The role of the two nanocomposites fullerenol/doxorubicin and fullerenol/iron in toxic effects caused by doxorubicin

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

Applications of Anti/Prooxidant Fullerenes in Nanomedicine along with Fullerenes Influence on the Immune System

Fullerenes are molecules that, due to their unique structure, have very specific chemical properties which offer them very wide array of applications in nanomedicine. The most prominent are protection from radiation-induced injury, neuroprotection, drug and gene delivery, anticancer therapy, adjuvant within different treatments, photosensitizing, sonosensitizing, bone reparation, and biosensing. However, it is of crucial importance to be elucidated how fullerenes immunomodulate human system of defense. In addition, the most current research, merging immunology and nanomedicine, results in development of nanovaccines, which may represent the milestone of future treatment of diseases

FADS2 gene variant rs174593 is associated with multiple sclerosis

Introduction: The hallmark pathogenic mechanisms of multiple sclerosis (MS) are proposed to be associated with long chain polyunsaturated fatty acids(LC-PUFA)-mediated neuroinflammation, through LC-PUFA-derived pro- and anti-inflammatory eicosanoids. Variants in genes coding for fatty acid desaturases (FADS), the key enzymes in LC-PUFA biosynthesis from essential fatty acids, are associated with changesin circulating LC-PUFA levels. The aim of thisstudy wasto investigate the FADS2 intronic variants, rs174576 (C/A), rs174593 (T/C) and rs174616 (G/A), in association with MS. Methods: The study involved 124 patients with relapsing-remitting form of MS and 83 healthy control subjects. The FADS2 gene variants were detected using TaqMan® SNP genotyping assays. Analysis of allele and genotype distributions in patients and controls was done by using the chi-square test. Results: According to the model of dominant effect of allele, genotypes containing the alternative, C, allele of FADS2 rs174593 variant were significantly less frequent in MS patients than in controls (MS: TT=57,26%, TC+CC=42,74%; controls: TT=42,17%, TC+CC=57,83%; p=0,03). In addition, the frequency of rs174593 C allele was significantly lower in patients, compared to controls (MS: T=0,76, C=0,24; controls: T=0,67, C=0,33; p=0,04). The frequency distributions of rs174576 and rs174616 alleles and genotypes were not significantly different between the study groups (p>0,05). Conclusion: The obtained resultssupply a rationale for further investigation of the association of FADS2 rs174593 with circulating LC-PUFA levels, in the context of MS. The genotype-LC-PUFA phenotype association could provide guidelinesfor personalized LC-PUFA supplementation, to potentially ameliorate the disease course and improve the effectiveness of therapyThe Second Congress of Molecular Biologists of Serbia; October 6-8, Belgrade, 2023

Identification of micro RNA from common copy number variants as risk factors for CAKUT

Introduction: Congenital anomalies of the kidney and urinary tracts(CAKUT) are a diverse spectrum of defects with complex etiology and not fully explained genetic background. miRNA-containing copy number variants (CNVs) are described as genetic risk factor for the disease development. We aimed to identify miRNAs with the maximum regulatory coverage of previously reported differentially expressed genes in CAKUT tissue compared to controls and bioinformatically characterize a set of these miRNAs which are located in common CNVs. Methods: Differentially expressed genes were identified from ureter tissue transcriptome open data GSE83946 from 15 CAKUT patients and 7 healthy controls, generated in house previously. miRPathDB v2.0 was used for identification of miRNAs with maximum coverage of DEGs(miRNAs which complimentarily regulate all DEGs). Mapping of maximum coverage miRNAs onto common CNVs (frequency >0.2) was performed using UCSC genome browser and gnomAD database. miRNA mapping common CNVs were further bioinformatically analyzed using miRPathDB v2.0. Results: In a maximum coverage set of 50 miRNAs interacting with DEGs in CAKUT, we have identified 3 miRNA geneslocated in the common CNVs(hsa-miR-663b, hsa-miR-3180-3p and hsa-miR-1302). Using Reactome database we identified all three miRNAsto be significantly enriched in the pathway Neuronal System: -log(p-value)>2.326 for hsa-miR-1302; -log(p-value)>1.556 for hsa-miR-3180-3p; and -log(pvalue)>1.703 for hsa-miR-663b. Conclusion: CAKUT is characterized with variable penetrability and expressivity and often followed with other comorbiditiessuch as neurodevelopmental disorders. miRNAsinvolved in DEG networks and prone to CNV effects could present modulating factors of the disease phenotype. Further studies should provide additional evidence about hsa-miR-1302, hsa-miR-3180-3p and hsa-miR-663b involvements in CAKUT etiologyThe Second Congress of Molecular Biologists of Serbia; October 6-8, Belgrade, 2023

