Author ORCID Identifier

https://orcid.org/0000-0002-9303-1740

Date Available

5-10-2023

Year of Publication

2023

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

First Advisor

Dr. Younsoo Bae

Abstract

Cancer remains a real and present threat to global health. In the United States, according to cancer statistics, almost 40% of people will be diagnosed with cancer at some point in their lifetime. Conventional chemotherapy has become the mainstay for cancer treatment option. However, chemotherapeutic agents are plagued with problems such as poor aqueous solubility, chemical degradation, Bio instability, and off-site toxicity due to non-specificity. New drug modalities are needed to tackle the ever-growing burden on cancer. In recent times, the promise of nanotechnology has aided to develop drug delivery vehicles to facilitate the administration of potent chemotherapeutics. Nanoformulations such as liposomes and polymeric micelles have been previously developed to tackle these pharmaceutical limitations, however, these modalities have inherently low drug loading, uncontrolled release and particle instability that ultimately produce sub-par outcomes. This work hypothesizes that the design of novel nanoparticles with attention paid to particle stability and controlled release will improve the performance of potent drugs. To this end, this work showcases the design and optimization of a novel self-assembled ternary polypeptide nanoparticle. For this study, we used a model drug carfilzomib (CFZ) already approved by the Food and Drug Association (FDA). Carfilzomib (CFZ) is a tetrapeptide epoxyketone proteasome inhibitor approved by the FDA for treatment of relapsed/refractory multiple myeloma (MM). Although it has been successful in treating patients with MM, CFZ has shown limited efficacy against solid tumors in the clinic. One pharmaceutical limitation of CFZ is its poor aqueous solubility. Additionally, CFZ is prone to peptide degradation and epoxyketone ring opening. Clinical limitations include low half-life, rapid clearance and poor targetability. To address these limitations, self-assembled ternary polypeptide nanoparticles (tPNPs) was designed comprising Heptakis(6-amino-6-deoxy)-βCD-heptahydrochloride (HaβCD) and Azido-poly (ethylene glycol)-block-poly (L-glutamic acid sodium salt) (N3-PEG-PLE). CFZ is entrapped within the core of HaβCD which then ionically interacts with N3-PEG-PLE to form CFZ/tPNPs. For further optimization, small molecule organic acids and polycations were used to enhance particle stability and controlled release. For targeted delivery of CFZ, epithelial cell adhesion molecule (EpCAM) antibody was conjugated to N3-PEG-PLE before drug loading to form (CFZ/EpCAM-tPNPs). All formulations (tPNPs, CFZ/tPNPs and CFZ/EpCAM-tPNPs) showed a uniform particle size of ~ 50 nm. Addition of cryoprotectants ensured the stability of these formulations after freeze drying. CFZ/tPNPs were also able to achieve high drug loading (> 1 mg/mL). These formulations were tested in vitro in DLD-1 CFZ resistant cells. The EpCAM-tPNPs had significantly better cellular uptake than tPNPs and the control. CFZ/tPNPs better sustained proteasome inhibition compared to free CFZ. The optimized formulation prepared with polycation stabilization also showed better proteasome suppression than free CFZ. Finally, CFZ/tPNPs and CFZ/EpCAM-tPNPs showed a greater cytotoxicity against DLD-1 CFZ resistant cells than free CFZ. These results suggest that CFZ/tPNPs and CFZ/EpCAM-tPNPs could be potentially useful for improving the efficacy of CFZ in solid tumors, hence, expanding its clinical utility. Biodistribution studies show particle accumulation in tumor as well as peripheral organs. This potentiates the development of different tumor models to better assess particle effect and treatment outcomes. Taken together, this thesis provides the basis for the development and optimization of a novel nanoparticle to tackle some of the limitations of current chemotherapy. It also paves the way for further exploration to expand our knowledge of the effect of such formulation modalities on in vivo systems.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2023.211

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