Fabrication of Chitosan/PEGDA Bionanocomposites for Enhanced Drug Encapsulation and Release Efficiency.

Drug delivery systems (DDS) have evolved in the last decades with the development of hydrogels and particles. However, challenges such as high systemic uptake, side effects, low bioavailability, and encapsulation efficiency continue to be significant hurdles faced by such DDSs. Particles and hydrogels can be specifically designed for targeted DDSs to mitigate some of these problems. This study developed chitosan (Cs) particles (Ps) and composite films using poly(ethylene glycol) diacrylate (PEGDA) as a copolymer to encapsulate gentamicin (GtS) for drug delivery. We demonstrated that lysozyme degrades the chitosan ß-1,4 glycosidic bonds to release GtS. PEGDA increased drug encapsulation efficiency by shielding the repelling forces of like charges between Cs and GtS. The data show that PEGDA does not hinder enzymatic degradation while increasing drug encapsulation efficiency and producing more homogeneous particles. Additionally, we utilized Michael's reaction to cross-link Cs, CsPs, and PEGDA to produce a film designed for drug delivery. The film is an anchor for CsPs to prevent premature drug release. The cross-linking of Cs and PEGDA does not affect lysozyme activity, and CsPs could successfully release GtS without affecting GtS activity.

Ultrafiltration Membranes Functionalized with Copper Oxide and Zwitterions for Fouling Resistance.

Polymeric membrane fouling is a long-standing challenge for water filtration. Metal/metal oxide nanoparticle functionalization of the membrane surface can impart anti-fouling properties through the reactivity of the metal species and the generation of radical species. Copper oxide nanoparticles (CuO NPs) are effective at reducing organic fouling when used in conjunction with hydrogen peroxide, but leaching of copper ions from the membrane has been observed, which can hinder the longevity of the CuO NP activity at the membrane surface. Zwitterions can reduce organic fouling and stabilize NP attachment, suggesting a potential opportunity to combine the two functionalizations. Here, we coated polyethersulfone (PES) ultrafiltration membranes with polydopamine (PDA) and attached the zwitterionic compound, thiolated 2-methacryloyloxyethyl phosphorylcholine (MPC-SH), and CuO NPs. Functionalized membranes resulted in a higher flux recovery ratio (0.694) than the unfunctionalized PES control (0.599). Copper retention was high (>96%) for functionalized membranes. The results indicate that CuO NPs and MPC-SH can reduce organic fouling with only limited copper leaching.

Microcystin-LR Removal from Water via Enzymatic Linearization and Ultrafiltration.

Microcystin-LR (MC-LR) is a toxin produced by cyanobacteria that can bloom in freshwater supplies. This study describes a new strategy for remediation of MC-LR that combines linearization of the toxin using microcystinase A, MlrA, enzyme with rejection of linearized byproducts using membrane filtration. The MlrA enzyme was expressed in Escherichia coli (E. coli) and purified via a His-tag with 95% purity. Additionally, composite membranes made of 95% polysulfone and 5% sulfonated polyether ether ketone (SPEEK) were fabricated and used to filter a solution containing cyclic and linearized MC-LR. Tests were also performed to measure the adsorption and desorption of MC-LR on polysulfone/SPEEK membranes. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the progress of linearization and removal of MC-LR. Results indicate that the MlrA was successful at linearizing MC-LR. Membrane filtration tests showed rejection of 97% of cyclic MC-LR and virtually all linearized MC-LR, with adsorption to the membranes being the main rejection mechanism. Adsorption/desorption tests indicated that methanol could be used to strip residual MC-LR from membranes to regenerate them. This study demonstrates a novel strategy of remediation of microcystin-tainted water, combining linearization of MC-LR to a low-toxicity byproduct along with removal by membrane filtration.

Development and Characterization of Membranes with PVA Containing Silver Particles: A Study of the Addition and Stability.

