Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells

Aim. To study the dynamics of lipophilic content release from nanocarriers of different types, organic molecular ensembles and inorganic nanoparticles (NPs) in vitro experiments. Methods. Two-channel ratiometric fluorescence detection method based on Forster Resonance Energy Transfer, fluorescent sp...

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Datum:	2014
Hauptverfasser:	Tkacheva, T.N., Yefimova, S.L., Klochkov, V.K., Sorokin, A.V., Malyukin, Yu.V.
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Sprache:	English
Veröffentlicht:	Інститут молекулярної біології і генетики НАН України 2014
Schriftenreihe:	Вiopolymers and Cell
Schlagworte:	Bioorganic Chemistry
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spelling	nasplib_isofts_kiev_ua-123456789-1546412025-02-23T18:29:28Z Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells Динаміка вилучення барвників з наноконтейнерів різного типу в модельних мембранах і живих клітинах Динамика высвобождения красителей из наноконтейнеров различного типа в модельных мембранах и живых клетках Tkacheva, T.N. Yefimova, S.L. Klochkov, V.K. Sorokin, A.V. Malyukin, Yu.V. Bioorganic Chemistry Aim. To study the dynamics of lipophilic content release from nanocarriers of different types, organic molecular ensembles and inorganic nanoparticles (NPs) in vitro experiments. Methods. Two-channel ratiometric fluorescence detection method based on Forster Resonance Energy Transfer, fluorescent spectroscopy and micro-spectroscopy have been used. Results. It has been found that the profiles of lipophilic dyes release from organic nanocarriers (PC liposomes and SDS micelles) and inorganic ones (GdYVO₄:Eu³⁺ and CeO₂ NPs) are well fitted by the first-order reaction kinetics in both model cell membranes and living cells (rat hepatocytes). The dye release constants (K) and half-lives (t1/2) were analyzed. Conclusions. GdYVO₄:Eu³⁺ and CeO₂ NPs have been shown to provide faster lipophilic content release in model cell membranes as compared to PC liposomes. Negatively charged or lipophilic compounds added into nanocarriers can decrease the rate of lipophilic dyes release. Specific interaction of GdYVO₄:Eu³⁺ NPs with rat hepatocytes has been observed. Мета. Вивчення динаміки вилучення ліпофильного вмісту з наноконтейнерів різного типу, органічних молекулярних ансамблів і неорганічних наночастинок (НЧ) в експериментах in vitro. Методи. Двоканальний ратіометричний метод реєстрації інтенсивності флуоресценції із застосуванням безвипромінювального перенесення енергії електронного збудження, метод флуоресцентної спектроскопії і мікроспектроскопії. Результати. Вивільнення ліпофильных барвників з органічних (ліпосоми і міцели) і неорганічних (на основі НЧ GdYVO₄:Eu³⁺ і CeO₂) наноконтейнерів може бути описано кінетичною реакцією першого порядку як у модельних клітинних мембранах, так і в живих клітинах. Отримано константи швидкості вивільнення (K) і час напіввиведення (t1/2) барвників. Висновки. Наноконтейнери на основі НЧ GdYVO₄:Eu³⁺ і CeO₂ забезпечують швидше вивільнення ліпофильного вмісту в модельних клітинних мембранах порівняно з ліпосомами. Проте додавання негативно заряджених або ліпофильних компонент у систему знижує швидкість вивільнення барвників. Виявлено специфічність взаємодії НЧ GdYVO₄:Eu³⁺ з гепатоцитами щурів. Цель. Изучение динамики высвобождения липофильного содержимого из наноконтейнеров различного типа, органических молекулярных ансамблей и неорганический наночастиц (НЧ) в экспериментах in vitro. Методы. Использовали двуканальный ратиометрический метод регистрации интенсивности флуоресценции на основе безызлучательного переноса энергии электронного возбуждения, а также метод флуоресцентной спектроскопии и микро- спектроскопии. Результаты. Выход липофильных красителей из органических (липосомы и мицеллы) и неорганических (на основе НЧ GdYVO₄:Eu³⁺ и CeO₂) наноконтейнеров может быть описан кинетической реакцией первого порядка как в модельных клеточных мембранах, так и в живых клетках. Получены константы скорости высвобождения (K) и время полувыведения (t1/2) красителей. Выводы. Наноконтейнеры на основе НЧ GdYVO₄:Eu³⁺ и CeO₂ обеспечивают более быстрое высвобождение липофильного содержимого в модельных клеточных мембранах по сравнению с липосомами. Однако добавление отрицательно заряженных или липофильных компонент в систему снижаает скорость высвобождения красителей. Обнаружена специфичность взаимодействия НЧ GdYVO₄:Eu³⁺ с гепатоцитами крыс. 2014 Article Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells / T.N. Tkacheva, S.L. Yefimova, V.K. Klochkov, A.V. Sorokin, Yu.V. Malyukin // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 314-320. — Бібліогр.: 24 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008A7 https://nasplib.isofts.kiev.ua/handle/123456789/154641 [547.97:577.115.7-022.532]:576.314 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Bioorganic Chemistry Bioorganic Chemistry
spellingShingle	Bioorganic Chemistry Bioorganic Chemistry Tkacheva, T.N. Yefimova, S.L. Klochkov, V.K. Sorokin, A.V. Malyukin, Yu.V. Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells Вiopolymers and Cell
description	Aim. To study the dynamics of lipophilic content release from nanocarriers of different types, organic molecular ensembles and inorganic nanoparticles (NPs) in vitro experiments. Methods. Two-channel ratiometric fluorescence detection method based on Forster Resonance Energy Transfer, fluorescent spectroscopy and micro-spectroscopy have been used. Results. It has been found that the profiles of lipophilic dyes release from organic nanocarriers (PC liposomes and SDS micelles) and inorganic ones (GdYVO₄:Eu³⁺ and CeO₂ NPs) are well fitted by the first-order reaction kinetics in both model cell membranes and living cells (rat hepatocytes). The dye release constants (K) and half-lives (t1/2) were analyzed. Conclusions. GdYVO₄:Eu³⁺ and CeO₂ NPs have been shown to provide faster lipophilic content release in model cell membranes as compared to PC liposomes. Negatively charged or lipophilic compounds added into nanocarriers can decrease the rate of lipophilic dyes release. Specific interaction of GdYVO₄:Eu³⁺ NPs with rat hepatocytes has been observed.
format	Article
author	Tkacheva, T.N. Yefimova, S.L. Klochkov, V.K. Sorokin, A.V. Malyukin, Yu.V.
author_facet	Tkacheva, T.N. Yefimova, S.L. Klochkov, V.K. Sorokin, A.V. Malyukin, Yu.V.
author_sort	Tkacheva, T.N.
title	Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells
title_short	Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells
title_full	Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells
title_fullStr	Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells
title_full_unstemmed	Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells
title_sort	dynamics of dye release from nanocarriers of different types in model cell membranes and living cells
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2014
topic_facet	Bioorganic Chemistry
url	https://nasplib.isofts.kiev.ua/handle/123456789/154641
citation_txt	Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells / T.N. Tkacheva, S.L. Yefimova, V.K. Klochkov, A.V. Sorokin, Yu.V. Malyukin // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 314-320. — Бібліогр.: 24 назв. — англ.
series	Вiopolymers and Cell
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first_indexed	2025-11-24T10:29:52Z
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fulltext	BIOORGANIC CHEMISTRY UDC [547.97:577.115.7-022.532]:576.314 Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells T. N. Tkacheva, S .L. Yefimova, V. K. Klochkov, A. V. Sorokin, Yu. V. Malyukin Institute for Scintillation Materials NAS of Ukraine 60, Lenin Ave., Kharkiv, Ukraine, 61001 ephimova@isma.kharkov.ua Aim. To study the dynamics of lipophilic content release from nanocarriers of different types, organic molecular ensembles and inorganic nanoparticles (NPs) in vitro experiments. Methods. Two-channel ratiometric fluores- cence detection method based on Forster Resonance Energy Transfer, fluorescent spectroscopy and micro- spectroscopy have been used. Results. It has been found that the profiles of lipophilic dyes release from organic