key: cord-0005494-iu4dq0pg
authors: Zarytova, V. F.; Zinov’ev, V. V.; Ismagilov, Z. R.; Levina, A. S.; Repkova, M. N.; Shikina, N. V.; Evdokimov, A. A.; Belanov, E. F.; Balakhnin, S. M.; Serova, O. A.; Baiborodin, S. I.; Malygin, E. G.; Zagrebel’nyi, S. N.
title: An examination of the ability of titanium dioxide nanoparticles and its conjugates with oligonucleotides to penetrate into eucariotis cells
date: 2009-10-20
journal: Nanotechnol Russ
DOI: 10.1134/s1995078009090158
sha: ffa4440314406bc74fafc1b39b06871b5949d7bf
doc_id: 5494
cord_uid: iu4dq0pg

In this study we examine the possibility that TiO(2) nanoparticles and their conjugates can penetrate into cultivated cells without any special transfection procedures. Oligonucleotides and their derivates were conjugated with the TiO(2) nanoparticles, which were obtained as colloidal solutions at a concentration of TiO(2) 0.3M by TiCl(4) hydrolysis. The electronic microscopy of various cell cultures (KCT, Vero, and MDCK) treated with nanoparticle solutions (20 µg/µl) showed that nanoparticles could enter the cells and accumulate in the vacuoles and phagosomes and form inclusions in cytoplasm. Thus, we demonstrated the penetration of TiO(2) nanoparticles and their oligonucleotide conjugates into intracellular space without any auxiliary operations. Most other researches used electroporation techniques for similar purposes [1, 2, 5].

The threat that pathogenic virus agents pose to mankind has considerably increased in recent years. One reason for this growth is antibioticotherapy (a widespread method for treating bacterial infections), which is ineffective in treating viral diseases because of the principal difference in the duplication biology of bacteria and virus in the human organism. In addition, many of virus pathogens very changeable, which sig nificantly decreases the efficiency of the vaccinal pre vention of virus infections. Viral genetic material damage, particularly with antisense oligonucleotides, is one of the most promis ing strategies for antiviral therapy. However, delivering olygonucleotides directly into the injured cell is the biggest problem in this case.

Because of the rising interest in nanotechnologies in recent years, several methods for using nanoparti cles of different nature-as a way to deliver drugs into cells-have appeared. Voloshchak et al. studied intra cellular distribution in the eukaryotic cells of TiO 2 nanoparticles conjugated with specific oligonucle otides [1, 2] . As was determined during the experi ments, these nanoconjugates interacted specifically with DNA localized in the cell nucleus and mitochon drion. The main method for the transformation of the cells was electroporation, which damages cell mem branes. Unfortunately, this technology of delivering nanoparticles conjugated with bioactive substances into the damaged cells is not acceptable in clinical conditions. The authors specified that nanoparticles penetrate into the cells during incubation, but they didn't detail the conditions of the incubation.

In this work we examine how the TiO 2 nanoparticles and their conjugates with specific oligonucleotides pene trate into the eukaryotic cells in more detail.

TiO 2 nanoparticles were obtained by TiCl 4 hydrol ysis as a colloidal solution with a TiO 2 concentration of 0.3M and pH 6.7 [3] . For the immobilization of the oligonucleotides on the nanoparticles, TiO 2 solutions were diluted to 0.0125M (1-2 mg/ml).

X ray analysis has indicated that titanium dioxide preparation is defined as a radioamorphous phase state. It is probably a superfine brookite with D < 5 nm. Methods of small angle X ray scattering (SARS) and atomic force microscopy (AFM) were used to estab lish that the titanium dioxide in a colloidal solution is present in separated particles 3.5-5 nm in size and preserves the sol's dispersion degree for six months (during the study).

Conjugates of nanoparticles with the oligonucle otides and their derivatives were obtained by a similar procedure described previously [4] .

Vero, KCT, MDCK, and MDBK cell lines were used for experiments. Cells were received from the cell culture collection at the Federal State Institute of Sci ence, Scientific Research Center (FSIS SRC) VB Vek tor, Rospotrebnadzor (Kol'tsovo, Novosibirsk oblast). Cells were cultivated in Igla DMEM media with 5% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml).