Developing technologies for the reduction of biofouling and enhancement of membrane functionality and durability are challenging but critical for the advancement of water purification processes. Silver (Ag) is often used in the process of purification due to its anti-fouling properties; however, the leaching of this metal from a filtration membrane significantly reduces its effectiveness. Our study was designed to integrate the positive characteristics of poly vinyl alcohol (PVA) with the controlled incorporation of nano-scale silver ions across the membrane. This approach was designed with three goals in mind: (1) to improve antifouling activity; (2) to prevent leaching of the metal; and (3) to extend the durability of the functionalized membrane. The fabrication method we used was a modified version of manual coating in combination with sufficient pressure to ensure impregnation and proper blending of PVA with cellulose acetate. We then used the spin coater to enhance the cross-linking reaction, which improved membrane durability. Our results indicate that PVA acts as a reducing agent of Ag+ to Ag0 using X-ray photoelectron spectroscopy analysis and demonstrate that the metal retention was increased by more than 90% using PVA in combination with ultraviolet-photo-irradiated Ag+ reduced to Ag0. The Ag+ ions have sp hybrid orbitals, which accept lone pairs of electrons from a hydroxyl oxygen atom, and the covalent binding of silver to the hydroxyl groups of PVA enhanced retention. In fact, membranes with reduced Ag displayed a more effective attachment of Ag and a more efficient eradication of E. coli growth. Compared to pristine membranes, bovine serum albumin (BSA) flux increased by 8% after the initial addition of Ag and by 17% following ultraviolet irradiation and reduction of Ag, whereas BSA rejection increased by 10% and 11%, respectively. The implementation of this hybrid method for modifying commercial membranes could lead to significant savings due to increased metal retention and membrane effectiveness. These enhancements would ultimately increase the membrane's longevity and reduce the cost/benefit ratio.

The roles of Rhodobacter sphaeroides copper chaperones PCu(A)C and Sco (PrrC) in the assembly of the copper centers of the aa(3)-type and the cbb(3)-type cytochrome c oxidases.

The α proteobacter Rhodobacter sphaeroides accumulates two cytochrome c oxidases (CcO) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa(3)-type CcO containing a di-copper Cu(A) center and mono-copper Cu(B), plus a cbb(3)-type CcO that contains Cu(B) but lacks Cu(A). Three copper chaperones are located in the periplasm of R. sphaeroides, PCu(A)C, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu(B) of the aa(3)-type but not the cbb(3)-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in Cu(A) assembly in mitochondria and bacteria, and with Cu(B) assembly of the cbb(3)-type CcO. PCu(A)C is present in many bacteria, but not mitochondria. PCu(A)C of Thermus thermophilus metallates a Cu(A) center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa(3)- and cbb(3)-type CcOs of R. sphaeroides has been examined in strains lacking PCu(A)C, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu(2+). The absence of PrrC has a greater effect than the absence of PCu(A)C and PCu(A)C appears to function upstream of PrrC. Analysis of purified aa(3)-type CcO shows that PrrC has a greater effect on the assembly of its Cu(A) than does PCu(A)C, and both chaperones have a lesser but significant effect on the assembly of its Cu(B) even though Cox11 is present. Scenarios for the cellular roles of PCu(A)C and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to Cu(A) of the aa(3)-type CcO and to Cu(B) of the cbb(3)-type CcO, while the predominant role of PCu(A)C may be to capture and deliver copper to PrrC and Cox11. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Mutagenic analysis of Cox11 of Rhodobacter sphaeroides: insights into the assembly of Cu(B) of cytochrome c oxidase.

The Cu(I) chaperone Cox11 is required for the insertion of Cu(B) into cytochrome c oxidase (CcO) of mitochondria and many bacteria, including Rhodobacter sphaeroides. Exploration of the copper binding stoichiometry of R. sphaeroides Cox11 led to the finding that an apparent tetramer of both mitochondrial and bacterial Cox11 binds more copper than the sum of the dimers, providing another example of the flexibility of copper binding by Cu(I)-S clusters. Site-directed mutagenesis has been used to identify components of Cox11 that are not required for copper binding but are absolutely required for the assembly of Cu(B), including conserved Cys-35 and Lys-123. In contrast to earlier proposals, Cys-35 is not required for dimerization of Cox11 or for copper binding. These findings, and the location of Cys-35 at the C-terminus of the predicted transmembrane helix and thereby close to the surface of the membrane, allow a proposal that Cys-35 is involved in the transfer of copper from the Cu(I) cluster of Cox11 to the Cu(B) ligands His-333 and His-334 during the folding of CcO subunit I. Lys-123 is located near the Cu(I) cluster of Cox11, in an area otherwise devoid of charged residues. From the analysis of several Cox11 mutants, including K123E, -L, and -R, we conclude that a previous proposal that Lys-123 provides charge balance for the stabilization of the Cu(I) cluster is unlikely to account for its absolute requirement for Cox11 function. Rather, consideration of the properties of Lys-123 and the apparent specificity of Cox11 suggest that Lys-123 plays a role in the interaction of Cox11 with its target.