Biophysics, Poster


N.Y. Lekanova, N.I. Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia
M.V.Shirmanova, Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia
I.V. Balalaeva, N.I. Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia
L.G. Klapshina, Nizhny Novgorod Institute of Organometallic Chemistry, Nizhny Novgorod, Russia
E.V. Zagaynova, Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia


Photodynamic therapy (PDT) is based on photosensitizers (PSs), selectively accumulating in pathological tissues and producing cytotoxic singlet oxygen under irradiation. No ideal PS, which would meet all the clinical requirements, has been found so far, therefore development of novel PSs is still actual. The purpose of our study was to test the functional properties of a potential photosensitizer based on biocompatible polymeric nanoparticles, dopped with ytterbium tetracyanotetraphenylporphyrazine (YPz).
Materials and methods
Ytterbium tetracyanotetraphenylporphyrazine (Fig. 1) was synthesized and converted into stable biocompatible water-soluble forms by forming polymeric nanoparticles [1].
Absorption and fluorescence spectra have been obtained for nanoparticles of different composition and fluorescence intensity dependence on polymer type and medium characteristics (water, blood serum, albumin solution) has been investigated. Ytterbium porphyrazine was also tested as the singlet oxygen producer. For 1O2 registration we used its ability to phosphoresce at 1270 nm.
For in vitro research of cellular uptake of the YPz nanoparticles we used SKBR-3 cell line (human breast carcinoma). Cells were incubated with the YPz (40 mkg/ml) for 30 minutes. Intracellular localization of the YPz was studied by confocal laser scanning microscopy.
Whole-body imaging experiment was carried out using fluorescence transilluminative imaging setup with low-frequency modulation and transilluminative configuration of scanning, created at the Institute of Applied Physics of RAS (Russia) [2]. In this setup synchronous scanning of the object in the transilluminative configuration is provided by a single pair of a source and a detector set. As a source of excitation light we employed semiconductor laser at 635 nm. As a detector of fluorescent light we used a high-sensitivity cooled photomultiplier tube Hamamatsu H7422-20. Emission signal was filtered using 685 to 735 nm band-pass filter. Image acquisition time per animal was 3–5 minutes. Previously, serial imaging of the same animal showed that this technique was able to estimate photosensitizer accumulation in transplantable tumor and washout over time in individual animals [3]. Quantification of the fluorescence in the tumor area provided an opportunity to define tumor uptake and retention kinetics.
The experiments were performed on 20 female CBA mice bearing cervical carcinoma. To investigate the YPz pharmacokinetics, the mice were imaged in vivo for 15 min and 1, 2, 3, 4, 6, and 24 hrs following the chemicals administration. The image obtained before injection was used as a control one. For verification of photosensitizer accumulation in tumor tissue, fluorescence was analyzed by standard methods — confocal microscopy and fluorescence spectroscopy ex vivo.
The synthesized YPz and its biocompatible nanoparticles demonstrated light absorption with maximum at 590 nm and strong fluorescence at around 640 nm. A large enhancement of red emission of the nanoparticles in serum and albumin was detected. This effect is supposed to be resulted from their bindings to proteins. A possibility of singlet oxygen generation by the YPz nanoparticles was confirmed. The YPz particles with polyethylene glycol (YPz-PEG) were chosen for testing of biological properties.
In experiments on cell culture we revealed that the YPz-PEG nanoparticles were internalized and accumulated in the tumor cells around a nucleus. We suppose they are localized in lysosomes preferably. Experiments on mice bearing transplanted cervical carcinoma demonstrated rather suitable pharmacokinetics of the YPz-PEG (Fig. 2). 3 hours after the intravenous injection of the YPz-PEG in 10 mg/kg dose it accumulated in the tumor tissue and retained there up to 6 hours. However, tumor selectivity of the potential photosensitizer was quite low. After 2 days complete elimination of the complex from the animal body was not observed. A prolonged (more than 6 days) retention of the YPz-PEG was typical for the tumor. The kinetics of tumor uptake of the YPz-PEG obtained by transillumination imaging in vivo agrees to the data of standard ex vivo methods.
Conclusions and perspectives
In general, the ytterbium porphyrazine nanoparticles are of interest as a photosensitizer for fluorescence diagnostics and/or PDT due to their optical and biomedical characteristics. Hereafter we plan to test other forms of the YPz with chemical modifications in porphyrazine structure as well as nanoparticles of different polymer composition in order to select one with absorption and fluorescence at longer wavelengths and selective accumulation in tumor.
This work was partly supported by the Ministry of Education and Science of the Russian Federation (project #02.740.11.0086), the Russian Foundation for Basic Research (projects ## 11-04-97100, 10-03-90006), and the Program of RAS Presidium “Fundamental Sciences to Medicine”.
1. L.G. Klapshina, W.E. Douglas, I.S. Grigoryev et al., J. Mater. Chem., 2010, 46(44), 8398-8400.
2. I. V Turchin, V.A. Kamensky, V.I. Plehanov et al., J. Biomed. Opt., 2008, 13(4), 041310.
3. M. V. Shirmanova, E.V. Zagaynova, M.A. Sirotkina et al., J. Biomed. Opt., 2010, 15(4), 048004.

Representing author


Ms. Natalia Yurievna Lekanova

N.I. Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia, junior research fellow
Nizhny Novgorod, Russia

Page views: 1489