nanocarriers (PC liposomes and SDS micelles) and inorganic ones (GdYVO 4 :Eu 3+ and CeO 2 NPs) are well fitted by the first-order reaction kinetics in both model cell membranes and living cells (rat hepatocytes). The dye release constants (K) and half-lives (t 1/2 ) were analyzed. Conclusions. GdYVO 4 :Eu 3+ and CeO 2 NPs have been shown to provide faster lipophilic content release in model cell membranes as compared to PC liposomes. Ne- gatively charged or lipophilic compounds added into nanocarriers can decrease the rate of lipophilic dyes re- lease. Specific interaction of GdYVO 4 :Eu 3+ NPs with rat hepatocytes has been observed. Keywords: nanocarries, Forster Resonance Energy Transfer, dye release, model cell membranes, living cells. Introduction. Nano-drugs (NDs) are the important pro- ducts of rapidly developing nanotechnologies in bio- logy and medicine field [1–4]. NDs are composed of a nano-scale matrix (carriers, platform) and therapeutic or any other active compounds (diagnostics or imaging agents) encapsulated in a carrier or adsorbed on its sur- face [2, 4, 5]. A nanocarrier serves as a delivery system providing targeted drug delivery (passive and active tar- geting) to the pathological area and their controlled re- lease that increases drastically the efficiency of therapy [1–5]. Such nanomaterials have unique physicochemi- cal properties, such as ultra small size, large surface area to mass ratio, and high reactivity, which can be used to overcome some of the limitations found in traditional therapeutic and diagnostic agents [6]. Today, a variety of nanocarriers such as polymeric micelles, liposomal vesicles, dendrimeres, inorganic nanoparticles, etc. ha- ve been attempted as drug-delivery systems for treat- ment of cancer, some infectious, inherited and incurab- le diseases [1–6]. Recently we have reported the method of GdYVO4:Eu3+ nanoparticles (NPs) synthesis with controlled size and shape [7]. NPs can be obtained as aqueous colloidal solutions which are transparent in incident light and brightly luminescent under the laser excitation with an appropriate wavelength. It was also found that cationic dye molecules interact with negatively charged NPs that causes neutralization of NPs surface charge and pro- vokes the formation of hybrid dye/NPs complexes [8, 9]. Using microspectroscopic technique, we found that spherical GdYVO4:Eu3+ NPs with the average diameter of 2 nm accumulate in rat hepatocytes nuclei in situ and in isolated nuclei of the cells and exhibit tropism to nuc- lear structural components [10]. Our findings are very promising for biomedical applications of orthovanadate NPs as a nano-scale platform for new anti-cancer NDs. In spite of amazing promises of NDs, many challen- ges remain in their clinical applications. One of the ob- stacles is a requirement of a better understanding of the interactions of nanomaterials with biological systems, which will facilitate the engineering of their properties specific to biomedical applications. 314 ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 4. P. 314–320 doi: http://dx.doi.org/10.7124/bc.0008A7 � Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014 Another important aspect in the NDs development is controlling the rate of active compound release from the carrier [11]. In the present paper, we report in vitro study of the dyes/GdYVO4:Eu3+ NPs complexes interaction with mo- del membranes and living cells (rat hepatocytes) and the dye release kinetics using fluorescent spectroscopy and microspectroscopic techniques. For comparison, we used other inorganic NPs (CeO2 NPs) and organic ones (liposome vesicles, surfactant micelles). The dyes used mimic hydrophobic drug molecules. To study the dye release in dynamics, we used a pair of dyes, so-cal- led FRET-pair (Forster Resonance Energy Transfer [12]), DiO and DiI dye molecules, encapsulated in or ad- sorbed on nanocarriers and a �-ratiometry method of fluorescence detection based on fluorescence recording at two wavelengths [13–15]. Materials and methods. Chemicals. Fluorescent hydrophobic dyes 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), 