Engineered nanoparticles for gene delivery

The development of safe and efficient carriers for in vivo gene transfer has been one of the key challenges in fulfilling the promises of gene therapy. DNA-compacting nanoparticles have been developed as a safer alternative to viral vectors, due to the ease of synthesis of these polycation/DNA nanoparticles, flexibility in the size of the transgene to be delivered, and minimal host immune responses induced by the carriers. However, these nanoparticles face serious challenges for in vivo applications, particularly when administered though intravascular administrations. Currently-available polymer/DNA nanoparticles, although showing excellent transfection efficiencies in vitro, often have negligible transfection activities in target tissue or cells of interest. This is mostly due to the severe aggregation of the complexes (poor colloidal stability) and in some cases dissociation of complexes in physiological media with high concentrations of serum and salts (poor complex stability). Specifically for liver-targeted delivery, the aggregated nanoparticles prevent the passage through sinusoid fenestrae and access to parenchymal cells, and lead to sequestration by the reticuloendothelial system. Therefore, developing nanoparticles with uniform sizes, high colloidal stability and high complex stability is the critical first step in developing an efficient liver-targeting strategy. We are working on several strategies towards this goal.

New polymer carrier design that allows for controlled self-assembly of DNA/RNA nanoparticles

We have developed a series of biocompatible and biodegradable polymers that self-assemble with DNA or RNA to form micellar nanoparticles. 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.  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. 

References:

• Jiang X, Dai H, Ke CY, Mo X, Torbenson MS, Li Z and Mao HQ. PEG-b-PPA/DNA micelles improve transgene expression in rat liver through intrabiliary infusion. Journal of Controlled Release. 122: 297–304 (2007).
• Sun TM, Du JZ, Yan LF, Mao HQ, Wang J. Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery. Biomaterials. 29(32): 4348–4355 (2008).
• Mao HQ, Leong KW, Design of poly(phosphoester) nanoparticles for nonviral gene therapy. Advances in Genetics. 53: 275-306 (2005).
• Zhang PC, Wang J, Leong KW, Mao HQ. Ternary complexes comprising polyphosphoramidate gene carriers with different types of charge groups improve transfection efficiency. Biomacromolecules. 6(1): 54-60 (2005).
• Chen HH, Ho YP, Jiang X, Mao HQ, Wang TH, Leong KW. Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET. Molecular Therapy. 16(2): 324-332 (2008).

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 and observed increasing DNA binding affinity as the molecular weight of PPA block increased from 3 kDa to 80 kDa.  Interestingly however, 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.[2] 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.

Figure 2. (a). Preparation of dual-sensitive micellar nanoparticles. Micelles are prepared in distilled water to yield compact nanoparticles, and then oxided to crosslink the core. The disulfide-crosslinked micelles are stable in blood, extracellular melieu and the endolysosomal compartment where the glutathione (GSH) concentration is in the micromolar range. These crosslinks can be reduced when they reach the cytosol and nucleus where GSH concentration is in the range of 1-10 mM; the reduced micelles become unstable due to the salt-sensitive nature of the PEG12K-b-PPA128K carrier, thus releasing unpacked DNA. (b) and (c). TEM images of dual-sensitive micelles prepared with 18.8% thiolated copolymer in deionized water (b) and after incubation with 0.15 M NaCl for 30 min (c).

References:

• Jiang X, Zheng Y, Chen HH, Leong KW, Wang TH, Mao HQ. Dual-sensitive micellar nanoparticles regulate DNA unpacking and enhance gene delivery efficiency. Advanced Materials.22(23): 2556-2560 (2010).

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.

References:

• Dai H, Jiang X, Tan GCY, Chen Y, Torbenson MT, Leong KW, Mao HQ. Chitosan-DNA Nanoparticles Delivered by Intrabiliary Infusion Enhance Liver-Targeted Gene Delivery. International Journal of Nanomedicine. 1(4): 507-522 (2006).
• Jiang X, Dai H, Ke CY, Mo X, Torbenson MS, Li Z and Mao HQ. PEG-b-PPA/DNA micelles improve transgene expression in rat liver through intrabiliary infusion. Journal of Controlled Release. 122: 297–304 (2007).

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