Walk the line: a systemic perspective on stress experienced by emergency medical personnel by comparing military and civilian prehospital settings.

Introduction: Emergency Medicine (EM) personnel in both military and civilian prehospital settings are often exposed to stressful and extreme events. Therefore, a cross-pollination between both contexts in terms of coping strategies may generate new information for purposes of training, prevention, and support programs. In the current study, we aimed at comparing both contexts to understand the type of stress events personnel experience; whether experience differs between civilian and military personnel; and how they cope with it. Methods: We used a mixed method approach, combining the results of a quantitative questionnaire and a thematic analysis of 23 in-depth semi-structured interviews to gain additional qualitative information. Results: Whereas the questionnaire pointed to a significant preference for task-oriented coping over avoidant and emotion-oriented coping, the interviews offered a more nuanced insight, showing a constant aim to position themselves on a continuum between emotional disconnection from the patient to preserve operationality on the one hand; and remaining enough empathic to preserve humanity on the other hand. We further identified an ambivalent awareness regarding emotions and stress, a vulnerable disbalance between an excessive passion for the job with the sacrifice of own's personal life (for a growing volatile and dangerous working environment) and a lack of recognition from both the patient and organizational environment. The combination of these factors may carry the risk for moral injury and compassion fatigue. Therefore, mutual trust between the organizational level and EM personnel as well as among team members is crucial. Discussion: The results are discussed from a systemic SHELL perspective, indicating how the specific profile of EM personnel relates to the software, hardware, environmental and liveware components of their professional and private life. Trainings on stress- and risk awareness should be approached both on an individual and systemic level, knowing that there is clearly no "one-size-fits-all" manner.

Liposome-gold nanorod hybrids for high-resolution visualization deep in tissues.

The design of liposome-nanoparticle hybrids offers a rich toolbox for the fabrication of multifunctional modalities. A self-assembled liposome-gold nanorod hybrid vesicular system that consists of lipid-bilayer-associated gold nanorods designed to allow deep tissue detection, therapy, and monitoring in living animals using multispectral optoacoustic tomography has been fabricated and characterized in vitro and in vivo.

Acid-triggered release via dePEGylation of fusogenic liposomes mediated by heterobifunctional phenyl-substituted vinyl ethers with tunable pH-sensitivity.

A new family of heterobifunctional phenyl-substituted vinyl ether (PIVE) coupling agents with tunable acid-sensitivity has been developed. The PIVE compounds are designed to hydrolyze under acidic conditions with hydrolysis rates that can be varied by rational selection of the phenyl ring substituent. These reagents were incorporated within 2-methoxypoly(ethylene glycol) PEG-conjugated 1,3-dioctadecyl-rac-glycerol lipids to produce the acid-cleavable lipopolymers mPEG-[H-PIVE]-DOG, mPEG-[F-PIVE]-DOG, mPEG-[Me-PIVE]-DOG, and mPEG-[MeO-PIVE]-DOG. These lipopolymers were hydrolyzed under acidic conditions (pH 3.5 or 4.5) at rates that were dependent on the electron donating or withdrawing character of the α-phenyl vinyl ether substituent, while remaining stable at pH 7.4. Blending of these compounds with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 10:90 mPEG-PIVE-Lipid:DOPE ratio produced stable liposomes at neutral pH; however, acidification of the solution led to dePEGylation and release of the liposomal cargo in a manner that correlated with the PIVE proton affinity. Specifically, we observed 70% calcein release within 12 h from mPEG-[MeO-PIVE]-DOG-containing liposomes at pH 4.5, whereas only 22% calcein release was observed from mPEG-[F-PIVE]-DOG:DOPE liposomes over this same time scale and pH. These results indicate that dePEGylation following acidification is a triggering mechanism that can be rationally designed and controlled through the appropriate selection of PIVE moieties.

Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery.

Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-ßGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. ß-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.

Chemical and biological mechanisms of pathogen reduction technologies.

