Endocytosis of titanium dioxide nanoparticles in prostate cancer PC-3M cells.

Nanotechnology has introduced many exciting new tools for the treatment of human diseases. One of the obstacles in its application to that end is the lack of a fundamental understanding of the interaction that occurs between nanoparticles and living cells. This report describes the quantitative analysis of the kinetics and endocytic pathways involved in the uptake of anatase titanium dioxide (TiO(2)) nanoparticles into prostate cancer PC-3M cells. The experiments were performed with TiO(2) nanoconjugates: 6-nm nanoparticles with surface-conjugated fluorescent Alizarin Red S. Results obtained by flow cytometry, fluorescence microscopy, and inductively coupled plasma-mass spectrometry confirmed a complex nanoparticle-cell interaction involving a variety of endocytic mechanisms. The results demonstrated that a temperature, concentration, and time-dependent internalization of the TiO(2) nanoparticles and nanoconjugates occurred via clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis. FROM THE CLINICAL EDITOR: The interaction and uptake of TiO(2) nanoparticles (6-nm) with prostate PC-3M cells was investigated and found to undergo temperature, time, and concentration dependent intracellular transport that was mediated through clathrin pits, caveolae, and macropinocytosis. These results suggest that nanoparticles may widely permeate through tissues and enter almost any active cell through a variety of biological mechanisms, posing both interesting opportunity and possible challenges for systemic use.

Labeling TiO2 nanoparticles with dyes for optical fluorescence microscopy and determination of TiO2-DNA nanoconjugate stability.

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO2) nanoparticles. These nanoparticles, when electronically linked to single-stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO2-DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO2 nanoparticles and TiO2-DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO2 nanoparticle uptake in a large population of living cells (>10(4) cells). X-ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO2-DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO2-DNA nanoconjugates in malignant cells.

Methods for assessing DNA hybridization of peptide nucleic acid-titanium dioxide nanoconjugates.

We describe the synthesis of peptide nucleic acid (PNA)-titanium dioxide (TiO(2)) nanoconjugates and several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes that hybridize to single-stranded DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double-stranded DNA (dsDNA), and form different PNA/DNA complexes. Previously, we developed a DNA-TiO(2) nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner with the ability to cleave DNA when excited by electromagnetic radiation but susceptible to degradation that may lower its intracellular targeting efficiency and retention time. PNA-TiO(2) nanoconjugates described in the current article hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid, inexpensive, sequence-specific concentration of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA-TiO(2) nanoconjugates are enhanced, providing essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO(2)-DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems.

Titantium Dioxide Nanoparticles Assembled by DNA Molecules Hybridization and Loading of DNA Interacting Proteins.

This work demonstrates the assembly of TiO(2) nanoparticles with attached DNA oligonucleotides into a 3D mesh structure by allowing base pairing between oligonucleotides. A change of the ratio of DNA oligonucleotide molecules and TiO(2) nanoparticles regulates the size of the mesh as characterized by UV-visible light spectra, transmission electron microscopy and atomic force microscopy images. This type of 3D mesh, based on TiO(2)-DNA oligonucleotide nanoconjugates, can be used for studies of nanoparticle assemblies in material science, energy science related to dye-sensitized solar cells, environmental science as well as characterization of DNA interacting proteins in the field of molecular biology. As an example of one such assembly, proliferating cell nuclear antigen protein (PCNA) was cloned, its activity verified, and the protein was purified, loaded onto double strand DNA oligonucleotide-TiO(2) nanoconjugates, and imaged by atomic force microscopy. This type of approach may be used to sample and perhaps quantify and/or extract specific cellular proteins from complex cellular protein mixtures affinity based on their affinity for chosen DNA segments assembled into the 3D matrix.