Hydrolase stabilization via entanglement in poly(propylene sulfide) nanoparticles: stability towards reactive oxygen species.

In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase's stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme's exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a 'sacrificial barrier' by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase-PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO(2) (100 ppm).

Encapsulation and enzyme-mediated release of molecular cargo in polysulfide nanoparticles.

Poly(propylene sulfide) nanoparticles (<150 nm) have been synthesized by an anionic, ring-opening emulsion polymerization. Upon exposure to parts per million (ppm) levels of oxidizing agent (NaOCl), hydrophobic polysulfide particles are oxidized to hydrophilic polysulfoxides and polysulfones. Utilizing this mechanism, the encapsulation of hydrophobic molecular cargo, including Nile red and Reichardt's dye, within polysulfide nanoparticles has been characterized by a variety of microscopic and spectroscopic methods and its release demonstrated via chemical oxidation. Moreover, release of cargo has been enzymatically driven by oxidoreductase enzymes such as chloroperoxidase and myeloperoxidase in the presence of low concentrations of sodium chloride (200 mM) and hydrogen peroxide (500 µM). This oxidation-driven mechanism holds promise for controlled encapsulation and release of a variety of hydrophobic cargos.

Understanding ligand distributions in modified particle and particlelike systems.

Chemical modification of nanoparticles or particlelike systems is ubiquitously being used to facilitate specific pharmaceutical functionalities or physicochemical attributes of nanocrystals, proteins, enzymes, or other particlelike systems. Often the modification process is incomplete and the functional activity of the product depends upon the distribution of functional ligands among the different particles in the system. Here, the distribution function describing the spread of ligands in particlelike systems undergoing partial modification reactions is derived and validated against a conjugated enzyme model system by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). The distribution function is shown to be applicable to describe the distribution of ligands in a wide range of particlelike systems (such as enzymes, dendrimers, or inorganic nanocrystals) and is used to establish guidelines for the synthesis of uniformly modified particle systems even at low reaction efficiencies.

Targeting microspheres and cells to polyethylene glycol-modified biological surfaces.

It has previously been demonstrated that damaged arterial tissue can be acutely modified with protein-reactive polyethylene glycol (PEG) to block undesirable platelet deposition. This concept might be expanded by employing PEG-biotin and its strong interaction with avidin for site-specific targeted delivery. Toward this end, cultured endothelial cells (ECs) were surface modified with PEG-biotin and the available biotin was quantified with flow cytometry. NeutrAvidin-coated microspheres and PEG-biotin modified ECs with NeutrAvidin as a bridging molecule were delivered under arterial shear stress to PEG-biotin modified ECs on a coverslip as well as scrape-damaged bovine carotid arteries. After incubation with a 10 mM solution for 1 min, 8 x 10(7) PEG-biotin molecules/EC were found and persisted for up to 120 h. Perfused microspheres adhered to NHS-PEG-biotin treated bovine carotid arteries with 60 +/- 16 microspheres/mm(2) versus 11 +/- 4 microspheres/mm(2) for control arteries (p < 0.015). Similarly, 22 +/- 5 targeted ECs/mm(2) adhered to NHS-PEG-biotin treated bovine carotid arteries versus 6 +/- 2 ECs/mm(2) for control arteries (p < 0.01). The targeting strategy demonstrated here might ultimately find application for drug delivery, gene therapy, or cell therapy where localization to specific labeled vascular regions is desired following catheter-based or surgical procedures.