Synthesis of Light-Responsive Pyrene-Based Polymer Nanoparticles via Polymerization-Induced Self-Assembly.

The use of an in situ, one-pot polymerization-induced self-assembly method to synthesize light-responsive pyrene-containing nanoparticles is reported. The strategy is based on the chain extension of a hydrophilic macromolecular chain transfer agent, poly(oligo(ethylene glycol) methyl ether methacrylate), using a light-responsive monomer, 1-pyrenemethyl methacrylate (PyMA), via a reversible addition-fragmentation chain transfer dispersion polymerization; yielding nanoparticles of various morphologies (spherical micelles and worm-like micelles). In this process, addition of comonomers, such as butyl methacrylate (BuMA) or methyl methacrylate (MMA), are required to obtain high PyMA monomer conversion (>80% in 24 h). The addition of comonomers reduces the π-π stacking of the pyrene moieties, which facilitates the diffusion of monomers in the nanoparticle core. The addition of BuMA (as a comonomer) offers P(PyMA-co-BuMA) core-forming chains with high mobility that enables the reorganization of chains and then the evolution of morphology to form vesicles. In contrast, when MMA comonomer is used, kinetically trapped spheres are obtained; this is due to the low mobility of the core-forming chains inhibiting in situ morphological evolution. Finally, the UV-light-induced dissociation of these light-responsive nanoparticles due to the gradual cleavage of the pyrene moieties and the subsequent hydrophobic-to-hydrophilic transitions of the core-forming blocks is demonstrated.

Ferrous ion as a reducing agent in the generation of antibiofilm nitric oxide from a copper-based catalytic system.

The work found that the electron-donating properties of ferrous ions (Fe2+) can be used for the conversion of nitrite (NO2-) into the biofilm-dispersing signal nitric oxide (NO) by a copper(II) complex (CuDTTCT) catalyst, a potentially applicable biofilm control technology for the water industries. The availability of Fe2+ varied depending on the characteristics of the aqueous systems (phosphate- and carbonate-containing nitrifying bacteria growth medium, NBGM and phosphate buffered saline, PBS at pH 6 to 8, to simulate conditions typically present in the water industries) and was found to affect the production of NO from nitrite by CuDTTCT (casted into PVC). Greater amounts of NO were generated from the CuDTTCT-nitrite-Fe2+ systems in PBS compared to those in NBGM, which was associated with the reduced extent of Fe2+-to-Fe3+ autoxidation by the iron-precipitating moieties phosphates and carbonate in the former system. Further, acidic conditions at pH 6.0 were found to favor NO production from the catalytic system in both PBS and NBGM compared to neutral or basic pH (pH 7.0 or 8.0). Lower pH was shown to stabilize Fe2+ and reduce its autoxidation to Fe3+. These findings will be beneficial for the potential implementation of the NO-generating catalytic technology and indeed, a 'non-killing' biofilm dispersal activity of CuDTTCT-nitrite-Fe2+ was observed on nitrifying bacteria biofilms in PBS at pH 6.

Exploiting the Versatility of Polydopamine-Coated Nanoparticles to Deliver Nitric Oxide and Combat Bacterial Biofilm.

In this study, an antimicrobial platform in the form of nitric oxide (NO) gas-releasing polydopamine (PDA)-coated iron oxide nanoparticles (IONPs) is developed for combating bacterial biofilms. NO is bound to the PDA-coated IONPs via the reaction between NO and the secondary amine moieties on PDA to form N-diazeniumdiolate (NONOate) functionality. To impart colloidal stability to the nanoparticles in aqueous solutions (e.g., phosphate buffered saline (PBS) and bacteria cell culture media M9), a polymer bearing hydrophilic and amine pendant groups, P(OEGMA)-b-P(ABA), is synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization and is subsequently grafted onto the PDA-coated IONPs by employing the Schiff base/Michael addition reaction between o-quinone and a primary amine. These nanoparticles are able to effectively disperse Pseudomonas aeruginosa biofilms (up to 79% dispersal) at submicromolar NO concentrations. In addition, the nanoparticles demonstrate excellent bactericidal activity toward P. aeruginosa planktonic and biofilm cells (up to 5-log10 reduction).

Polymerization of a Photocleavable Monomer Using Visible Light.

The polymerization of the photocleavable monomer, o-nitrobenzyl methacrylate (NBMA), is investigated using photoinduced electron/energy transfer reversible addition-fragmentation chain transfer polymerization. The polymerizations under visible red (λ max = 635 nm, 0.7 mW cm(-2) ) and yellow (λ max = 560 nm, 9.7 mW cm(-2) ) light are performed and demonstrate rational evidence of a controlled/living radical polymerization process. Well-defined poly(o-nitrobenzyl methacrylate) (PNBMA) homopolymers with good control over the molecular weight and polymer dispersity are successfully synthesized by varying the irradiation time and/or targeted degree of polymerization. Chain extension of a poly(oligo(ethylene glycol) methyl ether methacrylate) macro-chain transfer agent with NBMA is carried out to fabricate photocleavable amphiphilic block copolymers (BCP). Finally, these self-assembled BCP rapidly dissemble under UV light suggesting the photoresponsive character of NBMA is not altered during the polymerization under yellow or red light. Such photoresponsive polymers can be potentially used for the remote-controlled delivery of therapeutic compounds.