For electronic microscopy, cells were incubated with nanoparticles for 24 h, then washed from media and fixed with 2.5% glutaraldehyde in a phosphate salt buffer (PBS) with pH 7.2 for 1 h on ice. Rest fixation was made in 1% osmium tetroxide in PBS with pH 7.2 for 1 h at room temperature. One they were dehydrated with ethanol, the cells were put on Araldite epoxy resin. Ultrathin sections were prepared on a Leica Ultracut UCT ultratome, then they were contrasted with uranyl acetate and plumbum citrate and analyzed using an LEO 910 (Zeiss) electronic microscope.

MYCROSCOPY Cells growing on cover slips were incubated with nanocomposites TiO 2 PL(Flu) and TiO 2 PL(Flu) ON marked with a fluorescent label for 24 h. 1 Then cells were fixed with 0.4% formalin with 0.1% Triton X 100 (10 min) and treated with 0.5% Triton X 100 (10 min) and 2% formalin (10 min). Actin filaments were stained with a phalloidin TRITC complex (red signal) to visualize the border of the cells. All the solutions were prepared on PBS, pH 7.2. The glasses with cells were put upside down on the slide plates onto restricted media with antifade and DAPI. The samples were analyzed with a laser scanning microscope LSM 510 META (Zeiss). For label identification we used different laser wave lengths: 405 nm (for DAPI they were cellular nuclei), 488 nm (for nanocompos ites they were marked with flouroscein), and 543 nm (for actin filaments they were marked with a phalloi din TRITC complex). The thickness of optical sec tions was 0.3 mm.

Nanocomposites TiO 2 PL and TiO 2 PL oligo were marked with a fluorescent label by treatment with FITC in 0.2 M NaHCO 3 solution for 1 h at 60°C with following washing off of excess FITC. In addition, we obtained nanocomposits with dopamine (TiO 2 DA PL(Ac) and TiO 2 DA PL(Ac) oligo), because, accord ing to the published data, DA increases the effects of nanoparticles under radiation [5] . TiO 2 nanoparticles (1 mg) were sequentially treated with glycidylisopropyl ether (100 µl) and then with 1 M DA solution. Sam ples with DA were brick red color, which indicates the complex formation with charge transferring [6] . PL or PL oligo were added to washed nanoparticles in the proportion TiO 2 : PL = 1 : 1000.

The cells growing on cover glasses were incubated for the whole day with TiO 2 PL(Flu) nanoparticles labeled by FITC; then they were fixed with 0.4% for malin with 0.1% Triton for 10 min, treated with 0.5% Triton X 100 for 10 min, and fixation was finished with 2% formalin for 10 min. All solutions were prepared in PBS, pH 7.2. The glasses with cells were put upside down on the slide plates onto restricted media with antifade and DAPI. Samples were analyzed under an LSM 510 META laser scanning microscope (Zeiss). TiO 2 PL(Flu) nanocomposits were dyed green and localized in cytoplasm; cell nuclei were stained by DAPI (blue); and actin filaments were stained by actin phalloidin TRITC complex (red). The micro scopic lens was ×100. Scan conditions were as follows: laser lines 405, 488, and 543; filters BP 420 480, BP 505 530, and LP 560. The thickness of the optical sec tions was 0.3 µm.

Voloshchak et al. [2] obtained oligonucleotides conjugated with TiO 2 nanoparticles and detected that these conjugates specifically interact with DNA when introduced into the cells, according to the com plementary principles [2] . Nanoparticles with oligo nucleotides, which are complementary to the gene sequence of the NADH dehydrogenase's (ND2) sec ond sub particle on the mitochondrial genome, were detected in mitochondria, whereas conjugates with oligonucleotides, which are complementary to the gene fragment of 18S rRNA, were localized in nuclei. These interesting findings allow us to suppose the complementary interaction between conjugated oligo nucleotides and cell nucleic acids. The authors of [1, 2] used the electroporation technique to introduce conjugates into the cells.

In this study we examined the capability of the con jugates to penetrate the cells without the electropora tion technique, because this method isn't available for the cells of an organism.

