Theresa M. Reineke

Polymer beacons for luminescence and magnetic resonance imaging of DNA delivery

The delivery of nucleic acids with polycations offers tremendous potential for developing highly specific treatments for various therapeutic targets. Although materials have been developed and studied for polynucleotide transfer, the biological mechanisms and fate of the synthetic vehicle has remained elusive due to the limitations with current labeling technologies. Here, we have developed polymer beacons that allow the delivery of nucleic acids to be visualized at different biological scales. The polycations have been designed to contain repeated oligoethyleneamines, for binding and compacting nucleic acids into nanoparticles, and lanthanide (Ln) chelates [either luminescent europium (Eu3+) or paramagnetic gadolinium (Gd3+)]. The chelated Lns allow the visualization of the delivery vehicle both on the nm/μm scale via microscopy and on the sub-mm scale via MRI. We demonstrate that these delivery beacons effectively bind and compact plasmid (p)DNA into nanoparticles and protect nucleic acids from nuclease damage. These delivery beacons efficiently deliver pDNA into cultured cells and do not exhibit toxicity. Micrographs of cultured cells exposed to the nanoparticle complexes formed with fluorescein-labeled pDNA and the europium-chelated polymers reveal effective intracellular imaging of the delivery process. MRI of bulk cells exposed to the complexes formulated with pDNA and the gadolinium-chelated structures show bright image contrast, allowing visualization of effective intracellular delivery on the tissue-scale. Because of their versatility, these delivery beacons posses remarkable potential for tracking and understanding nucleic acid transfer in vitro, and have promise as in vivo theranostic agents.

A β-Cyclodextrin “Click Cluster” Decorated with Seven Paramagnetic Chelates Containing Two Water Exchange Sites

The development of novel macromolecular contrast agents that offer enhanced relaxivity profiles at high magnetic fields have the potential to greatly improve the diagnosis, understanding, and treatment of disease. To this end, we have designed a monodiperse paramagnetic beta-cyclodextrin click cluster decorated with seven paramagnetic arms. A novel alkyne-functionalized diethylenetriaminetetraacetic acid (DTTA) chelate (6) has been created and coupled to a per-azido-beta-cyclodextrin core (7) to yield the precursor macromolecule (8). After removal of the protecting groups and titrating with Gd (3+), the final paramagnetic click cluster, Gd10, was obtained. Luminescence measurements were carried out in H 2O and D 2O on an analogous structure, Eu10, and indicated that at each lanthanide has an average of 1.8 water exchange sites, which is important for enhancing relaxivity and MRI resolution. This discrete paramagnetic click cluster yields a high relaxivity profile (43.4 mM (-1) s (-1) per molecule and 6.2 mM (-1) s (-1) per Gd (3+) at 9.4 T) and enhanced contrast on a human MRI scanner as compared to a commercial agent, Magnevist (3.2 mM (-1) s (-1) at 9.4 T). Moreover, the useful inclusion properties exhibited by beta-cyclodextrin also make this an excellent host scaffold to functionalize via noncovalent assembly with receptor specific targeting moieties for biomolecular imaging.

Versatile supramolecular pDNA vehicles via click polymerization of β-cyclodextrin with oligoethyleneamines

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain beta-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1(46), Cd2(44), Cd3(49), and Cd4(47) (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n(w)=44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4(27), Cd4(47), Cd4(93), and Cd4(200) [n(w)=27, 47, 93, 200] via the click reaction. The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3(49) and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3(49) and Cd4(93) had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.

Poly(glycoamidoamine) vehicles promote pDNA uptake through multiple routes and efficient gene expression via caveolae-mediated endocytosis

The use of synthetic polymers for the delivery of nucleic acids holds considerable promise for understanding and treating disease at the molecular level. This work aims to decipher the cellular internalization mechanisms for a series of synthetic glycopolymer DNA delivery vehicles we have termed poly(glycoamidoamine)s (PGAAs). To this end, we have performed cellular delivery experiments in the presence of pharmacological endocytosis inhibitors. Confocal microscopy analysis showed colocalization of labeled pDNA in polyplexes with immunolabeled endocytic molecules to identify the cellular internalization pathways in HeLa cells. Direct membrane penetration was also investigated through various methods, including cellular energy depletion and leakage of a cytosolic enzyme from the cell. The data suggests that the cellular internalization of PGAA polyplexes occurs through a multifaceted internalization mechanism primarily involving caveolae, yet clathrin-coated vesicles and macropinosomes were also involved to a lesser degree. The primary mechanism that leads to efficient nuclear delivery and transgene expression appears to be caveolae/raft-mediated endocytosis. The cellular internalization pathways for PGAAs were not identical to those for polyethylenimine, illustrating that differences in the chemical structure of materials directly impacts the cellular internalization mechanisms.

Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing N-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000.

Interaction of poly(ethylenimine)-DNA polyplexes with mitochondria: implications for a mechanism of cytotoxicity.

Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure-function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F(0)F(1)-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F(0)F(1)-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

Toward targeting RNA structure: branched peptides as cell-permeable ligands to TAR RNA.

Rational design of RNA ligands continues to be a formidable challenge, but the potential powerful applications in biology and medicine catapults it to the forefront of chemical research. Indeed, small molecule and macromolecular intervention are attractive approaches, but selectivity and cell permeability can be a hurdle. An alternative strategy is to use molecules of intermediate molecular weight that possess large enough surface area to maximize interaction with the RNA structure but are small enough to be cell-permeable. Herein, we report the discovery of nontoxic and cell-permeable branched peptide (BP) ligands that bind to TAR RNA in the low micromolar range from on-bead high-throughput screening of 4,096 compounds. TAR is a short RNA motif in the 5-UTR of HIV-1 that is responsible for efficient generation of full RNA transcripts. We demonstrate that BPs are selective for the native TAR RNA structure and that branching in peptides provides multivalent interaction, which increases binding affinity to RNA.

Carbohydrate Polymers for Nonviral Nucleic Acid Delivery

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed significantly to progress in the field of non-viral DNA delivery, and these new discoveries are impactful for developing new vehicles and materials for treatment of human disease.

Poly(glycoamidoamine)s: a broad class of carbohydrate-containing polycations for nucleic acid delivery

In the era of nucleic acid therapeutics, there is an urgent need for non-viral delivery vehicles that can cross the extracellular and intracellular barriers and deliver nucleic acids to specific intracellular regions. This paper reviews the development of a subclass of polymer-based delivery vehicles termed poly(glycoamidoamine)s (PGAAs). The general design of this family consists of carbohydrate residues copolymerized with oligoethyleneamine units, which have proven to be an effective motif that promotes polyplex formation, efficient cellular internalization, high gene expression and low cytotoxicity with cultured cell lines and primary cell types. We then discuss the structure-property relationships of the PGAA class of delivery vehicles and studies aimed at understanding the mechanisms involved in cellular internalization and trafficking.

DNA delivery in vitro via surface release from multilayer assemblies with poly(glycoamidoamine)s.

Localized controlled release of nucleic acid therapeutics could be an effective way to reduce the extracellular barriers associated with systemic delivery. Herein, we have used the layer-by-layer film deposition approach to construct ultrathin multilayer assemblies for in vitro controlled release of plasmid DNA (pDNA). Layer-by-layer assemblies containing alternate layers of cationic poly(l-tartaramidopentaethylenetetramine) (T4), and anionic pDNA were fabricated. The film thickness and the absorbance at 260 nm for different T4/pDNA multilayer assemblies were characterized by ellipsometry and UV-vis spectrophotometry, respectively. The results indicated an increased loading capacity of pDNA with respect to an increase in the number of T4/pDNA bilayers deposited. For the controlled-release studies we incubated the bilayers coated on quartz slides in phosphate-buffered saline (PBS) at 37 degrees C and collected the media at different incubation time points. The collected PBS samples were characterized for pDNA release by complexing solutions containing the released pDNA with Lipofectamine 2000 and following cellular pDNA uptake via flow cytometry and GFP gene expression assays with HeLa cells. The study showed that the multilayer films started to release pDNA after 1 day of incubation and increased after 7 days of incubation. Assays monitoring green fluorescent protein (GFP) expression in HeLa cells indicated that about 20% of the cells were positive for GFP expression at all sample time points up to 11 days. Although an increase in cells positive for Cy5-pDNA was found as the incubation time increased, the number of cells positive for GFP expression remained constant over the same time frame.