User:Tgreynol/sandbox

Titanium dioxide, also known as titanium (IV) oxide or titania, is the naturally occurring oxide of titanium, chemical formula TiO2. Titanium dioxide is widely used in common household items such as sunscreen, tattoo pigments, toothpaste and skim-milk whitener.

Titanium dioxide nanoparticles come in many crystal systems depending on the synthetic parameters such as temperature, substrate and reaction time. Two of the most common forms, rutile and anatase, are used in solar cells as a photocatalyst under visible and ultraviolet (UV) light. For example, in dye- sensitized solar cells (DSSC or Graetzel cell), the TiO2 layer acts as a graft for the dye sensitizer to allow the efficient and rapid transfer of electrons through the solar cell circuit. In addition to its use in solar cells, TiO2 nanoparticles are being examined for uses in electronic data storage, environmental and medicinal chemistry.

When covalently tethered to a specific antibody with a bivalent linker, a TiO2-antibody hybrid has been shown to mediate the phototoxic death of cancer cells.

Synthesis
The Bio-TiO2 hybrid interface as a cancer therapy was first introduced in 2009 by Tijana Rajh’s group at Argonne National Laboratory Center for Nanoscale Materials and Chemical Sciences and Engineering in collaboration with the University of Chicago Brain Tumor Center.

Synthesis of therapeutic TiO2 nanoparticles involves two main processes:

First, “bare” TiO2 nanoparticles are synthesized via colloidal synthesis, followed by the formation of a TiO2-mAB interface. TiO2 nanoparticles are prepared by drop-wise addition of titanium (IV) chloride to chilled water at pH 4 controlled by NaOH or HCl in an inert atmosphere. The desired diameter for therapeutic applications is 5nm. This size control is achieved by adjusting the concentration of titanium precursor and solvent composition. This solution is enriched through dialysis against 2L of deionized water at 2,000 MW for 3 days. It is essential that the dialysis temperature is kept at 4&deg;C to prevent aggregation of the particles. Next, the TiO2 colloid is diluted twenty-fold to achieve a final concentration of ~0.015 M. The particles are then capped with 1,2-epoxy-3- isopropoxypropane (glycidyl isopropyl ether). The pH of the solution is then adjusted to 9-12 using LiOH. This solution is run through dialysis again with a buffer at the desired pH. This step can vary depending on the specific application of TiO2. For this application, pH is adjusted to 6.3 with phosphate buffer (PBS), as this is the optimal pH for carboxyl-group activation.

TiO2 nanoparticles are then covalently conjugated with a IL13α2R-targeting antibody (mAb) via 3,4-dihydroxyphenilaceitic acid (DOPAC), a bidentate surface linker. DOPAC is mixed with TiO2 until a ratio of 1:100 TiO2 nanoparticle:DOPAC is reached. Next, N-hydroxysulfosuccinimide (Sulfo-NHS) and 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) are added to the TiO2-DOPAC complex and run through dialysis (2,000 MW) against a phosphate buffer at pH 7.4 for one hour to complete the activation of TiO2-DOPAC-N-hydroxysulfosuccinimide ester. The IL13α2R-targeting antibody (mAb) is added and incubated for 4-6 hours at room temperature in the absence of light to complete the conjugation. The product is purified via dialysis under same condition (pH 7.4) for two hours. Any remaining active sites are quenched by adding glycine and incubating for 15 minutes. Final purification is done by spin-washing TiO2-mAb in a phosphate buffer (pH 7.4). TiO2-mAb complex has been shown to be usable for up to one month when stored in the dark.

This approach for tethering biomolecules to the TiO2 nanoparticle surface takes advantage of the ability of the oxygen-containing carboxyl, hydroxyl, and phosphate groups to bind to the surface of nanoparticles. The presence of two ortho-hydroxyl groups in DOPAC allows it to form a strong bidentate complex with the Ti atoms at the surface of the nanoparticle.

DOPAC serves two additional functions in this interface. First, when DOPAC is chemisorbed, the semiconducting surface of the TiO2 nanocrystals is enhanced and optimized for more effective and efficient charge-transfer, thus allowing it to absorb at the visible part of the spectrum, which is essential for its therapeutic purpose. Second, chemisorption of DOPAC functionalizes the particle surface with carboxylic functional groups allowing for further interaction with other biomolecules increasing the possibility for other TiO2-biomolecular effects.

Figure 2. This highly simplified scheme is presented in efforts to help the reader better understand the players involved in this photodynamic therapeutic process. Isolated TiO2 nanoparticles absorb a wavelength of light around 380 nm, however upon complexation with an organic linker molecule such as DOPAC, the maximum wavelength absorbed shifts to a range of 450-740 nm. TiO2 is inherently known to be a semiconductor—which means the metal’s valence electrons form a continuum of the highest molecular orbitals (HOMO) known as the valence band. Upon excitation with light from 450-750 nm, these electrons can be excited and travel to the continuum of lowest unoccupied molecular orbitals (LUMO) known as the conduction band. When electrons leave the filled valence band, they form positive holes in the valence band and negative charges in the conduction band—this is the property of conductance. These excited electrons then have the potential to excite intracellular oxygen molecules and form radical oxygen species which can go on to disrupt the normal processes of cellular mitochondria.

Mechanism of Action
Photoreactive inorganic titanium dioxide nanoparticles (Around 5nm) coupled with specific antibodies are introduced into the body and selectively bind to corresponding surface antigens on glioblastoma multiforme (GBM) cells, cells involved in a very treatment-resistive form of brain cancer. The nanoparticles are essentially tethered to the antibody through a linker molecule like DOPAC, a dopamine metabolite that is found in the body.

