinetics and pharmacodynamics. Because of its physico-chemical and biological activities,fullerenol C60(OH)24 represents a suitable platform for drug delivery. In the aqueous solution fullerenol is in the form of negatively charged polianionic nanoparticles (FNP) that possess a large surface area and have the ability of binding positively charged chemotherapeutic drug, such as doxorubicin (DOX), as well as positively charged metal ions, particularly Fe 2+ . The main biological activity of fullerenol is manifested through its antioxidant activity. In this study we used physico-chemical properties of fullerenol, doxorubicin and iron (Fe2+ ) in order to form stable fullerenol/doxorubicin (FNP/DOX) nanocomposite and fulerenol/iron (FNP/Fe 2+) nanocomposite. The nanocomposites were characterized by different physical- chemical methods which showed that FNP/DOX has particle size between 20 nm and 58 nm and charge of -6 mV, while FNP/Fe 2+ has particle size between 11 nm and 60 nm and charge of -30.8 mV. Both nanocomposites revealed discoidal morphology. The nanocomposites have also displeyed good stability in aqueous systems, which makethem biocompatible and suitable for use in biomedical applications. FNP decorated with DOX, in the form of FNP/DOX nanocomposite, allows significantly more extensive DOX uptake into cells and reduces the percentage of cells in the G2M phase of the cell cycle, compared to the independent application of DOX, in malignant breast cell lines. DOX has been used in the treatment of cancer for the last forty years and still represents an irreplaceable drug in oncology. Its longterm use leads to cardiomyopathy and hepatopathy, while its main mechanism of toxicity is induction of oxidative stress. Cardio and hepatotoxicity of FNP/DOX nanocomposite in comparison to DOX was tested on healthy adult male Wistar rats. Ultrastructural analysis of heart and liver tissues revealed that treatment with DOX induced injuries within different cell compartments, such as: mitochondria, myofibrils and nucleai. Compromised integrity of sarcolemma, hypercontraction of sarcomeres, interstitial edema, and vacuolization of the cytoplasm, presence of myelin figures, autophagosomes and microvascular fatty changes have also been observed. Contrary to these findings, FNP/DOX nanocomposite induced significantly less injury to the heart and liver tissues compared to DOX, with a dose of 4 mg /kg FNP/DOX achieving a better effect than a dose of 2 mg /kg. At the molecular level, the expression of the genes: Bax and Bcl-2, which participate in apoptosis, as well as catalase and manganese superoxide dismutase (MnSOD), which participate in the cell's response to oxidative stress, has been quantified. FNP/DOX nanocomposite changed the gene expression levels of the antioxidant enzymes indicating the presence of decreased oxidative stress in heart and liver tissues in comparison with DOX. Furthermore, FNP/DOX nanocomposite in heart tissue changed gene expression levels of enzymes involved in apoptosis, Bax and Bcl-2; it increased transcription levels of theantiapoptotic Bcl-2 gene, enabling cells to resist to the programmed cell death. Changes in mRNA expression for two antioxidant genes as well as for two genes involved in apoptosis, in the heart and liver tissues, cannot indicate a clear mechanism by which FNP/DOX causes less harmful effects in the heart and liver of healthy Wistar rats, compared to DOX applied alone. Cardiotoxicity of DOX is attributed to iron-based free radical oxidative stress. The hydroxy radical is directly responsible for DNA damage and is formed in the reaction of H2O2 and O2 • - which is catalyzed by iron ions (Fe 2 + ). Additionally, when DOX binds to mRNA encoding ferritin, ferritin expression is modified, which affects iron metabolism and homeostasis. Therefore, iron chelators can interfere with DOX in a much more complex way than the Fenton reaction. In this thesis, the chelation characteristics of FNP to bind iron ions in the form of FNP/Fe 2+ nanocomposite were used and its biological effects in vivo on Wistar rats were examined. At the ultrastructural level, FNP/Fe 2 + nanocomposite in pretreatment 1h before DOX administration was shown to cause less detrimental changes to the heart and liver tissues of healthy Wistar rats, compared to DOX applied alone. At the level of gene expression, FNP/Fe 2+ nanocomposite in pretreatment 1h before DOX administration, for two antioxidant genes in the heart and liver tissues, cannot indicate a clear mechanism through which FNP/Fe 2 + anocomposite causes less harmful effects in heart and liver. Overall, this research has enabled a more detailed insight into the complex interaction between fullerenol nanoparticles,doxorubicin, Fe 2+ and cells, both malignant and healthy. This experimental setup has provided an opportunity for a better understanding of the protective potential of fullerenol nanoparticles when combined with cytostatic DOX and Fe 2+ which may have positive implications during drug design.