XIX. WORKSHOP OF BIOPHYSICAL CHEMISTS AND …labifel/files/soubory/sborník_2019.pdf · XIX....

xix. Workshop of

biophysical chemists and

electrochemists

XIX. WORKSHOP OF

BIOPHYSICAL CHEMISTS AND

ELECTROCHEMISTS

BOOK OF ABSTRACTS

14th JUNE 2019

Brno

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Masaryk University Press

Brno 2019

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THE ORGANIZATION HOSTING THE CONFERENCE

Masaryk University

Faculty of Science

Department of Chemistry

Kotlářská 2

611 37 Brno

http://www1.sci.muni.cz

THE ORGANIZATIONAL SECURITY OF THE CONFERENCE

Libuše Trnková

[email protected]

(Department of Chemistry, Faculty of Science, Masaryk University)

The publication did not undergo the language control. All contributions are publicated in the

form, in which they were delivered by the authors. Authors are also fully responsible for the

material and technical accuracy of these contributions.

© 2019 Masaryk University

ISBN 978-80-210-9309-6

XIX. WORKSHOP OF BIOPHYSICAL CHEMISTS AND ELECTROCHEMISTS 2019

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The Workshop of Biophysical Chemists and Electrochemists was

supported by research organizations:


5

The sponsors of the Workshop of Biophysical Chemists and

Electrochemists:

The organizers thank a lot to all this year’s sponsors for the support, which enabled to

organize this traditional conference: Metrohm Czech Republic s.r.o., Institute of Biophysics

of the Czech Academy of Sciences in Brno, Eppendorf Czech & Slovakia s.r.o., Oncomed

manufacturing a.s., UNIMED Praha, s.r.o., Chromservis, s.r.o., MERCI, s.r.o. and Czech

Chemical Society, subdivision Brno.

The main sponsor

The main sponsor


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An introductory word...

On behalf of the Organizing Committee I am honored to welcome you to Brno, for the

19th

Workshop of (Bio)physical Chemists and Electrochemists which is held again in Masaryk

University (MU) Campus (Kamenice 5, Bohunice, building A11/room 205). This year´s

conference will be held to celebrate two events, the 100th

anniversary of Masaryk University

and 60th

years of awarding the Nobel Prize to Prof. Jaroslav Heyrovský, Czech physical

chemist. During the conference we will remember also Prof. Emil Paleček, the famous Czech

electrochemist who worked in Brno and devoted his career to electrochemistry of biologically

important substances, especially of nucleic acids.

According to the attached program you can see the schedule of plenary lectures, invited

lectures as well as oral presentations of young scientists. The conference will also include

presentations of posters and companies which supporting the event. Special thanks go to the

main sponsor, which, in addition to the Biophysical Institute, financially participates in the

Emil Paleček Award. All participants are invited not only to the “Young Scientists’ Session”

and the Poster Session but also to the gala party on Friday early in the evening (“On the

foodbridge”) where three winners of the “Young Scientists’ Session” and one winner of the

Poster Session will be announced.

We are confident that our 19th

Workshop of Biophysical Chemists and Electrochemists will

be memorable for its scientific quality, also thanks to your contributions. We do hope that you

will welcome the opportunity to present and discuss your scientific results with respect the

Heyrovsky´s logo: “Experimenting to knowledge, learning to progress“.

Welcome to Brno and enjoy this conference!

Libuše Trnková

Motto: “Experimenting to knowledge, learning to progress“

Jaroslav Heyrovský


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Remembering Prof. Emil Paleček

Na podzim roku 2018 nás navždy opustil významný český vědec, který jako první na

světě v roce 1960 objevil způsob, jak zkoumat DNA pomocí elektrochemie. S využitím

objevu Jaroslava Heyrovského – polarografie – výrazně přispěl k pokroku v oblasti chemické

reaktivity nukleových kyselin a studia lokálních struktur. Zasloužil se i o rozvoj

elektrochemie proteinů. Pomocí elektroanalytických metod studoval chování proteinů a jejich

komplexů s DNA na elektricky nabitých površích, zabýval se analýzou glykoproteinů se

zvláštním zřetelem na její budoucí uplatnění v lékařství.

Jeho celoživotní dílo je významné, mnohé jeho poznatky vešly do učebnic biofyziky a

molekulární biologie.

Věnujme tichou vzpomínku tomuto významnému českému vědci a držiteli mnoha

čestných ocenění.

Ocenění vědecké práce profesora Emila Palečka v posledních letech

2009 – Čestná medaile Akademie věd České republiky

De scientia Et Humanitate Optime Meritis

Profesor Jiří Drahoš, předseda Akademie věd ČR,

udělil 18. září 2009 Čestnou medaili AV ČR De

Scientia et Humanitate Optime Meritis prof. Emilu

Palečkovi z Biofyzikálního ústavu AV ČR v Brně.

Medaile je udělována za mimořádné aktivity

v oblasti vědy a úspěšné zajišťování infrastuktury

výzkumu a vývoje.

Foto: Zdeněk Tichý, Archiv KNAV


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2011 – Cena ministra školství, mládeže a tělovýchovy Cena ministra školství, mládeže a tělovýchovy za mimořádné

výsledky výzkumu, experimentálního vývoje a inovací byla v roce

2011 udělena Prof. RNDr. Emilu Palečkovi, DrSc. za výsledky

výzkumu v oblasti elektrochemických metod, vhodných k analýze

změn ve struktuře bílkovin a jejich aplikace v biomedicíně

(například při výzkumu Parkinsonovy choroby a rakoviny) a také za

dlouholetý významný přínos k rozvoji elektrochemie nukleových

kyselin, která má široké uplatnění při decentralizované analýze

DNA.

2011 – Plaketa Johanna Gregora Mendela Za mimořádné zásluhy o rozvoj vědy a vzdělání

v oblasti biologie, molekulární biologie a genetiky

byl prof. Emil Paleček spolu s Dr. Michaelem

Heyrovským poctěn plaketou Johanna Gregora

Mendela při příležitosti konání XI. Pracovního

setkání chemiků a elektrochemiků na MENDELU

v Brně.

2014 – Cena firmy Metrohm Prestižní cenu firmy Metrohm za celoživotní přínos k

rozvoji elektroanalytické chemie získal za rok 2013

prof. RNDr. Emil Paleček, DrSc. z Biofyzikálního

ústavu AV ČR v.v.i. v Brně. Založil v 60. letech

minulého století zcela novou oblast – elektrochemii

nukleových kyselin. V poslední době se zabýval i

výzkumem bílkovin, které mají souvislost se

vznikem rakoviny.

2014 – Národní cena vlády Česká hlava Národní cenu vlády Česká hlava za rok 2014 vyhlásil na tiskové konferenci 27. listopadu

2014 místopředseda vlády pro vědu, výzkum a inovace Pavel Bělobrádek. Oceněným pro

tento rok byl prof. RNDr. Emil Paleček, DrSc. přední český vědec v oblasti přírodních věd.

Svými pracemi položil základ pro obor elektrochemie nukleových kyselin. Na slavnostním

Galavečeru v Národním domě na Vinohradech v Praze pak cenu předal premiér vlády České

republiky Bohuslav Sobotka.


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2017 – Cena Neuron za přínos světové vědě Profesor Emil Paleček z Brna se stal prvním

vědcem na světě, který ukázal, že DNA lze

analyzovat elektrochemicky, a založil nové

vědecké pole, na kterém dnes pracují stovky

laboratoří ve světě. Patřil a ještě patří díky více

než třem stovkám vydaných vědeckých prací

mezi nejcitovanější vědce v zemi. Zaujal

tuzemskou odbornou veřejnost, byl hojně

citován i v zahraničí. Jeho dílo vešlo do učebnic

biofyziky a molekulární biologie.


10

Table of contents POLAROGRAPHY IN CONTEMPORARY MOLECULAR ELECTROCHEMISTRY .................... 12

SUPRAMOLECULAR PUZZLES: HOST-GUEST COMPLEXES IN METALLODRUG

RESEARCH .......................................................................................................................................... 13

SIMPLE AND RAPID ELECTROCHEMICAL ASSAY FOR DETECTION OF HPV IN CLINICAL

SAMPLES ............................................................................................................................................. 14

EFFECT OF THE LENGTH OF CYTOSINE OLIGONUCLEOTIDE BLOCKS ON THEIR

ELECTROREDUCTION AT THE MERCURY ELECTRODE .......................................................... 15

CHARGE TRANSPORT IN EXTENDED BIPYRIDINIUM SINGLE MOLECULE JUNCTIONS .. 16

„PRINTED CIRCUIT BOARD” ELECTRODES AS A PLATFORM FOR DISPOSABLE SENSING

PARTS ................................................................................................................................................... 18

SIMULTANEOUS ACTIVATION AND NANOMATERIAL MODIFICATION OF ELECTRODE

SURFACES USING SPARK DISCHARGES ...................................................................................... 20

STRUCTURAL STUDY OF 14-3-3 ζ MONOMERIC MUTANT ....................................................... 21

ELECTROCHEMICAL ASSAY FOR microRNA DETECTION USING SPECIFIC ANTIBODY

AND HYBRIDIZATION CHAIN REACTION: APPLICATION TO CLINICAL SAMPLES ........... 22

AMPEROMETRIC IMMUNOSENSOR FOR DIAGNOSIS OF EUROPEAN FOULBROOD ......... 24

STUDY OF CHARGE TRANSFER AND CHARGE TRANSPORT MECHANISM IN EXPANDED

PYRIDINIUM MOLECULES .............................................................................................................. 26 BIOPHYSICAL ANALYSIS OF SILVER NANOPARTICLES AND USE THEIR

ANTIMICROBIAL ACTIVITY IN 3D PRINTING ............................................................................ 28

POLYMER NANOSPHERE-ASSEMBLED SURFACE FOR BIOSENSING BASED ON

ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY ................................................................. 31

ELECTROCHEMICAL DETERMINATION OF INSULIN ON NiONPS MODIFIED CARBON

ELECTRODES ..................................................................................................................................... 32

AMINOFERROCENE: DETERMINATION OF THE ACIDITY OF UNSTABLE COMPOUNDS .. 34

EFFECT OF NEWLY DESIGNED STABLE CFTR-MESSENGER RNA ON TRANSFECTION OF

HUMAN CYSTIC FIBROSIS AIRWAY EPITHELIUM .................................................................... 36

ITP ANALYSIS OF SWEET BEVERAGES AND BEERS ................................................................. 38

APOFERRITIN NANOCAGE AS A PROMISING DOXORUBICIN NANOCARRIER AND ITS

EFFECTS ON NEUROBLASTOMA CELL LINES ............................................................................ 39

ANALYSIS OF METABOLISM AND DNA ADDUCT FORMATION BY ARISTOLOCHIC ACIDS I

AND II IN RATS IN VIVO .................................................................................................................... 41

DIRECT ELECTRODEPOSITION OF SILVER AMALGAM PARTICLES ON SCREEN PRINTED

SILVER ELECTRODES USING DOUBLE PULSE CHRONOAMPEROMETRY ........................... 43


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METABOLISM OF TYROSINE KINASE INHIBITOR CABOZANTINIB BY LIVER

MICROSOMES .................................................................................................................................... 44

COMPARISON OF EFFICIENCIES OF PEROXIDASES TO OXIDIZE THE ANTICANCER DRUG

ELLIPTICINE AND THEIR INFLUENCING BY VANDETANIB, LENVATINIB AND

CABOZANTINIB ................................................................................................................................. 46

DEVELOPMENT OF INSTRUMENTATION FOR COULOMETRIC TITRATIONS ....................... 48

ON ELECTROCHEMISTRY OF 1-PENTYL-3-(1-NAPHTOYL)INDOLE AND 1-PENTYL-3-(2-

METHOXYPHENYLACETYL)INDOLE ........................................................................................... 50

OXIDATION OF A TYROSINE KINASE INHIBITOR VANDETANIB BY RAT ENZYMATIC

SYSTEMS IN VITRO ............................................................................................................................ 51

APPLICATION OF LOW-FIELD

1H NMR SPECTROSCOPY IN ANALYTICAL CHEMISTRY ... 53

STUDY ON ENCAPSULATION OF LENVATINIB AND ELLIPTICINE INTO

NANOTRANSPORTERS; EXPERIMENTAL AND THEORETICAL APPROACHES .................... 55

METALLOTHIONEIN AND SELENITE IN BRDIČKA REACTION ............................................... 57

OXIDATION POTENTIALS OF GUANINE SPECIES ...................................................................... 59

APPLICATION OF CD SPECTROSCOPY IN ANALYTICAL CHEMISTRY .................................. 61

METABOLISM OF THE TYROSINE KINASE INHIBITOR LENVATINIB BY HUMAN HEPATIC

MICROSOMES AND CYTOCHROMES P450 ................................................................................... 62


12

POLAROGRAPHY IN CONTEMPORARY MOLECULAR

ELECTROCHEMISTRY

Jiří LUDVÍK

J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, 18223

Prague, Czech Republic

*[email protected]

Lecture dedicated to 60th anniversary of Nobel Prize for polarography with thanks to my

teachers, prof. Jiří Volke and prof. Petr Zuman

Polarography was awarded by the Nobel Prize originally as a new analytical method (by the

way the first fully automatic analytical method ever). After years of its theoretical and

experimental development in 40th and 50th, in 60th and later it was broadly used not only in

chemical research, but also in biology, medicine, pharmacy, archaeology, metallurgy, then

since 80th up to now in environmental sciences and in biochemistry. Today in practical

applications, polarography in its original form (DME) is often replaced by more fast, more

sophisticated (but also more expensive) analytical methods which could be operated by

laboratory assistants and which can be easily standardized.

However, polarography (and its derived methods based on mercury electrodes) is currently a

unique, irreplaceable and indispensable part of a spectrum of electrochemical methods namely

for fundamental electrochemical research. And the ban on the use of mercury electrodes in

chemical laboratories in several countries is unjustified and unwarranted.

Every day many new organic and organometallic compounds as well as complexes and

supramolecules are synthesized as promising pharmaceuticals, catalysts, agrochemicals, dyes,

organic semiconductors, liquid crystals, molecules for photovoltaics, etc.etc. and their

fundamental redox properties and reactivity in their oxidized or reduced state must be

characterized and elucidated. Therefore polarography is currently very important in so called

Molecular electrochemistry, discipline, where individual molecules in solution are

investigated in detail. For full understanding, polarography can be combined in-situ with UV-

vis, IR or EPR spectrometry and the acquired data should be checked by quantum chemical

calculations. For the further application the known thermodynamic, kinetic and structural data

should be correlated with expected activities.

For this presentation I selected one of the most recent investigations from our laboratory

based mainly on polarography - the systematic research of nitro-substituted calixarenes, their

electroreduction, stereoelectrochemistry and radical formation. In the second part some other

interesting topics will be presented and discussed, like e.g. diphenyl isobenzofurans for

singlet fission, Fischer carbenes or ferrocene derivatives.