1,1'-dioctadecyl-3,3,3',3'-tetrame thylindocarbocyanine perchlorate (DiI) (Fig. 1), L-�- phosphatidylcholine (PC) from egg yolk and sodium dodecylsulfate (SDS) were purchased from «Sigma-Al- drich» (USA) and used without purification. Chloro- form («Sigma-Aldrich») used to prepare lipid and dye stock solutions was a spectroscopic grade product. DMSO («Sigma-Aldrich») was also of spectroscopic grade. To prepare aqueous solutions of the dyes with na- nocarriers, double distilled water was used. NPs synthesis. The aqueous colloidal solutions of GdYVO4:Eu3+ and CeO2 were synthesized according to the method reported earlier [7, 9]. NPs were characteri- zed using Transmission Electron Microscopy (TEM- 125K electron microscope, «Selmi», Ukraine). Zeta po- tentials were measured using a ZetaPALS/BI-MAS ana- lyzer («Brookhaven Instruments Corp.», USA) opera- ted in phase analysis light scattering mode. In the pre- sent work the spherical GdYVO4:Eu3+ (d = 2 nm; � po- tential –20.94 ± 1.27 mV) and CeO2 (d = 2 nm; � po- tential –24 ± 1.07 mV) NPs were used. Preparation of SDS micelles with DiO and DiI dyes. The concentration of the surfactant in the solutions was 1 �10–2 M. The concentration of the dyes in the water-mi- celles solutions was 2 � 10–5 M. First, stock solution of each dye in chloroform of 1 �10–3 M concentration was prepared. To prepare the solutions for measurements, 3 mg of SDS were mixed in a flask with the required amount of the dye stock solution. After chloroform eva- poration the required amount of double distilled water was added. The solutions were heated to 80 oC to ensu- re the uniform distribution of the components and then cooled to room temperature. Preparation of lipid vesicles with DiO and DiI dyes. Unilamellar PC lipid vesicles containing DiO and DiI dy- es were prepared by the extrusion method [16]. Appro- priate amount of PC (40 mg/ml) and dyes (10–3 M) stock solutions in chloroform was mixed in a flask and dried until complete chloroform evaporation. The thin lipid- dyes film was then hydrated with 2 ml of double distilled water. Final concentration of PC was 1 �10–3 M. The obtai- ned lipid-dyes suspension was finally extruded through 100 nm pore size polycarbonate filter using a mini-extru- der («Avanti Polar Lipids, Inc.», USA). The concentra- tion of each dye in liposomal suspension was 2 �10–5 M. If PC liposomes contain SDS, 10 % of SDS (with respect to PC weight) were added at the stage of lipid- dyes film formation. Preparation of dyes/NPs complexes. Water colloi- dal solutions of GdYVO4:Eu3+ or CeO2 (0.5 g/l) NPs and DiO and DiI dyes (3 �10–3 M) stock solutions in iso- propyl were mixed in a flask. The mixture was careful- ly stirred using a rotary evaporator (Rotavapor R-3, «Buchi», Switzerland) during 1 h to complete evapora- tion of isopropyl. The final concentration of each dye was 2 � 10–5 M. If the complexes contain cholesterol, its solution in isopropyl (0.1 M) was added at the stage of mixture preparation. Cell labeling procedure. Isolated rat hepatocytes from male Wistar rats were obtained by the method des- 315 DYNAMICS OF DYE RELEASE FROM NANOCARRIERS OF DIFFERENT TYPES N + O N O ClO 4 N + N CH 3 CH 3 CH 3 CH 3 ClO4 C18H37 C18H37 - C18H37 C18H37 - A B Fig. 1. Structural formulas of the dyes: A – DiO; B – DiI cribed by Wang after dissociation of the liver with 2 mM EDTA [17]. Cell viability was assessed via the trypan blue exclusion test. The cell viability 95 % and yield of 1.5 � 107 cells g–1 are in good agreement with those pre- viously described [17]. The cell pellets (50 ml 107 cells/ ml) were incubated with suspension of dye-loaded lipo- somes or dyes/NPs complexes (50 ml) in 1 ml of Eagle’s medium with 10 % fetal calf serum at 37 °C for requi- red time intervals. Afterwards the non-bound liposomes or NPs were removed by centrifugation at 500 g and wa- shing-out by adding HBSS (HEPES buffered saline so- lution) buffer (pH 7.4) with 0.1 % BSA. Cell imaging, spectroscopy and microspectroscopy. Fluorescence spectra of the solutions were taken with a spectrofluorimeter Lumina («Thermo Scientific», USA). Cell visualization