Eric Michael Bratsolias Brown

Nanoparticles for applications in cellular imaging.

In the following review we discuss several types of nanoparticles (such as TiO2, quantum dots, and gold nanoparticles) and their impact on the ability to image biological components in fixed cells. The review also discusses factors influencing nanoparticle imaging and uptake in live cells in vitro. Due to their unique size-dependent properties nanoparticles offer numerous advantages over traditional dyes and proteins. For example, the photostability, narrow emission peak, and ability to rationally modify both the size and surface chemistry of Quantum Dots allow for simultaneous analyses of multiple targets within the same cell. On the other hand, the surface characteristics of nanometer sized TiO2allow efficient conjugation to nucleic acids which enables their retention in specific subcellular compartments. We discuss cellular uptake mechanisms for the internalization of nanoparticles and studies showing the influence of nanoparticle size and charge and the cell type targeted on nanoparticle uptake. The predominant nanoparticle uptake mechanisms include clathrin-dependent mechanisms, macropinocytosis, and phagocytosis.

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

UNLABELLED 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.

A Near Infrared Light Triggered Hydrogenated Black TiO2 for Cancer Photothermal Therapy

White TiO2 nanoparticles (NPs) have been widely used for cancer photodynamic therapy based on their ultraviolet light-triggered properties. To date, biomedical applications using white TiO2 NPs have been limited, since ultraviolet light is a well-known mutagen and shallow penetration. This work is the first report about hydrogenated black TiO2 (H-TiO2 ) NPs with near infrared absorption explored as photothermal agent for cancer photothermal therapy to circumvent the obstacle of ultraviolet light excitation. Here, it is shown that photothermal effect of H-TiO2 NPs can be attributed to their dramatically enhanced nonradiative recombination. After polyethylene glycol (PEG) coating, H-TiO2 -PEG NPs exhibit high photothermal conversion efficiency of 40.8%, and stable size distribution in serum solution. The toxicity and cancer therapy effect of H-TiO2 -PEG NPs are relative systemically evaluated in vitro and in vivo. The findings herein demonstrate that infrared-irradiated H-TiO2 -PEG NPs exhibit low toxicity, high efficiency as a photothermal agent for cancer therapy, and are promising for further biomedical applications.

Stability enhanced polyelectrolyte-coated gold nanorod-photosensitizer complexes for high/low power density photodynamic therapy

Photodynamic therapy (PDT) is a promising treatment modality for cancer and other malignant diseases, however safety and efficacy improvements are required before it reaches its full potential and wider clinical use. Herein, we investigated a highly efficient and safe photodynamic therapy procedure by developing a high/low power density photodynamic therapy mode (high/low PDT mode) using methoxypoly(ethylene glycol) thiol (mPEG-SH) modified gold nanorod (GNR)-AlPcS4 photosensitizer complexes. mPEG-SH conjugated to the surface of simple polyelectrolyte-coated GNRs was verified using Fourier transform infrared spectroscopy; this improved stability, reduced cytotoxicity, and increased the encapsulation and loading efficiency of the nanoparticle dispersions. The GNR-photosensitizer complexes were exposed to the high/low PDT mode (high light dose = 80 mW/cm(2) for 0.5 min; low light dose = 25 mW/cm(2) for 1.5 min), and a high PDT efficacy leads to approximately 90% tumor cell killing. Due to synergistic plasmonic photothermal properties of the complexes, the high/low PDT mode demonstrated improved efficacy over using single wavelength continuous laser irradiation. Additionally, no significant loss in viability was observed in cells exposed to free AlPcS4 photosensitizer under the same irradiation conditions. Consequently, free AlPcS4 released from GNRs prior to cellular entry did not contribute to cytotoxicity of normal cells or impose limitations on the use of the high power density laser. This high/low PDT mode may effectively lead to a safer and more efficient photodynamic therapy for superficial tumors.

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.

Dye Surface Coating Enables Visible Light Activation of TiO2 Nanoparticles Leading to Degradation of Neighboring Biological Structures

Biologically and chemically modified nanoparticles are gaining much attention as a new tool in cancer detection and treatment. Herein, we demonstrate that an alizarin red S (ARS) dye coating on TiO2 nanoparticles enables visible light activation of the nanoparticles leading to degradation of neighboring biological structures through localized production of reactive oxygen species. Successful coating of nanoparticles with dye is demonstrated through sedimentation, spectrophotometry, and gel electrophoresis techniques. Using gel electrophoresis, we demonstrate that visible light activation of dye-TiO2 nanoparticles leads to degradation of plasmid DNA in vitro. Alterations in integrity and distribution of nuclear membrane associated proteins were detected via fluorescence confocal microscopy in HeLa cells exposed to perinuclear localized ARS-TiO2 nanoparticles that were photoactivated with visible light. This study expands upon previous studies that indicated dye coatings on TiO2 nanoparticles can serve to enhance imaging, by clearly showing that dye coatings on TiO2 nanoparticles can also enhance the photoreactivity of TiO2 nanoparticles by allowing visible light activation. The findings of our study suggest a therapeutic application of dye-coated TiO2 nanoparticles in cancer research; however, at the same time they may reveal limitations on the use of dye assisted visualization of TiO2 nanoparticles in live-cell imaging.