13

SUPRAMOLECULAR PUZZLES: HOST-GUEST COMPLEXES IN

METALLODRUG RESEARCH

Radek MAREK1,2*

1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno CZ-62500, Czechia

2 CEITEC – Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, Czechia

*[email protected]

Supramolecular interactions govern many properties of systems ranging from the biological

effects of drugs to the function of novel materials. These interactions are responsible for

“communication” between individual molecules and formation of supramolecular host-guest

assemblies.

In this contribution, supramolecular metallocomplexes will be shown as prospective drug-

delivery systems for administrating the novel anticancer metallodrugs based on platinum or

ruthenium.1 Our recent investigations of the host-guest systems containing Pt(IV) or Ru(II)

anticancer cargo in cucurbit[n]uril carrier will be discussed.2,3

Further, unprecedented

paramagnetic NMR characterization of Ru(III) coordination compounds4-6

and their host-

guest complexes with macrocyclic cavitands (cyclodextrins or cucurbiturils)7 will be

demonstrated. Finally, perspectives of our research in the design and development of

supramolecular cages and metallodrugs will be outlined.

ACKNOWLEDGEMENT

This work has received support from the Czech Science Foundation (Grant No. 18-05421S)

and the Ministry of Education of the Czech Republic (Grant No. LQ1601).

REFERENCES

[1] Mjos K. D., Orvig C.: Chem. Rev., 114 (2014), 4540-4563.

[2] Chyba J. et al.: unpublished results.

[3] Sojka M. et al.: Inorg. Chem., submitted.

[4] Novotny J. et al.: J. Am. Chem. Soc., 138 (2016), 8432-8445.

[5] Novotny J. et al.: Inorg. Chem., 57 (2018), 641-652.

[6] Jeremias L. et al.: Inorg. Chem., 57 (2018), 8748-8759.

[7] Chyba J. et al.: Inorg. Chem., 57 (2018), 8735-8747.


14

SIMPLE AND RAPID ELECTROCHEMICAL ASSAY FOR

DETECTION OF HPV IN CLINICAL SAMPLES

Martin BARTOŠÍK1*

, Ludmila JIRÁKOVÁ1, Roman HRSTKA

1

1 RECAMO, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic

*[email protected]

Persistent infection with human papilloma virus (HPV) can cause malignant tissue

transformation and lead to various types of cancers, most often cervical cancer in women [1].

HPV16 and HPV18 are two most oncogenic high-risk HPV types which cause over 70% of

HPV-positive cervical carcinomas. Current detection methods [2] involve complex protocols,

need for skilled personnel and expensive reagents and instruments.

Electrochemical methods of detection may circumvent these challenges by offering rapid,

simple and inexpensive assays [3]. In our work, we focused on development of such assay by

combining loop-mediated isothermal amplification (LAMP) reaction to amplify DNA from

cancer cells, magnetic beads for improved selectivity and amperometric measurement

performed on carbon electrode arrays for parallel measurements to speed up the protocol. We

were able to determine and discriminate HPV16 and HPV18 types not only in cervical cancer

cell lines, but more importantly in clinical samples isolated from cervical smears obtained

during gynecological examinations [4].

Currently, we are adapting this method also for detection of oncoviral mRNAs, E6 and E7

mRNAs, which better reflect virus activity and may thus help to reveal disease progression

before cellular changes become visible in classical cytological examination.

ACKNOWLEDGEMENT

The work has been supported by GACR 17-08971S and MEYS-NPS I-LO1413.

REFERENCES

[1] Schiffman M., Castle P. E., Jeronimo J., Rodriguez A. C., Wacholder S.: Lancet 370 (2007), 890–907.

[2] Kurian E. M., Caporelli M. L., Baker S., Woda B., Cosar E. F, Hutchinson L.: Am. J. Clin. Pathol. 136

(2011), 808–816.

[3] Palecek E., Bartosik M.: Chem. Rev. 112 (2012), 3427-3481.

[4] Bartosik M., Jirakova L., Anton M., Vojtesek B., Hrstka R.: Anal. Chim. Acta 1042 (2018), 37-43.


15

EFFECT OF THE LENGTH OF CYTOSINE OLIGONUCLEOTIDE

BLOCKS ON THEIR ELECTROREDUCTION AT THE MERCURY

ELECTRODE

Miroslav FOJTA1,2

, Hana PIVONKOVA1, Stanislav HASON

1, Zuzana BABKOVA

1,

1Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, CZ-61265 Brno, Czech

Republic

2 Central European Institute of Technology, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech

Republic

*[email protected]

Behavior of nucleic acids (NA) at electrodes is influenced by their nucleobase composition,

sequence and secondary structure. Mercury electrodes are still most frequently used tool for

the studies of electrochemical reduction of natural nucleobases. Among them, cytosine and

adenine are irreversibly electroreduced while reduction of guanine gives a product that can be

electrooxidized back to guanine, giving a well-developed anodic signal. Moreover, signals of

polyanionic NAs interacting with the negatively charged atomically smooth mercury

electrode surface are strongly influenced by the NA structure, allowing sensitive detection of

helix-coil transitions, DNA damage, interactions of DNA with small molecules as well as G-

quadruplex formation.

Our recent data indicate that interactions of NA chains with the mercury electrode surface are

remarkably influenced by the presence of homonucleotide blocks. Homopyrimidine

oligonucleotides exhibit 2D condensation at negatively charged electrode surface and stronger

adsorption than homopurine ones. Cytosine blocks showed a tighter adsorption, compared to

other homonucleotide blocks, as indicated by the dominance of C-E curves characteristic for

homocytosine stretches obtained in competition experiments. Moreover, cytosine block

exhibited an anomalous reduction behavior. Longer homocytosine stretches gave well

developed reduction signals at unusually high pH values (above pH 8) at which random (but

containing cytosine at levels corresponding to at least ¼ of all nucleobases) yielded no

reduction peaks. Maximum pH values at which cytosine reduction was observed were

dependent on the length of homocytosine blocks separated by other nucleotide sequences and

followed similar trends as the propensity of the same nucleotides to formation cytosine

tetraplexes - i-motifs (as assessed by CD spectra measurements).

ACKNOWLEDGEMENT

This work has been supported by the SYMBIT project reg. no.

CZ.02.1.01/0.0/0.0/15_003/0000477 financed from the ERDF.


16

CHARGE TRANSPORT IN EXTENDED BIPYRIDINIUM SINGLE

MOLECULE JUNCTIONS

Magdaléna HROMADOVÁ1*

, Viliam KOLIVOŠKA1, Jakub ŠEBERA

1, Táňa

SEBECHLEBSKÁ1,2

, Štěpánka NOVÁKOVÁ LACHMANOVÁ1, Jindřich GASIOR

1, Pavel

MORENO GARCIA3, Gábor MÉSZÁROS

4, Michal VALÁŠEK

5

1 J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, 18223 Prague,

Czech Republic

2 Department of Physical and Theoretical Chemistry, Faculty of Natural Sciences, Comenius University in

Bratislava, Mlynská dolina, Ilkovičova 6, 84215 Bratislava, Slovak Republic

3 Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland

4 Research Centre for Natural Sciences, HAS, Magyar Tudósok krt. 2, H-1117, Budapest, Hungary

5 Karlsruhe Institute of Technology, Institute of Nanotechnology, P. O. Box 3640, 76021 Karlsruhe, Germany

*[email protected]

Aim of this contribution is to compare charge transport properties of two photochemically

addressable single molecule switches (see Figure below), which have the same length but

differ in a torsion angle between individual aromatic rings of the conductance path. Single

molecule conductance was obtained by scanning tunneling microscopy break junction

technique that was complemented by theoretical analysis based on the density functional

theory and non-equilibrium Green’s function approach [1].

Figure: Chemical structures of extended bipyridinium cations 12+

and 22+

.

The conductance measurements were complemented by UV-VIS spectroscopy and

electrochemical studies. Both molecules accept four electrons. Molecule 12+

is reduced in two

two-electron steps, whereas molecule 22+

in three steps involving first transfer of two

electrons, followed by two one electron waves [2,3]. We will show that the energy and shape

of the LUMO is insensitive to the value of θ, but the difference in torsion angle θ leads to a

sizable shift of the LUMO energy and single molecule conductance value in the metal-

molecule-metal junction arrangement. Single molecule conductance of cation 22+

is 3.2 times

higher than that for cation 12+

and an increase in the charge transport magnitude upon the

photochemical cyclization of 12+

to 22+

is related to an enhanced electronic communication

between pyridine and pyridinium moieties. The experimental conductance ratio is somewhat

smaller than that obtained from the DFT/NEGF analysis, but DFT/NEGF is in a perfect

agreement with the value calculated from torsion angles demonstrating that the investigated

extended 4,4’-bipyridinium system follows the cos2θ law [3,4].


17

ACKNOWLEDGEMENT

This work has been supported by the Czech Science Foundation (18-04682S).

REFERENCES

[1] Hromadová M., Kolivoška V.: Studying the Electrical Properties of Single Molecules by Break Junction

Techniques. In: Encyclopedia of Interfacial Chemistry: Surface Science and Electrochemistry. (Ed. K.

Wandelt), Elsevier, 5, (2018) 271–280.

[2] Nováková Lachmanová Š., Šebera J., et al.: Electrochimica Acta, 264 (2018) 301–311.

[3] Šebera J., Sebechlebská T., et al.: Electrochimica Acta, 301 (2019) 267–273.

[4] Venkataraman L., Klare J. E., et al.: Nature, 442 (2006) 904–907.


18

„PRINTED CIRCUIT BOARD” ELECTRODES AS A PLATFORM FOR

DISPOSABLE SENSING PARTS

Karel LACINA

Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech

Republic *[email protected]

Printed circuit boards (PCBs) are utilised in production of electronics. It combines mechanical

support with conductive copper tracks for electronic elements in circuits. All employed

methods and techniques are well-developed, robust, optimised and cheap. Sometimes, the

contact pads are galvanically plated with gold to enhance their long-term stability compared

to bare copper. These exposed gold pads can be used as sensing electrodes [1-3].

A routine, large scale monitoring, such as point-of-care (POC) systems, requires inexpensive

and simple analytical platforms. If specifically designed, disposable and very cheap electrodes

can be produced using PCB techniques (Figure 1). If the dimensions are optimized, estimated

price for one piece can be $ 0.091 (1.8 Kč).

The crucial parameter for all electrochemical methods is the quality of a reference electrode.

In fact, the reference electrode appeared to be the Achilles’ heel of electroanalytical

disposable applications, where cheap and simple parts should be integrated. Use of external

reference, additional plating of electrode surfaces with silver or combination with screen-

printing was necessary.

One of the possible solutions for this issue is to use biamperometry – measuring with the

electrode system consisting of two identical electrodes (e.g. two gold electrodes) [4]. An

electroanalytical concept of biamperometry has been overlooked due to the widespread

potentiostat-controlled experiments. In this case, no reference electrode is needed as the

sensing electrodes are referenced to each other throughout the well-designed electrolyte. So,

well-defined electrochemistry can be performed even with such simple and cheap setup.

This system – combination of PCB electrodes with biamperometry - has already been utilised

in biosensing applications employing both enzymes and antibodies as biorecognition

elements. The possible utilization of the introduced concept was proven on an exemplar

determination of glucose by means of immobilized glucose oxidase. Affinity-based

biosensing was performed with biamperometric electrode systems as well. Determination of

the antibody (anti-Human Serum Albumin) binding on the antigen-modified (HSA) surface

was followed with measurement of electrochemical impedance. Interesting behavior of the

signal was observed – decrease of the impedance upon binding of the analyte in the

environment of 5 mM ferro/ferricyanide. The behavior was explained as charge-dependent

[3].


19

Figure: Photography of a set of 240 disposable biamperometric electrode systems prepared using Printed Circuit

Board approach (left). Various architectures of electrodes can be designed – example of biamperometric

electrode system with different electrode areas (right).

ACKNOWLEDGEMENT

The work has been financially supported by the Ministry of Education, Youth and Sports of

the Czech Republic under the project CEITEC 2020 (LQ1601) and by the Czech Science

Foundation, grant nr. 19-16273Y.

REFERENCES

[1] La Belle J. T., Shah M, Reed J, Nandakumar V, Alford T. L., Wilson J. W., Nickerson C. A. and Joshi L.:

Electroanalysis, 21 (2009), 2267–2271.

[2] Cui H, Xiong X, Gao B, Chen Z, Luo Y, He F, Deng S. and Chen L.: Electroanalysis, 28 (2016), 2000–2006.

[3] Lacina K, Sopoušek J, Čunderlová V, Hlaváček A, Václavek T and Lacinová V.: Electrochem. Commun.,

93(2018), 183–186.

[4] Lacina K, Vanýsek P, Bednář P, Trnková L and Skládal P.: ChemElectroChem, 3 (2016), 877–882.


20

SIMULTANEOUS ACTIVATION AND NANOMATERIAL

MODIFICATION OF ELECTRODE SURFACES USING SPARK

DISCHARGES

Jan HRBÁČ1

1Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

*[email protected]

The sensors capable to sensitively determine diverse analytes important in various research

and industry applications often utilize electrochemical principles. Novel strategies to develop

cheap and easy to use sensors, preferentially disposable and mass producible are intensely

studied. Often, these sensors use inorganic nanoparticles (mostly metallic) or nanomaterials

(graphene, nanotubes etc.) The fabrication of nanoparticle based sensors can be tedious,

encompassing a number of steps, including nanoparticle synthesis, purification, embedding in

a suitable matrix, application onto sensor surface, temperature stabilization etc. The specific

problem of mass produced sensors is the surface poorly performing from the electrochemical

point of view, exhibiting sluggish electron transfer between the electrode surface and analyte

molecules or electrode surface and modifying nanomaterial, triggering the need to activate the

electrodes prior to modification and use. As an alternative to mechanical polishing, often

impractical with mass produced sensors, chemical activation (e.g. exposing to oxidiying

agents), electrochemical activation (e.g. potential cycling) or low temperature plasma is used.

A novel approach based on application of spark discharges offers simultaneous activation and

nanomaterial modification of electrode surfaces. The lecture aims at presenting an overview

of current work on spark discharge modified disposable electrodes [1-7].

REFERENCES

[1] Riman D, Jirovsky D, Hrbac J, Prodromidis M.I.: Electrochem. Commun. 50 (2015), 20.

[2] Bartosova Z, Riman D, Halouzka V, Vostalova J, Simanek V, Hrbac J, Jirovsky D.: Anal Chim Acta, 935

(2016), 82-89.

[3] Riman D, Avgeropoulos A, Hrbac J, Prodromidis M.I.: Electrochim. Acta, 165 (2015), 410.

[4] Riman D, Spyrou K, Karantzalis A.E, Hrbac J, Prodromidis M.I.: Talanta, 165 (2017), 466.

[5] Trachioti M.G., Hrbac J, Prodromidis M.I.: Sens. Actuators B, 260 (2018), 1076.

[6] Trachioti M.G., Karantzalis A.E., Hrbac J, Prodromidis M.I.: Sensor. Actuat. B-Chem., 281 (2019), 273.