Insight into serum protein interactions with functionalized magnetic nanoparticles in biological media.

Surface modification with linear polymethacrylic acid (20 kDa), linear and branched polyethylenimine (25 kDa), and branched oligoethylenimine (800 Da) is commonly used to improve the function of magnetite nanoparticles (MNPs) in many biomedical applications. These polymers were shown herein to have different adsorption capacity and anticipated conformations on the surface of MNPs due to differences in their functional groups, architectures, and molecular weight. This in turn affects the interaction of MNPs surfaces with biological serum proteins (fetal bovine serum). MNPs coated with 25 kDa branched polyethylenimine were found to attract the highest amount of serum protein while MNPs coated with 20 kDa linear polymethacrylic acid adsorbed the least. The type and amount of protein adsorbed, and the surface conformation of the polymer was shown to affect the size stability of the MNPs in a model biological media (RPMI-1640). A moderate reduction in r(2) relaxivity was also observed for MNPs suspended in RPMI-1640 containing serum protein compared to the same particles suspended in water. However, the relaxivities following protein adsorption are still relatively high making the use of these polymer-coated MNPs as Magnetic Resonance Imaging (MRI) contrast agents feasible. This work shows that through judicious selection of functionalization polymers and elucidation of the factors governing the stabilization mechanism, the design of nanoparticles for applications in biologically relevant conditions can be improved.

Assembly of polyethylenimine-based magnetic iron oxide vectors: insights into gene delivery.

The use of a nonviral magnetic vector, comprised of magnetic iron oxide nanoparticles (MNP), polyethylenimine (PEI), and plasmid DNA, for transfection of BHK21 cells under a magnetic field is presented. Four different vector configurations were studied by systematically varying the mixing order of MNP, PEI, and DNA. The assembly of the vector has significant effects on its vector size, surface charge, cellular uptake, and level of gene expression. Mixing MNP with PEI first improved MNP stability, giving a narrow aggregate size distribution and positive surface charge at physiological pH, which in turn facilitated DNA binding onto MNP. The presence of serum in culture media improves vector dispersion and alters the surface charge of all vectors to negative charge, indicating serum protein adsorption. Cellular uptake was greater for larger vectors than the smaller vectors due to enhanced gravitational and magnetic aided sedimentation onto the cells. High MNP uptake by the cells, however, does not inevitably lead to increase gene expression efficiency. It can be shown that besides vector uptake, gene expression is affected by extracellular factors such as premature DNA release from MNP and DNA degradation by serum as well as intracellular factors such as vector lysosomal degradation, inability of DNA to detach from MNP, and cytotoxic effects of MNP at high uptake. Some of these extra- and intracellular properties are shown to be mediated by the presence of PEI.

Controlled fabrication of polyethylenimine-functionalized magnetic nanoparticles for the sequestration and quantification of free Cu2+.

Presented herein is a detailed study into the controlled adsorption of polyethylenimine (PEI) onto 50 nm crystalline magnetite nanoparticles (Fe(3)O(4) NPs) and how these PEI-coated Fe(3)O(4) NPs can be used for the magnetic capture and quantification of ultratrace levels of free cupric ions. We show the ability to systematically control the amount of PEI adsorbed onto the Fe(3)O(4) magnetic nanoparticle surfaces by varying the concentration of polymer during the adsorption process. This in turn allows for the tailoring of important colloidal properties such as the electrophoretic mobility and aggregation stability. Copper adsorption tests were carried out to investigate the effectiveness of PEI-coated Fe(3)O(4) NPs in copper remediation and detection. The study demonstrated that the NPs ability to bind with copper is highly dependent on the amount of PEI adsorbed on the NP surface. It was found that PEI-coated Fe(3)O(4) NPs were able to capture trace levels (approximately 2 ppb) of free cupric ions and concentrate the ions to allow for detection via ICP-OES. More importantly, it was found that due to the amine-rich structure of PEI, the PEI-coated Fe(3)O(4) NPs selectively adsorb toxic free cupric ions but not the less toxic EDTA complexed copper. This unique property makes PEI-coated Fe(3)O(4) NPs a novel solution for the challenge of separating and quantifying toxic cupric ions as opposed to the total copper concentration of a sample.

Polyethylenimine based magnetic iron-oxide vector: the effect of vector component assembly on cellular entry mechanism, intracellular localization, and cellular viability.

