Anti-EGFR aptameric construct GR20HH for controllable delivery of doxorubicin into glioblastoma cells

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Abstract

This publication describes research on a possibility of controllable delivery of doxorubicin (DOX) into glioblastoma (GB) cells, being inside non-covalent construct with anti-EGFR DNA aptamer by intercalating into artificially created duplex. The construct has been made with previously described DNA aptamer GR20 (46 nucleotides), with 3’-end 18 nucleotides extension (GR20h), which was hybridized with the complementary DNA oligonucleotides (h).

The duplex assembly is effective, the construct GR20hh is stable at 37 ºС, Tm = 59 ºС. DOX is intercalated into the construct. By applying xCelligence Real-Time Cell Analysis (RTCA) combined with self-created data processing, it has been shown that during a treatment of cell culture DOX, inside the non-covalent construct GR20hh – DOX, saves cytotoxic ability, though a kinetics of toxic action of the complex on GB cells is completely different from the kinetics of DOX along.

The unique approach and the data are the bases for a development of both a regulation and a targeting of DOX cytotoxic activity toward specific GB cells.

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About the authors

B. M. Ivanov

Lomonosov Moscow State University

Author for correspondence.
Email: ivanovb661@yandex.ru

Chemistry Department

Russian Federation, Moscow

O. M. Antipova

Lomonosov Moscow State University

Email: ivanovb661@yandex.ru

Chemistry Department

Russian Federation, Moscow

Y. A. Sliman

Burdenko National Medical Research Center of Neurosurgery, Ministry of Healthcare of Russia; Moscow institute of physics and technology, national research university

Email: ivanovb661@yandex.ru
Russian Federation, Moscow; Dolgoprudny

N. S. Samoylenkova

Burdenko National Medical Research Center of Neurosurgery, Ministry of Healthcare of Russia

Email: ivanovb661@yandex.ru
Russian Federation, Moscow

I. N. Pronin

Burdenko National Medical Research Center of Neurosurgery, Ministry of Healthcare of Russia

Email: ivanovb661@yandex.ru
Russian Federation, Moscow

G. V. Pavlova

Burdenko National Medical Research Center of Neurosurgery, Ministry of Healthcare of Russia; Institute of Higher Nervous Activity and Neurophysiology of Russian Academy of Sciences; Sechenov First Moscow State Medical University

Email: ivanovb661@yandex.ru
Russian Federation, Moscow; Moscow; Moscow

A. M. Kopylov

Lomonosov Moscow State University

Email: ivanovb661@yandex.ru

Chemistry Department

Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Spectrophotometric melting of the double-stranded duplex ahh (а), the GR20 aptamer (б), and the construct of the extended GR20h aptamer hybridized with complementary oligonucleotide h (в). Each graph shows the melting curves at 260 nm and their differential appearance (bottom).

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3. Fig. 2. Сhromatogramm of the GR20 aptamer (red line), its extended variant GR20h (blue line), and the construction of the extended GR20h aptamer hybridized with its complementary oligonucleotide h (green line) (а) – Sensogramms of binding of the construction GR20hh with the recombinant EGFR protein (б). Scheme of interaction of immobilized aptamer construction GR20hh with protein EGFR (в).

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4. Fig. 3. Fluorescence emission spectra of DOX (СDOX = 1 μМ) solution upon addition of the GR20 (а) and GR20hh construction (б). Dependence of the intensity of the DOX fluorescence maximum on the ratio of the concentrations of the oligonucleotide and DOX (в).

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5. Fig. 4. Cellular index alteration for cells of the continuous GB cell culture Sus/fP2 when cells were exposed to DOX at concentrations of 0.05 μM and 1 μM (blue and blue lines) and DOX intercalated into the GR20hh complex (orange line): intact Sus/fP2 cells without exposure (gray line). Data in classical representation (а) and the same data in differentiated form, representing the rate of cell growth/death (б).

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6. Fig. 5. Possible secondary structure of aptamer GR20 (a) and GR20hh construction (б).

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