Fullerenol nanoparticles as a new delivery system for doxorubicin

Doxorubicin is a very potent chemotherapeutic drug, however its side effects limit its clinical use. The aim of this research was to investigate the properties of a fullerenol/doxorubicin nanocomposite, its potentially cytotoxic and genotoxic effects on malignant cell lines, as well as its toxicity towards zebra fish embryos. Chromatographic, NMR and mass spectral analysis of the nanocomposite imply that interactions between doxorubicin and fullerenol are non-covalent bonds. The stability of the nanocomposite was confirmed by the use of atomic force microscopy, dynamic light scattering and transmission electron microscopy. The nanocomposite, compared to the free doxorubicin at equivalent concentrations, significantly decreased the viability of MCF-7 and MDA-MB-231 cells. The flow cytometry results indicated that doxorubicin-loaded fullerenol could remarkably increase the uptake of doxorubicin suggesting that fullerenol might be a promising intracellular targeting carrier for the efficient delivery of antitumor drugs into tumor cells. The nanocomposite also affected cell cycle distribution. A genotoxicity test showed that the nanocomposite at all examined concentrations on MCF-7 and at lower concentrations on MDA-MB-231 cells caused DNA damage. Consequently, cell proliferation was notably reduced when compared with controls. Results of the zebrafish embryotoxicity assay showed a decreased overall toxicity, particularly cardiotoxicity and increased safety of the nanocomposite in comparison to doxorubicin alone, as manifested by a higher survival of embryos and less pericardial edema

The Puzzling Potential of Carbon Nanomaterials: General Properties, Application, and Toxicity

Being a member of the nanofamily, carbon nanomaterials exhibit specific properties that mostly arise from their small size. They have proved to be very promising for application in the technical and biomedical field. A wide spectrum of use implies the inevitable presence of carbon nanomaterials in the environment, thus potentially endangering their whole nature. Although scientists worldwide have conducted research investigating the impact of these materials, it is evident that there are still significant gaps concerning the knowledge of their mechanisms, as well as the prolonged and chronic exposure and effects. This manuscript summarizes the most prominent representatives of carbon nanomaterial groups, giving a brief review of their general physico-chemical properties, the most common use, and toxicity profiles. Toxicity was presented through genotoxicity and the activation of the cell signaling pathways, both including in vitro and in vivo models, mechanisms, and the consequential outcomes. Moreover, the acute toxicity of fullerenol, as one of the most commonly investigated members, was briefly presented in the final part of this review. Thinking small can greatly help us improve our lives, but also obliges us to deeply and comprehensively investigate all the possible consequences that could arise from our pure-hearted scientific ambitions and work

Fullerenol/iron nanocomposite modulates doxorubicin-induced hepatotoxicity

Introduction: Doxorubicin is the most prominent chemotherapeutic, but its clinical use is limited by its severe systemic toxicity. An iron overload aggravates anthracycline toxicity. Fullerenol in aqueous solutions is in the form of polyanionic nanoparticles, serving as a good carrier of positively charged ions, such as Fe2+. Fullerenol’s antioxidant activity has already been proved in different biological systems. The aim of our study was to investigate the effects of the fullerenol/iron nanocomposite on the rat liver as a pretreatment to doxorubicin application. Methods: After the 24h-treatment, adult male Wistar rats were sacrificed and livers were collected for ultrastructural and qRT-PCR analysis. Considering the ability of doxorubicin to induce oxidative stress, and the fullerenol’s capability to mitigate it, gene expression of enzymes involved in antioxidant defense was measured. Results: Ultrastructural analysis revealed that liver tissue was mainly preserved after the nanocomposite was applied prior to doxorubicin. However, the hepatocytes of animals treated with doxorubicin, presented significantly damaged morphology. Apoptosis of hepatocytes and endothelial cells, mitochondria of irregular size and with disruption of cristae, diffuse injury of capillaries were observed. RT-PCR results have shown that treatment with doxorubicin alone significantly increase the mRNA levels of catalase (p=0.008) and superoxide-dismutase (p=0.000003), while the pretreatment with the nanocomposite prior the doxirubicine treatment, dramaticly downregulated the mRNA levels of catalase (p=0.0004) and superoxide-dismutase (p=0.0001). Conclusion: Our results suggest that the fullerenol/iron nanocomposite applied as pretreatment to doxorubicin, demonstrated protection to the liver tissue and induced less damage to the hepatocytes in comparison to doxorubicin alone

