Biodegradable poly(ethylenimine) for plasmid DNA delivery

Poly(ethylenimine) (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on the molecular weight. Synthesis of cationic copolymers derived from the low molecular weight of PEI and hydrophilic poly(ethylene glycol) (PEG), which are water soluble and degradable under physiological conditions, was investigated for plasmid delivery. Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PEI and DNA complexes, and help to introduce degradable bonds by reaction with the primary amines in the PEI. Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PEI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the introduction of PEG and the degradation of the copolymer into low molecular weight PEIs. Reaction conditions were carefully controlled to produce water soluble copolymers. Results from a gel retardation assay and zetapotentiometer indicated that complete neutralization of the complexes was achieved at the charge ratios of copolymer/pSV-β-gal plasmid from 0.8 to 1.0 with the mean particle size of the polyplexes ranging from 129.8±0.9 to 151.8±3.4 nm. In vitro transfection efficiency of the synthesized copolymer increased up to three times higher than that of starting low molecular weight PEI, while the cell viability was maintained over 80%.     

Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration.

Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions. INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation. MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT. RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition. CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics.     

A simple and rapid assay to evaluate purity of foot-and-mouth disease vaccine before animal experimentation

Currently, foot-and-mouth disease (FMD) vaccine purity is tested in cattle to detect antibodies against the non-structural protein (NSP) after repeated immunization with the final vaccine product. In case of vaccine failure, the manufacturing company would suffer significant economic loss. To prevent such unfortunate losses with the final vaccine product, in vitro testing is required to quantitate an NSP antigen during the manufacturing process prior to animal experiments. A novel lateral-flow assay device was developed using a monoclonal antibody (MAb) against the 3B NSP. To determine the minimal amount of NSP required to elicit antibodies in livestock, goats were immunized several times with various concentrations of either the recombinant 3AB (rec.3AB) protein or FMD virus culture supernatant. Antibodies against 3AB were elicited after a second immunization with 10.6 ng to 42.5 ng of rec.3AB and a third immunization with a 10-fold diluted FMD virus culture supernatant in goats. The lateral-flow assay device detected the minimal amount of rec.3AB and native NSP in FMD virus culture supernatant required to induce NSP antibodies in goats. The in vitro assay device is simple and economical, provides rapid results, and should be useful for FMD vaccine-manufacturing companies prior to conducting animal experiments to test the vaccine purity.     

牛磺酸的合成新工艺

目的研究牛磺酸的合成新工艺。方法以乙醇胺(MEA)为原料,在改性HZSM-5分子筛催化剂作用下,经分子内脱水和与亚硫酸氢胺开环加成两步反应制备牛磺酸。结果分子内脱水的适宜工艺条件是:反应温度为425℃、反应压力为常压、反应器空速为2 300 h 1、反应原料配比为N2/MEA=10。在该条件下,乙醇胺的转化率93.2%,选择性84.6%,牛磺酸的总收率为73%(以乙醇胺计)。结论新工艺优于乙醇胺酯化法(>50%)和环氧乙烷法(>60%),具有很好的工业化前景。     

Off-target effects of an insect cell-expressed influenza HA-pseudotyped Gag-VLP preparation in limiting postinfluenza Staphylococcus aureus infections

Clinical and historical data underscore the ability of influenza viruses to ally with Staphylococcus aureus and predispose the host for secondary bacterial pneumonia, which is a leading cause of influenza-associated mortality. This is fundamental because no vaccine for S. aureus is available and the number of antibiotic-resistant strains is alarmingly rising. Hence, this leaves influenza vaccination the only strategy to prevent postinfluenza staphylococcal infections. In the present work, we assessed the off-target effects of a Tnms42 insect cell-expressed BEI-treated Gag-VLP preparation expressing the HA of A/Puerto Rico/8/1934 (H1N1) in preventing S. aureus superinfection in mice pre-infected with a homologous or heterologous H1N1 viral challenge strain. Our results demonstrate that matched anti-hemagglutinin immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection. This effect was observed even when employing a single low antigen dose of 50 ng HA per animal. However, induction of anti-hemagglutinin immunity alone was not helpful in inhibiting heterologous viral replication and subsequent bacterial infection. Our results indicate the potential of the VLP vaccine approach in terms of immunogenicity but suggest that anti-HA immunity should not be considered as the sole preventive method for combatting influenza and postinfluenza bacterial infections.     