[7] Trachioti M.G., Tzianni E.I., Riman D, Jurmanova J, Prodromidis M.I., Hrbac J.: Electrochim. Acta, 304

(2019), 292.


21

STRUCTURAL STUDY OF 14-3-3 ζ MONOMERIC MUTANT

Tomáš BROM1,2

, Aneta KOZELEKOVÁ1,2

, Petr LOUŠA1, Norbert GAŠPARIK

1,2,

1 Central European Institute of Technology, Masaryk university, Kamenice 5, 625 00, Brno, Czech Republic

2 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00

Brno, Czech Republic

3 Center for Interdisciplinary Biosciences, Technology and Innovation Park P.J. Šafárik University, Jesenná 5,

041 54 Košice, Slovakia

*[email protected]

The dimeric character of 14-3-3 proteins is often crucial for their functions and for their

ability to bind phosphorylated binding partners [1]. 14-3-3s form homo/heterodimers that are

in dynamic equilibrium with monomers [2]. The dimeric form of 14-3-3 proteins has been

heavily studied, however, the structure and properties of the monomeric form remain

insufficiently described. The monomerization of 14-3-3ζ is regulated in different ways,

including phosphorylation of Ser58 located at the dimer interface [3]. The mutant of 14-3-3ζ

with a double mutation was designed [4] that exhibits complete monomerization over a wide

range of concentrations while retaining the characteristic α-helical structure based on the

far-UV CD measurements. The monomeric character of the designed mutant was proved by

analytical ultracentrifugation and small angle X-ray scattering. Differential scanning

calorimetry showed that the stability of monomeric mutant is lower than 14-3-3ζ WT. Nuclear

magnetic resonance provided partial insight into the structure. NMR HN-HSQC experiments

revealed that the monomeric mutants possess very similar overall NMR fingerprints as

14-3-3ζ WT that could indicate similar fold of monomeric mutants. Taken together, these

results offer additional insight into the monomeric form stability and structure of this

important family of regulatory proteins.

ACKNOWLEDGEMENT

This work was supported by the research grant from the Czech Science Foundation, grant no.

GA. 15-34684L. The results of this research have been acquired within the CEITEC 2020

(LQ1601) project. This work was supported by Ministry of Education, Youth and Sports

within program INTER-ACTION (LTAUSA18168).

REFERENCES

[1] Aitken, A., Semin. Cancer Biol., 2006, 16, 162-172

[2] X. Yang et al., Proc. Natl. Acad. Sci., 2006, 103, 17237–17242

[3] Sluchanko, N. N., Gusev, N. B., Arch. Biochem. Biophys., 2008, 477, 305-312

[4] Jandova, Z. and Hritz, J., Biochim. Biophys. Acta, 2018,1866(3), 442-450


22

ELECTROCHEMICAL ASSAY FOR microRNA DETECTION USING

SPECIFIC ANTIBODY AND HYBRIDIZATION CHAIN REACTION:

APPLICATION TO CLINICAL SAMPLES

Ludmila JIRÁKOVÁ1*

, Martin BARTOŠÍK1

1RECAMO, Masaryk Memorial Cancer Institute, Žlutý kopec 7, 656 53 Brno, Czech Republic

*[email protected]

MicroRNAs (miRNAs) are small non-coding RNA molecules with many regulatory functions,

including cell differentiation, proliferation or apoptosis. Recent research demonstrated that

miRNAs can be associated with onset and progression of various types of cancer and are thus

considered to be interesting biomarkers or therapeutic targets. Current detection methods involve

long protocols, often with reverse transcription step or fluorescent labeling and require expensive

and very sensitive fluorescent readout instruments.

Aim of our project is to develop rapid, reliable and inexpensive electrochemical (EC) method for

up-regulated miRNAs detection. Our EC assay comprised miRNA-specific hybridization probe

and two biotinylated auxiliary probes (Figure). Target miRNA separation from the sample was

facilitated by protein G-magnetic beads, which were modified with special S9.6 antibody

specifically binding RNA/DNA heteroduplexes [1]. EC signal, generated by enzymatic reaction

catalyzed by horseradish peroxidase (HRP) conjugated to streptavidin [2], was measured by

chronoamperometry.

As a target miRNA model, we used miR-21, which has already been shown to be up-regulated in

a wide range of tumors and is thus well detectable in real samples. Following the optimization, we

then detected other up-regulated miRNAs on a wide panel of cancer cell lines as well as in total

RNA samples isolated from precancerous cervical tissues. These results make the assay

potentially useful in treatment response prediction or early cancer diagnostics.

Figure: Detection system for miRNA, including magnetic beads modified with S9.6 antibody and three different

DNA probes to increase the sensitivity of the measurement in so-called hybridization chain reaction.


23

ACKNOWLEDGEMENT

The work has been supported by GAČR 17-08971S and MEYS-NPS I-LO1413.

REFERENCES

[1] Boguslawski S., Smith D. et al.: Journal of Immunological Methods, 89 (1986), 1, 123-130

[2] Torrente-Rodríguez R., Ruiz-Valdepeñas Montiel V. et al: ACS Sensors, 1 (2016), 7, 896-903


24

AMPEROMETRIC IMMUNOSENSOR FOR DIAGNOSIS OF

EUROPEAN FOULBROOD

Zuzana MIKUŠOVÁ1,2 *

, Matěj PASTUCHA1,2

, Veronika POLÁCHOVÁ1,2

, Radka

OBOŘILOVÁ1, Petr SKLÁDAL

1,2, Zdeněk FARKA

2

1 Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech

Republic

2 Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

* [email protected]

Western honeybee is an important pollinator and therefore, an invaluable part of agriculture

and biodiversity. Serious losses of honeybee colonies in recent years are attributed mostly to

climate changes and various diseases. One of the most important microbial diseases of

honeybees is European foulbrood (EFB), which is caused by pathogen Melissococcus

plutonius [1]. From the economic and environmental point of view, the prevention of EFB

spreading is crucial to prevent losses of honeybee colonies. Therefore, the development of an

effective method for the detection of M. plutonius is necessary, ideally in the point-of-care

(POC) format with sensitivity high enough to detect the pathogen before the clinical

symptoms develop.

Amperometry and electrochemical impedance spectroscopy (EIS) are highly sensitive and

robust approaches compatible with POC testing [2]. Due to their low cost, portability and

mass production capabilities, the electrochemical biosensors are typically based on screen-

printed electrodes (SPEs). For proper functionality, the immunosensors require antibodies

with high affinity and low cross-reactivity. Since there were no antibodies against M.

plutonius available, we have prepared them in-house. Purified bacterial cell wall fraction was

prepared for rabbit immunization. After 45 days, rabbit blood was collected and serum was

prepared. Subsequently, the immunoglobulin G fraction was separated from the serum by

liquid chromatography with protein G column. The final antibody in PBS was stored at

−30 °C for further use.

Functionality of the prepared antibodies was verified using enzyme-linked immunosorbent

assay (ELISA). The sandwich assay provided a limit of detection (LOD) of

1.4×105 CFU·mL

−1. The ELISA was used to detect M. plutonius in real samples of bees,

larvae and bottom hive debris, which are the matrices where this bacterium is typically

present in the case of EFB infection [3].

For the electrochemical biosensing, the anti-Melissococcus antibody was immobilized on the

surface of SPE and allowed specific capture of bacteria. Non-specific binding was evaluated

by incubating the sensor with Paenibacillus alvei instead of M. plutonius. The label-free EIS

allowed to detect M. plutonius, however, the level of non-specific binding was very high,

which was limiting for real samples analysis. Thus, better performance was obtained with

amperometric sandwich assay, where the antibodies were conjugated with horseradish

peroxidase (HRP). The Ab-HRP conjugate was binding to surface-captured immunocomplex

and provided oxidation of 3,3´,5,5´-tetramethylbenzidine (TMB) in presence of H2O2. The

electrochemical detection of current was based on the reduction of the enzymatically oxidized

TMB on working electrode. For pure bacterial culture in buffer, the LOD was

6.6×104 CFU·mL

−1. After optimization of amperometric immunosensor, real samples of bees


25

and larvae were tested with LODs 2.4×105 CFU·mL

−1 and 7.0×10

5 CFU·mL

−1, respectively.

Time of analysis was only 2 hours compared to time-consuming laboratory assays such as

ELISA [4]. The analysis of real bee and larvae samples confirmed the suitability of the

developed immunosensor for in-field M. plutonius detection.

Figure: (A) EIS response of antibody-modified SPE electrode to M. plutonius; (B) detection of M. plutonius

based on amperometry in presence of H2O2 after addition of TMB. The bacteria concentrations are expressed in

CFU·mL−1

.

ACKNOWLEDGEMENTS

We thank Dr. Martin Faldyna and Dr. Lubomír Janda from the Veterinary Research Institute

(Brno, Czech Republic) for collaboration on the development of polyclonal antibodies. This

research has been financially supported by the Technology Agency of the Czech Republic

(project TJ01000386) and Ministry of Education, Youth and Sports of the Czech Republic

under the project CEITEC 2020 (LQ1601).

REFERENCES

[1] Bailey L., Collins M. D.: J. Appl. Bacteriol., 53 (1982), 215–217

[2] Mistry K.K., Layek K., Chell T.N., Chaudhuri C.R., Saha H.: Anal. Methods, 8 (2016), 3096–3101

[3] Poláchová V., Pastucha M., Mikušová Z., Mickert M. J., Hlaváček A., Gorris H. H., Skládal P., Farka Z.:

Nanoscale, 11 (2019), 8343–8351

[4] Mikušová Z., Farka Z., Pastucha M., Poláchová V., Obořilová R., Skládal P.: Electroanalysis, 2019,

submitted.


26

STUDY OF CHARGE TRANSFER AND CHARGE TRANSPORT

MECHANISM IN EXPANDED PYRIDINIUM MOLECULES

Štěpánka NOVÁKOVÁ LACHMANOVÁ1*

, Jakub ŠEBERA1, Jindřich GASIOR

1, Gábor

MÉSZÁROS2, Grégory DUPEYRE

3, Philippe P. LAINÉ

3, Magdaléna HROMADOVÁ

1

1 J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, 182 23

Prague 8, Czech Republic

2 Research Centre for Natural Sciences, HAS, Magyar Tudósok krt. 2, H-1117, Budapest, Hungary

3 Univ Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR CNRS 7086, 15 rue J-A de Baïf, 75013, Paris,

France

*[email protected]

Development of the daily used electronic devices is limited by the miniaturization of single

electronic components. One of the possibilities, how to reach even smaller component size, is

the exchange of traditional silicon-based technologies by the specialized molecules.

Such molecules could serve as molecular wires, diodes or switches [1].

Expanded pyridinium derivatives rank among the promising candidates for the purpose

of molecular electronics. Expanded pyridinium molecules are due to their electroactivity and

relatively high single molecule conductance suitable model compounds for the study of

correlation between the characteristics of electron transfer (electrochemical activity) and

electron transport (single molecule conductance). Even though the linear dependence between

the charge transfer heterogeneous rate constant and conductance was predicted by theoretical

works, [2, 3] the conclusive experimental confirmation is missing [4, 5].

Electron transfer mechanism of four expanded pyridinium derivatives (for structure see figure

below) were studied by various electrochemical techniques. Compounds 1 and 3 are reduced

by two electrons in two separate one-electron steps. The charge transfer rate constants of both

reduction steps were obtained and higher values were observed for the first electron transfer.

On the other hand, only one two-electron signal was obtained for molecules 2 and 4. Only the

charge transfer rate constant for the first electron transfer was measurable by the used

methods. The contrast between the reduction mechanism is caused by the ability of the

molecules to undergo the structural changes after the first electron reduction [6, 7].

The presence of pyridyl anchoring groups enabled the single molecule conductance

measurements by the scanning tunneling microscopy break junction (STM BJ) technique.

Statistical analysis of measured conductance curves showed the highest conductance value for

the molecule 1 [6].

Figure: Chemical structure of compounds 1 to 4.


27

The obtained values of charge transfer rate constants for the first electron reduction were

ploted against the values of single molecule conductance. The linear dependence was

observed for the molecules 1, 2 and 4. The obtained correlation experimentally confirms the

theoretical relationship of charge transfer and charge transport characteristics [2, 3].

The compound 3 deviated from this dependence. Considerably higher value of charge transfer

rate constant was observed. The electrochemical behaviour of compound 3 is influenced by

the locked conformation caused by the chemical structure of molecule 3.

ACKNOWLEDGEMENT

The work has been supported by the Czech Science Foundation (18-04682S).

REFERENCES

[1] Metzger R. M.: Nanoscale, 10 (2018), 10316-10332

[2] Nitzan A.: Isr. J. Chem. 42 (2002), 163-166

[3] Berlin Y.-A.; Ratner M. A.: Radiat. Phys. Chem. 74 (2005), 124–131

[4] Venkatramani R.; Wierzbinski E.; Waldeck D. H.; Beratan D. N.: Faraday Discuss. 174 (2014), 174, 57–78

[5] Zhou X. S.; Liu L.; Fortgang P.; Lefevre A. S.; Serra-Muns A.; Raouafi N.; Amatore C.; Mao B. W.;

Maisonhaute E.; Schöllhorn B.: J. Am. Chem. Soc. 133 (2011), 7509–7516

[6] Nováková Lachmanová Š.; Šebera J.; Kolivoška V.; Gasior J.; Mészáros G.; Dupeyre G.; Lainé P. P.;

Hromadová M.: Elchim. Acta 264 (2018), 301–311

[7] Lachmanová Š.; Dupeyre G.; Tarábek J.; Ochsenbein P.; Perruchot C.; Ciofini I.; Hromadová M.; Pospíšil

L.; Lainé P. P.: J. Am. Chem. Soc. 137 (2015), 11349–11364


28

BIOPHYSICAL ANALYSIS OF SILVER NANOPARTICLES AND USE

THEIR ANTIMICROBIAL ACTIVITY IN 3D PRINTING

Karel SEHNAL1,3*

, Martina STANKOVA2, Michaela DOCEKALOVÁ

2,3, Zuzana

TOTHOVA2, Dagmar UHLIROVA

2, Branislav RUTTKAY-NEDECKÝ

1,3, Augustine

Enakpodia OFOMAJA4, Marta KEPINSKA

5, Rene KIZEK

1,2,3

1 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and

Pharmaceutical Sciences Brno, Palackeho 1946/1, 612 42 Brno, Czech Republic

2 Department of Research and Development, Prevention Medicals, Tovární 342, 742 1, Czech Republic

3 Institute of Viticulture and Wine Production, Faculty of Horticulture, Valtická 337,691 44 Lednice, Czech

Republic

4 Biosorption and Wastewater Treatment Research Laboratory, Department of Chemistry, Faculty of Applied

and Computer Sciences, Vaal University of Technology, P. Bag X021, Vanderbijlpark, 1900, South Africa,

5 Department of Biomedical and Environmental Analyses, Faculty of Pharmacy with Division of Laboratory

Medicine, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland

*[email protected]

In the 21st century there is a steady increase in the resistance of microorganisms to antibiotics.

