The aim of this study is to investigate the structure-transfection efficiency relationship of PPA gene carriers; and to investigate the effect of PPA structure on DNA compaction ability of PPA, stability of PPA/DNA nanoparticles in physiological medium, cellular uptake efficiency, intracellular trafficking, DNA unpacking, and nuclear translocation.
The development of safe and efficient vectors or carriers for in vivo gene transfer has been one of the key challenges in fulfilling the promises of gene therapy. Viral vectors, while efficient in many gene transfer applications in vivo, pose safety concerns that are unlikely to abate in the near future, rendering synthetic carriers attractive alternatives. The synthetic vectors including cationic liposomes and polycations, while offering better safety profile, continue to suffer from low gene transfer efficiency. Mechanistic studies are essential to identify the rate-limiting steps in the non-viral gene transfer process. Controlled and systematic studies are needed in order to reveal the structure-function relationship. We have designed and synthesized a series of biodegradable polyphosphoramidate (PPA) gene carriers with the same backbone but different structural parameters (type and structure of charge group, charge density, side chain spacer, etc.) in an effort to elucidate the structure-activity relationship. The aim of this study is to investigate the structure-transfection efficiency relationship of PPA gene carriers; and to investigate the effect of PPA structure on DNA compaction ability of PPA, stability of PPA/DNA nanoparticles in physiological medium, cellular uptake efficiency, intracellular trafficking, DNA unpacking, and nuclear translocation. More importantly, the biodistribution of PPA/DNA nanoparticles and transport of nanoparticles in the liver will be correlated to the structure and gene transfer efficiency of the PPA/DNA nanoparticles.
1. Controlled self-assembly of polymer/DNA or SiRNA micellar nanoparticles
We have developed a series of biocompatible and biodegradable polymers that self-assemble with DNA or RNA to form micellar nanoparticles (Fig 1). These nanoparticles consist of a DNA/polyphosphoramidate complex core and a polyethylene glycol (PEG) corona rendering particles more stable in aqueous medium. These nanoparticles are formed by self assembly, thus have uniform size ranging from tens to a couple hundreds of nanometers. With small and uniform size, good colloidal stability and longer circulation time, these nanoparticles offer opportunity for passive targeting to fenestrated tissues, for example solid tumor and liver sinusoid due to the “enhanced permeability and retention” effect. The size, morphology and stability can be controlled by adjusting PEG-b-PPA polymer structure and self-assembly conditions. In addition, cell-specific ligands can be introduced to micelle surface to improve cell binding and targeting. Our micelle system offers unique advantages on versatile structure, biophysical properties, transfection efficiency and safety. These favorable characteristics make this micelle system an ideal platform for systematic investigation of delivery mechanism and optimization of tissue-targeted delivery system. An ability to control nanoparticle stability, DNA or RNA release, cell binding, and intracellular trafficking will be the key to understanding the rate-limiting steps for gene delivery and to optimizing transgene expression efficiency in vivo. We are currently investigating the effect of PEG-b-PPA carrier structure on DNA compaction ability, nanoparticle assembly and stability, transport properties at cell and tissue levels, and delivery efficiency.
2. Dual-sensitive micellar nanoparticles to regulate DNA unpacking
In the context of in vivo delivery, it is important to enhance the colloidal and complex stability of the nanoparticles. On the other hand, making nanoparticles too stable will likely impede the DNA unpacking after they reach cytosol and nucleus. Hence a balanced colloidal and complex stability should be tailored by considering specific tissue and administration factors. We have synthesized a series of PEG-b-PPA carriers with different PPA block lengths. Interestingly, micelles formed with PEG12K-b-PPA128K (the molecular weight of PPA block is 128 KDa) exhibited instability in solution with physiological salt concentration and released most of encapsulated plasmid DNA 4 h after transfection in HEK293 cells. Taking advantage of this quick DNA unpacking ability of these micelles, we incorporated a microenvironment sensitive, reversible disulfide crosslinks in micelle core so that these micelles remain stable in circulation where glutathione (GSH) concentration is low, but the release/unpacking of DNA can be triggered by high level of GSH in cytosol and cell nucleus. We have shown that these dual-sensitive nanoparticles exhibited significantly enhanced complex and colloidal stability, yielded more sustained DNA unpacking in cytosols, and mediated enhanced and more prolonged transgene expression for at least 10 days.
3. Liver-targeting strategies for nanoparticle delivery
The liver is an important target for gene medicine applications. Successful liver-targeted gene delivery methods will be critical for treating liver-associated metabolic genetic disorders, viral infection and malignancies, and will also enable the expression of therapeutic products in the liver for treating systemic or off-site diseases. Intravenous and intraportal injections have been the primary routes for delivering nanoparticles or complexes to the liver. Even though intraportal injection provides a more direct delivery of nanoparticles to the liver, both methods face serious challenges in terms of particles being scavenged by Kupffer cells lining the sinusoidal endothelium and poor access to liver parenchymal cells. Retrograde intrabiliary infusion (RII), on the other hand, offers several unique advantages including direct access to hepatocytes and limited exposure to Kupffer cells. The biliary system consists of 7 to 10 orders of cholangiographically visible bile ducts spanning the expanse of the hepatic parenchyma (Fig. 3) with a surprisingly large and distensible volume (~29 ml for human liver). The ultrastructural surface of an entire normal biliary tree for a human liver would be around 3,000 cm2. This large surface area and broad distribution of the biliary system provide great access to nearly all hepatocytes in liver parenchyma through bile canaliculi, before the delivered drug or gene is exposed to KCs.
Due to these advantages, we have been evaluating the feasibility of using RII as an effective administration route for liver-targeted gene delivery of nanoparticles. We have demonstrated that RII delivery strategy can overcome several key limitations of intravenous delivery and yield significantly higher levels of transgene expression in the liver. We have been focusing on optimization of administration parameters for RII of DNA nanoparticles, characterization of nanoparticle transport kinetics following RII as compared with intravenous injection, and understanding the mechanism by which DNA nanoparticles transfect liver parenchymal cells and other cells and tissue.
The uniform size and high stability of PEG-b-PPA/DNA micellar nanoparticles in bile-containing medium make them excellent candidate for delivering genes through RII. We have been evaluating the efficiency and mechanism of gene delivery to rat and mouse livers using these micellar nanoparticles. Micelle formulations and delivery parameters will be optimized to achieve high and persistent gene expression. This will demonstrate the broad utility of this delivery strategy for expression of proteins intended for systemic distribution and for localized liver-specific diseases.