Date of Award
Doctor of Philosophy (PhD)
Molecular Science and Nanotechnology
Halloysite nanoclay is a natural material which is interesting for biomedical applications. Its unique physiochemical structure that forms the tubular shape originates from rolled sheets of aluminosilicate 15-20 times, such that alumina is inside and silica on the outside of the nanotubes, thus defining inner positive and outer negative charges. The dimensions of these tubules vary from 500-1,000 nm in length, external diameter 50-60nm, and inner lumen 12-15nm. Halloysites are biocompatible, cheap, and available in large quantities which suggests that these tubules can be used as effective nanocarriers. In this work, we exploited halloysite as a “nano-torpedo” for targeted drug delivery through the membranes in two ways. We discovered new “larger” nanotubes of 80 nm diameter and 1.6 μm length and used them for skin protection with in depth halloysite drug delivery. In the second part of the work, the use of traditional “smaller” halloysite as a carrier for drug delivery through the blood-brain barrier was developed, both in vitro and in vivo. In this work, we made all drug nanoformulations, and collaborate with Dr. DeCoster lab and Dr. Murray lab in cell and mice experiments.
We invented “new” larger halloysite nanotubes and utilized their potential for skin care applications. Analyzing the structure of the halloysite nanotubes provided by NorthStar LLC., we found an increase in dimensions when compared to traditional halloysite. The larger tubes have a length of 1.6 μm and a diameter of 80 nm. Visualization of halloysite dispersibility in water was possible with these larger tubules confirming favorable aqueous dispersibility and structural organization are comparable traditional smaller halloysite. We suggested that the larger tubes formation mechanism could be similar to smaller halloysite (rolling), but initial aluminosilicate sheets were of larger area, thus forming larger tubes. We theorized that these “new” nanotubes would be more efficient in penetration though strong barriers, like skin, for drug delivery due to their size and increased drug loading capacity. Encapsulating vitamin B-12, a known skin protecting agent inside these nanotubes create a new formulation with sustained B-12 release when admixed to traditional oil-based skin cream. Pig skin was used for initial testing and analysis due to it consisting of a similar epidermis to human skin. Initial results were promising with massaging of such a formulation on skin which provides perpendicular penetration of the halloysite nanotubes.
Additionally, we found that for penetration though cell membranes around the brain, smaller halloysite formulations could be more effective. Current treatments for brain disorders such as epilepsy are not effective due to the selectivity of the blood-brain barrier (BBB) preventing drug delivery. Utilizing traditional small halloysite with dimensions of 500-800 nm in length and external diameter of 50-60 nm and inner lumen 12-15 nm, we conducted in vitro experiments of halloysite formulations as a “nano-torpedo” penetrating brain microvascular endothelial cell (BMVECs). Through fluorescent and real time calcium imaging techniques we proved a prolonged gradual drug delivery mechanism by the encapsulation of rhodamine isothiocyanate and ionomycin within the nanotube. With delayed diffusion, the nanotubes effectively delivered the drug to the primary BMVECs without causing any toxic effects or killing them, by binding and penetration in time periods of 1 to 24 h.
Furthermore, constructing a two layer (astrocytes and endothelial) synthetic blood-brain model barrier with a 0.4 um porous transwell support enabling us to study structures closer to the BBB. The confluence of the bilayer of astrocyte and endothelial cells was examined using a non-inverted microscope after Diff-Quik staining of the cells. The halloysite rhodamine formulations were tested on this barrier by treating the samples to the dual layer of endothelial / astrocyte cells and allowing them to pass through the barrier on their own. Proving its penetration by measuring the fluorescence at the bottom of the membrane after 24 h. The fluorescence signal grew moderately in the first hour but by 24 h it had increased to 85 %.
In the last section of this work, in vivo experiments on halloysite delivery to the brains of mice via intranasal administration were conducted in collaboration with Y. Yanamadala. Halloysite formulations with encapsulation of rhodamine isothiocyanate to track halloysite as it moves throughout the brain were developed. We produced the nanoclay loading of diazepam and xylazine which are two commonly used drugs for their anxiolytic and sedation effects. Wild type C57BL/6NHsd were purchased from Jackson Laboratory and these three halloysite formulations were intranasally delivered to mice. Analysis of the olfactory bulb and cortex were accomplished through multiple microscopy techniques and the overall condition of the mice were measured by behavioral studies such as rotarod, novel object recognition, and open field tests over the course of seven days. In this collaborative work we proved not only delivery of halloysite to the brain by passing the blood-brain barrier, but also have shown that its presence in the brain was not harmful to the behavior or well-being of the mice. Therefore, potentially, such halloysite-drug delivery may be used for urgent brain disease treatment with deposition / of this biocompatible nanoclay residues.
Saleh, Mahdi, "" (2023). Dissertation. 993.