RESUMO
Bis(diphenylphosphino)alkanes quantitatively react with excess 1-bromododecane to prepare novel phosphonium gemini surfactants with spacer lengths ranging from 2 to 4 methylenes (12-2/3/4-12P). Dodecyltriphenylphosphonium bromide (DTPP), a monomeric surfactant analog, was readily water soluble, however, in sharp contrast, phosphonium gemini surfactants were poorly soluble in water due to two hydrophobic tails and relatively hydrophobic cationic head groups containing phenyl substituents. Isothermal titration calorimetry did not reveal a measurable critical micelle concentration for the 12-2-12P phosphonium gemini surfactant in water at 25 °C. Subsequent studies in 50/50 v/v water-methanol at 25 °C showed a CMC of 1.0 mM for 12-2-12P. All phosphonium gemini surfactants effectively complexed nucleic acids, but failed to deliver nucleic acids in vitro to HeLa cells. The solution behavior of phosphonium gemini surfactants was investigated in chloroform, which is an organic solvent where reverse micellar structures are favored. Solution rheology in chloroform explored the solution behavior of the phosphonium gemini surfactants compared to DTPP. The 12-2-12P and 12-3-12P gemini surfactants were successfully electrospun from chloroform to generate uniform fibers while 12-4-12P gemini surfactant and DTPP only electrosprayed to form droplets.
RESUMO
Reversible-addition fragmentation chain transfer (RAFT) polymerization enabled the synthesis of novel, stimuli-responsive, AB and ABA block copolymers. The B block contained oligo(ethylene glycol) methyl ether methacrylate (OEG) and was permanently hydrophilic in the conditions examined. The A block consisted of diethylene glycol methyl ether methacrylate (DEG) and [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TMA). The A block displayed both salt- and temperature-response with lower critical solution temperatures (LCSTs) dependent on the molar content of TMA and the presence of salt. Higher TMA content in the AB diblock copolymers increased the critical micelle temperatures (CMT) in HPLC-grade water due to an increased hydrophilicity of the A block. Upon addition of 0.9 wt% NaCl, the CMTs of poly(OEG-b-DEG95TMA5) decreased from 50 °C to 36 °C due to screening of electrostatic repulsion between the TMA units. ABA triblock copolymers displayed excellent hydrogel properties with salt- and temperature-dependent gel points. TMA incorporation in the A block increased the gel points for all triblock copolymers, and salt-response increased with higher TMA composition in the A block. For example, poly(DEG98TMA2-b-OEG-b-DEG98TMA2) formed a hydrogel at 40 °C in HPLC-grade water and 26 °C in 0.9 wt% NaCl aqueous solution. These salt- and temperature-responsive AB diblock and ABA triblock copolymers find applications as drug delivery vehicles, adhesives, and hydrogels.
RESUMO
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 ß-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.
Assuntos
DNA , Terapia Genética/métodos , Vetores Genéticos , Compostos Organofosforados/química , Polímeros , Amantadina/farmacologia , Anticarcinógenos/farmacologia , Cavéolas/química , Cavéolas/metabolismo , DNA/química , DNA/farmacologia , Dopaminérgicos/farmacologia , Endocitose/efeitos dos fármacos , Vetores Genéticos/química , Vetores Genéticos/farmacologia , Genisteína/farmacologia , Células HeLa , Humanos , Polímeros/química , Polímeros/farmacologia , beta-Ciclodextrinas/farmacologiaRESUMO
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.
Assuntos
Metacrilatos/química , Compostos Organofosforados/química , Fosforilcolina/análogos & derivados , Polietilenoglicóis/química , Transfecção , Animais , Células COS , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Coloides , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Luciferases/biossíntese , Luciferases/genética , Metacrilatos/toxicidade , Peso Molecular , Compostos Organofosforados/toxicidade , Tamanho da Partícula , Fosforilcolina/química , Fosforilcolina/toxicidade , Polietilenoglicóis/toxicidade , Polimerização , TransgenesRESUMO
Sulfonium macromolecules displayed for the first time nucleic acid binding and transfection in vitro. Conventional and controlled radical polymerization techniques coupled with subsequent alkylation generated a sulfonium homopolymer, poly(DMSEMA), and a sulfonium diblock copolymer, poly(OEG-b-DMSEMA). DNA gel shift assays probed the ability of sulfonium macromolecules to complex nucleic acids, and luciferase assays examined the transfection efficiency and cytotoxicity of both sulfonium macromolecules. Poly(DMSEMA) and poly(OEG-b-DMSEMA) bound pDNA at a charge ratio of 1, and both induced significant luciferase expression in HeLa cells under serum-free conditions. Colloidal stability studies using dynamic light scattering highlighted the excellent colloidal stability of poly(OEG-b-DMSEMA) under salt and serum conditions due to the sterically stabilizing OEG block. Sulfonium macromolecules offer an alternate route to design cationic macromolecules for nonviral nucleic acid delivery, and future work will aim to add functionality to create more efficient delivery vehicles.
RESUMO
Nucleic acids and proteins, two of nature's 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.