was carried out using fluorescent mic- roscope Olympus IX 71 supplied with a digital camera Olympus C-5060 with the magnification of �1000 in the conditions of oil immersion. Fluorescence was exci- ted by a xenon lamp 75 W using BP 460–490 and BP 510–550 nm filters to excite DiO and DiI, respectively. To study FRET, BP 460–490 filter was used. Micro- spectroscopy in the area of interest was carried out using spectral detector USB 4000 (Ocean Optics) connected with Olympus IX71. Results and discussion. Nanocarriers interaction with model cell membranes. Several research groups, including ours, use the FRET based methods to in vivo and in vitro study on the release of lipophilic agents from polymeric micelles [18, 19], kinetic and dynamic stability of polymeric micelles [20], liposomal vesicles interaction with cells of different types in dynamics [15]. A FRET pair, DiO as the energy donor and DiI as the energy acceptor, was used for these purposes. When both FRET molecules were encapsulated in one nano- carrier (liposome, micelle, nanoparticle), and excited at the appropriate wavelength, the energy transfer occur- red due to the close proximity between the dyes: excita- tion at 460 nm (donor excitation) resulted in a very strong emission at 565 nm (acceptor emission), Fig. 2, curve 1. When nanocarriers are broken down for any reason (for instance, in case of organic solvent DMF ad- dition), the donor and acceptor molecules are released and diffuse apart, eliminating the energy transfer [12, 14, 15]. In such a case, a redistribution of the donor (�max = 501 nm) and acceptor (�max = 565 nm) peaks was observed (Fig. 2, curves 2, 3). The�-ratiometry method is based on the analysis of the FRET ratio IDiI/(IDiO + IDiI) where IDiI and IDiO are the fluorescence intensities mea- sured at DiI and DiO maxima, respectively [13]. At the first stage of our research, we studied the ki- netics of lipophilic dyes release under the nanocarriers interaction with the model cell membranes. A concent- rated suspension of PC liposomes (1 �10–2 M), which do not contain dye molecules, was used as a model system of cell membranes [21]. Colloidal solutions of nanocar- riers (PC liposomes, SDS micelles, GdYVO4:Eu3+ or CeO2 NPs) containing FRET-dyes were mixed with the concentrated suspension of liposomes (1:1 ratio) and kept at room temperature during the desired time inter- vals (from 30 min up to 170 h). Schematic representa- tion of the experiment is presented on Fig. 3. A decrea- se of FRET ratio was observed over time for all nanocar- ries under study, but with different efficiency (Fig. 4). At the same time, in the solutions of nanocarries diluted at the ratio 1:1 with double distilled water without li- pids we did not observe any redistribution in time of the donor and acceptor fluorescence. These results indicate the nanocarriers interaction with the lipid bilayers of model membranes and lipophilic dyes release as well as dilution in lipid phase that leads to an increase in the do- nor-acceptor distance and, consequently, the FRET ratio decrease (Fig. 4). The SDS micelles exhibit high stability in liposo- mal suspension. The FRET ratio IDiI/(IDiO + IDiI) chan- ged very slowly (Fig. 4, curve 1). Curve 1 reached a pla- teau only after 120 h. Meanwhile, the FRET ratio chan- 316 TKACHEVA T. N. ET AL. 500 550 600 650 700 F lu o re sc e n c e in te n si ty , a rb . u n . Wavelength, nm 0 200 400 600 800 1000 1 2 3 Fig. 2. Redistribution of the donor and acceptor fluorescence relative intensities in solutions containing dyes-loaded PC liposomes and dif- ferent amounts of DMF: 1 – without DMF; 2 – 30 % of DMF; 3 – 80 % of DMF; � exc = 460 nm ged from 0.98 to 0.82 that indicates a very low effici- ency of hydrophobic cationic dyes release from the ani- onic nanocarriers into the lipid phase. The process of redistribution of the dyes between na- nocarriers and the lipid phase can be described by first- order reaction kinetics in the following form [20, 22]: I I I I I I eDiI DiO DiI t DiI DiO DiI � �� ( ) ( )0 � Kt , (1) where K – is the release rate constant (the dye leakage coefficient). First-order reaction kinetics can be assumed when the reaction rate depends on the concentration of only one reactor, in our case, on the lipid phase concent- ration. A nonlinear fit (eq. 1) of the FRET ratio changes was generated by the method of last-squares and allows K = 0.008 h–1 to be obtained (Table 1). For first-order reactions one can also use the dye release half-life (in our case, time for the initial FRET ratio to be reduced by 1/2), which can be obtained as [20]: t K 1 2 2 / ln .