Author ORCID Identifier

https://orcid.org/0000-0001-8471-4200

Date Available

10-14-2019

Year of Publication

2019

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Engineering

Department/School/Program

Chemical and Materials Engineering

Advisor

Dr. Barbara L. Knutson

Co-Director of Graduate Studies

Dr. Stephen E. Rankin

Abstract

Mesoporous silica nanoparticles (MSNPs) possess large surface areas and ample pore space that can be readily modified with specific functional groups for targeted binding of bioactive materials to be transported through cellular barriers. Engineered silica nanoparticles (ESNP) have been used extensively to deliver bio-active materials to target intracellular sites, including as non-viral vectors for nucleic acid (DNA/RNA) delivery such as for siRNA induced interference. The reverse process guided by the same principles is called “nanoharvesting”, where valuable biomolecules are carried out and separated from living and functioning organisms using nano-carriers. This dissertation focuses on ESNP design principles for both applications.

To investigate the bioactive materials loading, the adsorption of antioxidant flavonoids was investigated on titania (TiO2) functionalized MSNPs (mean particle diameter ~170 nm). The amount of flavonoid adsorbed onto particle surface was a strong function of active group (TiO2) grafting and a 100-fold increase in the adsorption capacity was observed relative to nonporous particles with similar TiO2 coverage. Active flavonoid was released from the particle surface using citric acid-mediated ligand displacement. Afterwards, nanoharvesting of flavonoids from plant hairy roots is demonstrated using ESNP in which TiO2 and amine functional groups are used as specific binding sites and positive surface charge source, respectively. Isolation of therapeutics was confirmed by increased pharmacological activity of the particles. After nanoharvesting, roots are found to be viable and capable of therapeutic re-synthesis. In order to identify the underlying nanoparticle uptake mechanism, TiO2 content of the plant roots was quantified with exposure to nanoparticles. Temperature (4 or 23 °C) dependent particle recovery, in which time dependent release of ESNP from plant cells showed a similar trend, indicated an energy independent process (passive transport).

To achieve the selective separation and nanoharvesting of higher value therapeutics, amine functionalized MSNPs were conjugated with specific functional oligopeptides using a hetero-bifunctional linker. Fluorescence spectroscopy was used to confirm and determine binding efficiency using fluorescently attached peptides. Binding of targeted compounds was confirmed by solution depletion using liquid chromatography–mass spectrometry. The conjugation strategy is generalizable and applicable to harvest the pharmaceuticals produced in plants by selecting a specific oligopeptide that mimic the appropriate binding sites.

For related gene delivery applications, the thermodynamic interaction of amine functionalized MSNPs with double-stranded (ds) RNA was investigated by isothermal titration calorimetry (ITC). The heat of interaction was significantly different for particles with larger pore size (3.2 and 7.6 nm) compared to that of small pore particles (1.6 nm) and nonporous particles. Interaction of dsRNA also depended on molecular length, as longer RNA (282 base pair) was unable to load into 1.6 nm particles, consistent with previous confocal microscopy observations. Calculated thermodynamic parameters (enthalpy, entropy and free energy of interaction) are essential to design pore size dependent dsRNA loading, protection and delivery using MSNP carriers. While seemingly diverse, the highly tunable nature of ESNP and their interactions with cells are broadly applicable, and enable facile nano-harvesting and delivery based on a continuous uptake-expulsion mechanism.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2019.419

Funding Information

United States National Institutes of Health (NIH Grant nos. R41AT008312 and 2R44AT008312-02)

National Science Foundation sponsored Center for Arthropod Management Technologies (CAMTech) (NSF Industry-University Cooperative Research Center award no. 1238087)

National Science Foundation Experimental Program to Stimulate Competitive Research (EPSCoR) grant (award no, 1355438)

Department of Energy (DOE Grant No. DE-FG02-07-ER46375)

Kentucky Science and Engineering Foundation (KSEF-2929-RDE-016)

X-ray scattering experiments at the Advanced Photon Source of Argonne National Laboratory were conducted by using resources supported by United States Department of Energy (DOE) Office of Science User Facility operated under Contract No. DE-AC02-06CH11357.

Block Funding received from the University of Kentucky College of Engineering and Department of Chemical and Materials Engineering for multiple travel to Argonne National Laboratory, IL.

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