Hemocompatibility assessment of poly(2-dimethylamino ethylmethacrylate) (PDMAEMA)-based polymers

Poly(2-dimethylamino-ethylmethacrylate) (PDMAEMA), a cationic polymer, has been widely reported as a nonviral carrier. Despite the fact that the cytotoxicity of this polymer has been extensively studied, there is a lack of information about its blood compatibility. Hence, this work evaluates the hemocompatibility of free-form PDMAEMA homopolymers differing in molecular weight (Mw) with or without a poly(ethylene glycol) (PEG) sequence in the form of a palm tree-like structure. Poly(ethylenimine) (PEI) was used as a reference in order to compare its hemoreactivity. Hemagglutination, hemolysis, platelet number, blood coagulation, and the complement systems were assessed in normal human whole blood according to the ISO 10993-4. Results showed that Mw, concentration, and incubation time strongly affected the hemocompatibility of the polymers evaluated. Our in vitro observations highlight that PDMAEMA homopolymers interacted strongly with the surface of the red blood cells but not with the inner structure of the membrane, while PEI behaved in the opposite way. No clear correlation has been evidenced between PDMAEMA-induced hemagglutination, PEI-induced hemagglutination, and hemolysis. Interestingly, if these polyelectrolytes strongly affect the platelets and blood coagulation cascades in a dose dependent way, none of them significantly affects the complement system. Our work reveals new knowledge on the toxicology of 2 families of polycations largely explored for gene delivery and on their mechanisms of cellular and humoral interactions.     

Cyclophosphamide, 2,2-dimethylaziridines and other alkylating agents as inhibitors of serum cholinesterase.

The effects of cyclophosphamide (CTX) and of alkylating agents containing aziridine or 2,2-dimethylaziridine moieties on the procaine esterase activity of horse serum cholinesterase were investigated. The results indicated that CTX is a competitive, reversible inhibitor of the enzyme, while all the other agents studied caused irreversible inhibition. However, there was no over-all correlation between the cholinesterase inhibitory activities of these agents and their alkylating reactivities toward the model nucleophile 4-(p-nitrobenzyl)pyridine (NBP). The kinetics of inhibition were consistent with the formation of a reversible enzyme alkylating agent complex prior to the irreversible inactivation of the enzyme. In the case of the ring-C-unsubstituted aziridines (TEM. TEPA and AB-100), the inactivation process could be described by the Main equation from which a dissociation constant (K_d) and a reaction rate constant (k₂)wcre calculated. The 2,2-dimethylaziridines(AB-132. AB-163 and TEPA-132) readily hydrolyzed. with rapid loss of alkylating activity (vs NBP). Simultaneously, the cholinesterase inhibitory activities of AB-132 and AB-163 significantly increased, reached a maximum and then gradually decreased on further hydrolysis; in contrast, TEPA-132 showed progressive loss of inhibitory activity. These results indicate that both AB-132 and AB-163 (but not TEPA-132) hydrolyze with the formation of an unstable intermediate(s) having little or no alkylating activity but acting as a potent, irreversible cholinesterase inhibitor(s).     

Fabrication and characterization of poly (ethylenimine) modified poly (l-lactic acid) nanofibrous scaffolds

Abstract

Bone tissue engineering aims to construct biological substitutes for repairing bone defects. Nanofibrous (NF) scaffolds are commonly utilized to mimic the extracellular matrix (ECM) environment and promote tissue regeneration in tissue engineering process. Poly (lactic acid) (PLA) has attracted much attention in the field of tissue engineering because of its biocompatibility, biodegradability and so on. However, the intrinsic hydrophobicity and the lacking of active functional groups limit its practical application to some extent. In this study, poly(ethylenimine) (PEI) modified PLLA nanofibrous scaffolds were fabricated in a one step process by aminolysis combined with thermally induced phase separation technique for introducing more functional groups, PEI acting as the modifier. The morphology of PEI-modified PLLA scaffolds prepared under different experimental conditions was analyzed by scanning electron microscope (SEM). The suitable conditions to fabricate scaffolds with a homogeneous nanofibrous structure, good hydrophilicity and excellent mechanical properties were determined according to the results of SEM, water contact angle (WCA) and mechanical properties testing. Besides, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (¹H NMR), X-ray Photoelectron Spectroscopy (XPS) and gel permeation chromatography (GPC) were used to confirm the occurrence of the ammonolysis reaction between PLLA and PEI. The in vitro biomineralization study showed that the PEI-modified PLLA scaffolds had a greater ability to induce the formation of apatite in 1.5SBF than PLLA scaffolds, indicating that the bone-bioactivity of PLLA scaffolds was significantly improved after modification with PEI. Furthermore, cell culture assay revealed that MC3T3-E1 osteoblasts exhibited better proliferation performance on the PEI-modified PLLA scaffolds. All the results implied that the synthesized modified PLLA nanofibrous scaffolds may provide promising applications in bone tissue engineering.     

Antibacterial quaternary ammonium compounds in dental materials: A systematic review

Objective

Quaternary ammonium compounds (QACs) represent one of the most effective classes of disinfectant agents in dental materials and resin nanocomposites. This reviews aims to give a wide overview on the research in the field of antibacterial QACs in dental materials and nanocomposites.