This item is under embargo and not available online per the author's request. For access information, please visit http://libanswers.wustl.edu/faq/5640.
Date of Award
Doctor of Philosophy (PhD)
The synthesis and design of biocompatible nanoparticles for targeted drug delivery and bioimaging requires knowledge of both their potential toxicity and their transport. For both practical and ethical reasons, evaluating exposure via cell studies is a logical precursor to in vivo tests. As a step towards clinical trials, this work extensively investigated the toxicity of gold nanoparticles (Au NPs) and carbon dot (CD) nanoparticles as a prelude to their in vivo application, focusing specifically on ocular cells. As a further step, it also evaluated their whole-body transport in mice. The research pursued two approaches in assessing the toxicity of engineered nanoparticles and the suitability of their use in targeted delivery and bioimaging applications: (1) In vitro (using retinal pigment epithelial, corneal, and lens epithelial cells (2) In vivo (mouse whole body studies).
Part. 1. In the in vitro assessments of Part 1, the biocompatibilities of spherical, rod, and cubic shaped Au NPs were compared for different exposure concentrations. Spherical Au NPs were evaluated in particular detail, and a possible toxicity mechanism was proposed, based on the findings of a colorimetric assay, electrical impedance measurements, and confocal imaging analysis. The assay measured the activity of succinate hydrogenase, a mitochondrial enzyme, while electrical impedance spectroscopy quantified the strength of cell-cell and cell-substrate attachment, a proxy of viability. Finally, confocal imaging analysis verified that the NPs were internalized and confirmed the degree of their toxicity. Collectively, the data indicated that surface area concentration was the critical toxicity parameter. Subsequently, to create biocompatible Au NPs, a unique end-thiolation of hyaluronic acid was adapted to create homogenously coated Au NPs. The end-thiolated hyaluronate (HS-HA) coating not only improved the biocompatibility of the Au NPs but also enhanced the internalization rate of the larger Au NPs, which could not enter the cells otherwise.
The first part of this research also studied the synthesis of biocompatible deep red-emissive CDs for bioimaging applications. For this purpose, a central-composite design response surface methodology (CCD-RSM) was utilized. A scalable isolation-free microwave pyrolysis method for synthesizing deep red-emissive nitrogen-doped carbon dots (nCDs) from citric acid and ethylenediamine was successfully developed and optimized. The formation of C‒N and the presence of pyrrolic N content proved to be keys to creating red-emissive nCDs. Confocal images demonstrated that the nanoparticles could enter healthy corneal, retinal, and lens epithelial ocular cells, as well as cancerous Chinese Hamster Ovary cells.
Part 2. Building on the results of in-vitro testing of the engineered Au NPs and nCDs, in Part 2 we developed protocols for injecting both types of NPs in-vivo. Prior to any intravenous or intravitreal injections, a preliminary study tested the ability of Au NPs to cross the tight junctions between retinal pigment epithelial cells. Transwell® permeable supports were used to simulate the blood-retinal barrier (BRB). The results showed that 20 nm Au NPs successfully crossed the permeable supports covered with confluent retinal pigment epithelial cells. Based on this finding, both intravitreal and intravenous injections of nascent and HS-HA coated Au NPs were tested. The intravitreal injections caused retinal detachment, very probably due to the mechanical intrusion of the injection needle and the volume, albeit small, of the injected NPs. Far more significant and encouraging, intravenously injected coated and uncoated NPs successfully crossed the BRB. As a result of the intravenous injections, it was observed that both coated and uncoated Au NPs were able to cross the blood-retinal barrier. As expected, the numbers of HS-HA-coated Au NPs were significantly higher in specific parts of the retina that contain more CD44 expressing cells, which have cell surface receptors for internalizing HA. Finally, based on the confocal imaging analysis, the NP concentration in each retinal layer was quantified as a function of time, post-injection. The NPs reached the retina in less than 5 minutes and reached a maximum concentration within approximately 20 minutes. Due to the enhanced retention and permeability effect of NPs, 8.5% of the uncoated and 12.1% of the HA-coated NPs that reach the retina remained after 24 hours.
Next, nCDs with and without the HA coating were injected subcutaneously into post-mortem mouse and porcine eye globes. Ex-vivo porcine eye images showed that intravitreally injected nCDs had effectively diffused through the vitreous to the cornea, and post-mortem whole-body mouse images also demonstrated that the nCDs are suitable for bioimaging, excitable in the NIR region with the sensitivity of 15%.
Cumulatively, our observations indicate that HA coated NPs could potentially deliver other payloads such as DNAs, mRNAs, proteins, siRNAs, and drugs into the cells which overexpress CD44 receptors, for example, cancerous and inflammatory cells, thus providing a platform for targeted treatment and imaging of many severe vision-threatening diseases and degenerative conditions.
Nathan Pratim . Ravi Biswas
Rajendra S. Apte, Palghat Ramachandran, Fuzhong Zhang,
Available for download on Saturday, July 27, 2019