The order of assembly of a magnetic nanoparticle (MNP) vector comprised of the same components (MNP, PEI, and plasmid DNA) on entry mechanism, intracellular localization, and viability of BHK21 cells was investigated. Cellular uptake measurements under four different uptake inhibiting conditions, such as low temperature, depleted cellular ATP, nystatin treatment, and hypertonic environment, show that the cellular entry mechanism of the MNP vector was mediated via clathrin endocytosis. Despite different vector component assembly, all MNP vectors were taken up by the cells through the same mechanism. Labeling and intracellular tracking of the MNP vectors using epi-fluorescence and confocal laser scanning microscopy showed localization of MNP vector within the lysosomes when DNA was assembled on the outer layer of vector. Conversely, when PEI was on the surface of the vector, such that it enclosed both magnetic nanoparticles and the DNA, vector localization in the cell nucleus was observed. The microscopy results demonstrated that the configuration of the MNP vectors dictate the vector's final intracellular target location, and thus the efficiency of transfection. The cellular viability assessment using three different assays further showed that the cellular viability of MNP vector was dose-dependent and varied with the assembly of vector component. All viability assays found negligible toxicity when DNA was on the outer layer of MNP vector except at the highest vector loading. In contrast, attachment of PEI on MNP vector surface induced a significant decrease in cellular viability, due to the ability of PEI on the MNP vector to rupture the lysosomal vesicles.

Copper Complex in Poly(vinyl chloride) as a Nitric Oxide-Generating Catalyst for the Control of Nitrifying Bacterial Biofilms.

In this study, catalytic generation of nitric oxide by a copper(II) complex embedded within a poly(vinyl chloride) matrix in the presence of nitrite (source of nitric oxide) and ascorbic acid (reducing agent) was shown to effectively control the formation and dispersion of nitrifying bacteria biofilms. Amperometric measurements indicated increased and prolonged generation of nitric oxide with the addition of the copper complex when compared to that with nitrite and ascorbic acid alone. The effectiveness of the copper complex-nitrite-ascorbic acid system for biofilm control was quantified using protein analysis, which showed enhanced biofilm suppression when the copper complex was used in comparison to that with nitrite and ascorbic acid treatment alone. Confocal laser scanning microscopy (CLSM) and LIVE/DEAD staining revealed a reduction in cell surface coverage without a loss of viability with the copper complex and up to 5 mM of nitrite and ascorbic acid, suggesting that the nitric oxide generated from the system inhibits proliferation of the cells on surfaces. Induction of nitric oxide production by the copper complex system also triggered the dispersal of pre-established biofilms. However, the addition of a high concentration of nitrite and ascorbic acid to a pre-established biofilm induced bacterial membrane damage and strongly decreased the metabolic activity of planktonic and biofilm cells, as revealed by CLSM with LIVE/DEAD staining and intracellular adenosine triphosphate measurements, respectively. This study highlights the utility of the catalytic generation of nitric oxide for the long-term suppression and removal of nitrifying bacterial biofilms.

Bi-functional gold-coated magnetite composites with improved biocompatibility.

The effect of gold attachment on the physical characteristics, cellular uptake, gene expression efficiency, and biocompatibility of magnetic iron oxide (MNP) vector was investigated in vitro in BHK21 cells. The surface modification of magnetite with gold was shown to alter the morphology and surface charge of the vector. Nonetheless, despite the differences in the surface charge with and without gold attachment, the surface charge of all vectors were positive when conjugated with PEI/DNA complex, and switched from positive to negative when suspended in cell media containing serum, indicating the adsorption of serum components onto the composite. The cellular uptake of all MNP vectors under the influence of a magnetic field increased when the composite loadings increased, and was higher for the MNP vector that was modified with gold. Both bare magnetite and gold-coated magnetite vectors gave similar optimal gene expression efficiency, however, the gold-coated magnetite vector required a 25-fold higher overall loading to achieve a comparable efficiency as the attachment of gold increased the particle size, thus reducing the surface area for PEI/DNA complex conjugation. The MNP vector without gold showed optimal gene expression efficiency at a specific magnetite loading, however further increases beyond the optimum loading decreased the efficiency of gene expression. The drop in efficiency at high magnetite loadings was attributed to the significant reduction in cellular viability, indicating the bare magnetite became toxic at high intracellular levels. The gene expression efficiency of the gold-modified vector, on the other hand, did not diminish with increasing magnetite loadings. Intracellular examination of both bare magnetite and gold-coated magnetite vectors at 48h post-magnetofection using transmission electron microscopy provided evidence of the localization of both vectors in the cell nucleus for gene expression and elucidated the nuclear uptake mechanism of both vectors. The results of this work demonstrate the efficacy of gold-modified vectors to be used in cellular therapy research that can function both as a magnetically-driven gene delivery vehicle and an intracellular imaging agent with negligible impact on cell viability.