Synthesis of a new potential biodegradable disulfide containing poly(ethylene imine)-poly(ethylene glycol) copolymer cross-linked with click cluster for gene delivery

Poly(ethylene glycol)-grafted-polyethylenimine (PEG-PEI) are promising non-viral gene delivery systems. Herein, we aimed to synthesize a biodegradable disulfide containing PEGylated PEI to attempt to reduce its cytotoxicity and enhance the gene transfer activity. Using click chemistry, low Mw PEI (br. 2 kDa) and short chain length PEG (tetraethylene glycol, TEG) were cross-linked to a high Mw PEG-PEI copolymer (～ 22 kDa). The chemical structure of the copolymer was characterized using (1)H NMR and GPC. The degradation behavior was investigated under in vitro conditions in the presence of 1,4-dithiothreitol (DTT). The gel retardation assay, dynamic light scattering and atomic force microscopy showed good DNA condensation ability by forming polyplexes with small particle size and positive zeta potential. In particular, MTT assay indicated that this PEG-PEI polymer is about 22-fold less toxic than PEI 25k and only 2-fold more toxic than PEI 2k in L929 cell line. After coupling of small PEG chains and cross-linking by disulfide bridges, the transfection efficiency is increased approximately 6-fold in comparison to PEI 2k and still reaches approximately 17% of PEI 25k. Hence, this click cluster cross-linked disulfide containing PEG-PEI copolymer could be an attractive cationic polymer for non-viral gene delivery.     

Cellular uptake mechanism and knockdown activity of siRNA-loaded biodegradable DEAPA-PVA-g-PLGA nanoparticles

Efficient downregulation of gene expression depends on the uptake, intracellular distribution and efficient release of siRNA from their carrier. Therefore, the cellular uptake behavior and mechanism and intracellular localization of siRNA-loaded biodegradable nanoparticles were investigated.A biodegradable polymer, composed of poly(vinyl alcohol) (PVA) modified with diamine moieties and grafted with PLGA, abbreviated as DEAPA-PVA-g-PLGA, was used for the preparation of siRNA-loaded nanoparticles by solvent displacement. Particle sizes and morphology were determined by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The dependence of particle uptake into H1299-EGFP cells (lung cancer cells expressing green fluorescent protein) on both incubation time and temperature was studied by flow cytometry. Inhibition experiments focusing on clathrin- or caveolae-mediated uptake or uptake by macropinocytosis were performed. The intracellular localization was investigated by confocal laser scanning microscopy. The GFP knockdown efficiency was determined in vitro to establish the potential of the nanoparticles for the downregulation of gene expression.Nanoparticles with diameters of 120-180 nm were successfully generated. In contrast to the uptake of standard PEI-polyplexes, which increased continuously over a period of 4 h, nanoparticle uptake was complete within 2 h. A decrease in particle uptake at 4 °C (in comparison with 37 °C) suggests an active uptake process. Inhibition experiments revealed the predominance of clathrin-mediated uptake for siRNA-loaded nanoparticles. The siRNA-loaded nanoparticles could be clearly located within cells, mainly in intracellular vesicles. Particle uptake could be increased by the addition of lung surfactant to the formulation. Bioactivity in terms of successful GFP knockdown in vitro was demonstrated and could be further optimized by the use of surfactant-modified particles.In conclusion, a high and rapid cellular uptake was shown for siRNA-loaded nanoparticles. Cell internalization is based on an energy-dependent and predominantly clathrin-mediated process. Particle localization in endosomes and lysosomes was demonstrated. Evidence for the efficient delivery of bioactive siRNA and specific GFP knockdown provides a solid basis for the application of DEAPA-PVA-g-PLGA-based particles for gene silencing in vivo.     

Inflammatory responses to pulmonary application of PEI-based siRNA nanocarriers in mice

Polymeric non-viral vector systems for pulmonary application of siRNA are promising carriers, but have failed to enter clinical trials because of safety and efficiency problems. Therefore, improving their transfection efficiency, as well as their toxicological profile, is the subject of intensive research efforts. Six different poly(ethylene imine) (PEI)-based nanocarriers, with hydrophilic and hydrophobic PEG modifications, were toxicologically evaluated for pulmonary application in mice. Nanocarriers were intratracheally instilled to determine their toxicological profile, with particular focus on the inflammatory response in the lungs. Nanocarriers from both groups caused an acute inflammatory response in the lungs, albeit with different resolution kinetics and cytotoxicity. Hydrophobic modifications caused a severe inflammatory response with increased epithelial barrier permeability, accompanied by an acute antioxidant response. Hydrophilic modifications, with high PEG-grafting degrees, induced less proinflammatory effects without depleting macrophages and disrupting the epithelial/endothelial barrier in the lungs, while showing only a minor oxidative stress response. For pulmonary applications, local proinflammatory effects should be optimized by further development of nanocarriers with highly grafted PEG-PEI-based carriers or Jeffamine-modified hydrophobic PEI modifications.     