Poorly understood mechanisms have remained a major problem. Prevention of these

infections is not easy [1]. According to the NIH (National Institutes of Health), 99,987 people

die in the United States as a result of these infections which translates to an average of 271

deaths per day, accounting for more loss of human lives than from HIV AIDS, car accidents

and breast cancer put together [2, 3]. Metal nanoparticles have shown significant antibacterial

activity similar to the plant/animal materials used in traditional medicine. The study focuses

on the preparation of silver nanoparticles (AgNPs) by green synthesis which utilizes plant

extracts as reducing agents and their use in 3D printing.

AgNPs obtained by this type of synthesis contain biomolecules bound to their surface. AgNPs

and used plant extracts were studied in detail by physico-chemical methods (Table). A

concentration of total protein in plant extract from S. officinalis prepared at different

temperatures was determined by both biuret (BM) and pyrrogallol red (PM) methods. We also

optimized a methodology for measuring of flavones using aluminium method. For

determination of fenolic compounds Folin Ciocalteatu method was used. Antioxidant activity

was measured using both ABTS (2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid) and

DPPH ((N,N-dimethyl)-1,4-diaminobenzene) method.

Dried plant homogenized mixture was stirred in water at different temperatures (20, 40, 60,

80 °C) for 60 minutes in a ratio of 1:20 followed by centrifugation (15 min, 4000 g). The

extract was mixed with 0.1 M AgNO3 (1:1), and the prepared nanoparticles were purified with

methanol (1:1). After precipitation, methanol was removed and the AgNPs were dried. In

addition, the purified AgNPs were dispersed in 18 MΩ of water and acetone (1:1, c = 3

mg/mL). AgNPs in this form were applied to the 3D fiber by means of a brush and a material

was printed (Figure).


29

Silver nanoparticles have pronounced antibacterial properties. However, there is a concern

about their persistence in the environment. An alternative is the surface treatment by

biomolecules form plant extracts of such nanoparticles. We created unique material modified

by AgNPs which has a potential antimicrobial activity. This material can be use in hospitals to

reduce the number of infected patients by nosocomial infections.

Figure: Optimalization of a methodology of a 3D filament modification. A) printed material without AgNPs, B)

printed material with AgNPs (c = 3 mg/ml), C) printed material with AgNPs (c = 12 mg/ml), D) nonmodified

filament E) modified filament by AgNPs (l filament = 40 cm; V AgNPs = 2 ml), F) modified filament by AgNPs (l

filament = 120 cm; V AgNPs = 6 ml).

Table: Characterization of plant extracts from S.officinalis by physico-chemical methods.

Plant extract

Color

scheme

Total

protein

(BM)

Total

protein

(PM)

Fenolic

compounds Flavones

Antioxidant

activity

ABTS

Antioxidant

activity

DPPH

Recovery

(D) (g/l) (g/l) (g/l) (g/l) (GA g/l) (GA g/l) (%)

Extraction

temperature 20 °C

S. officinalis (S) 0.27±0.01 93.5±18.3 115.6±2.7 702.9±12.5 1.9±0.05 861±13.5 10.5±2.7 146.4

Extraction

temperature 40 °C

S. officinalis (S) 0.27±0.01 89.6±26.7 60.1±1.0 763.2±5.7 2.2±0.05 805±13.5 9.4±2.7 133.6

Extraction

temperature 60 °C

S. officinalis (S) 0.30±0.01 91.5±26.4 63.8±2.1 736.2±73.9 2.4±0.05 733±13.5 9.5±2.7 134.6

Extraction

temperature 80 °C

S. officinalis (S) 0.31±0.01 81.6±15.3 33.6±1.9 1046.1±49.5 2.6±0.06 588±13.5 8.1±2.7 115.8

ACKNOWLEDGEMENT

The work has been supported by H2020 CA COST Action CA15114, and INTER-COST

LTC18002.

REFERENCES

[1] Bailey, L. and M.D. Collins, Reclassification of Streptococcus pluton (White) in a new genus

Melissococcus, as Melissococcus pluton nom. rev.; comb. nov. J. Appl. Bacteriol., 1982. 53(2): p. 215-

217.

[2] Heger, Z., et al., A Novel Insight into the Cardiotoxicity of Antineoplastic Drug Doxorubicin. Int. J. Mol.

Sci., 2013. 14(11): p. 21629-21646.

[3] Arora, A. and E.M. Scholar, Role of tyrosine kinase inhibitors in cancer therapy. J. Pharmacol. Exp.

Ther., 2005. 315(3): p. 971-979.


30

[4] Reibenwein, J. and M. Krainer, Targeting signaling pathways in ovarian cancer. Expert Opin. Ther.

Targets, 2008. 12(3): p. 353-365.

[5] Meyer, M.R., New psychoactive substances: an overview on recent publications on their toxicodynamics

and toxicokinetics. Archives of Toxicology, 2016. 90(10): p. 2421-2444.

[6] Wintermeyer, A., et al., In vitro phase I metabolism of the synthetic cannabimimetic JWH-018.

Analytical and Bioanalytical Chemistry, 2010. 398(5): p. 2141-2153.

[7] Grigoryev, A., et al., Gas and liquid chromatography–mass spectrometry studies on the metabolism of

the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking

mixtures. Journal of Chromatography B, 2011. 879(25): p. 2519-2526.

[8] Fujita, K., Cytochrome P450 and anticancer drugs. Curr. Drug Metab., 2006. 7(1): p. 23-37.

[9] He, D.D., et al., Structural characterization of encapsulated ferritin provides insight into iron storage in

bacterial nanocompartments. Elife, 2016. 5.


31

POLYMER NANOSPHERE-ASSEMBLED SURFACE FOR

BIOSENSING BASED ON ELECTROCHEMICAL IMPEDANCE

SPECTROSCOPY

Jakub SOPOUŠEK1*

, Karel LACINA1

1 Central European Institute of Technology, Masaryk University, Kamenice 753/5 625 00, Brno, Czech

Republic

*[email protected]

Electrochemical impedance spectroscopy (EIS) emerges as the promising method for

biosensing due to its label-free detection capabilities together with the high sensitivity. But

despite all the praise, EIS has difficulties in differentiation of particular (bio)chemical

processes as the output signal is affected by impedance of a bulk and by impedance at the

solution/electrode interface. Moreover, bio-related surfaces are very complex, and it is thus

highly challenging to design a reliable biosensing system where only an analyte generates

measurable signal specifically. We develop an analyte-sensitive surface that provides

dominant output signal changes upon interaction. Its modification layer consists mainly of

spherical polystyrene nanoparticles, homopolymer poly-L-lysine, and protein human serum

albumin (HSA). Immobilized HSA protein specifically interacts with the analyte, anti-HSA

antibody present in a liquid sample. The biosensing system is designed in a way that it

contains the assembly of spherical nanoparticles forming thus specifically large nanopores. As

the nanoparticles are coated with the protein ligand (HSA), an interaction with specific

antibodies results in the blockage of nanopores. This effect results in radical changes of

impedance as the analytical response comes from differentiation between permeable and

isolated state of the modification layer.

Such surfaces can be applied for testing of blood sera to detect immunological response of a

patient’s pathologic state – antigen will be immobilized onto the assembly of nanoparticles

and the specific antibodies, present in patient’s blood, will be detected.

ACKNOWLEDGEMENT

This research has been financially supported by the Ministry of Education, Youth and Sports

of the Czech Republic under the project CEITEC 2020 (LQ1601).


32

ELECTROCHEMICAL DETERMINATION OF INSULIN ON NiONPS

MODIFIED CARBON ELECTRODES

Ivana ŠIŠOLÁKOVÁ1*

, Jana HOVANCOVÁ1, Renáta ORIŇAKOVÁ

1, Libuše TRNKOVÁ

2,

Andrej ORIŇAK1

1 Department of Physical Chemistry, Faculty of Science, Pavol Jozef Šafárik University, Moyzesova 11, 040 01

Košice, Slovak Republic

2Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech

Republic

*[email protected]

Diabetes mellitus can be considered as the one of the most widespread disease in the world

[1]. Thus a construction of highly sensitive, small, rapid, selective, accurate and cost-effective

electrochemical sensor for glucose or insulin determination is called for [2]. This work deals

with electrochemical determination of insulin on nanomodified screen printed carbon

electrodes (SPCEs). Mentioned electrodes have significantly smaller size of working

electrode (4 mm in diameter) compared to classic electrodes, achieving miniaturization of the

system. SPCEs were modified by combination of multi walled carbon nanotubes (MWCNTs),

chitosan and nickel oxide nanoparticles (NiONPs). NiONPs were used because of high

electrical conductivity, low cost and excellent catalytic activity towards insulin oxidation

because of the presence of NiO(OH) active species formed in alkaline solution. The

correlation coefficient, limit of detection and sensitivity of prepared electrode was determined

and compared to unmodified SPCE. NiONPs/chitosan-MWCNTs/SPCE displayed wide linear

concentration range for insulin determination (0.25 µM – 0.5 µM) with R2 = 0.997, low limit

of detection (94 nm) and excellent sensitivity of 0.021 µA/µM. Having in mind the possible

interaction of substances that can be contained in real samples, the influence of interferences

(sucrose, glucose, ascorbic acid and lactic acid) on insulin determination at NiONPs/chitosan-

MWCNTs/SPCE was studied. The cyclic voltammograms of above mentioned compounds

did not show any measurable interference in insulin detection. Due to the small size and

appropriate analytical characteristics, NiONPs/chitosan-MWCNTs/SPCE can be considered

as the suitable candidate for electrochemical determination of insulin in real samples.

Figure: Prepration of NiONPs/chitosan-MWCNTs/SPCE


33

ACKNOWLEDGEMENT

The work has been supported by the projects VEGA 1/0074/17 of the Slovak Scientific Grant

Agency, APVV-16-0029 of the Slovak Research and Development Agency, VVGS-PF-2018-

795 and VVGS-PF-2018-794 of Pavol Jozef Šafárik University in Košice.

REFERENCES

[1] Kanaka-Gantenbein C., Mastorakos G., Chrousos G.P., Ann. N. Y. Acad. Sci., 997 (2003) 150–157

[2] Prasad B.B., Madhuri R., Tiwari M.P., Sharma P.S., Electrochim. Acta. 55 (2010) 9146–9156.


34

AMINOFERROCENE: DETERMINATION OF THE ACIDITY OF

UNSTABLE COMPOUNDS

Jakub VĚŽNÍK1,2*

, Libuše TRNKOVÁ1, Karel LACINA

2

1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

2 Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

*[email protected]

Ferrocenes are quite popular in electrochemistry, due to the simplicity of redox reactions of

iron core. While ferrocene is nonpolar and thus of limited use, its cyclopentadienyl ring opens

way for many interesting modifications. One of which is aminoferrocene (FcNH2), the -NH2

group increases solubility in polar solvents, introduces chemical reactivity and most

importantly pH sensitivity to the ferrocene complex.

Due to the proximity of NH2 group and Fe2+

ion in FcNH2, the redox potential of FcNH2 is

influenced by protonation and vice versa the Fe3+

ion changes acidity of the complex upon

oxidation. Electrochemical observation of this interplay can be used to deduce the acidity of

the Fe3+

complex. Such approach is useful for complexes that are unstable in their oxidized

form.

pKa constants determined using electrochemical methods are obtained through kinetic

processes, therefore great care needs to be taken when interpreting these results. While the

limiting redox potential for FcNH2 is easily obtained from electrochemical results (Figure),

the limiting potential for FcNH3+ strongly varies depending on the type of electrode used.

This is anomalous from other ferrocene derivates, namely ferroceneboronic acid [1] and

ferrocenecarboxylic acid [2], where both limiting redox potentials are easily obtainable.

In our contribution, we tried to uncover the correct pKa value of aminoferrocene through

different electrochemical methods both in buffered and unbuffered solutions.

Spectrophotometry was used along to validate our findings.


35

Figure: Redox potential of 1 mM aminoferrocene (FcNH2) in dependence on pH. Standard three electrode setup

was used with different working electrodes: Gold, platinum, glassy carbon and boron doped diamond (BDD).

The intercepts of the slope and the limiting values of redox potential equates to pKa of FcNH2 (pKa = 5.8).

Determination of pKa of FcNH3+ was affected by the type of working electrode (glassy carbon and boron doped

diamond) and was not obtainable for gold and platinum electrodes.

ACKNOWLEDGEMENT

This research has been financially supported by the project CEITEC 2020 (LQ1601),

MUNI/A/1359/2018 and by the Czech Science Foundation, grant nr. 19-16273Y.

REFERENCES

[1] Moore A.N.J., Wayner D.D.M.: Can. J. Chem., 77 (1999) 681–686

[2] De Santis G, Fabbrizzi L, Licchelli M, Pallavicini P: Inorganica Chim. Acta, 225 (1994) 239–244.


36

EFFECT OF NEWLY DESIGNED STABLE CFTR-MESSENGER RNA

ON TRANSFECTION OF HUMAN CYSTIC FIBROSIS

AIRWAY EPITHELIUM

Lucie BOŘEK-DOHALSKÁ1*, Tomáš KOBLAS

2, Kateřina PECKOVÁ

1, Jan KRÁL

1, Marie

STIBOROVÁ1, Pavel DŘEVÍNEK

3, Petr HODEK

1

1 Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 40 Prague 2,

Czech Republic

2 Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, 140 21 Prague 4, Czech Republic

3 Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University and University Hospital

Motol, V Úvalu 84, 150 06 Prague 5, Czech Republic

* [email protected]

Cystic fibrosis (CF) is the most frequent lethal autosomal recessive disease within the

Caucasian population. This disorder results from mutations in the gene for “cystic fibrosis

transmembrane conductance regulator” (CFTR). The F508del mutation of CFTR allele gives

raise a misfolded protein causing functional abnormalities in chloride ion secretion in the

apical membrane of epithelial cells. The major clinical manifestation of the CF disorder

occurs in respiratory tract. Thick and sticky mucus blocks the airways. Consequently, the

ability for clearance of microorganisms is impaired. Thus, airways of CF patients are

susceptible to chronic microbial infections predominantly with Pseudomonas aeruginosa and

Burkholderia cepacia complex which significantly contribute to morbidity and mortality in

CF patients. The gene therapy seems to be a promising way for CF patients. Attempts for the

gene replacement in clinical applications are focused on mRNA approaches as the mRNA

transfection is effective without need of reaching the nucleus [1]. In addition, using this

approach the risk of mutagenesis does not need to be considered.

