Light Scattering Analysis of Mono- and Multi-PEGylated Bovine Serum Albumin in Solution: Role of Composition on Structure and Interactions.

The effect of polymer conjugation on the interactions between proteins in solution is evaluated by systematic analysis of the second virial coefficient (A2) for the particular example of single- and double-PEGylated bovine serum albumin (PEG-BSA) in dilute PBS solution. The effect of PEGylation on A2 is found to sensitively depend on both the composition and the distribution of PEG segments within the conjugate. Most importantly, at a given PEG volume fraction, A2 significantly increases with the degree of polymerization of tethered chains. Hence, a lesser number of long chains is more effective in solubilizing BSA than a correspondingly larger number of short chains. Analysis of the hydrodynamic radii of protein-PEG conjugates suggests that the increased solubility is concurrent with a structural transition in the case of high molecular PEG grafts that results in compact core-shell-type structures. The results reveal a link between the composition, structure, and solubility of polymer conjugates that might benefit the understanding of their biochemical characteristics and their design for functional material applications.

Analysis of heterogeneity in nonspecific PEGylation reactions of biomolecules.

The compositional heterogeneity associated with polymer conjugation reactions of biomolecules is analyzed for the particular case of nonspecific PEGylation reactions. It is shown that the distribution of the number of PEG moieties grafted to biomolecules such as proteins is a binomial-type function of two parameters-the reaction efficiency as well as the number of binding sites per biomolecule. The nature of this distribution implies that uniform compositions are favored for increasing number of coupling sites per biomolecule as well as for increasing efficiency of the modification process. Therefore, the binomial distribution provides a rationale for the pronounced heterogeneity that is observed for PEGylated small enzyme systems even at high coupling efficiencies. For the particular case of PEGylated trypsin it is shown that the heterogeneity results in a broad distribution of deactivation times that is captured by a stretched exponential decay model. The presented analysis is expected to apply to general modification processes of compounds in which partial functionalization of a fixed number of reactive sites is achieved by means of a nonspecific coupling reaction.

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.

Enabling Thermoreversible Physically Cross-Linked Polymerized Colloidal Array Photonic Crystals.

We physically cross-linked a thermoreversible poly(vinyl alcohol) (PVA) hydrogel (TG) within a crystalline colloidal array (CCA) to form an enabling photonic crystal material. The TG consists of a physically cross-linked network formed in a process reminiscent of the well-known freeze-thaw physically cross-linking process, but which avoids solvent freezing which invariably disorders the CCA. These TGCCA can be inexpensively fabricated in any large volume and shape by avoiding the previous covalently polymerized CCA constraints that required thin sheet geometries to enable penetration of the UV light used to photopolymerize the system. This TG hydrogel enables rigidificaton of CCA crystals and subsequent chemical functionalization. In addition, an additional interpenetrating hydrogel can be polymerized within the TGPCCA. The TG can then be dissolved away by simply increasing the temperature. The TGCCA photonic crystal diffraction is highly efficient and similar to previously demonstrated PCCA with covalent cross-links. These TGCCA are stable for weeks or longer at room temperature and can be utilized as photonic crystal materials. They also can be irreversibly covalently cross-linked by using gluteraldehyde. These gluteraldehyde cross-linked TGCCA can be made into chemically responsive sensor photonic crystals by functionalizing the PVA hydroxyl groups with chemical recognition agents. We demonstrate low and high pH sensing by functionalizing with carboxylates and phenol derivatives, respectively.

Photonic crystal sensor for organophosphate nerve agents utilizing the organophosphorus hydrolase enzyme.

We developed an intelligent polymerized crystalline colloidal array (IPCCA) photonic crystal sensing material which reversibly senses the organophosphate compound methyl paraoxon at micromolar concentrations in aqueous solutions. A periodic array of colloidal particles is embedded in a poly-2-hydroxyethylacrylate hydrogel. The particle lattice spacing is such that the array Bragg-diffracts visible light. We utilize a bimodular sensing approach in which the enzyme organophosphorus hydrolase (OPH) catalyzes the hydrolysis of methyl paraoxon at basic pH, producing p-nitrophenolate, dimethylphosphate, and two protons. The protons lower the pH and create a steady-state pH gradient. Protonation of the phenolates attached to the hydrogel makes the free energy of mixing of the hydrogel less favorable, which causes the hydrogel to shrink. The IPCCA's lattice constant decreases, which blueshifts the diffracted light. The magnitude of the steady-state diffraction blueshift is proportional to the concentration of methyl paraoxon. The current detection limit is 0.2 micromol methyl paraoxon per liter.

Progress toward the development of a point-of-care photonic crystal ammonia sensor.

We have developed an ammonia-sensitive material by coupling the Berthelot reaction to our polymerized crystalline colloidal array (PCCA) technology. The material consists of a periodic array of highly charged colloidal particles (110 nm diameter) embedded in a poly(hydroxyethyl acrylate) hydrogel. The particles have a lattice spacing such that they Bragg-diffract visible light. In the Berthelot reaction, ammonia, hypochlorite, and phenol react to produce the dye molecule indophenol blue in an aqueous solution. We use this reaction in our sensor by covalently attaching 3-aminophenol to the hydrogel backbone, which forms cross-links through the Berthelot mechanism. Ammonia reacts with hypochlorite, forming monochloramine, which then reacts with a pendant aminophenol to form a benzoquinone chlorimine. The benzoquinone chlorimine reacts with another pendant aminophenol to form a cross-link. The creation of new cross-links causes the hydrogel to shrink, which reduces the lattice spacing of the embedded colloidal array. This volume change results in a blue-shift in the diffracted light proportional to the concentration of NH3 in the sample. We demonstrate that the NH3 photonic crystal sensing material is capable of quantitative determination of concentrations in the physiological range (50-350 micromol NH3 L(-1)) in human blood serum.

Acetylcholinesterase-based organophosphate nerve agent sensing photonic crystal.

We developed a polymerized crystalline colloidal array (PCCA) photonic crystal sensing material that senses the organophosphorus compound parathion at ultratrace concentrations in aqueous solutions. A periodic array of colloidal particles is embedded in a hydrogel network with a lattice spacing such that it Bragg diffracts visible light. The molecular recognition agent for the sensor is the enzyme acetylcholinesterase (AChE), which binds organophosphorus compounds irreversibly, creating an anionic phosphonyl species. This charged species creates a Donnan potential, which swells the hydrogel network, which increases the embedded particle array lattice spacing and causes a red-shift in the wavelength of light diffracted. The magnitude of the diffraction red-shift is proportional to the amount of bound parathion. These AChE-PCCAs act as dosimeters for parathion since it irreversibly binds. Parathion concentrations as low as 4.26 fM are easily detected.