In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers

Complement activation by polymeric gene and drug delivery systems has been overlooked in the past. As more reports appear in the literature concerning immunogenicity of polymers and their impact on gene expression patterns, it is important to address possible immune side effects of polymers, namely complement activation. Therefore, in this study the activity of low and high molecular weight poly(ethylene imine) and two PEGylated derivatives to induce complement activation were investigated in human serum. These in vitro results revealed that PEI 25 kDa caused significant and concentration dependent complement activation, whereas none of the other polymers induced such effects at their IC(50) concentrations determined by MTT-assays. To verify these in vitro results, additionally, studies were carried out in a swine model after intravenous administration, showing complement activation-related pseudoallergy (CARPA), reflected in symptoms of transient cardiopulmonary distress. Injections of PEI 25 kDa or PEI(25k)-PEG(2k)(10) at a dose of 0.05 and 0.1 mg/kg caused strong reactivity, while PEI 5 kDa and with PEI(25k)-PEG(20k)(1) were also reactogenic at 0.1 mg/kg. It was found that PEI 25 kDa caused both self- and cross-tolerance, whereas the PEG-PEIs were neither self- nor cross-reactively tachyphylactic. As a result of this study, it was shown that PEGylation of polycations with PEG of 20 kDa or higher molecular weight may be favorable. However, potential safety concerns in the development of PEI-based polymeric carriers for drugs and nucleic acids and their translation from bench to bedside need to be taken into consideration for human application.     

Amphiphilic and biodegradable hy-PEI-g-PCL-b-PEG copolymers efficiently mediate transgene expression depending on their graft density

Novel biodegradable amphiphilic copolymers hy-PEI-g-PCL-b-PEG were prepared by grafting PCL-b-PEG chains onto hyper-branched poly(ethylene imine) as non-viral gene delivery vectors. Our investigations focused on the influence of graft densities of PCL-b-PEG chains on physico-chemical properties, DNA complexation and transfection efficiency. We found that the transfection efficiencies of these polymers increased at first towards an optimal graft density (n=3) and then decreased. The buffer-capacity-test showed almost exactly the same tendency as transfection efficiency. Cytotoxicity (MTT-assay) depended on the cooperation of PEG molecular weight and graft density of PCL-b-PEG chains. With increasing the graft density, cytotoxicity, zeta-potential, affinity with DNA, stability of the polyplexes and CMC-values were reduced strongly and regularly. Increasing the excess of polymer over DNA was shown to result in a decrease of the observed particle size to 100-200 nm.     

Conservative management of symptomatic Carpal Bossing in an elite hockey player: a case report

Objective:

To present the characteristics and create awareness of symptomatic carpal bossing and discuss potential etiologies and the role of conservative management through the presentation of an athlete with traumatic onset of symptomatic carpal bossing.

Clinical features:

This case report outlines the presentation and conservative management of an elite eighteen year old hockey player with symptomatic carpal bossing after a traumatic on ice collision. Carpal bossing is a bony, dorsal prominence in the quadrangular joint of the wrist that is inconsistently symptomatic.

Intervention and outcome:

A conservative treatment plan consisting of education, reassurance, avoidance of aggravation, and soft tissue therapy allowed return to play in two weeks without restrictions or need for surgical consultation.

Conclusion:

With inconsistent recurrence rates and surgical complications, the role of conservative management for symptomatic carpal bossing deserves further exploration. The conservative practitioner should be aware of the signs and symptoms of symptomatic carpal bossing to institute suitable treatment.     

An analysis of disease severity based on SMN2 copy number in adults with spinal muscular atrophy

To evaluate the effect of SMN2 copy number on disease severity in spinal muscular atrophy (SMA), we stratified 45 adult SMA patients based on SMN2 copy number (3 vs. 4 copies). Patients with 3 copies had an earlier age of onset and lower spinal muscular atrophy functional rating scale (SMAFRS) scores and were more likely to be non‐ambulatory. There was, however, no difference between the groups in quantitative muscle strength or pulmonary function testing. Functional scale may be a more discriminating outcome measure for SMA clinical trials. Muscle Nerve, 2009