Matthew D. Green

Designing Imidazole-Based Ionic Liquids and Ionic Liquid Monomers for Emerging Technologies

Imidazolium-based ionic liquids and ionic liquid monomers are becoming increasingly popular in a variety of areas including biphasic reaction catalysis, electromechanical actuator membranes and diluents, separation science membranes, and water purification agents. Ionic liquids first incorporated the imidazole ring in 1984 and this heterocyclic ring has emerged as the focal point of the ionic liquid field. Imidazole was targeted for its ability to form cationic compounds, which are molten salts at low molar mass. Ionic liquids offer several beneficial attributes including fixed charge, potential as green solvents, and relatively high thermal stability. Due to an ionic liquids ability to facilitate electron or ion motion, they are now enabling electroactive devices. Commercially available conductive membranes are swollen with ionic liquids to enhance their conductivity; alternatively, conductive membranes are synthesized from novel ionic liquid monomers, also termed polymerizable ionic liquids. The imidazole ring has gained much attention for its ability to tune the properties of the resulting ionic liquid. Careful selection of substituents on any of the positions in the ring and exchange of the counteranion influences many physical properties such as the melting point, the boiling point, and the viscosity. Finally, imidazolium ionic liquids utilize two of their unique properties in biphasic catalysis, i.e. their ability to coordinate transition metals and their hydrophilic ionic nature. Several imidazolium-based ionic liquid molecules have displayed the ability to catalyze atom transfer radical polymerization and facilitate the synthesis of polymers with narrow molecular weight distributions. This manuscript reviews some of the more recent advances that are associated with these unusual liquids.

Influence of polycation molecular weight on poly(2-dimethylaminoethyl methacrylate)-mediated DNA delivery in vitro.

Establishing clear structure-property-transfection relationships is a critical step in the development of clinically relevant polymers for nonviral gene therapy. In this study, we determined the influence of poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) molecular weight on cytotoxicity, DNA binding, and in vitro plasmid DNA delivery efficiency in human brain microvascular endothelial cells (HBMEC). Conventional free radical polymerization was used to synthesize PDMAEMA with weight-average molecular weights ranging from 43,000 to 915,000 g/mol. MTT and LDH assays revealed that lower molecular weight PDMAEMA (M(w) = 43,000 g/mol) was slightly less toxic than higher molecular weights (M(w) > 112,000 g/mol) and that the primary mode of toxicity was cellular membrane destabilization. An electrophoretic gel shift assay revealed that all PDMAEMA molecular weights completely bound with plasmid DNA. However, heparin competitive binding experiments revealed that higher molecular weight PDMAEMA (M(w) = 915,000 g/mol) had a greater binding affinity toward plasmid DNA than lower molecular weight PDMAEMA (M(w) = 43,000 g/mol). The molecular weight of PDMAEMA was found to have a dramatic influence on transfection efficiency, and luciferase reporter gene expression increased as a function of increasing molecular weight. However, cellular uptake of polyplexes was determined to be insensitive to PDMAEMA molecular weight. In addition, our data did not correlate polyplex size with transfection efficiency. Collectively, our data suggested that the intracellular fate of the polyplexes, which involves endosomal release and DNase resistance, is more important to overall transfection efficiency than barriers to entry, such as polyplex size.

Phosphonium-containing polyelectrolytes for nonviral gene delivery.

Nonviral gene therapy focuses intensely on nitrogen-containing macromolecules and lipids to condense and deliver DNA as a therapeutic for genetic human diseases. For the first time, DNA binding and gene transfection experiments compared phosphonium-containing macromolecules with their respective ammonium analogs. Conventional free radical polymerization of quaternized 4-vinylbenzyl chloride monomers afforded phosphonium- and ammonium-containing homopolymers for gene transfection experiments of HeLa cells. Aqueous size exclusion chromatography confirmed similar absolute molecular weights for all polyelectrolytes. DNA gel shift assays and luciferase expression assays revealed phosphonium-containing polymers bound DNA at lower charge ratios and displayed improved luciferase expression relative to the ammonium analogs. The triethyl-based vectors for both cations failed to transfect HeLa cells, whereas tributyl-based vectors successfully transfected HeLa cells similar to Superfect demonstrating the influence of the alkyl substituent lengths on the efficacy of the gene delivery vehicle. Cellular uptake of Cy5-labeled DNA highlighted successful cellular uptake of triethyl-based polyplexes, showing that intracellular mechanisms presumably prevented luciferase expression. Endocytic inhibition studies using genistein, methyl β-cyclodextrin, or amantadine demonstrated the caveolae-mediated pathway as the preferred cellular uptake mechanism for the delivery vehicles examined. Our studies demonstrated that changing the polymeric cation from ammonium to phosphonium enables an unexplored array of synthetic vectors for enhanced DNA binding and transfection that may transform the field of nonviral gene delivery.

