Date of Award

2021

Degree Type

Thesis

Degree Name

Master of Science in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Jyothi Menon

Abstract

Idiopathic Pulmonary Fibrosis (IPF) is a chronic lung disease which causes scarring of the lung tissue and makes breathing more difficult [1]. Pirfenidone (PFD), one of only two FDA-approved therapies for IPF, is clinically shown to reduce the rate of scarring of lung tissue; however, it is limited by the need for daily oral administration of chronic doses which can lead to off-target side effects [2]. Inhalable nanoparticle-based (NP) drug delivery systems allow for targeted, controlled drug release in the deep lung tissue while increasing contact with and uptake by lung cells [3]. Research has found that Galectin-3 plays an important role in the pathogenesis of human IPF [4]. The goal of this project is to develop, characterize, and evaluate a dual-drug nanoparticle formulation that can be simultaneously used for targeted Galectin-3 inhibition and sustained PFD release, to provide an effective combination therapy to treat IPF.

The drug-loaded polymeric NPs were prepared using a standard double emulsion technique followed by ultracentrifugation to purify the particles and lyophilization for long term storage. Surface decoration with citrus pectin (CP) was carried out on the freeze-dried (lyophilized) particles to yield citrus pectin coated NPs (CP-NPs). Particle size, size distribution, and zeta potential (ZP) were measured using a dynamic light scattering (DLS) instrument. A UV-Vis spectrophotometric method was developed to determine the encapsulation efficiency of the PFD-loaded NPs as well as quantify amount of CP coated on final CP-NPs formulation. Fourier transform infrared spectroscopy (FTIR) was carried out to confirm the surface coating of the CP-NPs. For stability studies, CP-NPs were incubated at 37 degrees Celsius for 7 days in both phosphate-buffered saline (PBS) and simulated lung fluid (SLF), with particle sizes of the NPs measured daily. Cytocompatibility of the CP-NPs was determined by treating MRC5, an immortalized human fetal lung fibroblast cell line, with CP-NPs at varying concentrations.

The following NP characterization data suggests that a uniform and high-concentration NP batch was formulated: Particle size of 190.1 d.nm with 100% peak intensity, ZP of -8.21, PFD encapsulation efficiency of 71.95%, and CP conjugation efficiency of 40.3%. The FTIR spectra captured for CP-NPs displayed bands indicative of O-H bond stretching of carboxyl groups, confirming presence of CP. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) images confirmed uniform size and spherical morphology of both coated and un-coated NPs. TEM images displayed core-shell structure of CP-NPs. Stability studies showed NP diameter to have minimal variation for up to 7 days in PBS and SLF, and zeta potentials greater than -10mV for 7 days, indicating highly stable NPs. MRC5 fibroblasts treated with CP concentrations of 100-2000 ug/ml demonstrated nearly 100% viability at all concentrations when compared to untreated cells (0 ug/ml). Cellular uptake studies of CP-coated nanoparticles in MRC5 cells viewed under EVOS microscope for fluorescence showed uptake of 500 ug/ml concentration for both non-activated and TGF-β1-activated cells, with quantitative data of fluorescence intensity showing greater uptake in the TGF-β1-activated group. Western blot data showed decreased α-SMA and Galectin-3 expression in MRC5 cells treated with CP-coated NPs for 24 hours after TGF-β1 activation. Overall, the results presented in this thesis show that CP is an attractive candidate with potential for targeted inhalation delivery via nanoparticles to the lungs to treat IPF as it is found to be easily coated to polymeric nanoparticle surface, cytocompatible with lung cells, taken up more in a fibrotic state, and reduces TGF-β1-induced α-SMA expression in vitro.

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Available for download on Tuesday, August 13, 2024

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