Date of Award

5-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmaceutics

Research Advisor

George C. Wood, Ph.D.

Committee

Himanshu Bhattacharjee, Ph.D. Leonard Lothstein, Ph.D. Timothy D. Mandrell, Ph.D. Duane D. Miller, Ph.D.

Abstract

Hematological malignances of the B cells affect almost 130,000 people in the United States every year of which approximately 44,000 lose their lives. Therapies for B cell malignancies such as doxorubicin, have limitations due to dose related adverse effects such as neutropenia and cardiomyopathy. AD 198 is a novel PKC-delta activating agent that has cytotoxic superiority over doxorubicin by being able to circumvent resistance mechanisms developed by the cancer cells towards doxorubicin and being cadioprotective from the damage caused to cardiomyocytes by doxorubicin. Targeted delivery of AD 198 is crucial to moderate the non-specific interactions of the chemotherapeutic agent with healthy cells. The objective of this work was to design, develop and evaluate AD 198 loaded liposomal formulations for targeted delivery to CD22 overexpressing B cell cancers. Liposomes were prepared by the classical Bangham method followed by size reduction by extrusion. Anti-CD22 Fab’ were generated and conjugated to the long circulating AD 198 liposomes by thioether bonds. Physicochemical parameters of the CD22 targeted liposomal formulation such as size, zeta-potential, drug encapsulation, drug release and drug targeting were optimized to maximize efficacy and stability of the nanoparticles. In vitro studies established that uptake of the targeted liposomes was via an endocytotic pathway independent of the conventional proteins involved in clathrin and caveolae dependent endocytosis. Uptake was preferentially higher in CD22 overexpressing malignant B cells (Daudi) compared to cells devoid of CD22 (Jurkat). Intracellular localization of the targeted liposomes was observed to be in endolysosomes which suggests that drug release was via lysosomal enzymatic breakdown of the liposomal structure which would release encapsulated AD 198. Cytotoxicity was also observed to be higher in Daudi cells compared to Jurkat cells whereas free solution AD 198 had comparable cytotoxicity in both cell types. The mechanism of cell death was deduced to be apoptosis by the activation of apoptotic proteins such as caspase-3, and by the inhibition of oncoproteins such as c-myc. Stability of the dispersed liposomal formulation was inadequate for long term storage. To address this concern, lyophilized formulations were developed and optimized to maximize long term storage as displayed by short term stability studies. It was determined that targeted drug delivery with liposomal AD 198 was more potent and specific compared to other untargeted formulations.

DOI

10.21007/etd.cghs.2015.0214

Comments

One year embargo expired May 2016

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