Meredith A. Mintzer

Nonviral Vectors for Gene Delivery

The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,Llactide-co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (~200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less

Exploiting dendrimer multivalency to combat emerging and re-emerging infectious diseases.

The emergence and re-emergence of bacterial strains that are resistant to current antibiotics reveal the clinical need for new agents that possess broad-spectrum antibacterial activity. Furthermore, bacteriophobic coatings that repel bacteria are important for medical devices, as the lifetime, reliability, and performance of implant devices are hindered by bacterial adhesion and infection. Dendrimers, a specific class of monodisperse macromolecules, have recently shown potential to function as both antibacterial agents and antimicrobial surface coatings. This review discusses the limitations with currently used antibacterial agents and describes how various classes of dendrimers, including glycodendrimers, cationic dendrimers, anionic dendrimers, and peptide dendrimers, have the potential to improve upon or replace certain antibiotics. Furthermore, the unexplored areas in this field of research will be mentioned to present opportunities for additional studies regarding the use of dendrimers as antimicrobial agents.

Triazine dendrimers as nonviral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity.

A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this nonviral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents.

Triazine dendrimers as nonviral gene delivery systems: effects of molecular structure on biological activity.

A family of generation 1, 2, and 3 triazine dendrimers differing in their core flexibility was prepared and evaluated for their ability to accomplish gene transfection. Dendrimers and dendriplexes were analyzed by their physicochemical and biological properties such as condensation of DNA, size, surface charge, morphology of dendriplexes, toxic and hemolytic effects, and ultimately transfection efficiency in L929 and MeWo cells. Flexibility of the backbone was found to play an important role with generation 2 dendrimer displaying higher transfection efficiencies than 25 kDa poly(ethylene imine) or SuperFect at a lower cytotoxicity level. This result is surprising, as PAMAM dendrimers require generations 4 or 5 to become effective transfection reagents. The ability to delineate effects of molecular structure and generation of triazine dendrimers with biological properties provides valuable clues for further modifying this promising class of nonviral delivery system.

The 8 year thicket of triazine dendrimers: strategies, targets and applications

This manuscript focuses on the routes, methods and reagents used to synthesize triazine-based dendrimers. Our pursuit of macromolecular architectures for drug delivery—dendrimers based on triazines—has been an ongoing effort for 8 years. To date, we have produced complex dendrimers with diverse peripheries as proof-of-concept, less complex molecules tailored for specific applications including DNA and RNA delivery and drug-decorated dendrimers for potential therapeutic applications including infectious disease and cancer. These syntheses have been executed at scales that range from high milligrams to over a kilogram. The routes, reagents and diversity displayed by a target anchors it in time. Early targets derive from convergent synthetic routes while later targets are prepared using divergent syntheses. The core of early dendrimers was a simple diamine, including piperazine, yielding the so-called bow-tie structures, middle period targets boast either a trispiperazinyltriazine core or a ‘super-core’ with six piperazine groups. Later targets return to the trispiperazinyltriazine core. The choice of linking diamine has also changed. Over time, p-aminobenzylamine was replaced by piperazine and then by aminomethylpiperidine with more exotic diamines sprinkled in throughout. Peripheral group choice has undergone similar variations: from AB2 to AB4 to, more recently, AB3. The diversity communicated by these groups yields dendrimers ranging from those with a common surface to examples where two groups were presented to those where four orthogonally reactive groups appear. Over time, these groups have grown in complexity from protected amines to tags for biodistribution and drugs like paclitaxel. Herein, strategies adopted and lessons learned are reviewed, intuitions relayed and future directions forecast.

Synthesis of Odd Generation Triazine Dendrimers Using a Divergent, Macromonomer Approach

Using a macromonomer, first, third, and fifth generation triazine dendrimers can be prepared using a divergent approach. The nine-step process to the fifth generation target relies on an iterative two-reactions-per-generation strategy to yield the desired material in approximately 48% overall yield. This target displays 96 surface groups. NMR spectroscopy and mass spectrometry show that exceptionally narrow polydispersity is achieved using this strategy.