The aim of our study was to synthesize a stable CFTR mRNA containing 3'-0-Me-

m7G(5')ppp(5')G cap (ARCA cap), 25% pseudouridine triphosphate, 25% 5-methylcytidine

triphosphate and a 200poly(A) tail. This ARCA cap should allow better in vitro translation of

mRNA than that with a standard cap analog. In order to study the delivery and expression of

an introduced gene, human airway cells with the CF genetic disorder, CuFi, and airway cells

derived from a healthy subject, NuLi, were employed. The modified mRNA construct

containing a marker gene coding for GFP in a combination with Lipofectamine™

MessengerMAX™ was initially used for the cell transfection. The production of GFP was

detected by immunofluorescence assay and Western blotting. Then, the cells were transfected

with CFTR mRNA construc and the CFTR expression at the protein level was assayed using

immunostaining techniques. The CFTR function, as a chloride channel, was studied using a

fluorescent probe MQAE sensitive to halides [2]. Finally, the effect of CFTR mRNA

transfection on the adhesion of P. aeruginosa (ST 966) on CF cells was assessed in a

fluorescent adherence assay [3].

To prove our concept of human NuLi and CuFi cell transfection the mRNA construct

containing a marker gene coding for GFP was tested. The efficient GFP expression (based on


37

GFP fluorescence) was detected 48 hrs after the cell line treatment. Under optimized

conditions CuFi cells were transfected with CFTR mRNA construct. Immunofluorescence

staining revealed that the levels of total CFTR protein are similar to that detected in non-

transfected NuLi cells. Next, the iodide efflux mediated by CFTR protein was measured in

CuFi cells treated with CFTR mRNA. After 24 hrs from the transfection the iodide efflux was

clearly stimulated by forskolin and reduced by the specific inhibitor CFTRinh-172 to the level

similar in NuLi cells. Finally, the adhesion of P. aeruginosa to CuFi cells after transfection

with CFTR mRNA was significantly lowered compared to untreated CuFi cells.

In conclusion the present data suggest that the transfection of non-polarized CF human

epithelial cells with novel CFTR-mRNA increased expression of the CFTR protein up to the

level of healthy cells and their functional restoration occurred, too. Moreover, transfected

cells are more resistant to adhesion of P. aeruginosa.

ACKNOWLEDGEMENT

The financial support from the grant UNCE 204025/2012 is highly acknowledged.

REFERENCES

[1] Bangel-Ruland N, Tomczak K, Fernández Fernández E, Leier G, Leciejewski B, Rudolph C., Rosenecker J,

Weber W.M.: J. Gene Med, 15 (2013), 414-26.

[2] Mahlangu D.A., Dix J.A.:Anal. Biochem., 325 (2004), 28-34.

[3] Nosková L., Kubíčková B., Vašková L., Bláhová B., Wimmerová M., Stiborová M., Hodek P.: Sensors

(Basel), 15 (2015), 1945-53.


38

ITP ANALYSIS OF SWEET BEVERAGES AND BEERS

Sandra BUGDOLOVÁ1*

, Michaela BAHELKOVÁ1, Přemysl LUBAL

1, Marta FARKOVÁ

1


*[email protected]

Isotachophoresis (ITP) is a modern analytical method which provides information about

qualitative and quantitative composition of the sample [1]. The sample is injected between

leading (LE) and terminating (TE) electrolyte which ions have higher (LE) and lower (TE)

mobility than all ions present in the sample. When the voltage is applied on inert Pt electrodes

placed in solution, the ions are separated into individual zones according to their decreasing

electrophoretic mobility[2,3].

A B

Figure: ITP records of selected drinks: Sprite light beverage (A), beer “Staropramen Smíchov” (B)

The ITP method was applied for analysis of samples of sweet beverages, alcoholic and non-

alcoholic beers when the content of artificial sweeteners (e.g. acesulfame, cyclamate,

saccharin) and some anions (e.g. sulphate, malate, succinate, citrate, phosphate) was

determined (see Fig. 1). The analyses were carried out on electrophoretic analyzer EA 102

(Villa Labeco, Slovakia) with conductivity detector.

ACKNOWLEDGEMENT

The work has been supported by Masaryk University (MUNI/A/1359/2018).

REFERENCES

[1] Křivánková L, Herrmannová M, Bartoš M, Vytřas K.: J. Sep. Sci., 29 (2006), 1132–1137.

[2] Boček P, et al.: Analytická kapilární izotachoforéza. Academia, Praha 1987. [3] Wilson I. D. (Ed.): Encyclopedia of Separation Science – ITP chapter. Academic Press, San Diego 2000.

1-cyclamate sodium, 2-saccharin 1-sulphate, 2-malate, 3-succinate, 4-citrate,

5-phosphate

mailto:[email protected]


39

APOFERRITIN NANOCAGE AS A PROMISING DOXORUBICIN

NANOCARRIER AND ITS EFFECTS ON NEUROBLASTOMA CELL

LINES

Tereza ČERNÁ1,2*

, Radek INDRA1, Katarína VAVROVÁ

1, Jan HRABĚTA

2, Tomáš

ECKSCHLAGER2, Zbyněk HEGER

3, Vojtěch ADAM

3, Marie STIBOROVÁ

1

1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40, Prague, Czech

Republic

2 Department of Pediatric Hematology and Oncology, 2nd

Medical Faculty, Charles University and University

Hospital Motol, V Úvalu 84, 150 06, Prague, Czech Republic

3 Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel

University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic

*[email protected]

Abstract

Although doxorubicin (DOX) is an efficient anticancer agent, it exerts several adverse effects.

One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable

nanocarrier. Apoferritin with encapsulated DOX (APODOX) was prepared and tested.

APODOX was compared with free DOX in field of cytotoxicity, apoptosis, histone

phosphorylation and localization in neuroblastoma UKF-NB-4 cells.

1. INTRODUCTION

Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most common

solid extracranial tumor in children and a major cause of neoplastic death in infancy [1].

Prognosis of high risk neuroblastoma is poor because of gradually developing tumor cell

chemoresistance [2]. Conventional cancer treatment based on cytostatic therapy is highly

toxic not only for cancer cells, but also for normal ones [1-3]. Currently nanocarriers are

promising agents to improve drug therapeutic index, divert ABC-transporter mediated drug

efflux mechanism and selectively target tumor cells [4]. Apoferritin is a protein composed of

24 polypeptide subunits, structurally arranged to create an internal cavity, which is naturally

used for storage of iron ions; but artificially it can be employed for carrying of any molecule

of interest [4-6]. To enhance the targeting ability of apoferritin to cancer cells, it is possible to

modify its surface with antibodies. To evaluate potential application of this technology for

cancer therapy, the aim of this study was to compare the cytotoxic effects of anthracycline

antibiotic doxorubicin encapsulated into apoferritin (APODOX) [5,6] and free doxorubicin

(DOX) on neuroblastoma cells and chemoresistant subline derived from this cell line in vitro.

Moreover, the surface of apoferritin was modified with targeting antibodies against CD133

and GD2, antigens expressed on neuroblastoma cells.

2. MATERIALS AND METHODS

Apoferritin with encapsulated DOXO (APODOX) was prepared [5,6] and its effect on tumour

cells was analysed. The nanocarrier was compared with free doxorubicin in field of

cytotoxicity, apoptosis, histone phosphorylation and localization in cells. Using the Western

blot analysis, expression of TfR 1 and SCARA5 receptors in tested neuroblastoma cells

(UKF-NB-4) was also analysed [5].


40

3. RESULTS AND DISCUSSION

Cell viability was assessed using Alamar Blue assay, the real time impedance based platform

(xCELLigence) and apoptosis rates were measured by flow cytometer using Annexin V and

DAPI staining. DNA double-strand breaks (phosphorylation of H2A.X) and caspase-3

activation were detected by flow cytometry. Subcellular location of cytostatic loaded

apoferritin and free cytostatic was analyzed by confocal microscopy. Here, we have shown

that apoferritin can carry and deliver a high dose of doxorubicin into UKF-NB-4

neuroblastoma cancer cells. The effect of APODOX on sensitive and resistant neuroblastoma

cells is similar to that of free DOX, but percentage of sensitive neuroblastoma cells with DNA

double-strand breaks after APODOX treatment was higher than after free cytostatic. In

addition, apoferritin loaded DOX are cytotoxic in hypoxic conditions (1% O2). Further, using

fluorescence microscopy, we have shown that apoferritin can deliver drugs inside cancer cells

and the drug exerts their effect thereof. Entry of APODOX and free DOX into sensitive and

resistant cells was similar. We suppose that apoferritin is targeted to the several cancer cells

(i.e. neuroblastoma) through TfR 1 and/or SCARA5 which are overexpressed in a

neuroblastoma cell line. To enhance the specificity of APODOX, we tested modification of its

surface with different antibodies (anti-CD133 and anti GD2) targeted to neuroblastoma cells.

The specificity of targeted nanocarrier is dependent on a type and concentrations of

antibodies. The results found in this study seem to be promising, because encapsulation does

not affect toxicity of cytostatic and improves drug stability. Moreover, entry of APODOX is

significantly lower into non-malignant cells than into cancer cells.

4. ACKNOWLEDGEMENT

This work was supported by GACR (17-12816S) and Charles University (GAUK 998217)

5. REFERENCES [1] Brodeur G.M.: Nat Rev Cancer, 3 (2003), 203–216

[2] Maris J.M., et al.: Lancet, 369 (2007), 2106–2120

[3] Cerna T., et al.: Int J Mol Sci. 19 (2018), article E164

[4] Dostalova S., et al.: ACS Appl. Mater. Interfaces 8 (2017), 14430-14441 [5] Indra R., et al., Toxicology 419 (2019), 40-54

[6] Dostalova S., et al..: Scientific Reports, 8 (2018), 1-13


41

ANALYSIS OF METABOLISM AND DNA ADDUCT FORMATION BY

ARISTOLOCHIC ACIDS I AND II IN RATS IN VIVO

Alena DEDIKOVA1, Frantisek BARTA

1, Petr HODEK

1, Jaroslav MRÁZ

2, Šárka

DUŠKOVÁ2, Eva FREI

1, Heinz H. SCHMEISER

3, Volker M. ARLT

4, Marie STIBOROVA

1*

1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2

2 Centre of Occupational Health, The National Institute of Public Health, Srobarova 48, 100 42, Prague 10

3 Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer

Feld 280, 69120 Heidelberg, Germany

4 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, King’s

College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK

*[email protected]

Abstract

A plant extract aristolochic acid (AA) is a mixture of AAI and AAII that causes Aristolochic

acid nephropathy, Balkan endemic nephropathy, and urothelial malignancies. The impact of

exposure of rats to AAI or AAII alone and their combination on metabolism of AA and their

genotoxicity was investigated. The higher formation of the nitroreduction metabolites of AAII

than AAI, aristolactam II (AlacII) than aristolactam I (AlacI), respectively, was found in rats

exposed to AAs. Likewise, the higher levels of AAII- than AAI-derived DNA adducts were

produced. The results demonstrate that induction of NAD(P)H:quinone oxidoreductase

(NQO1) reducing AAs to species forming AA-DNA adducts, which prevails the induction of

cytochromes P450 (CYP) oxidatively detoxifying AA, potentiates AA-derived genotoxicity.

1. INTRODUCTION

Exposure to AA, a natural mixture of plant alkaloids aristolochic acid I (AAI) and II (AAII),

causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), which

are both diseases associated with a high risk of urothelial malignancy (UUC) [1]. Nitro-

reduction of AAs is required to exert their carcinogenic properties (i.e. UUC development)

[1,2]. One of the most efficient enzymes reductively activating AA to species forming AA-

DNA adducts [predominantly 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI) which causes

specific ATTA transversions in the TP53 tumor suppressor gene in tumors from AAN and

BEN patients] is NAD(P)H:quinone oxidoreductase 1 (NQO1) [1,2]. AAI is reductively

activated, too, but also oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by

cytochromes P450 (CYP) 1A1 and 1A2 [2,3]. Besides CYP1A/2, rat CYPs of the 2C

subfamily also oxidize AAI [2,3]. A balance between activation and detoxification reactions

of individual AA components, AAI and AAII, can influence the AA-induced AAN or

BEN/UUC development. Therefore, the aim of this study was to investigate the impact of

exposure of rats to AAI or AAII alone and to combination of both compounds on expression

and activities of enzymes dictating AA metabolism, on formation of individual metabolites of

AAI and AAII and on AA-derived DNA adducts in rats in vivo.


42

2. MATERIAL AND METHODS

Rats were treated with AAI or AAII or AAI together with AAII (a ratio of 1:1). Expression of

enzymes was determined by Western blotting and their activities with marker substrates [2,3].

AA metabolites were determined in urine of rats by HPLC and mass spectrometry. DNA

adducts were analyzed by 32

P-postlabeling [2,3].


Strong induction of NOQ1 activity in liver, up to more than 10-fold, was caused by exposure

of rats to AAs, mainly by exposure to the combination of both compounds. However, the

combined treatment leads to higher NQO1 activity only in the liver, but not in kidney.

Compared to control (untreated) rats, oxidation of Sudan I, 7-ethoxyresorufin-O-deethylase

and 7-methoxyresorfin-O-demethylase, marker activities of CYP1A1, 1A1/2 and 1A2,

respectively, were increased in livers of exposed animals, while they were almost not

influenced in kidney. The diclofenac 4'-hydroxylation reaction, a marker for CYP2C6, was

also elevated by treatment of rats with AAs, while 16-hydroxylation of testosterone, a

marker for CYP2C11, decreased by this treatment. Elevated activities of CYP1A1/2 and 2C6

correlated with an increase in oxidation of AAI to its detoxification metabolite, AAIa. This O-

demethylated metabolite of AAI, AAIa, was found to be the major metabolite of AAI found

in rat urine in vivo, while aristolactam II (AlacII) is the major metabolite of AAII. AlacIa is

formed both from AAI and AAII, being the final metabolite of both compounds. Up to three

AA-derived DNA adducts were formed in liver, kidney and lung of rats; dA-AAI, 7-

(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI), 7-(deoxyadenosin-N

6-yl)-aristolactam II

(dA-AAII) and 7-(deoxyguanosin-N2-yl)-aristolactam II (dG-AAII). The highest levels of

AA-DNA adducts were formed in rat kidney, followed by those in liver and lung. Compared

to rats treated with AAI or AAII alone, the sum of total levels of AA-DNA adducts were

higher in all tested organs of rats exposed to a mixture of AAs. The results demonstrate the

additive effects of exposure of rats to AAI combined with AAII on AA-genotoxicity.

4. ACKNOWLEDGEMENT

The work was supported by GACR (17-12816S).

5. REFERENCES [1] Gökmen M.R., et al.: Annals of Internal Medicine, 158 (2013), 469-477

[2] Stiborova M., et al.: Archives of Toxicology, 90 (2016), 2595-2615

[3] Stiborova M., et al.: International Journal of Molecular Sciences 8 (2017), pii: E2144


43

DIRECT ELECTRODEPOSITION OF SILVER AMALGAM

PARTICLES ON SCREEN PRINTED SILVER ELECTRODES USING

DOUBLE PULSE CHRONOAMPEROMETRY

Pavlína HAVRANOVÁ1*

, Lukáš FOJT1, Aleš DAŇHEL

1

1 Institute of Biophysics of the Czech Academy of Sciences, v.v.i., Královopolská 135, 61265 Brno, Czech

Republic

*[email protected]

Searching for alternatives to mercury electrodes exhibiting an analogous electrochemical

behavior represents an important contribution to the research in electroanalytical chemistry.

