RESUMEN
The creation of polymer nanoparticles with protein functionality is of great interest to many applications such as targeted drug or gene delivery, diagnostic imaging, cancer theranostics, delivery of protein therapeutics, sensing chemical and biomolecular analytes in complex environments, and design of protective clothing resembling a second skin. Many approaches to achieving this goal are being explored in the current literature. In this chapter, we describe a relatively simple and flexible approach of conjugating the protein to an amphiphilic block copolymer and creating polymer nanoparticles with protein functionality by taking advantage of the intrinsic self-assembly behavior of the amphiphilic block copolymer. The commercially available and biocompatible polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer is used as the polymer building block. For demonstrative purposes, bovine serum albumin was chosen as the protein. We determine the molecular weight of the protein-polymer conjugate and thereby the degree of conjugation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry measurements. Retention of protein secondary structure in the conjugate was determined by circular dichroism spectroscopy, and the biological activity of the protein in the conjugated state has been evaluated by kinetic assay involving hydrolysis of an organophosphate compound. Dynamic light scattering and zeta potential measurements were used to characterize the size and charge of the protein-polymer conjugate micelle. Precise control of the size of the micelle and surface number density of the proteins on the micelle surface by coassembling with a second block copolymer have been demonstrated. These studies document a rational approach to armor the protein by conjugation with a block copolymer micelle, as a general approach.
Asunto(s)
Proteínas Inmovilizadas/química , Polietilenglicoles/química , Glicoles de Propileno/química , Albúmina Sérica Bovina/química , Sistemas de Liberación de Medicamentos , Hidrólisis , Micelas , Nanopartículas/química , Tamaño de la Partícula , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
Electrospun membranes were studied for the chemical deactivation of threat agents by means of enzymatic proteins. Protein loading and the surface chemistry of hybrid nanofibers influenced the efficacy by which embedded enzymes could digest the substrate of interest. Bovine serum albumin (BSA), selected as a model protein, was electrospun into biologically active fibers of poly(vinyl alcohol), PVA. Single-walled carbon nanotubes (SWNTs) were blended within these mixtures to promote protein assembly during the process of electrospinning and subsequently the ester hydrolysis of the substrates. The SWNT incorporation was shown to influence the topography of PVA/BSA nanofibers and enzymatic activity against paraoxon, a simulant for organophosphate agents and a phosphorus analogue of p-nitrophenyl acetate (PNA). The esterase activity of BSA against PNA was uncompromised upon its inclusion within nanofibrous membranes because similar amounts of PNA were hydrolyzed by BSA in solution and the electrospun BSA. However, the availability of BSA along the fiber surface was shown to affect the ester hydrolysis of paraoxon. Atomic force microscopy images of nanofibers implicated the surface migration of BSA during the electrospinning of SWNT filled dispersions, especially as greater weight fractions of protein were added to the spinning mixtures. In turn, the PVA/SWNT/BSA nanofibers outperformed the nanotube free PVA/BSA membranes in terms of paraoxon digestion. The results support the development of electrospun polymer nanofiber platforms, modulated by SWNTs for enzyme catalytic applications relevant to soldier protective ensembles.
Asunto(s)
Membranas Artificiales , Nanotubos de Carbono/química , Alcohol Polivinílico/química , Albúmina Sérica Bovina/química , Animales , Bovinos , Ésteres , Hidrólisis , Nitrofenoles/químicaRESUMEN
A simple and robust protocol to maintain the structural feature of polymer-protein core-shell nanoparticles (PPCS-NPs) is developed based on the synergistic interactions between proteins and functional polymers. Using the self-assembly method, a broad range of proteins can be assembled to the selective water-insoluble polymers containing pyridine groups. The detailed analysis of the PPCS-NPs structure was conducted using FESEM and thin-sectioned TEM. The results illustrated that the protein molecules are located on the corona of the PPCS-NPs. While proteins are displacing between water and polymer to minimize the interfacial energy, the polymer offers a unique microenvironment to maintain protein structure and conformation. The proposed mechanism is based on a fine balance between hydrophobicity and hydrophilicity, as well as hydrogen bonding between proteins and polymer. The PPCS-NPs can serve as a scaffold to incorporate both glucose oxidase (GOX) and horseradish peroxidase (HRP) onto a single particle. Such a GOX-HRP bienzymatic system showed a ~20% increase in activity in comparison to the mixed free enzymes. Our method therefore provides a unique platform to preserve protein structure and conformation and can be extended to a number of biomolecules.
