Graduation Year

2017

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Chemical Engineering

Major Professor

Vinay K. Gupta, Ph.D.

Co-Major Professor

Venkat Bhethanabotla, Ph.D.

Committee Member

Piyush Koria, Ph.D.

Committee Member

Nathan Crane, Ph.D.

Committee Member

David Mitchell, Ph.D.

Keywords

osteoclast, osteoblast, pamidronate, alendronate, remodeling

Abstract

The use of nanoparticles for disease treatment is an increasingly popular area of research. The potential for multi-functionality allows nanoparticles to be used as transport and delivery vehicles for drugs and as diagnostic aides, among other applications, to address the unmet needs of many disease treatments. One such class of disease is osteoporosis including severe disorders, like Paget’s disease, Osteogenesis Imperfecta and Legg Calve Perthes disease. In this dissertation, we discuss a nanoparticle system consisting of gold nanoparticles surface functionalized with primary amine bisphosphonates, which is a classification of pharmaceuticals that is common in the treatment of osteoporosis. Functionalized nanoparticles allow for greater intracellular concentrations of pharmaceutical, while the properties of the gold nanoparticles provide the ability to track the pharmaceutical and enhance imaging.

We have synthesized and characterized bisphosphonate functionalized gold nanoparticles of controlled size of approximately 15 nm, which are suitable for cellular uptake, and functionalized the surface using self-assembly with pamidronate and alendronate. In one major finding of this study, inductively coupled plasma mass spectrometry was used to estimate approximate surface density of the bisphosphonates on the gold nanoparticles. This resulted in concentrations of approximately 0.65 molecules per nm2 (approximately 154 Å2/molecule) for pamidronate functionalized on gold, and approximately 2.6 molecules per nm2 (approximately 39 Å2/molecule) for alendronate functionalized on gold. This allows for more accurate estimates of pharmaceutical concentrations, during in vitro and in vivo studies.

Additionally, we investigated the effects of bisphosphonate functionalized gold nanoparticles on the viability and morphology of osteoclast and osteoblast cells in vitro. We found that attaching the bisphosphonates to the surface of the nanoparticles leads to increased apoptotic effects of the bisphosphonates on the osteoclast cells compared to free bisphosphonates. Further, we showed bisphosphonate functionalized gold nanoparticles may have an effect on nuclei morphology that may provide an additional means of modulating bone resorption rather than just through influencing viability. Further we showed that it may be possible to target concentrations that are safe for osteoblasts, which is critical in determining potential treatment concentrations. These viability results bring to light a number of potential considerations into the optimization of potential treatments, such as dosing concentrations.

Finally, detailed results are given on effects of bisphosphonate functionalized gold nanoparticles on important behavior and activity of osteoclast and osteoblast cells in vitro. We showed that while using concentrations below the toxicity threshold, some of the normal activity of the cells could be maintained. RANKL and ALP expression in osteoblasts were maintained when removing viability as a variable. Additionally, bone nodule formation was also maintained for osteoblasts and co-cultured in vitro systems. Finally, we showed that the introduction of bone in the in vitro studies adds a new degree of consideration as to the interaction of the bisphosphonates with the hydroxyapatite surface. This strong interaction with bone is an important consideration in further developing potential treatments for osteoporotic disease.

This dissertation provides insights into the use of bisphosphonate functionalized gold nanoparticles as a potential treatment and means of study for bone remodeling disorders.

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