Different solid composite electrodes can serve as substitutes for liquid mercury. Silver

amalgam was found to be one of the most suitable alternative electrode material with its

electrochemical behavior very close to mercury electrodes [1,2]. The silver solid amalgam

electrodes (AgSAE) are usually prepared by mixing silver powder with liquid mercury [2].

According to its composition based on ratio of silver and mercury, resulting material can be

liquid (<10% Ag), paste (10 – 15% Ag), solid (15 – 80% Ag), or overamalgamed metal

(>80% Ag), what increase its application variability. However, silver amalgam can be

prepared by its direct electrodeposition on various conductive materials from the solution of

both Ag+ and Hg

2+ ions [3].

This work is focused on the preparation of silver amalgam particles (AgAP) by its direct

electrodeposition on disposable screen printed silver electrodes (SPAgE) using double pulse

chronoamperometry from the solution containing soluble Ag+ and Hg

2+ salts. Different

parameters of the chronoamperometry (pulse and grow potentials and times) and various

Ag/Hg ratios in the solution were optimized during electrodepositions of silver amalgam

particles.

This work confirmed the SPAgE as suitable platform for direct electrodeposition of silver

amalgam particles from small volume of the solution decreasing its consumption.

Simultaneous electro-reduction of silver and mercury ions could be applied for preparation of

nanostructured and sufficiently stable silver amalgam particles with controlled distribution

and surface coverage. Its electroanalytical application was proved by detection of model

organic nitrocompound, 4-nitrophenol.

ACKNOWLEDGEMENT

This work was supported by The Czech Science Foundation (grant 17-23634Y) and a part of

the work was carried out with the support of CEITEC Nano Research Infrastructure (MEYS

CR, 2016–2019).

REFERENCES

[1] Yosypchuk B, Novotny L.: Crit. Rev. Anal. Chem., 32 (2002), 2, 141-151.

[2] Yosypchuk B., Barek J.: Crit. Rev. Anal. Chem. ,39 (2009), 3, 189-203

[3] Danhel A., Ligmajer F., at al.: J. Electroanal. Chem., 821 (2018), 53-59


44

METABOLISM OF TYROSINE KINASE INHIBITOR CABOZANTINIB

BY LIVER MICROSOMES

Radek INDRA1*

, Tomas JURECKA1, Katarina VAVROVA

1, Petr POMPACH

1, Zbynek

HEGER2,3

, Vojtech ADAM2,3

, Marie STIBOROVA1

1Department of Biochemistry, Faculty of Science, Charles University, Albertov 6, 128 00 Prague 2, Czech

Republic

2Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel

University in Brno, Zemedelska 1, 61300 Brno, Czech Republic

3Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00 Brno, Czech

Republic

* [email protected]

Abstract

The metabolism of cabozantinib was studied by microsomes isolated from human livers and

livers of several animal models (rat, rabbit and mice). The metabolites were separated and

identified by LC/MS. Microsomes of all tested animal models generate six metabolites. On

the contrary only three metabolites were generated by human microsomes. The predominant

metabolite with all tested liver microsomes is cabozantinib N-oxide.

1. INTRODUCTION

Cabozantinib is oral drug that was approved by Food and Drug Administration (FDA) and

European Medicines Agency (EMA) for treatment of medullary thyroid cancer in 2012 and

2014, respectively. In 2016, both agencies approved the drug also for treatment of kidney

cancer [1, 2]. It is an inhibitor of tyrosine kinases affecting vascular endothelial growth factor

receptor-2 (VEGFR-2), hepatocyte growth factor receptor (HGFR) and “rearranged during

transfection” (RET) [3]. Although cabozantinib is used for treatment, only limited information

is known about its metabolism and efficiency of its potential metabolites. 17 individual

metabolites were identified after single oral dose of cabozantinib to healthy volunteers in

plasma, urine and faeces, but individual enzymes responsible for their formation are still

mystery [4].

2. MATERIALS AND METHODS

Cabozantinib (50 μM) were incubated with liver microsomes from human livers and livers of

several animal models (rat, rabbit and mice at 37 °C in open tubes. The microsomal protein

concentration was 0.5 mg/ml. After 20 minutes of incubation the reaction was stopped by

ethyl acetate and extraction with this solvent was done. Organic phases of individual samples

were evaporated, dissolved in methanol (50 μl) and analysed on HPLC. Individual

metabolites were separated using C18 column by acetonitrile/acetate buffer mobile phase and

identified by mass spectrometry.


Because only limited information is known about efficiencies of cabozantinib metabolites and

available data indicate their lower efficiency, the knowledge of cabozantinib metabolic


45

pathway is crucial for improvement of treatment and prognoses. Microsomes isolated from

mice, rabbits and rats generate six metabolites. The predominant metabolite was identified as

cabozantinib N-oxide. Other metabolites such as desmethyl cabozantinib,

monohydroxycabozantinib and cabozantinib amide cleavage product were also identified to

be formed. Pre-treatment of rats with inducers of cytochromes P450 (CYP) enzymes

influences the amount of metabolites formed. Inducers of CYPs of a family 1 and CYP2E1

decrease the amount of cabozantinib metabolites, in contrast to an inducer of CYP2B that

slightly increases the amount of formed metabolites. Pregnenolone carbonitrile (PCN, an

inducer of CYP3A) causes a significant increase in cabozantinib metabolism. These results

demonstrate the importance of CYP3A in cabozantinib metabolism. In contrast to animal

samples, human liver microsomes were less efficient in cabozantinib metabolism. Human

microsomes generate only three metabolites. Cabozantinib N-oxide was the predominant

metabolite and the two other metabolites were identified as monohydroxy cabozantinibs. On

the base of activity of individual cytochrome P450 enzymes in human liver microsomes from

single donors, the main isoform responsible for cabozantinib transformation seems to be

CYP3A4. Nevertheless, the identification of individual enzymes and the mechanisms of their

action in the cabozantinib biotransformation need further investigation. Therefore, they are

the subjects of additional studies of our laboratory

4. ACKNOWLEDGEMENT

This work was supported by GACR (18-10251S).

5. REFERENCES [1] Food and Drug Administration (FDA)

[2] European Medicines Agency (EMA)

[3] Song E-K., et al.: International journal of cancer ,136 (2015), 8, 1967-1975

[4] Lacy S., e al.: Drug Metabolism and Disposition, 43 (2015), 8, 1190-1207


46

COMPARISON OF EFFICIENCIES OF PEROXIDASES TO OXIDIZE

THE ANTICANCER DRUG ELLIPTICINE AND THEIR

INFLUENCING BY VANDETANIB, LENVATINIB AND

CABOZANTINIB

Matúš KOLÁRIK1, Radek INDRA

1, Vojtěch ADAM

2, Zbyněk HEGER

2, Marie

STIBOROVÁ1*

1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech

Republic



*[email protected]

Abstract

The plant alkaloid ellipticine and inhibitors of tyrosine kinases (TKIs) vandetanib, lenvatinib

and cabozantinib are efficient drugs suitable for treatment of several cancers, including

tumors of thyroid glands. Here, ellipticine oxidation by several peroxidases was investigated

in detail and the effects of the tested TKIs on this oxidation were evaluated. Horseradish

peroxidase, followed by lactoperoxidase, myeloperoxidase and cyclooxygenase I are capable

of oxidizing ellipticine. Moreover, oxidation of ellipticine by thyreoperoxidase, the enzyme

playing role in thyroid glands, was found for the first time. The TKIs had essentially no effect

on ellipticine oxidation; only cabozantinib stimulates the reaction catalyzed by horseradish

peroxidase, while the ellipticine oxidation by lactoperoxidase is inhibited by lenvatinib.

1. INTRODUCTION

The drugs utilized for cancer chemotherapy have usually a narrow therapeutic index, and

often the produced responses are only palliative as well as unpredictable [2]. The targeted

therapy is directed against cancer-specific targets and signaling pathways and thus provides

more limited nonspecific mechanisms [3]. The most promising drugs to target the cancer cells

are inhibitors of receptor tyrosine kinases (TKIs) [4] and DNA-damaging drugs targeted to

cancer cells due to their metabolism [5-7]. Ellipticine and its derivatives are the DNA-targeted

anticancer agents effective against certain tumors of the thyroid gland (i.e. anaplastic thyroid

carcinoma), ovarian carcinoma, breast cancer and osteolytic breast cancer metastasis [5-7].

TKIs vandetanib, lenvatinib and cabozantinib are inhibitors targeting VEGFR subtypes 1 and

2, EGFR and the RET-tyrosine kinase, thus considered as multiple TKIs. These TKIs have

already been approved for treating patients suffering from thyroid cancer and renal cell

carcinoma [8-10]. In cancer chemotherapy, serious clinical consequences may occur from

small alterations in drug metabolism affecting drug pharmacokinetics. Ellipticine anticancer

efficiencies are dependent on its metabolism leading both to the activation metabolites

causing DNA damage (covalent DNA adducts) and their detoxification to products that are

excreted. Ellipticine is oxidized by cytochromes P450 (CYP) and peroxidases. The CYP

enzymes generate up to five metabolites, 9-hydroxy-, 12-hydroxy-, 13-hydroxy-, 7-

hydroxyellipticine and N2-oxide of ellipticine. 12-Hydroxy-, 13-hydroxyellipticine and


47

ellipticine N2-oxide are metabolites that form reactive species binding to DNA. Peroxidases

mediate formation of ellipticine dimer that is considered as a detoxification metabolite and

ellipticine N2-oxide activating this drug to species generating ellipticine-DNA adducts [5-7].

In contrast to ellipticine, the knowledge on metabolism of TKIs is scarce.


HPLC was utilized for separation of ellipticine metabolites formed by peroxidases.


Oxidation of ellipticine by horseradish peroxidase, lactoperoxidase, myeloperoxidase and

cyclooxygenase I leads to formation of dimer and ellipticine N2-oxide [5-7]. However, the

efficacy of thyreoperoxidase, the enzyme playing a role in proper function of thyroid glands,

to catalyze the reactions has not been investigated, yet. Therefore, the aim of this study was to

investigate this feature. Here, we found for the first time that thyreoperoxidase oxidizes

ellipticine. Its efficiency to oxidize ellipticine is comparable to that of lactoperoxidase,

myeloperoxidase, and cyclooxygenase I in the presence of hematin. A number of studies

testing the effectiveness of individual anticancer drugs alone or in combination with other

cytostatics demonstrated that such combination can have additive and/or even synergistic

effects on treatment regimen. The tested TKIs vandetanib, lenvatinib and cabozantinib had

low, if any, effects on oxidation of ellipticine by peroxidases; only cabozantinib slightly

stimulates oxidation of ellipticine by horseradish peroxidase, while lenvatinib inhibits the

reaction catalyzed by lactoperoxidase. The study might provide a rationale for the clinical

evaluation of the combination of DNA-damaging anticancer drugs and TKIs.

4. ACKNOWLEDGEMENT

The work was supported by GACR (grant 18-10251S).

5. REFERENCES [1] Heger Z., et al.: International Journal of Molelcular Sciences, 14 (2013), 21629-21646

[2] Arora A., et al.: Journal of Pharmacology and Experimental Therapeutics, 315 (2005), 971-979

[3] Reibenwein J., et al.: Expert Opinon on Therapeutic Targets, 12 (2008), 353-365

[4] Hartmann, J.T., et al., Current Drug Metabolism, 10 (2009), 470-481

[5] Stiborova M., et al.: International Journal of Cancer, 120 (2007), 243-251

[6] Stiborová M., et al.: Biochimica et Biophysica Acta, 1814 (2011), 175-185

[7] Stiborova M. and Frei E.: Current Medicinal Chemistry, 21 (2014), 575-591

[8] Greenhill C.: Nature Reviews Endocrinology, 13 (2017), 688

[9] Roviello G., et al.: Expert Opinion on Investigational Drugs, 27 (2018), 507-512

[10] Abdelaziz A., Vaishampayan U.: Expert Review in Anticancer Therapy, 17 (2017), 577-584


48

DEVELOPMENT OF INSTRUMENTATION FOR COULOMETRIC

TITRATIONS

Matěj KUČERA1*

, Jiří VOLÁNEK1, Pavel KRÁSENSKÝ

1, Přemysl LUBAL

1, Zdeněk

FARKA2, Marta FARKOVÁ

1


2CEITEC MU, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

*[email protected]

This contribution is focused on the development and testing of coulometric analyzer, which

was employed for in situ generation of analytical agent. The goal of the project was

construction of a new coulometer, which ensures the exact and precise automatic coulometric

titration.

Coulometry is one of the basic electroanalytical methods. Two main approaches are direct and

indirect coulometry, based on monitoring of the current required to convert the substance of

interest into a precisely defined product (direct), and/or the amount of current required to

produce an equivalent amount of reagent which takes place in the reaction with selected

analyte (indirect). The main advantage of indirect coulometry is the possibility of in situ

generation of reactive unstable ions that may serve as titration agents [1,2].

The newly constructed device is based on an integrated circuit with a connected screen and

electrode connection inputs. The measurement set-up consists of a beaker in which the

coulometric titration takes place, and a pair of generator Pt electrodes connected to a

controllable DC source for coulometric analyzer. Reference and Pt indicator electrodes are

utilized for potentiometric detection of equivalence point.

The titrations in this work are based on oxidization of analyte by iodine, which was

coulometrically generated from alkaline potassium iodide. Other additives in solution were

oxalic acid, which ensures the stability of analyzed ascorbic acid, and starch, which serves as

an indicator forming blue-colored inclusive compounds with iodine.

Firstly, the sample solution of ascorbic acid was titrated and its oxidation over time was

recorded. Oxidation of ascorbic acid occurs due to oxygen in the air, even in solution, where

metal ions have the same effect. All titrations of ascorbic acid for different concentrations

were evaluated by observing the blue color of starch-iodine inclusion complex. However, the

color of titration solution is very weak at the equivalence point, and thus the end of titration is

subjective [3].

Secondly, the purity of sodium thiosulphate was checked using two ways of equivalence point

estimation – (i) again using visual detection with starch, and (ii) by potentiometric indication

using the second-derivative curve [4] (Figure), which allowed to achieve significantly

improved results under the optimized experimental conditions.


49

Figure: The potentiometric titration of thiosulphate by iodine generated by coulometer. (A) Normal record of

titration curve; (B) the second derivation of the titration curve.

ACKNOWLEDGEMENT

This research has been financially supported by the Ministry of Education, Youth and Sports

of the Czech Republic CEITEC 2020 (LQ1601) and by Masaryk University

(MUNI/A/1359/2018).

REFERENCES

[1] Padilla Mercado J. B., Coombs E. M., De Jesus J. P., Bretz S. L., Danielson N. D.: J. Chem. Educ., 95

(2018), 5, 777.