� (2) For SDS micelles, the dye release half-life was esti- mated to be 86 h that is rather large value indicating a low efficiency of the dye molecules release from SDS micel- les. We suggest that this fact can be explained by the key role of electrostatic interactions, which hold cationic dy- es in oppositely charged micelles and prevent their rele- ase into the lipid bilayers of the model cell membranes. For other nanocarriers under study the dye release process was much faster (Table 1). The FRET ratio chan- ged within about 5 h and then all the curves reached a plateau (Fig. 4). For PC liposome nanocarriers the dye leakage coefficient was obtained to be 0.82 h–1, while t1/2 is 0.85 h (Fig. 4, curve 3, Table 1). To test a role of electrostatic interactions, we added 10 % of SDS in the lipid bilayers of liposomal containers that provided a ne- gative charge of liposome without changing its proper- ties. As seen in Table 1, t1/2, becomes about three times higher that confirms the role of electrostatic interactions in the dye–to–nanocarrier binding. 317 DYNAMICS OF DYE RELEASE FROM NANOCARRIERS OF DIFFERENT TYPES 0 20 40 60 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Time, hours 6 5 4 3 2 1 I D iI /( I D iO + I D iI ) Time, h Fig. 4. Time traces of FRET ratio I DiI /(I DiO + I DiI ) : 1 – SDS micelles; 2 – liposomes + 10 % of SDS; 3 – liposomes; 4 – GdYVO 4 :Eu 3+ ; 5 – GdYVO 4 :Eu 3+ + cholesterol; 6 – CeO 2 Stable container (No dye release) Green emission. No red emission No green emission. Red emission Mixing Nanocarrier - DIO + DII DIO (FRET donor) DII (FRET acceptor) No green emission. Red emission Model cell membrane (“empty” liposome vesicle) h�` h� h� h�` h� h�`` Leaky container (Dye release) FRET Fig. 3. Schematic represen- tation of the experiment In case of GdYVO4:Eu3+ and CeO2 NPs, a very fast release of dyes from the dyes/NP complexes and their transition into the lipid phase were observed (Fig. 4, curves 4, 6). The K values are large, whereas the dye release half-lives are less than 30 min (Table 1). Such effects could be explained by the appearance of lipo- philic gradient and fast transition of lipophilic dyes DiO and DiI into the lipophilic environment. If nanocarriers contain encapsulated cholesterol, the process of dye re- lease slowed down by almost in three times by reducing the lipophilic gradient in such a solution (Table 1). Nanocarriers interaction with living cells in vitro.To test nanocarriers in the experiments with living cells, freshly isolated rat hepatocytes were used. Fig. 5, A, shows the fluorescent images of rat hepatocytes ob- tained at different time intervals of cell incubation with the dyes/CeO2 complexes. A gradual increase in cell brightness over time was observed that points to the dy- es accumulation in a cell membrane. At the same time, we observed a decrease in the FRET ratio, IDiI/(IDiO + + IDiI), over time (Fig. 6, curve 3) from 0.7 to 0.2 that points to an increase of the distance between the donor and acceptor molecules due to their diffusion in the li- pid bilayers of cell membranes. Similarly to the model cell membranes, the process of the donor and acceptor fluorescence redistribution took about five hours and then curve 3 reached the plateau (Fig. 6). The dye relea- se kinetic parameters are K = 3.1 h–1 and t1/2 = 0.22 h that agrees well with those obtained for the model cell membranes (Table 2). Features of the GdYVO4:Eu3+ nanocarriers interac- tion with the living cells differs from that for the CeO2 nanocarriers. As shown in Fig. 5, b, at the short time in- cubation, 10 min and 1 h, the dyes are located mainly 318 TKACHEVA