Asunto(s)
Nanopartículas/química , Polímeros/química , Proteínas/química , Glucosa Oxidasa/química , Peroxidasa de Rábano Silvestre/química , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Molecular , Agua/químicaRESUMEN
A simple approach to enhancing the activity and stability of organophosphorus hydrolase (OPH) is developed based on interactions between the hydrophobic poly(propylene oxide) (PPO) block of amphiphilic Pluronics and the enzyme. This strategy provides an efficient route to new formulations for decontaminating organophosphate neurotoxins.
Asunto(s)
Arildialquilfosfatasa/química , Arildialquilfosfatasa/metabolismo , Poloxámero/metabolismo , Polímeros/metabolismo , Glicoles de Propileno/metabolismo , Tensoactivos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Poloxámero/química , Polímeros/química , Glicoles de Propileno/química , Tensoactivos/químicaRESUMEN
Enhancing the stability of enzymes under different working environments is essential if the potential of enzyme-based applications is to be realized for nanomedicine, sensing and molecular diagnostics, and chemical and biological decontamination. In this study, we focus on the enzyme, organophosphorus hydrolase (OPH), which has shown great promise for the nontoxic and noncorrosive decontamination of organophosphate agents (OPs) as well as for therapeutics as a catalytic bioscavanger against nerve gas poisoning. We describe a facile approach to stabilize OPH using covalent conjugation with the amphiphilic block copolymer, Pluronic F127, leading to the formation of F127-OPH conjugate micelles, with the OPH on the micelle corona. SDS-PAGE and MALDI-TOF confirmed the successful conjugation, and transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed â¼100 nm size micelles. The conjugates showed significantly enhanced stability and higher activity compared to the unconjugated OPH when tested (i) in aqueous solutions at room temperature, (ii) in aqueous solutions at higher temperatures, (iii) after multiple freeze/thaw treatments, (iv) after lyophilization, and (v) in the presence of organic solvents. The F127-OPH conjugates also decontaminated paraoxon (introduced as a chemical agent simulant) on a polystyrene film surface and on a CARC (Chemical Agent Resistant Coating) test panel more rapidly and to a larger extent compared to free OPH. We speculate that, in the F127-OPH conjugates (both in the micellar form as well as in the unaggregated conjugate), the polypropylene oxide block of the copolymer interacts with the surface of the OPH and this confinement of the OPH reduces the potential for enzyme denaturation and provides robustness to OPH at different working environments. The use of such polymer-enzyme conjugate micelles with improved enzyme stability opens up new opportunities for numerous civilian and Warfighter applications.
Asunto(s)
Arildialquilfosfatasa/química , Descontaminación , Estabilidad de Enzimas , Organofosfatos/química , Poloxámero/química , Arildialquilfosfatasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Micelas , Microscopía Electrónica de Transmisión , Paraoxon/química , Poliestirenos/química , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TemperaturaRESUMEN
N-(2-Hydroxypropyl)methacrylamide (HPMA) co-polymers containing disulfide and carbonyl thiazolidine-2-thione (TT) reactive groups in their side-chains (pHPMA-TT) were used as reductively removable chemical modification of the surface of cowpea mosaic viruses (CPMV). CPMV was used in this study as a model particle for viral gene delivery. The co-polymer reaction with CPMV surfaces carried out in aqueous solution was evaluated by monitoring the changes in the weight-average molecular weight and hydrodynamic size of the viruses using light scattering methods. The reaction conditions were optimized. The surface modification of CPMV with pHPMA-TT under selected conditions (concentrations of a coating polymer (c(p)) and NaCl) has not influenced the size distribution of the viral particles. The uncharged polymers bound to the viral surface via biodegradable S-S bonds can be fully removed by the exchange reaction with reductive dithiothreitol. To achieve optimal covering of viral surfaces, the positively charged reactive polymers (with or without biodegradable S-S bonds) should be applied at low concentrations (c(p)=0.1-0.5 mg/ml) and in presence of NaCl (0.15 M).
Asunto(s)
Comovirus/química , Metacrilatos/química , Polímeros/química , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Biomimética , Disulfuros/química , Interacciones Hidrofóbicas e Hidrofílicas , Espacio Intracelular/metabolismo , Polímeros/síntesis química , Propiedades de Superficie , Tiazolidinas/químicaRESUMEN
A practical method to assemble rodlike tobacco mosaic virus and bateriophage M13 with polymers was developed, which afforded a 3D core-shell composite with morphological control.