University of Delaware NER: Rational Design of Biodegradable Nanoparticles for Gene Delivery
Gene therapy holds the potential to revolutionize disease treatment, but depends upon successful DNA transport and intracellular DNA delivery. Nanoparticle (NP) DNA formulations are in principle ideal for gene therapy: NPs are small enough to be ingested by cells and to access molecular-scale transport mechanisms, but large enough to contain full-length genes as well as cellular and intracellular targeting moieties. Unfortunately, common electrostatic NP assembly techniques are problematic: frequently, they result in toxic formulations that aggregate under physiological conditions. NP unpackaging and intracellular DNA release are also highly inefficient. The goal of this project is to design and demonstrate a novel assembly strategy for physiologically stable, environmentally responsive NPs for site-directed gene delivery. Intellectual merit: The gene delivery pathway is inherently hierarchical. For example, once an NP has reached its target cell, it must sequentially cross the plasma membrane, exit the endosome, traverse the cytoplasm, enter the nucleus, and unpackage. The proposed delivery system will be designed to mimic this hierarchy: (i) modules for NP protection/targeting will be introduced step-wise, at the site/time of use; (ii) modules will be removed following use to avoid hindering both further NP transport and DNA release. This rational assembly strategy has important consequences. For example, the introduction and removal of functional modules is easily altered in the proposed design. This enables the systematic analysis of each active transport step on the overall effectiveness of delivery: a critical function for elucidating the parameters (e.g., physical, chemical, or biological) that govern NP transport through each gene delivery barrier. To balance feasibility with novelty, well-established chemistries and biomodules will be employed. Specifics: The NPs will be constructed as a series of sheddable, functional "shells" surrounding a core of reversibly-packaged plasmid DNA (pDNA). Each shell will be incorporated via biodegradable peptide linkers engineered to degrade in response to environmental cues at the target degradation site.
The objectives of this Project:
1) To formulate "minimal" NPs and characterize their physical, chemical, and biological properties as a function of the design parameters. The properties of NPs have a strong influence on their interactions with cells and the efficiency of their transport. This objective will explore the relationship between design parameters and NP properties. For example, NP size will be controlled in part by the degree of pDNA condensation; NP size will in turn affect cellular uptake and intracellular transport. The controlling parameters for NP size, mechanical properties, and interactions (with proteins and cells) will be determined.
2) To demonstrate the capacity of the NPs for hierarchical, targeted degradation. The novelty of this platform depends upon the capacity of the NPs to degrade in response to site-specific cues. NPs containing a single degradable "shell" will be formulated. A fluorescence resonance energy transfer (FRET) system for monitoring biodegradation will be validated and used to determine the rate and (cellular) localization of biodegradation.
Broader impacts: This proposal provides an exceptional opportunity to engage and encourage participation in engineering by the application of chemical engineering fundamentals to a cutting-edge biological problem. The following strategies are targeted. Outreach: With colleagues at the University of Delaware, an exposure/recruitment program will be developed to increase understanding/interest in engineering at the K 12 level. Curriculum: A new elective course will be developed to educate undergraduate/graduate students on the application of chemical engineering concepts to problems in drug delivery and tissue engineering. Research: Multidisciplinary interactions at the Delaware Biotechnology Institute and with the university's biotechnology IGERT program will be used as a framework for the active recruitment of underrepresented groups such as women to engineering.
This proposal will address the theme of active nanostructures.