T. N. ET AL. Type of nanocarriers K, h–1 t1/2, h SDS micelles 0.008 86 Liposomes 0.82 0.85 Liposomes + 10 % SDS 0.29 2.4 GdYVO4:Eu 3+ 1.96 0.36 GdYVO4:Eu 3+ + cholesterol 0.75 0.93 CeO2 2.86 0.24 Table 1 Dye release constant (K) and half-life (t1/2) in model cell membranes Type of nanocarriers K, h–1 t1/2, h CeO2 3.1 0.22 GdYVO4:Eu 3+ 0.54 1.28 Liposomes 0.9 0.77 Table 2 Dye release constant (K) and half-life (t1/2) in rat hepatocytes Autofluorescence 10 min 1 h 3 h 24 h A B C Fig. 5. Fluorescent images of rat hepatocytes taken at different times of cell in- cubation with dyes/CeO 2 (A), dyes/GdYVO 4 :Eu 3+ (B) complexes and dyes-loaded liposomes (C). Excitation with BP 460–490 filter on the cell membrane surface (yellow spots). At the sa- me time, even after 24 h incubation of the cells with the dyes/GdYVO4:Eu3+ complexes, the FRET ratios chan- ged not sufficiently (from 0.7 to 0.5) and the yellow spots were still observed. The dye release half-life was almost six times higher than in case of the CeO2 nano- carriers (Table 2) and the obtained kinetic parameters K and t1/2 also differed from those obtained in the model cell membranes (Table 1). We can ascribe these facts to the specificity of the GdYVO4:Eu3+ NPs interaction with living cells. As it was mentioned above, spherical GdYVO4:Eu3+ NPs (d = 2 nm) exhibit the penetration into cells and accumulation in cell organelles [10]. We suppose the yellow spots to be the dyes/GdYVO4:Eu3+ complexes accumulated either on a cell membrane sur- face or inside the cells similarly to the dyes accumula- tion in the cells endocytosis vesicles after their interna- lization revealed by Chen and co-workers [18, 19]. For comparison, the kinetic parameters of dye-loa- ded PC liposomes interaction with rat hepatocytes were also analyzed. The fluorescent images of cells taken in different time periods show a gradual increase in cell brightness over time, no spots on the cell membranes were observed that points to the effective dye release from the liposomes (Fig. 5, c). The FRET ratio changed and the kinetic parameters K and t1/2 were similar to those obtained for the model cell membranes (Fig. 6, c, Table 2). Controlled release of drugs and other bioactive agents is a key point in drug formulation pharmacokinetic stu- dy and attract many researches [11, 21, 23]. Controlled drug delivery applications include both a one-time or sustained targeted delivery [21–24]. Controlled-release systems are designed to enhance drug therapy due to reducing the amount of drug necessary to cause the sa- me therapeutic effect in patient. Over the years of cont- rolled release research, different systems have been ex- plored to get predesigned release profile [11, 21–24]. In certain drug administration strategies, a fast release of drug immediately upon placement in the release me- dium is required [11]. We have shown that NPs as a plat- form for drug delivering can provide fast lipophilic content release from the nanocarrier, Table 1. How- ever, specificity of GdYVO4:Eu3+ NPs interaction with rat hepatocytes affect the kinetics of this process in- creasing the value (Table 2). Meanwhile, the rate of li- pophilic content release can be controlled by decrea- sing lipophilic gradient and providing Coulombic inter- actions between the nanocarrier and the encapsulated substance. Conclusions. The FRET-based method was used to study the nanocarriers interaction with the model cell membranes and living cells in dynamic experiments in vitro. The dye release constants (K) and half-lives (t1/2) we- re analyzed for different nanocarriers (SDS micelles, PC liposomes, GdYVO4:Eu3+ and CeO2 NPs). GdYVO4:Eu3+ and CeO2 NPs were shown to provide a faster lipophi- lic dyes release in the model cell membranes as com- pared to PC liposomes, the dye release half-life t1/2 is less than 30 min, that can be explained by the appea- rance of a lipophilic gradient. The negatively charged or lipophilic compounds can decrease the rate of lipo- philic agents release from nanocarriers i. e. allow this process to be controlled. Our experiments confirm the specificity of the GdYVO4:Eu3+ NPs interaction with li- ving cells. We suppose the intracellular uptake of the dyes/GdYVO4:Eu3+ NPs complex with a slower dye re- lease, whereas for the dyes/CeO2 complex the dye re- lease pattern is similar to that in case of the model cell membranes. Äèíàì³êà âèëó÷åííÿ áàðâíèê³â ç íàíîêîíòåéíåð³â ð³çíîãî òèïó â ìîäåëüíèõ ìåìáðàíàõ ³ æèâèõ êë³òèíàõ Ò. Ì. Òêà÷îâà, Ñ. Ë. ªô³ìîâà, Â. Ê. Êëî÷êîâ, À. Â. Ñîðîê³í, Þ. Â. Ìàëþê³í Ðåçþìå Ìåòà. Âèâ÷åííÿ äèíàì³êè âèëó÷åííÿ ë³ïîôèëüíîãî âì³ñòó ç íàíî- êîíòåéíåð³â ð³çíîãî òèïó, îðãàí³÷íèõ ìîëåêóëÿðíèõ àíñàìáë³â ³ íåîðãàí³÷íèõ íàíî÷àñòèíîê (Í×) â åêñïåðèìåíòàõ in vitro. Ìåòî- 319 DYNAMICS OF DYE RELEASE FROM NANOCARRIERS OF DIFFERENT TYPES 0 5 10 15 20 25 0,2 0,3 0,4 0,5 0,6 0,7 0,8 3 2 1 Time, hours I D iI /( I D iO + D iI ) I D iI /( I D iO + I D iI ) Time, h Fig. 6. Time traces of FRET ratio I DiI /(I DiO + I DiI ) in red hepatocytes: 1 – GdYVO 4 :Eu 3+ ; 2 – PC liposomes; 3 – CeO 2 äè. Äâîêàíàëüíèé ðàò³îìåòðè÷íèé ìåòîä ðåºñòðàö³¿ ³íòåíñèâ- íîñò³ ôëóîðåñöåíö³¿ ³ç çàñòîñóâàííÿì áåçâèïðîì³íþâàëüíîãî ïå- ðåíåñåííÿ åíåðã³¿ åëåêòðîííîãî çáóäæåííÿ, ìåòîä ôëóîðåñöåíò- íî¿ ñïåêòðîñêîï³¿ ³ ì³êðîñïåêòðîñêîï³¿. Ðåçóëüòàòè. Âèâ³ëüíåí- íÿ ë³ïîôèëüíûõ áàðâíèê³â ç îðãàí³÷íèõ (ë³ïîñîìè ³ ì³öåëè) ³ íåîð- ãàí³÷íèõ (íà îñíîâ³ Í× GdYVO4:Eu 3+ ³ CeO2) íàíîêîíòåéíåð³â ìî- æå áóòè îïèñàíî ê³íåòè÷íîþ ðåàêö³ºþ ïåðøîãî ïîðÿäêó ÿê ó ìî- äåëüíèõ êë³òèííèõ ìåìáðàíàõ, òàê ³ â æèâèõ êë³òèíàõ. Îòðèìàíî êîíñòàíòè øâèäêîñò³ âèâ³ëüíåííÿ (K) ³ ÷àñ íàï³ââèâåäåííÿ (t1/2) áàðâíèê³â. Âèñíîâêè. Íàíîêîíòåéíåðè íà îñíîâ³ Í× GdYVO4:Eu 3+ ³ CeO2 çàáåçïå÷óþòü øâèäøå âèâ³ëüíåííÿ ë³ïîôèëüíîãî âì³ñòó â ìîäåëüíèõ êë³òèííèõ ìåìáðàíàõ ïîð³âíÿíî ç ë³ïîñîìàìè. Ïðîòå äîäàâàííÿ íåãàòèâíî çàðÿäæåíèõ àáî ë³ïîôèëüíèõ êîìïîíåíò ó ñèñòåìó çíèæóº øâèäê³ñòü âèâ³ëüíåííÿ áàðâíèê³â. Âèÿâëåíî ñïå- öèô³÷í³ñòü âçàºìîä³¿ Í× GdYVO4:Eu 3+ ç ãåïàòîöèòàìè ùóð³â. Êëþ÷îâ³ ñëîâà: íàíîêîíòåéíåðè, áåçâèïðîì³íþâàëüíå ïåðå- íåñåííÿ åíåðã³¿, íàíî÷àñòèíêè, âèâ³ëüíåííÿ áàðâíèêà, ìîäåëüí³ êë³òèíí³ ìåìáðàíè, æèâ³ êë³òèíè. Äèíàìèêà âûñâîáîæäåíèÿ êðàñèòåëåé èç íàíîêîíòåéíåðîâ ðàçëè÷íîãî òèïà â ìîäåëüíûõ ìåìáðàíàõ è æèâûõ êëåòêàõ Ò. Í. Òêà÷åâà, Ñ. Ë. Åôèìîâà, Â. Ê. Êëî÷êîâ, À. Â. Ñîðîêèí, Þ. Â. Ìàëþêèí Ðåçþìå Öåëü. Èçó÷åíèå äèíàìèêè âûñâîáîæäåíèÿ ëèïîôèëüíîãî ñîäåð- æèìîãî èç íàíîêîíòåéíåðîâ ðàçëè÷íîãî òèïà, îðãàíè÷åñêèõ ìîëå- êóëÿðíûõ àíñàìáëåé è íåîðãàíè÷åñêèé íàíî÷àñòèö (Í×) â ýêñïåðè- ìåíòàõ in vitro. Ìåòîäû. Èñïîëüçîâàëè äâóêàíàëüíûé ðàòèîìåò- ðè÷åñêèé ìåòîä ðåãèñòðàöèè èíòåíñèâíîñòè ôëóîðåñöåíöèè íà îñíîâå áåçûçëó÷àòåëüíîãî ïåðåíîñà ýíåðãèè ýëåêòðîííîãî âîçáóæ- äåíèÿ, à òàêæå ìåòîä ôëóîðåñöåíòíîé ñïåêòðîñêîïèè è ìèêðî- ñïåêòðîñêîïèè. Ðåçóëüòàòû. Âûõîä ëèïîôèëüíûõ êðàñèòåëåé èç îðãàíè÷åñêèõ (ëèïîñîìû è ìèöåëëû) è íåîðãàíè÷åñêèõ (íà îñíîâå Í× GdYVO4:Eu 3+ è CeO2) íàíîêîíòåéíåðîâ ìîæåò áûòü îïèñàí êèíåòè÷åñêîé ðåàêöèåé ïåðâîãî ïîðÿäêà êàê â ìîäåëüíûõ êëåòî÷- íûõ ìåìáðàíàõ, òàê è â æèâûõ êëåòêàõ. Ïîëó÷åíû êîíñòàíòû ñêîðîñòè âûñâîáîæäåíèÿ (K) è âðåìÿ ïîëóâûâåäåíèÿ (t1/2) êðàñè- òåëåé. Âûâîäû. Íàíîêîíòåéíåðû íà îñíîâå Í× GdYVO4:Eu 3+ è CeO2 îáåñïå÷èâàþò áîëåå áûñòðîå âûñâîáîæäåíèå ëèïîôèëüíî- ãî ñîäåðæèìîãî â ìîäåëüíûõ êëåòî÷íûõ ìåìáðàíàõ ïî ñðàâíåíèþ ñ ëèïîñîìàìè. Îäíàêî äîáàâëåíèå îòðèöàòåëüíî çàðÿæåííûõ èëè ëèïîôèëüíûõ êîìïîíåíò â ñèñòåìó ñíèæààåò ñêîðîñòü âûñâî- áîæäåíèÿ êðàñèòåëåé. Îáíàðóæåíà ñïåöèôè÷íîñòü âçàèìîäåé- ñòâèÿ Í× GdYVO4:Eu 3+ ñ ãåïàòîöèòàìè êðûñ. Êëþ÷åâûå ñëîâà: íàíîêîíòåéíåðû, áåçûçëó÷àòåëüíûé ïåðå- íîñ ýíåðãèè, íàíî÷àñòèöû, âûñâîáîæäåíèå êðàñèòåëÿ, ìîäåëü- íûå êëåòî÷íûå ìåìáðàíû, æèâûå êëåòêè. REFERENCES 1. Hunziker P. Nanomedicine: shaping the future of medicine. Eur J Nanomed. 2012;2(1):4. 2. Bamrungsap S, Zhao Z, Chen T, Wang L, Li C, Fu T, Tan W. Na- notechnology in therapeutics: a focus on nanoparticles as a drug delivery system. Nanomedicine (Lond). 2012;7(8):1253–71. 3. Parveen S, Misra R, Sahoo SK. Nanoparticles: a boon to drug de- livery, therapeutics, diagnostics and imaging. Nanomedicine. 2012;8(2):147–66. 4. Torchilin VP. 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Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells

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