Synthesis and Biological Evaluation of Acid-Degradable Polymeric Materials for Pulmonary Gene Delivery
- Author(s): Cohen, Jessica Lynn
- Advisor(s): Fréchet, Jean M. J.
- et al.
RNA interference (RNAi) represents a promising method for the treatment and prevention of disease. Due to the tremendous potential of RNAi as a therapeutic strategy, there has been significant interest in the development of delivery vehicles which could efficiently deliver siRNA and reduce the expression of a protein of interest. Unfortunately, translation of the potential of siRNA into the clinic is limited by the lack of safe and effective delivery systems. Because of their tunability and synthetic addressability, acid–degradable polymeric materials represent a promising approach for the delivery of siRNA and are the subject of this dissertation.
Acid–sensitive systems have particularly desirable characteristics, as payload release can be triggered in response to endosomal acidification upon uptake. In particular, we describe the development, functionalization, and biological evaluation of biodegradable polymer systems which can efficiently deliver siRNA therapeutics to non–phagocytic cells. Specific emphasis is placed on the development of materials that have optimal physicochemical properties for pulmonary delivery.
Chapter 1 introduces various delivery strategies for siRNA therapeutics and discusses the basics of RNA interference. Additionally, the field of polymeric particulate siRNA carriers is reviewed, with an overview of relevant design criteria for materials intended for pulmonary administration. The advantages of systems capable of triggered payload release are discussed, with an emphasis on acid–sensitive carrier systems. Two acid–sensitive particle systems developed by our group are described.
In Chapter 2, the synthesis of acid–sensitive, acrylamide–based microparticles containing a cell–penetrating peptide (CPP) is discussed. Particles functionalized with a polyarginine CPP are prepared and evaluated for their ability to deliver a model therapeutic payload to non–phagocytic cells. The importance of CPP incorporation on cellular uptake in vitro is investigated in lung epithelial cells. The modification of the microparticles with CPPs greatly improved their uptake by non–phagocytic cells in culture without inducing any cytotoxic effects.
Chapter 3 discusses the role of particle size and functionalization with cationic CPPs on pulmonary delivery using hydrogel particles. The optimal particle design parameters for pulmonary delivery are investigated and the fate of model particles following intratracheal administration is studied by several techniques. Particles are characterized to determine their in vivo behavior in terms of lung retention, localization within specific cell types, and potential for inducing inflammatory responses. We found that altering both size and surface functionalization significantly affected the in vivo behavior of the particle system.
In Chapter 4, we describe the synthesis of a second–generation biocompatible, acid–sensitive particle system for siRNA delivery. The synthesis of spermine–modified acetalated–dextran (Spermine–Ac–DEX) is presented and the preparation of particles with high siRNA loading efficiency is described. The ability of these particles to silence protein expression in vitro is investigated. Spermine–Ac–DEX particles demonstrated efficient gene knockdown in a model cell line with minimal toxicity.
Chapter 5 investigates the synthesis and biological evaluation of functionalizable, acetal–modified dextran, a modular system with tunable functionality, degradation rate, and degree of modification. The preparation of acid–degradable, amine–functionalized dextran is described and the role of type of amine modification (primary, secondary, or tertiary amine) and degradation rate on the efficacy of siRNA delivery is studied in vitro. Altering both the type of amine as well as the degradation rate significantly affected the transfection efficiency. We found that two of the amine–dextrans were able to efficiently deliver siRNA and lead to gene silencing in HeLa–luc cells.