Tailoring charge density and hydrogen bonding of imidazolium copolymers for efficient gene delivery.

Conventional free radical polymerization with subsequent postpolymerization modification afforded imidazolium copolymers with controlled charge density and side chain hydroxyl number. Novel imidazolium-containing copolymers where each permanent cation contained one or two adjacent hydroxyls allowed precise structure-transfection efficiency studies. The degree of polymerization was identical for all copolymers to eliminate the influence of molecular weight on transfection efficiency. DNA binding, cytotoxicity, and in vitro gene transfection in African green monkey COS-7 cells revealed structure-property-transfection relationships for the copolymers. DNA gel shift assays indicated that higher charge densities and hydroxyl concentrations increased DNA binding. As the charge density of the copolymers increased, toxicity of the copolymers also increased; however, as hydroxyl concentration increased, cytotoxicity remained constant. Changing both charge density and hydroxyl levels in a systematic fashion revealed a dramatic influence on transfection efficiency. Dynamic light scattering of the polyplexes, which were composed of copolymer concentrations required for the highest luciferase expression, showed an intermediate DNA-copolymer binding affinity. Our studies supported the conclusion that cationic copolymer binding affinity significantly impacts overall transfection efficiency of DNA delivery vehicles, and the incorporation of hydroxyl sites offers a less toxic and effective alternative to more conventional highly charged copolymers.

Thermal, rheological, and ion-transport properties of phosphonium-based ionic liquids.

Phosphonium-based ionic liquids with varying counteranions from commercially available ionic liquid precursors enabled tunable viscosity, ionic conductivity, and thermal stability. Thermogravimetric analysis revealed a relationship between thermal stability and anion composition where anions with lower basicity remained stable to higher temperatures. Determination of glass transition temperatures and melting temperatures using differential scanning calorimetry revealed supercooling, crystallization, and dependence on anion composition. Rheological and ionic conductivity measurements determined the temperature-dependence of the viscosity and ionic conductivity of the phosphonium-based ionic liquids. Arrhenius analyses of conductivity and viscosity provided activation energies, which showed a decrease toward larger, more delocalized anions. An assessment according to the Walden plot displayed their efficacy relative to other ionic liquids.

Meta-analysis of ionic liquid literature and toxicology.

A meta-analysis was conducted to compare the total amount of ionic liquid (IL) literature (n = 39,036) to the body of publications dealing with IL toxicity (n = 213) with the goal of establishing the state of knowledge and existing information gaps. Additionally, patent literature pertaining to issued patents utilizing ILs (n = 3358) or dealing with IL toxicity (n = 112) were analyzed. Total publishing activity and patent count served to gauge research activity, industrial usage and toxicology knowledge of ILs. Five of the most commonly studied IL cations were identified and used to establish a relationship between toxicity data and potential of commercial use: imidazolium, ammonium, phosphonium, pyridinium, and pyrrolidinium. Toxicology publications for all IL cations represented 0.55% ± 0.27% of the total publishing activity; compared with other industrial chemicals, these numbers indicate that there is still a paucity of studies on the adverse effects of this class of chemical. Toxicity studies on ILs were dominated by the use of in vitro models (18%) and marine bacteria (15%) as studied biological systems. Whole animal studies (n = 87) comprised 31% of IL toxicity studies, with a subset of in vivo mammalian models consisting of 8%. Human toxicology data were found to be limited to in vitro analyses, indicating substantial knowledge gaps. Risks from long-term and chronic low-level exposure to ILs have not been established yet for any model organisms, reemphasizing the need to fill crucial knowledge gaps concerning human health effects and the environmental safety of ILs. Adding to the existing knowledge of the molecular toxicity characteristics of ILs can help inform the design of greener, less toxic and more benign IL technologies.