The electron microphotography of Vero cells incu bated with TiO 2 nanoparticles are shown in Fig.1 . The same pictures were made for KCT, MDCK, and MDBK cell cultures (data not shown).

The electron microscopy of different cell cultures (KCT, Vero, and MDCK) treated with 20 µg/µl showed that nanoparticles could enter the cells and accumulate in vacuoles and phagosomes and form inclusions in cytoplasm. Nanoparticles previously treated with ultrasound formed small inclusions or localized as separated particles and weren't detected in nuclei.

The penetration of nanoparticles into the cells were examined by confocal laser microscopy (Fig. 2) . As you can see in Fig. 2 , these data confirm the results of electron microscopy. Essentially, we didn't detect the oligonucleotides marked with fluoresceine and unbound to oligonucliotides in the cells (data not shown). Nanoparticles probably protect oligonucle otides from intracellular nucleases, which efficiently destroyed exposed oligonucleotides [7] . As was previ ously reported, Voloshchak et al. used the electropora tion technique to introduce nanoparticles into the cells [1, 2] . Moreover, other researchers examined the penetrations of gold nanoparticles (which were conju gated with the anticancer drug herceptin) into the cells [8] . Herceptin is a humanized monoclonal antibody to receptor HER2/neu (erbB2), which is exposed on the cancer cells of the mammary gland. The authors dem onstrated the dependency of the nanoparticles sizes and the penetrating efficiency of the conjugates. The optimal sizes of gold nanoparticles was from 40 to 50 nm. The authors supposed that, due to the binding of nanocojugates with their specific receptors, recep tor mediated endocytosis occurs in this case. Hercep tin binds to several specific receptors and formed clus ters. Such multipoint binding is necessary to form a high affinity bond.

These findings are convincing, but we think that maybe this is possible only when the ligand, for instat nce herceptin, is a large protein molecule which binds ineffectively with smaller nanoparticles. In our case the sizes of ligand molecules are smaller (20-30 bp oligonucleotides) than the herceptin molecule, which facilitates the penetration of nanocojugates into the cells. Thus, we suggested that the optimal size of our nanoparticles may be smaller and the nanoconjugate's internalization occurs without interaction with a spe cific receptor.

Zhang and Sun examined the effects of TiO 2 nano particles on the rectal carcinoma LS 147 t cell line and found that cells treated with nanoparticles and UV radiation died [9] . The cells weren't exposed to any other special transfection procedures. The authors wondered whether nanoparticles accumulate on the surface of the cells or penetrate into the cells by endocytosis, but they didn't try to show the nanopar ticles in the cells with direct methods.

Huojin et al. used nanodaimond to deliver doxoru bicine antibiotic (apoptotic agent/stimulator) into the cells [10] . The authors detected that nanodaimonds penetrate into the cells without any special trasfection procedures. This evidence and our results allow us to suggest that using such a way to inject drugs into the cells is a general (not a specific) method. Thus, in this study we detected the penetration of TiO 2 nanoparticles and their conjugates into the culti vated cells without any special transfection proce dures; however, how they penetrate is not yet clear. 

Intracellular Distribu tion of TiO 2 -DNA Oligonucleotide Nanoconjugates Directed to Nucleolus and Mitochondria Indicates Sequence Specificity

DNA-TiO 2 Nanoconjugates Labeled with Magnetic Resonance Contrast Agents

Influence of Conditions Providing Precipitation of Titanium Diox ide Hydrogel on the Porous Xerogel Structure

Oligonucleotide Probes Containing Polylysine Residues for Fabrication of DNA Chips on Various Solid Surfaces

Biology of TiO 2 -Oligonu cleotide Nanocomposites

Surface Restructuring of Nanopar ticles: An Efficient Route for Ligand-Metal Oxide Crosstalk

Cell and Tissue Distribution of Synthetic Oligonucleotides in Healthy and Tumor Bearing Nude Mice: An Autoradiographic, Immuno histological, and Direct Fluorescence Microscopy Study

Nanoparticle Mediated Cellular Response Is Size Dependent

Photocatalytic Killing Effect of TiO 2 Nanoparticles on Ls 174 t Human Colon Carcinoma Cells

Active Nanodiamond Hydrogels for Chemotherapeutic Deliv ery