The TiO2 nanoparticles employ the two ortho-positioned hydroxyl groups of DOPAC molecules to serve as a bidentate ligand. A consequence of this extension through a tethered ligand is that the absorption shifts from around 380 nm (for bare, untethered TiO2 nanoparticles) to 450-750 nm. This important shift allows for absorption in the visible part of the spectrum. Furthermore, the carboxylic acid group on the DOPAC molecule serves as a reactive domain that allows for further bridging to various biomolecules depending on the specific therapeutic application.

The antibodies serve as an active transport mechanism, and once situated, the nanoparticles are excited by a focused light source (generally introduced through surgery as brain tumors cannot be exposed to light otherwise) and subsequently releases free oxygen radicals, including reactive oxygen species (ROS), as described in Figure 2. Generally a threat to living systems due to their cytotoxic reactivity, these radicals work by initiating the process of programmed cellular death through mitochondria and damaging the cell membrane. This localized signaling is key to the selective killing of the malignant cells and preservation of the organism’s healthy cells. Further, cancer cells are thought to infect healthy cells through their “invasive feet” or invadopia, small extra-cellular protrusions from the surface that communicate with neighboring cells. In X-ray fluorescence studies, the nanoparticles were observed attacking the invadopia.

Medical Uses
Current methods of treatment for tumors of the brain are incredibly limited, and in most cases require invasive surgical techniques. Currently, the three most commonly administered methods of treatments are surgery, radiation, and chemotherapy.

Surgery involves craniotomy (surgical opening of the skull) and physical removal of part or the entire tumor. Patients must have their scalp removed and their skulls opened with a medical saw. During the surgical process, surgeons must be wary of not damaging brain tissue or the stem. One major disadvantage of the surgical method is that post-surgery regeneration of the tumor is possible. Additionally, as with many brain procedures, edema or seizures are common in patients post-surgery. Patients are also left with large scars and metal plates which are cosmetically unattractive and uncomfortable.

Another method of tumor therapy is radiotherapy. This method uses high-energy radiation to eliminate tumor cells. Radiation used in tumor therapy includes x-rays, gamma rays and photons. Radiotherapy is typically either coupled with invasive surgery as a maintenance procedure or used as primary treatment for patients who are not suited for the surgical procedure. However, exposure to high-energy radiation on healthy cells is detrimental to the rest of the body, including the spinal cord and healthy brain cells. Commonly with radiation therapy, patients experience weakness and hair loss.

A third common method for tumor treatment is chemotherapy. This treatment involves the injection/ingestion of drugs into the body which are specifically designed to attack tumor cells. While this method is less invasive than the previous two treatments, general exposure to drugs is associated with serious side effects. Common side effects include nausea, chills, vomiting, loss of appetite and weakness. Patients often take series of additional drugs to alleviate these side effects, which are often costly and leave the possibility for unexpected drug interactions.

Very often, the tumors are too small to detect and remove via surgical methods. Ideally, as with other forms of cancer treatment, doctors and researchers would like to be able to treat these tumors with a less invasive, more targeted approach. Researchers have increasingly explored the use of biologically-activated nanotechnology to engineer a new technique that not only eliminates the need for surgery, but is able to target only cancer cells while leaving normal cells untouched. The ability of the TiO2/antibody complex to be coupled with neuroreceptors such as dopamine gives the complex the ability to cross the blood-brain barrier. Once in the brain, the biologically-conjugated particle passively seeks out GBM cells, among other tumors. These cells can then be treated with light, triggering the mechanism described in Figure 2 and resulting in programmed cell death of the targeted cancer cells. Because of the specificity of binding, this complex has the potential for use in various forms of cancer treatment and researchers at the University of Chicago are preparing for pre-clinical trials.

Adverse Effects
There are several drawbacks to the use of TiO2 as a cancer-seeking agent, however. One major problem is the necessity of the complex to be directly exposed to light in order to induce reaction. Many brain tumors are difficult to access even during the most invasive of surgeries, making light-based techniques difficult. Even if the tumor is accessible, a small hole will still be necessary in order to provide the light needed. Another major obstacle with the use of TiO2 nanoparticles are recent findings that show a possible carcinogenic behavior of the complexes.

Toxicity
Concerns are raised regarding the toxicity levels of TiO2 nanoparticles. Although the nanoparticles are typically coupled to a targeting agent, they may disrupt other healthy functions in the body. Once in the body, the free nanoparticles cannot be directed to a specific location, which allows them to accumulate in various organs with no method of removal. Although titanium is inherently inert unless activated by light, the small size of the particle can allow TiO2 to enter cells, while the large relative surface area to volume ratio of nanoparticles creates the potential to cause oxidative stress to cells and even become lodged in the lungs--thus contributing to the death of healthy, normal cells. Research has shown evidence that TiO2 nanoparticles tend to accumulate in higher amounts in liver, spleen, lung, and peritoneal tissues.

Research at Institut für Umweltmedizinische Forschung (IUF) in Dusseldorf, Germany, has shown evidence that nanoparticles (such as SiO2 and TiO2) may be able to penetrate into the nuclei of healthy cells, and interfere directly with the DNA contained there. There is evidence of a direct chemical interaction between the TiO2 nanoparticle surface and the phosphate groups of the DNA strands, but no links to mutation formation has been proven. Other studies show that TiO2 particles can cause indirect damage to DNA strands through inflammation or creation of ROS. While this is a beneficial characteristic of the nanoparticles acting in cancerous cells, genetic mutation in healthy cells is undesirable and may, in fact, result in tumor growth in an entirely different area of the body.

A recent study showed that mice exposed to TiO2-doped drinking water showed evidence of genetic damage in under a week, and began developing tumors as a possible result. The researchers are quick to note that it is not everyday exposure that has been linked to TiO2 exposure, but for individuals who have high exposure levels, the risk may be serious.