Light-Mediated Activation of siRNA Release in Diblock Copolymer Assemblies for Controlled Gene Silencing

Controllable release is particularly important for the delivery of small interfering RNA (siRNA), as siRNAs have a high susceptibility to enzymatic degradation if release is premature, yet lack silencing activity if they remain inaccessible within the cytoplasm. To overcome these hurdles, novel and tailorable mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) diblock copolymers containing light-sensitive o-nitrobenzyl moieties and pendant amines are employed to provide both efficient siRNA binding, via electrostatic and hydrophobic interactions, as well as triggered charge reversal and nucleic acid release. In particular, siRNA/mPEG-b-P(APNBMA)23.6 polyplexes show minimal aggregation in physiological salt and serum, and enhanced resistance to polyanion-induced unpackaging compared to polyethylenimine preparations. Cellular delivery of siRNA/mPEG-b-P(APNBMA)23.6 polyplexes reveals greater than 80% cellular transfection, as well as rapid and widespread cytoplasmic distribution. Additionally, UV irradiation indicates ≈70% reduction in targeted gene expression following siRNA/mPEG-b-P(APNBMA)23.6 polyplex treatment, as compared to 0% reduction in polyplex-treated cells without UV irradiation, and only ≈30% reduction for Lipofectamine-treated cells. The results here highlight the potential of these light-sensitive copolymers with a well-defined on/off switch for applications including cellular patterning for guided cell growth and extension, and cellular microarrays for exploring protein and drug interactions that require enhanced spatiotemporal control of gene activation.

Catch and release: photocleavable cationic diblock copolymers as a potential platform for nucleic acid delivery

Binding interactions between DNA and cationic carriers must be sufficiently strong to prevent nuclease-mediated degradation, yet weak enough to permit transcription. We demonstrate cationic diblock copolymers containing PEG and o-nitrobenzyl moieties that facilitated tailorable DNA complexation and light-activated release. This design unlocks a new approach to advance non-viral gene packaging.

Anomalous isoelectronic chalcogen rejection in 2D anisotropic vdW TiS3(1−x)Se3x trichalcogenides

Alloying in semiconductors has enabled many civilian technologies in electronics, optoelectronics, photonics, and others. While the alloying phenomenon is well established in traditional bulk semiconductors owing to a vast array of available ternary phase diagrams, alloying in 2D materials still remains at its seminal stages. This is especially true for transition metal trichalcogenides (TMTCs) such as TiS3 which has been recently predicted to be a direct gap, high carrier mobility, pseudo-1D semiconductor. In this work, we report on an unusual alloying rejection behavior in TiS3(1-x)Se3x vdW crystals. TEM, SEM, EDS, and angle-resolved Raman measurements show that only a miniscule amount (8%) of selenium can be successfully alloyed into a TiS3 host matrix despite vastly different precursor amounts as well as growth temperatures. This unusual behavior contrasts with other vdW systems such as TiS2(1-x)Se2x, MoS2(1-x)Se2x, Mo1-xWxS2, WS2(1-x)Se2x, where continuous alloying can be attained. Angle-resolved Raman and kelvin probe force microscopy measurements offer insights into how selenium alloying influences in-plane structural anisotropy as well as electron affinity values of exfoliated sheets. Our cluster expansion theory calculations show that only the alloys with a small amount of Se can be attained due to energetic instability above/below a certain selenium concentration threshold in the ternary phase diagrams. The overall findings highlight potential challenges in achieving stable Ti based TMTCs alloys.

Effect of Crosslinker Length and Architecture on the Thermomechanical Properties of CNT-Loaded Elastomeric Polymer Matrix Composites

An evolving understanding of elastomeric polymer nanocomposites continues to expand commercial, defense, and industrial products and applications. This work explores the thermomechanical properties of elastomeric nanocomposites prepared from bisphenol A diglycidyl ether and three amine-terminated poly(propylene oxides) (Jeffamines). The Jeffamines investigated include difunctional crosslinkers with molecular weights of 2000 and 4000 g mol-1 and a trifunctional crosslinker with a molecular weight of 3000 g mol-1 . Additionally, carbon nanotubes (CNTs) are added, up to 1.25 wt%, to each thermoset. The findings indicate that the T g and storage modulus of the polymer nanocomposites can be controlled independently within narrow concentration windows, and that effects observed following CNT incorporation are dependent on the crosslinker molecular weight. Finally, the impact of crosslinker length and architecture as well as CNT addition on the molecular weight between crosslink points in the glassy and rubbery states are discussed.