Sean T. Hemp

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.

Phosphonium-containing diblock copolymers for enhanced colloidal stability and efficient nucleic acid delivery.

RAFT polymerization successfully controlled the synthesis of phosphonium-based AB diblock copolymers for nonviral gene delivery. A stabilizing block of either oligo(ethylene glycol(9)) methyl ether methacrylate or 2-(methacryloxy)ethyl phosphorylcholine provided colloidal stability, and the phosphonium-containing cationic block of 4-vinylbenzyltributylphosphonium chloride induced electrostatic nucleic acid complexation. RAFT polymerization generated well-defined stabilizing blocks (M(n) = 25000 g/mol) and subsequent chain extension synthesized diblock copolymers with DPs of 25, 50, and 75 for the phosphonium-containing block. All diblock copolymers bound DNA efficiently at ± ratios of 1.0 in H(2)O, and polyplexes generated at ± ratios of 2.0 displayed hydrodynamic diameters between 100 and 200 nm. The resulting polyplexes exhibited excellent colloidal stability under physiological salt or serum conditions, and they maintained constant hydrodynamic diameters over 24 h. Cellular uptake studies using Cy5-labeled DNA confirmed reduced cellular uptake in COS-7 and HeLa cells and, consequently, resulted in low transfection in these cell lines. Serum transfection in HepaRG cells, which are a predictive cell line for in vivo transfection studies, showed successful transfection using all diblock copolymers with luciferase expression on the same order of magnitude as Jet-PEI. All diblock copolymers exhibited low cytotoxicity (>80% cell viability). Promising in vitro transfection and cytotoxicity results suggest future studies involving the in vivo applicability of these phosphonium-based diblock copolymer delivery vehicles.

Phosphonium ionenes from well-defined step-growth polymerization: thermal and melt rheological properties

Step-growth polymerization of ditertiary phosphines with dibromoalkanes enabled the synthesis of novel phosphonium ionenes. In situ FTIR spectroscopy monitored the increase in absorbance as a function of time at 1116 cm−1, which corresponded to the polymeric P+–Ph stretch. Aqueous size-exclusion chromatography (SEC) provided absolute molecular weights and confirmed expected molecular weight growth for difunctional, step-growth polymerization. Phosphonium ionenes exhibited improved thermal and base stability compared to ammonium ionenes, which was attributed to the propensity of the ammonium cation towards Hofmann elimination. Melt rheology examined phosphonium ionene viscous flow and the influence of charge density on melt viscosity as a function of shear rate and temperature. Time–temperature superposition (TTS) resulted in both master curves and pseudomaster curves depending on phosphonium ionene composition. Two primary relaxations occurred: (1) onset of long-range segmental motion at Tg, and (2) relaxation attributed to electrostatic interactions. Higher charge densities shifted these two relaxations to longer time scales and increased flow activation energies. Phosphonium ionenes also readily bound pDNA effectively (± ratios of 1), and base stability suggested applications in energy generation.

Water-dispersible cationic polyurethanes containing pendant trialkylphosphoniums

Novel trialkylphosphonium ionic liquids chain extenders enabled the successful synthesis of poly(ethylene glycol)-based, cationic polyurethanes with pendant phosphoniums in the hard segments (HS). Aqueous size exclusion chromatography (SEC) confirmed the charged polyurethanes, which varied the phosphonium alkyl substituent length (ethyl and butyl) and cationic HS content (25, 50, 75 mol%), achieved high absolute molecular weights. Dynamic mechanical analysis (DMA) demonstrated the triethylphosphonium (TEP) and tributylphosphonium (TBP) polyurethanes displayed similar thermomechanical properties, including increased rubbery plateau moduli and flow temperatures. Fourier transform infrared spectroscopy (FTIR) emphasized the significance of ion–dipole interaction on hydrogen bonding. Atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), and wide-angle X-ray diffraction (WAXD) supported microphase separated morphologies in the trialkylphosphonium polyurethanes, despite the presence of ionic interactions. Sorption isotherm experiments revealed the TEP polyurethane exhibited the highest water vapor sorption profile compared to the TBP, which displayed similar water sorption profiles to the noncharged analogue. The phosphonium polyurethanes displayed significantly improved tensile strain; however, lower tensile stress of the TEP polyurethane was presumably due to absorbed water. In addition to physical characterizations, we also explored the trialkylphosphonium polyurethanes as nucleic acid delivery vectors. The phosphonium polyurethanes bound DNA at low charge ratios, and the polyplexes exhibited enhanced colloidal stability under physiological salt conditions.

DNA-inspired hierarchical polymer design: electrostatics and hydrogen bonding in concert.

Nucleic acids and proteins, two of natures biopolymers, assemble into complex structures to achieve desired biological functions and inspire the design of synthetic macromolecules containing a wide variety of noncovalent interactions including electrostatics and hydrogen bonding. Researchers have incorporated DNA nucleobases into a wide variety of synthetic monomers/polymers achieving stimuli-responsive materials, supramolecular assemblies, and well-controlled macromolecules. Recently, scientists utilized both electrostatics and complementary hydrogen bonding to orthogonally functionalize a polymer backbone through supramolecular assembly. Diverse macromolecules with noncovalent interactions will create materials with properties necessary for biomedical applications.

Synthesis and characterization of 4-vinylimidazole ABA triblock copolymers utilizing a difunctional RAFT chain transfer agent

Reversible addition–fragmentation chain transfer (RAFT) polymerization strategies enabled the unprecedented synthesis of 4-vinylimidazole (4VIM)-containing ABA triblock copolymers in glacial acetic acid. The synthesis of a novel, difunctional trithiocarbonate RAFT chain transfer agent (CTA) controlled the divergent RAFT polymerization of methacrylic and 4VIM monomers with controlled molecular weights and narrow polydispersity indices (PDIs). The triblock copolymers consisted of a low-Tg di(ethylene glycol) methyl ether methacrylate (DEGMEMA) center block (Mn = 26 000 g mol−1) and an amphoteric 4VIM external, mechanically reinforcing block (Mn = 6500–16 500 g mol−1). Varying the 4VIM content probed the influence of the triblock copolymer composition on the macromolecular thermomechanical and morphological properties. Dynamic mechanical analysis (DMA) of the triblock copolymers exhibited a rubbery plateau region over a wide temperature range (∼200 °C), which confirmed the establishment of microphase-separated morphologies with flow temperatures above 200 °C. Transmission electron microscopy (TEM), atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS) collectively probed the solid state morphologies of the triblock copolymers; all techniques revealed phase separation at nanoscale dimensions. The triblock copolymers with 40 wt% 4VIM formed lamellar morphologies. Well-defined, amphoteric, 4VIM ABA triblock copolymers (PDIs < 1.10) with microphase-separated morphologies now permit imidazole-containing macromolecules of controlled architectures for emerging applications.

Polymeric Imidazoles and Imidazoliums in Nanomedicine: Comparison to Ammoniums and Phosphoniums

Polymerized ionic liquids and polyelectrolytes play a major role in a broad range of biological applications including antimicrobials, nonviral gene delivery, synthetic enzymes, metal chelation, and drug delivery. Ammonium- and phosphonium-containing macromolecules will be reviewed with a focus on the literature that examines structure–property relationships of these polyelectrolytes for biological applications. Imidazole-containing macromolecules also receive significant focus due to their natural occurrence in nature and their ubiquitous use in nanomedicine. Special attention to imidazole- and imidazolium-containing polymers will focus on vinylimidazole regioisomers in particular.