Versatile applications of fullerenol nanoparticles

Nanomaterials have become increasingly important over time as research technology has enabled the progressively precise study of materials at the nanoscale. Developing an understanding of how nanomaterials are produced and tuned allows scientists to utilise their unique properties for a variety of applications, many of which are already incorporated into commercial products. Fullerenol nanoparticles C60(OH)n, 2 ≤ n ≤ 44 are fullerene derivatives and are produced synthetically. They have good biocompatibility, low toxicity and no immunological reactivity. In addition, their nanometre size, large surface area to volume ratio, ability to penetrate cell membranes, adaptable surface that can be easily modified with different functional groups, drug release, high physical stability in biological media, ability to remove free radicals, magnetic and optical properties make them desirable candidates for various applications. This review comprehensively summarises the various applications of fullerenol nanoparticles in different scientific fields such as nanobiomedicine, including antibacterial and antiviral agents, and provides an overview of their use in agriculture and biosensor technology. Recommendations are also made for future research that would further elucidate the mechanisms of fullerenols actions

Ekspresija nukleozidnih transportera u primarnoj kulturi astrocita pacova

Mnoge ćelije u CNS-u preuzimaju nukleozide iz vanćelijske teĉnosti i koriste ih za sintezu nukleotida u putevima uštede. Nukleozidi se kroz membranu astrocita transportuje putem dve genske familije nukleozidnih transportera: ekvilibrativnih (ENT1 i ENT2) koji omogućavaju jednosmernu olakšanu difuziju i koncentrativnih (CNT2) koji omogućavaju jednosmerni sekundarno aktivni transport kroz ćelijsku membranu. Cilj ovog istraţivanja je bio ispitivanje ekspresije razliĉitih klasa nukleozidnih transportera na primarno kultivisanim astrocitima pacova, kao i uticaj hroniĉno povećane koncentracije adenozina u vanćelijskom medijumu na stepen njihove ekspresije. Eksperimenti su raĊeni u primarnoj kulturi astrocita pacova soja Wistar. Ćelije su u toku 21 dana kontinuirano tretirane adenozinom u koncentracijama od 25µM i 100µM. Kvantifikacija genske ekspresije je vršena RT-qPCR analizom na svakih 7 dana i izraţena je kao ΔCt vrednost u odmosu na vrednost koamplifikovanog β-aktina. Rezultati su pokazali da se u primarnim astrocitima pacova eksprimiraju transporteri i ekvilibrativnog i koncentrativnog tipa. Najveću ekspresiju nakon 7-og dana u kulturi je pokazao koncentrativni transporter CNT 2 (ΔCt=7,13±0,42), dok je stepen ekspresije za ekvilibrativne transportere bio nešto niţi za ENT 1 ΔCt=9,48±0,29, a za ENT 2 ΔCt=8,67±0,24. TakoĊe je uoĉeno da se tokom kultivisanja u vremenskom periodu od 21-og dana postepeno smanjuje genska ekspresija svih ispitivanih transportera: za CNT 2 ΔCt=12,75±0,51; za ENT 1 ΔCt=12,78±0,41; i za ENT 2 ΔCt=12,37±0,44. U uslovima kontinuiranog prisustva adenozina u primarnoj kulturi astrocita (25µM, odnosno 100µM) nakon 7,14 i 21 dana, nije došlo do znaĉajne promene u ekpresiji ni jednog od ispitivanih transportera u odnosnu na populaciju ćelija koja nije bila izloţena tretmanu adenozinom.VII/XIII Kongres neurologa Srbije : Septembar 11-14, Kragujevac, 2008

Applications of Anti/Prooxidant Fullerenes in Nanomedicine along with Fullerenes Influence on the Immune System

Fullerenes are molecules that, due to their unique structure, have very specific chemical properties which offer them very wide array of applications in nanomedicine. The most prominent are protection from radiation-induced injury, neuroprotection, drug and gene delivery, anticancer therapy, adjuvant within different treatments, photosensitizing, sonosensitizing, bone reparation, and biosensing. However, it is of crucial importance to be elucidated how fullerenes immunomodulate human system of defense. In addition, the most current research, merging immunology and nanomedicine, results in development of nanovaccines, which may represent the milestone of future treatment of diseases