[2] Beilby A. L., Landowski C. A.: J. Chem. Educ., 47 (1970), 3, 238.

[3] Bertotti M., Vaz J. M., Telles R.: J. Chem. Educ., 72 (1995), 5, 445.

[4] Tanaka T., Hayashi H., Komiya Y., Nabekawa H., Hayashi H. Bunseki Kagaku 56 (2007), 5, 327.

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

340 350 360 370 380 390 400

E (V

)

Q (mC)

A

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

360 365 370 375 380 385 dE2

(V

)

Q (mC)

B


50

ON ELECTROCHEMISTRY OF 1-PENTYL-3-(1-NAPHTOYL)INDOLE

AND 1-PENTYL-3-(2-METHOXYPHENYLACETYL)INDOLE

Michaela OBLUKOVÁ1,2

, Romana SOKOLOVÁ1, Radomír ČABALA

2, Ilaria DEGANO

3

1 J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, 182 23

Prague 8, Czech Republic, E-mail: [email protected]

2 Charles University, 1st Faculty of Medicine, Kateřinská 1660/32, 12108 Prague 2

3 Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13, 56124 Pisa, Italy

The electrochemistry properties of 1-pentyl-3-(1-naphtoyl)indole (hereinafter referred to as

“JWH-018”) and 1-pentyl-3-(2-methoxyphenylacetyl)indole (hereinafter referred to as “JWH-

250”) were studied. These compounds belong to heterogenous group of synthetic

cannabinoids, which have been appeared in the drug market recently. These substances are

abused for their psychoactive effect and serve as alternative to classical drugs (heroin,

cocaine, ecstasy) due to their legal status [5]. In human organism, JWH-018 and JWH-250 are

rapidly and completely metabolized and thus parent compounds are often undetectable. To

detection of these compounds is necessary to know their metabolism pathways [6, 7].

In view of the fact that the electron transfer reactions play an important role in metabolic

pathways, this study is focused on electrochemical investigation of oxidation and reduction of

JWH-018 and JWH-250.

This report is based on cyclic voltammetry, UV/Vis and IR spectroelectrochemistry in non-

aqueous media combined with HPLC-ESI-MS/MS detection of reactions products.

JWH-018 JWH-250

Figure: Chemical structure of JWH-018 and JWH-250

ACKNOWLEDGEMENT

The work has been supported by the Czech Science Foundation (GAČR 19-03160S).

REFERENCES

[1] Meyer MR.: Archives of Toxicology, 90 (2016), 10,2421-44.

[2] Wintermeyer A et al.: Analytical and Bioanalytical Chemistry, 398 (2010), 5, 2141-53.

[3] Grigoryev A et al.: Journal of Chromatography B., 879 (2011), 25, 2519-26.


51

OXIDATION OF A TYROSINE KINASE INHIBITOR VANDETANIB

BY RAT ENZYMATIC SYSTEMS IN VITRO

Marie STIBOROVÁ1*

, Radek INDRA1, Petr POMPACH

1, Zbyněk HEGER

2, Vojtěch

ADAM2


Republic



*[email protected]

Abstract

Oxidation of vandetanib, an inhibitor of tyrosine kinases, by rat enzymatic systems in vitro

was investigated. The metabolites of vandetanib were identified to be N-desmethylvandetanib

and vandetanib N-oxide. Rat CYP2C11 is the most efficient enzyme oxidizing vandetanib to

N-desmethylvandetanib, whereas rat FMO1 and 3 are responsible for generation of

vandetanib N-oxide in rat liver microsomes.

1. INTRODUCTION

The drugs utilized for cancer chemotherapy have usually a narrow therapeutic index, and

often the produced responses are only palliative as well as unpredictable [2]. One of the most

promising drugs are inhibitors of receptor tyrosine kinases (TKIs) [4]. Vandetanib is a TKI

indicated for the treatment of symptomatic or progressive medullary thyroid cancer in patients

with unresectable locally advanced or metastatic disease. In cancer chemotherapy, serious

clinical consequences may occur from small alterations in drug metabolism affecting drug

pharmacokinetics [8]. Although metabolism of vandetanib has been partially studied, its

oxidation by enzymatic systems in detail is still missing [5]. Therefore, the target of this study

was to investigate the metabolism of vandetanib; enzymatic systems of rats, which were

considered to mimic the fate of vandetanib in humans [5], were used as a model for such a

study.


HPLC was utilized for separation of vandetanib metabolites and mass spectroscopy for their

structural characterization. Expression of rat hepatic cytochromes P450 (CYP) and flavin-

containing monooxygenases (FMO) was analyzed by Western blotting.


Rat liver microsomes oxidized vandetanib to N-desmethylvandetanib and vandetanib N-oxide.

Their formation was dependent on NADPH, which serves as cofactor for both POR-mediated

CYP catalysis and FMO-mediated oxidative reactions in liver microsomes. These results

indicate that vandetanib oxidation in hepatic microsomes is mediated by CYPs and/or FMOs.

In rat liver, CYPs of the 2C subfamily were identified to be mainly expressed [6] and

confirmed by Western blotting in our study. Further, FMO1 and 3 were found by Western

blotting to be expressed in rat liver microsomes.


52

In order to identify individual CYPs and/or FMOs oxidizing vandetanib and estimate their

contribution to the oxidation process, three approaches were utilized: (i) use specific inducers

of individual CYPs in a rat model; (ii) use selective CYP and FMO inhibitors in rat

microsomes; and (iii) use recombinant rat CYPs.

Under the experimental conditions used, rat microsomes oxidized vandetanib to N-

desmethylvandetanib most efficiently when those were isolated from livers of rats pretreated

with PCN (rich in CYP3A), followed by those isolated from livers of PB-pretreated rats (rich

in CYP2B and 2C) and those isolated from livers of control (uninduced) rats in which CYP2C

enzymes are highly expressed. Microsomes from BaP-pretreated rats and ethanol-pretreated

rats were also capable of oxidizing vandetanib, but to lesser extent. These findings suggest

that CYPs of the 3A subfamily, followed by those of the 2B/2C subfamilies, might play a role

in vandetanib oxidation to N-desmethylvandetanib. On the contrary, vandetanib N-oxide was

mainly formed by microsomes of control (uninduced) rats. The generation of N-

desmethylvandetanib was attenuated by inhibitors of CYP3A and 2C subfamilies in rat

microsomes, while the FMO inhibitor methimazol decreased the formation of vandetanib N-

oxide in this rat subcellular system (microsomes). These results indicate that CYP3A and/or

2C are mainly responsible for the formation of N-desmethylvandetanib and FMO1 and 3

mainly for the generation of vandetanib N-oxide. Indeed, rat recombinant

CYP2C11>>3A1>3A2 were most efficient to oxidize vandetanib. Based on the results

showing the velocities of vandetanib oxidation to N-desmethylvandetanib in experimental

systems containing recombinant CYP enzymes and the relative amounts of CYP enzymes

expressed in rat livers, the contributions of individual CYPs to this reaction in rat livers were

evaluated. The highest contribution to vandetanib oxidation to N-desmethylvandetanib in rat

livers was attributed to CYP2C11 (~79%), followed by CYP3A (~20.3%).

4. ACKNOWLEDGEMENT


5. REFERENCES

[1] Heger Z., et al.: International Journal of Molecular Sciences, 14 (2013), 21629-21646



[4] Fujita K.: Current Drug Metabolism, 7 (2006), 23-37

[5] Martin P., et al., Clinical Therapeutics, 34 (2012), 221-237

[6] Nedelcheva V. and Gut I.: Xenobiotica, 2 (1994), 1151-1175


53

APPLICATION OF LOW-FIELD 1H NMR SPECTROSCOPY IN

ANALYTICAL CHEMISTRY

Libuše SYCHROVÁ1, Přemysl LUBAL

1*,


*[email protected]

NMR spectroscopy is mostly applied for structural analysis of organic compounds, but less

for quantitative analysis, however, it is absolute analytical method [1]. A benchtop NMR

spectrometer Spinsolve Ultra 60 (Magritek, Germany) is low-field equipment suitable for fast

measurements of 1H NMR spectra of aqueous solutions of compounds because it efficiently

enables to suppress analytical signal of water [2].

This work is focused on testing of this instrumentation for both qualitative and quantitative

analysis of selected analytes. The quantitative analysis of primary alcohols (methanol, ethanol

- see Figure) and polyalcohols (propylene-glycol, 1,3-propanediol, glycerol, ethylene- glycol)

in binary and ternary mixtures with water was carried out The analytical procedure was

verified for determination of alcohols in wine, plum brandy and cooling liquid used in

automobiles.

Figure: NMR spectrum of selected analytes: ethanol (upper), methanol (middle), water (lower)

CH3

CH2

CH3

OH

OH

OH


54

ACKNOWLEDGEMENT


REFERENCES

[1] ROUESSAC, Francis a Annick ROUESSAC. Chemical analysis: modern instrumentation methods and

techniques. Chichester: John Wiley. 2007.

[2] http://www.magritek.com/ Downloaded April 28th,

2019.

http://www.magritek.com/


55

STUDY ON ENCAPSULATION OF LENVATINIB AND ELLIPTICINE

INTO NANOTRANSPORTERS; EXPERIMENTAL AND

THEORETICAL APPROACHES

Paulína TAKÁCSOVÁ1, Radek INDRA

1, Zbyněk HEGER

2, Vojtěch ADAM

2, Ivan

BARVÍK3, Marie STIBOROVÁ

1*


Republic



3 Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles

University, Ke Karlovu 5, 121 16 Prague 2, Czech Republic

*[email protected]

Abstract

Although lenvatinib, a tyrosine kinase inhibitor, is an efficient anticancer drug, it also

produces several adverse effects. One approach to decrease the adverse effects of drugs is

their encapsulation inside a suitable nanocarrier. The procedure for construction of an

apoferritin nanoparticle with encapsulated lenvatinib, similar to that for construction of

apoferritin-ellipticine particles, did not lead to encapsulation of lenvatinib into

apoferritin. Theoretical approaches investigating the encapsulation processes for lenvatinib

and ellipticine into apoferritin nanoparticles explained this phenomenon.

1. INTRODUCTION

The receptor tyrosine kinases (TKs) are the enzymes that selectively phosphorylate the

hydroxyl moieties of tyrosine residues on signal transduction molecules with a phosphate

moiety from adenosine triphosphate [4]. Inhibitors of these enzymes are one of the most

promising anticancer drugs used for treatment of several cancers in a last decade [4].

Lenvatinib, belonging to this group of inhibitors, is used for treatment of certain tumours of

thyroid glands [3] and metastatic renal cell carcinoma [4]. Lenvatinib is an oral multitargeted

TK inhibitor signaling networks implicated in tumor angiogenesis. Ellipticine is another

anticancer drug that exhibits high efficiencies in antineoplastic action [5,6]. DNA damage is

responsible for ellipticine’s biological effects. There are, however, several phenomena that

can cause a limited usage of lenvatinib and ellipticine and/or their limited anticancer

efficiencies [3,6]. Thus, we are aimed to develop efficient and reliable methods for targeted

delivery of these anticancer drugs and to prepare the drugs in forms that exhibit lower side

effects and lead to an increase in their anticancer effects. One of the aims is to develop

nanocarriers containing this drug. Apoferritins, which are responsible for the storage and

transfer of iron [9] can provide the much needed properties of nanocarriers. Indeed, a

nanotransporter with encapsulated ellipticine was prepared and found to be capable of

inhibiting the growth of cancer cells, while is less efficient to healthy cells [9]. The aims of

this study were to prepare nanocarriers based on apoferritin as well as on lipids bearing

lenvatinib. Besides the experimental procedures, theoretical approaches investigating the


56

encapsulation processes for ellipticine and lenvatinib into apoferritin nanoparticles were

utilized.


The procedure for preparation of apoferritin nanoparticles with encapsulated lenvatinib were

essentially the same as for preparation of nanotransporters bearing ellipticine [9]. The

solvents used to dissolve of both drugs were only the difference. Lenvatinib dissolved in

ethylene glycol, ethanol and DMA with ratio of 1:2 with water or ethanol were used.


The theoretical model of ellipticine and lenvatinib interactions with an apoferritin cavity, as

well as the model of their encapsulation obtained by computer modeling indicated that in

contrast to ellipticine, lenvatinib seems not to be suitable for preparation of apoferritin

nanoparticles. The differences in values of pKa of ellipticine (pKa = 7.05) and lenvatinib

(pKa = 5.4) and the experimental conditions used for preparation of nanoparticles of these

drugs were suitable only for preparation of ellipticine-loaded apoferritin nanoparticles.

Ellipticine exists during the procedure of apoferritin nanoparticles predominantly in a

protonated state. Therefore, it easily interacts with the core of apoferritin rich in acidic amino

acid residues, being properly distributed in the nanoparticle. On the contrary, since lenvatinib

occurs in its neutral form during preparation of nanoparticles, it does not properly interact

with a cavity of apoferritin nanoparticle. The neutral molecules of lenvatinib are precipitated

in the particle. The unsuccessful experimental preparation of lenvatinib-loaded apoferritin

nanoparticles confirmed that lenvatinib is not suitable for its preparation. Since the

experimental preparation of apoferritin nanoparticles was not successful, we used liposomes

as lenvatinib nanocarriers in further experiments. Even though the construction of liposome

nanoparticle with encapsulated lenvatinib was successful, the amount of prepared

nanoparticles was very low and, therefore, not relevant for cancer therapy. The results of this

study indicate that the theoretical model elaborated to follow the encapsulation procedure can

serve for screening of potentially suitable drugs before the experimental apoferritin

nanoparticle preparation.

4. ACKNOWLEDGEMENT


5. REFERENCES



[3] Greenhill C.: Nature Reviews Endocrinology, 13, (2017), 688

[4] Roviello G., et al.: Expert Opinion on Investigational Drugs 27 (2018), 507-512

[5] Stiborová M., et al.: Biochimica et Biophysica Acta, 1814 (2011), 175-185

[6] Stiborová M. and Frei E.: Current Medicinal Chemistry, 21 (2014), 575-591

[7] Dostalova S., Cerna T., Hynek D., et al.: ACS Applied Materials and Interfaces, 8 (2016), 14430-14441

[8] Liang MM, Fan KL, Zhou M, et al.: Proceedings of the National Academy of Sciences of the United States

of America, 111 (2014), 14900-14905 [9] Indra R., et al., Toxicology 419 (2019), 40-54


57

METALLOTHIONEIN AND SELENITE IN BRDIČKA REACTION

Libuše TRNKOVÁ1*

, Jan SLAVÍK2, Jaromír HUBÁLEK

2,3


2 Central European Institute of Technology, Brno University of Technology, Purkyňova 123, 616 00 Brno,

Czech Republic

3 Faculty of Electrical Engineering and Communication, Brno University of Technology, Technická 10, 616 00

Brno, Czech Republic

*[email protected]

Metallothionein II (MT II) is a cysteine-rich, low-molecular weight protein with a high

affinity to heavy metals; it is a single-polypeptide chain with the molecular weight 6-7 kDa,

consisting of 61-68 amino acid residues structured into two domains; α-domain with four and

β-domain with three binding sites for bivalent ions (Figure 1a). The higher MT concentration

in tissues reflects a higher amount of toxic heavy metals, oxidative stress, and DNA damage.

