Date of Award

5-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

George C. Wood, Ph.D.

Committee

Himanshu Bhattacharjee, Ph.D. James R. Johnson, Ph.D. Timothy D. Mandrell, Ph.D. Duane D. Miller, Ph.D.

Abstract

The objective of this work was to design, develop and evaluate erlotinib-loaded coreshell type lipid albumin hybrid nanoparticles (CSLAHNPs) for targeted drug delivery to nonsmall cell lung cancer (NSCLC). Erlotinib (ETB) is a highly selective, potent and reversible inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR) which is overexpressed (50-90%) in NSCLC. ETB is marketed as film coated tablets for oral delivery. However, poor survival rate along with life-threatening adverse effects were reported from oral administration. Nanoparticulate delivery system of ETB might be advantageous to target the tumor cells, thereby increasing therapeutic efficacy and reducing off-targeting toxicities of ETB to healthy cells. In this work, a unique nanoparticulate carrier termed as CSLAHNPs was used for targeted delivery of ETB. The CSLAHNPs system was composed of albumin core and phospholipid bilayer shell. For active targeting to EGFR positive NSCLC, anti-EGFR half-antibodes (hAbs) were conjugate to EGFR expressing NSCLC. Overall hypothesis was to improve the efficacy of ETB in EGFR positive NSCLC using the targeted hAb-ETB- CSLAHNPs and untargeted ETB-CSLAHNPs. Blank CSLAHNPs were prepared by two-step method using bovine serum albumin and lipid mixture composed of 60:30:10 molar ratio of dipalmitoyl-phosphatidylcholine (DPPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) respectively. ETB was loaded into CSLAHNPs by incubation method. Murine anti-EGFR monoclonal antibody (mAb) was reduced to hAb using tris(2- carboxyethyl)phosphine and conjugated to maleimide terminated ETB- CSLAHNPs via maleimide-thiol conjugation reaction. CSLAHNPs were further characterized for physicochemical properties including mean size, polydispersity index, zeta potential, drug loading efficiency, in vitro drug release, and in vitro serum stability. The optimized ETBCSLAHNPs and hAb-ETB-CSLAHNPs were evaluated for their in vitro biological properties including cellular association cellular uptake, endolysosomal trafficking, cell viability, colony formation assay and western blots in two human lung adenocarcinoma cells; A549 ( having wildtype EGFR) and HCC827 (having an acquired mutation in EGFR) cells. The mean size of hAbETB-CSLAHNPs (targeted) and ETB-CSLAHNPs (untargeted) was between 190-210 nm, suitable for intravenous delivery. The zeta potential, drug loading, and drug entrapment efficiency were about -13 mV, 2 % w/w, and 31% w/w respectively. CSLAHNPs exhibited sustained drug release profiles over 72-96 h in PBS pH 7.4. Fluorescent lipid tagged hAb-ETBCSLAHNPs showed enhanced uptake and accumulated in the cells. Significant reduction in % cell viability was observed for targeted hAb-ETB- CSLAHNPs compared to control groups in HCC827 cells after 72 h. The analysis of IC50 demonstrated that both targeted hAb-ETBCSLAHNPs and untargeted ETB-CSLAHNPs could be more effective than ETB alone in both EGFR- positive NSCLC cells. Short-term stability data at refrigerator condition demonstrated that the lyophilized form of CSLAHNPs containing 16-fold sucrose (lyoprotectant) significantly improved the physical and chemical stability compared to liquid dispersion for 60 days of storage. Overall, the results indicated that hAb-ETB- CSLAHNPs and ETB-CSLAHNPs would be promising ETB delivery systems for EGFR-overexpressing NSCLC.

DOI

10.21007/etd.cghs.2015.0196

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