Encapsulation of phosphorylated chemotherapeutics in calcium phosphosilicate nanoparticles and the translational perspective to nanoparticle drug delivery
Open Access
- Author:
- Loc, Welley Siu
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 01, 2016
- Committee Members:
- James Hansell Adair, Dissertation Advisor/Co-Advisor
James Hansell Adair, Committee Chair/Co-Chair
Thomas E Mallouk, Committee Member
David D Boehr, Committee Member
Peter J Butler, Outside Member - Keywords:
- nanomedicine
drug delivery
nanoparticles
CPSNPs
chemotherapeutics
phospho-drug
gold - Abstract:
- Cancer retains negative associations to chemotherapy even as treatments evolve. Alopecia, neuropathy, nail loss, and invasive surgical procedures, can arguably make a more detrimental experience than having the disease at early stages. On the other hand, patients cannot benefit from the best treatment outcome when symptoms surface later, so early detection is the key to maximize chances of survival. However, patients are often present at a certain stage of cancer when tumors are detectable by current imaging capabilities. This is the case for those with pancreatic ductal adenocarcinoma (PDAC). Being the fourth most lethal type of all cancers with a ≤10% 5-year survival, there is no treatment to effectively manage the condition. The motive of this dissertation emphasizes the possibilities and importance of enhancing the efficacy of existing medicine for pancreatic cancer. The common antimetabolite, 5-fluorouracil (5-FU), is amongst the most effective drug agents when first discovered in 1957 and is still part of the modern treatment regimen. Because 5-FU disrupts DNA and RNA replication, there are many toxic side effects from drug metabolism, so the ability to target cancer is a pivotal characteristic that 5-FU and other first generation chemotherapeutics lack. Advances in nanotechnology has opened a new era in this area by offering meticulous ways to engineer nanoparticles to encapsulate and deliver drugs to specific sites. Given the known mechanisms of 5-FU in cells from years of basic research and clinical trials, drug reformulation is a valuable alternative to the conventional route of drug discovery. Calcium phosphosilicate nanoparticles (CPSNPs) offer an effective drug delivery platform that is bioresorbable with a narrow particle size distribution to enable penetration of dense fibrotic tumors such as PDAC. Developed by the Adair, Kester, and Matters teams, both gastrin (T. Morgan, 2009 and E. Altinoglu, 2010 dissertations) and aptamer-modified (X. Tang, 2016 dissertation) CPSNPs doped with indocyanine green (ICG) dye have demonstrated accumulation and specific targeting at the pancreas of orthotopically transplanted BxPC-3 and PANC-1 athymic mice cancer models. The reports herein are a continuation of these investigations that focus on the therapeutic capabilities of CPSNPs. This involves the significance of active phosphorylated agents on drug load optimization in CPSNPs through adsorption-mediated encapsulation. These active metabolites include fluorodeoxyuridine monophosphate (FdUMP) for 5-FU and gemcitabine monophosphate (GemMP; dFdCMP) for gemcitabine (Gem; dFdC). Conversion from the prodrugs (biologically inactive 5-FU and gemcitabine) to the active forms is an enzyme-catalyzed event in cells. Without targeting abilities, majority of 5-FU and gemcitabine become catabolized before the agents can carry out mechanisms of action. Concepts behind adsorption-mediated encapsulation based on zeta potential evaluations and the role of potential-determining ions (hydroxyls, protons, and phosphates) from solution to the surface of calcium phosphate were examined. The relationship between adsorption and speciation diagrams of the different ions was also interpreted. These experiments were conducted to show that compounds with phosphate and other charged groups can adsorb to calcium phosphate, which was indicated by shifts in surface polarity. Brushite was synthesized and subjected to various concentrations of potassium hydroxide (KOH), hydrochloric acid (HCl), sodium citrate (Cit), adenosine triphosphate (ATP), 5-FU, and FdUMP, to analyze trends in the zeta potential curve vs. pH. The isoelectric point (IEP) of brushite in water at room temperature was pH ~7 and the addition of OH− and H+ ions influenced the solubility products of calcium phosphate at the solid-solution interface. At pH >7, the zeta potential was negative due to the net anionic species produced. The opposite effect was observed at pH <7. Both Cit and ATP were found to robustly adsorb to calcium phosphate and shifted the zeta potential to increasingly negative values at increasing concentrations. The effects of adsorption by FdUMP were not as profound as Cit and ATP even though FdUMP was expected to make the zeta potential more negative. The presence of FdUMP shifted the isoelectric point of the calcium phosphate and the event was not present with 5-FU. Results on ATP and FdUMP raised implications that a higher uptake of phosphorylated compounds can be achieved with CPSNPs by taking advantage of the affinity between calcium ions on calcium phosphate and the phosphate group on the compound. The non-phosphorylated drug analogs were not well encapsulated into CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. In Chapter 5, a novel synthesis of gold nanoparticles using sodium thiophosphate (TP) was introduced in the development of gold-calcium phosphosilicate (Au-CPS) core-shell nanoparticles for theranostic applications (combined imaging and therapeutic capabilities). Thiophosphates were the highlight of this work to induce gold core encapsulation through calcium ion immobilization at the gold surface to seed CPS growth. The kinetics of the Au-TP reduction was studied with the Johnson-Mehl-Avrami-Kolmogorov (JMAK) model and analyzed alongside with the gold particle number concentration as reactions progressed for 60 min. The reaction was conducted at various molar ratios of chloroauric acid to TP at room temperature. The values for the Avrami reaction rate constant, k, implied that TP was significant for inducing the supersaturation of gold-thiophosphate monomers into nuclei. The Avrami parameter, n, suggested a diffusion controlled mechanism. With additional excess thiophosphate, both k and particle concentration increased during the nucleation period (within 10 min). Overall particle sizes were smaller as TP concentration increased and the nanoparticles absorb within 500-600 nm in the surface plasmon resonance (SPR) band region. While the JMAK model has limitations such that the constants can only be interpreted within the Avrami definitions, the study offered insights into possible mechanisms and ways to control colloidal stability and size by comparing the reaction at different reactant ratios. The first example of Au-CPS gold cores was also presented with suggestions for future refinement work. Lastly, this entire work is described within the context of translational science in Chapter 6. To reach an understanding of the drug discovery cycle and the challenges is important for those conducting research to impact global healthcare. In STEM research, the need to generate data on the fundamental sciences can often overshadow the purpose to provide applicable solutions to improve patient outcome. Here, encountered experimental scenarios for nanoparticles were presented and discussed to identify components that are critical for advancing these benchtop findings into clinical trials.