The MT II has recently been considered as one of the potential cancer markers (Figure 1b).

Figure 1: a) Metallothionein and b) its functions in tissues of mammals and non-mammals

Selenium (Se) pertains to the essential part of the human diet and a low Se level in the diet is

generally associated with various diseases (e.g., Kashin–Beck disease). Selenium is

incorporated into selenoproteins through selenocysteine having the active selenol group (–

SeH). Selenium in the form of amino acids is suitable for any food supplementation and it can

be easily absorbed compared to inorganic Se, as is sodium selenite (a potential

chemotherapeutic agent).

The aim of this research was the study of interaction of MT II and selenite by means of the

catalytic reaction of hydrogen evolution at a mercury electrode, known under the name of the

Brdička reaction. The Brdička reaction has been widely used for protein analysis, even though

its mechanism was not completely elucidated. In addition, the mechanism of Brdička reaction

may vary from the case to the case. But what matters is that this reaction is usually performed

in a solution of ammonia buffer and Co(NH3)6Cl3 in differential pulse voltammetric mode at

mercury or amalgam electrodes.

We investigated the electrode processes of MT II (rabbit liver metallothionein) at a mercury

electrode in the presence of sodium selenite (Na2SeO3) by using differential pulse

voltammetry. The interaction between MT II and Na2SeO3 was analyzed via the hydrogen

evolution catalytic peaks Cat2.

https://www.sciencedirect.com/topics/chemistry/protein

https://www.sciencedirect.com/topics/chemistry/ammonia

https://www.sciencedirect.com/topics/chemistry/buffer-solution

https://www.sciencedirect.com/topics/chemistry/behavior-as-electrode


58

It was found that with the increasing selenite concentration, cobalt in MT is replaced by

selenium. When concentration of Na2SeO3 increases above the MT binding capacity, only

selenite ions are responsible for Cat2 signals.

Figure 2: The schematic dependence of the Cat2 peak height on the Na2SeO3 concentration of with respect to all

participating components. At lower selenite concentrations both components (Co-MT and Se-MT) contribute to

the processes resulting in the peak Cat2. The concentration of selenite (25 µg/cm3) indicates the binding capacity

of MT for metal ions in this experiment.

We have answered the questions: (i) how selenite participates in Brdička reaction, (ii) which

competitive behavior of selenium against cobalt should be expected and (iii) what is the

sequence of reaction processes in the modified Brdička reaction. Our new interpretation

leading to complete description of the Brdička´s mechanism is presented. Our results can be

helpful in biochemical and clinical studies involving selenium compounds as potential

chemotherapeutics.

ACKNOWLEDGEMENT

The work has been supported by the project CEITEC Nano Research Infrastructure (ID

LM2015041, MEYS CR,2016–2019) and SIX Research Center in the Czech Republic (the

grant LO1401 INWITE is gratefully acknowledged).

REFERENCES

[1] Brdička R.: Collect Czech Chem Commun, 5 (1933) 112.

[2] Heyrovsky J., Kůta J.: Principle of polarography. Publishing House of the CAS, Prague 1965.

[3] Palmiter R.D: Proc Natl Acad Sci USA, 91 (1994) 1219.

[4] Raspor B., Paic M., Erk M.: Talanta, 55 (2001)109 and J. Electroanal. Chem., 503 (2001) 159.

[5] Adam V., Baloun J., Fabrik I., Trnkova L., Kizek R.: Sensors, 8 (2008) 2293.

[6] Trnkova L., Kizek R., Vacek J.: Bioelectrochemistry, 56 (2002) 57.

[7] Ganther H.E.: Biochemistry 7 (1968) 2898 and Biochemistry, 10 (1971) 4089.

[8] Misra S., Boylan M., Selvam A., Spallholz J.E., Björnstedt M.: Nutrients, 7 (2015) 3536.


59

OXIDATION POTENTIALS OF GUANINE SPECIES

Iveta TŘÍSKOVÁ1, Alan LIŠKA

2, Jiří LUDVÍK

2, Libuše TRNKOVÁ

1


2 J. Heyrovsky Institute of Physical Chemistry of the CAS, Dolejškova 3/2155, CZ-182 23 Prague, Czech

Republic

*[email protected]

Guanine (G) belongs to the most easily oxidizable nucleobases which is well known from

various experimental and theoretical studies. Our contribution is directed towards studies, in

which we compare theoretical oxidation potentials with their experimentally measured values

for guanine (G), guanosine (Guo), deoxyguanosine (dGuo), guanosine -5´- monophosphate

(GMP) and 2´- deoxyguanosine -5´- monophosphate (dGMP).

Figure 1: Guanine derivatives G – guanine: 2-amino-1H-purin-6(9H)-one, Guo –guanosine: 2-amino-

1,9-dihydro-9-β-D-ribofuranosyl-6H-purin-6-one, GMP – guanosine-5´-monophosphate.

For determination of experimental oxidation potentials of all G species linear sweep

voltammetry was applied using polymer pencil graphite electrodes (pPeGEs). It was found

that the oxidation process for all studied derivatives is irreversible and strongly pH dependent.

Anodic peak potentials increase in the order G << dGMP < GMP < dGuo < Guo and correlate

well with the calculated thermodynamic redox potentials. For the determination of theoretical

oxidation potentials the structures of G and its derivatives were optimized and the identities of

minima were verified by vibration frequency calculations. Redox equilibria were modeled in

terms of corresponding thermochemical cycles. The changes in free energy were calculated at

DFT level using two different functionals: (a) general purpose B3LYP functional, and (b) a

more specific ωB97X-D functional, both based on a 6-31+G(d) set. Conformity of theoretical

and experimental data for radicals (cationic or neutral, respectively) indicates that the

deprotonation process of G differs from its analogues whereas the oxidation process of all

species takes place on the imidazole ring.


60

Figure 2: The linear sweep voltammograms of G (black), Guo (green full line), GMP (red), dGuo

(green dashed line) and dGMP (red dashed line) on the pPeGE. The scan rate 400 mV/s; the

concentration of G and its derivatives 50 µmol·L-1

; phosphate – acetate buffer (pH 5).

ACKNOWLEDGEMENT

Access to computing and storage facilities owned by parties and projects contributing to the

National Grid Infrastructure MetaCentrum provided under the programme "Projects of Large

Research, Development, and Innovations Infrastructures" (CESNET LM2015042), is greatly

appreciated. The authors A.L. and J.L. are grateful to the institutional support RVO 61388955

and the authors I.T. and L.T. thank Bc. M. Bosakova for her technical assistance in

voltammetric experiments.

REFERENCES

[1] Steenken S.: Chem. Rev., 89 (1989) 503.

[2] Li Q., Batchelor-McAuley C., Compton R.G.: J. Phys. Chem. B, 114 (2010) 7423.

[3] Brett A.M.O., Matysik F.M.: Bioelectrochem. Bioenerg., 42 (1997) 111.

[4] Ferapontova E.E., Electrochim. Acta, 49 (2004) 1751.

[5] Navratil R., Kotzianova A., Halouzka V., Opletal T., Triskova I., Trnkova L., Hrbac J.: J. Electroanal.

Chem., 783 (2016) 152.

[6] Shields G.C., Seybold P.G.: Computational approaches for the prediction of pKa values, CRC Press, 2013.

[7] Becke A.D.: J. Chem. Phys., 98 (1993) 5648.

[8] Chai J.D., Head-Gordon M.: Phys.Chem. Chem. Phys., 10 (2008) 6615.

[9] Frisch M., Trucks G., Schlegel H., Scuseria G., Robb M., Cheeseman J., Scalmani G., Barone V., Petersson

G., Nakatsuji H., Gaussian 16, revision A. 03, Gaussian Inc., Wallingford CT (2016).

[10] Tomasi J., Mennucci B., Cammi R.: Chem. Rev., 105 (2005) 2999.


61

APPLICATION OF CD SPECTROSCOPY IN ANALYTICAL

CHEMISTRY

Anna VACULÍKOVÁ1*

, Marta FARKOVÁ1, Přemysl LUBAL

1


*[email protected]

Many biological compounds exhibit optical activity, which is due to asymmetric carbon on

which four different substituents are attached. This configuration leads to two structurally

different forms of one substance, when both forms have the ability to rotate the plane of

polarized light, one turns to the left side (L-) and the second one to the right one (D-). This

phenomena can be monitored by circular dichroism (CD) spectroscopy when the ellipticity is

measured in the region of absorption band and this parameter can be used for structural

analysis as well as for quantitative determination. However, if L- and D- forms are present in

solution in ratio 1:1, i.e. racemic optically inactive mixture, they does not exhibit any

rotatation.

In our case, the L- and D- forms of tryptophan in aqueous solution were measured The

spectroscopic measurements were done at Jasco J-810 spectropolarimeter (Jasco, Japan) in the

wavelength range of 200-300 nm. CD-spectra of pure L- and D-tryptophan and their racemic

mixture are presented (see Fig. 1). It was demonstrated, that CD spectroscopy can be

employed for quantitative analysis and determination of purity of chiral aminoacid

(Tryptophan).

Figure: CD-spectra of aqueous solution of L- and D-tryptophan and mixure 1:1 (c = 0,05 mM, pH = 4)

ACKNOWLEDGEMENT


-10

-8

-6

-4

-2

0

2

4

6

8

10

200 210 220 230 240 250 260 270 280 290 300

θ (

md

eg

)

λ (nm)

D-tryptofan L-tryptofan Racemic mixture


62

METABOLISM OF THE TYROSINE KINASE INHIBITOR

LENVATINIB BY HUMAN HEPATIC MICROSOMES AND

CYTOCHROMES P450

Katarina VAVROVÁ1, Radek INDRA

1, Petr POMPACH

1, Zbyněk HEGER

2, Vojtěch

ADAM2, Tomáš ECKSCHLAGER

3, Kateřina KOPEČKOVÁ

3, Marie STIBOROVÁ

1*


Republic



3 Department of Pediatric Hematology and Oncology, 2nd Medical Faculty, Charles University and University

Hospital Motol, 150 06 Prague, Czech Republic

*[email protected]

Abstract. Using human hepatic microsomes containing cytochrome P450 (CYP) enzymes

and human recombinant CYPs, the oxidation of the tyrosine kinase inhibitor lenvatinib was

investigated. The metabolites of lenvatinib were found to be O-desmethyllenvatinib,

N-depropylated lenvatinib and the lenvatinib N-oxide. Of all tested human recombinant CYP

enzymes, the CYP1A1, 1A2, 2A6, 2B6, 2C19, 3A4 and 3A5, oxidize lenvatinib to

O-desmethyllenvatinib. CYP2B6, 3A4 and 3A5 are also responsible for oxidation of

lenvatinib to N-descyclopropyllenvatinib, while the only CYP3A4 generates N-oxide of

lenvatinib.

1. INTRODUCTION

The receptor tyrosine kinases (TKs) are the enzymes that selectively phosphorylate the

hydroxyl moieties of tyrosine residues on signal transduction molecules with a phosphate

moiety from adenosine triphosphate [4]. Inhibitors of these enzymes are one of the most

promising anticancer drugs used for treatment of several cancers in a last decade [4].

Lenvatinib is drug that belongs to this group of inhibitors that is used for treatment of certain

tumors of the thyroid gland [3] and metastatic renal cell carcinoma [4]. Lenvatinib is an oral,

multitargeted tyrosine kinase inhibitor (TKI) of vascular endothelial growth factor receptors

(VEGFR1-VEGFR3), fibroblast growth factor receptors (FGFR1-FGFR4), platelet-derived

growth factor receptor (PDGFR)α, rearranged during transfection (RET), and v-kit (KIT)

signaling networks implicated in tumor angiogenesis. Overall, in cancer chemotherapy,

serious clinical consequences may occur from small alterations in drug metabolism affecting

drug pharmacokinetics. Nevertheless, there is only little insight in the metabolism of TKIs at

this point, which is surprising since they are used on a daily basis in hundred thousand of

patients. The target of this study was to investigate the the in vitro metabolism of lenvatinib in

detail; human hepatic microsomes and human recombinant cytochromes P450 (CYPs)

expressed in SupersomesTM

were utilized for such a study.


HPLC was utilized for separation of lenvatinib metabolites formed by enzymatic systems and

mass spectrometry for their structural characterization.


63


Based on preliminary studies using human hepatic microsomes, lenvatinib was suggested to

be oxidized by cytochromes P450 (CYPs), mainly by CYP3A4, to its O-demethylated

metabolite, a desmethylated form of lenvatinib. However, no direct prove of this suggestion

was demonstrated. Therefore, the aim of this study was to investigate the metabolism of

lenvatinib by human microsomal enzymes in vitro in detail.

Two major lenvatinib metabolites, O-desmethyllenvatinib and N-descyclopropyllenvatinib

were formed by incubation of lenvatinib with human hepatic microsomes. The generation of

both these metabolites was attenuated by inhibitors of the CYP1A and 3A subfamilies in these

subcellular systems. These results suggest that CYP3A and 1A are mainly responsible for the

oxidation of lenvatinib in human livers. Of all tested human recombinant CYP enzymes, the

CYP1A1, 1A2, 2A6, 2B6, 2C19, 3A4 and 3A5, oxidize lenvatinib to O-desmethyllenvatinib.

CYP2B6, 3A4 and 3A5 are also responsible for oxidation of lenvatinib to N-descyclopropyl-

lenvatinib. Another metabolite lenvatinib N-oxide, which is a minor oxidation product of

lenvatinib, was generated only by CYP3A4. The presence of cytochrome b5, which serves as

electron donor to CYP enzymes, plays an essential role in lenvatinib oxidation, catalyzed by

the CYP3A4-catalyzed formation of all metabolites. The results found in this study approved

the knowledge showed by the preliminary studies, suggesting that lenvatinib is oxidized to O-

desmethyllenvatinib, N-descyclopropyllenvatinib and lenvatinib N-oxide. Further, they

specified the efficiencies of individual CYPs in lenvatinib oxidation reactions, and

demonstrated an essential role of cytochrome b5 in oxidation of lenvatinib by CYP3A4. The

study of the enzyme kinetics of lenvatinib oxidation by the tested enzymatic systems is under

way in our laboratory.

4. ACKNOWLEDGEMENT


5. REFERENCES



[3] Greenhill C.: Nature Reviews Endocrinology, 13 (2017), 688

[4] Roviello G., et al.: Expert Opinion on Investigational Drugs 27 (2018), 507-512


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80

XIX. Workshop of Biophysical Chemists and Electrochemists

Book of abstracts

Editor: Libuše Trnková

Technical adjustment: Iveta Třísková

Published by Masaryk University, Brno 2019

1st edition

ISBN 978-80-210-9039-6

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