Within the last decade new technologies have been developed and implemented which employ light, often in the presence of a photosensitizer, to inactivate pathogens that reside in human blood products for the purpose of transfusion. These pathogen reduction technologies attempt to find the proper balance between pathogen kill and cell quality. Each system utilizes various chemistries that not only impact which pathogens they can inactivate and how, but also how the treatments affect the plasma and cellular proteins and to what degree. This paper aims to present the various chemical mechanisms for pathogen reduction in transfusion medicine that are currently practiced or in development.

The effect of artificial lipid envelopment of Adenovirus 5 (Ad5) on liver de-targeting and hepatotoxicity.

Human Adenovirus type 5 (Ad5) has been extensively explored in clinical gene therapy, but its immunogenicity dramatically affects the kinetics and toxicity profile of the vector. We previously designed a variety of artificial lipid bilayer envelopes around the viral capsid to develop safer hybrid vectors. Here, we studied the interaction of enveloped Ad in cationic (DOTAP:Chol) or anionic (DOPE:CHEMS) lipid bilayers with different blood components. When Ad was enveloped by cationic lipids, significantly high levels of viral uptake in HepG2 cultured cells were achieved, independent of blood coagulation factors present. In vitro experiments also showed that artificial envelopment of Ad completely altered the affinity towards both human and murine red blood cells. After intravenous administration in BALB/c mice, real-time PCR and transgene expression studies indicated that cationic lipid envelopes significantly reduced hepatocyte transduction significantly increasing virus lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. ALT/AST serum levels and liver histology showed that envelopment also improved hepatotoxicity profiles compared to naked Ad. This study suggests that artificial envelopes for Ad significantly alter the interactions with blood components and can divert viral particles from their natural liver tropism resulting in reduced hepatotoxicity.

Autophagy and formation of tubulovesicular autophagosomes provide a barrier against nonviral gene delivery.

Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5 (-/-) cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors.

Purified graphene oxide dispersions lack in vitro cytotoxicity and in vivo pathogenicity.

Prompted by the excitement from the description of single layer graphene, increased attention for potential applications in the biomedical field has been recently placed on graphene oxide (GO). Determination of the opportunities and limitations that GO offers in biomedicine are particularly prone to inaccuracies due to wide variability in the preparation methodologies of GO material in different laboratories, that results in significant variation in the purity of the material and the yield of the oxidation reactions, primarily the Hummers method used. Herein, the fabrication of highly pure, colloidally stable, and evenly dispersed GO in physiologically-relevant aqueous buffers in comparison to conventional GO is investigated. The purified GO material is thoroughly characterized by a battery of techniques, and is shown to consist of single layer GO sheets of lateral dimensions below 500 nm. The cytotoxic impact of the GO in vitro and its inflammation profile in vivo is investigated. The purified GO prepared and characterized here does not induce significant cytotoxic responses in vitro, or inflammation and granuloma formation in vivo following intraperitoneal injection. This is one of the initial steps towards determination of the safety risks associated with GO material that may be interacting with living tissue.

pH-responsive biodegradable assemblies containing tunable phenyl-substituted vinyl ethers for use as efficient gene delivery vehicles.

Novel pH-responsive assemblies (PEG-lipid:DOPE liposomes) containing tunable and bifunctional phenyl-substituted vinyl ether (PIVE) cross-linkers were prepared. The assemblies consisted of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), acid-cleavable poly(ethylene glycol) (PEG)-conjugated lipids, pDNA, and protamine sulfate (PS). The PIVE linkage was designed to hydrolyze under acidic conditions, and the hydrolysis studies of PEG-lipid compounds containing PIVE at pH 4.2, 5.4, and 7.4 indicated that the hydrolysis rates of PIVE linker were influenced by the substitution of electron withdrawing or electron donating groups on the phenyl ring. Acid-catalyzed hydrolysis of PIVE leads to destabilization of the acid labile PEG-PIVE-lipid:DOPE liposomes via dePEGylation, thereby triggering content release. Content release assays showed that dePEGylation was highly pH-dependent and correlated with the PIVE proton affinity of the phenyl group. These results indicated that the dePEGylative triggering based on a new pH-sensitive PIVE linkage can be controlled. In vitro transfection studies on the pH-responsive assemblies containing mPEG-(MeO-PIVE)-conjugated 1,3-dioctadecyl-rac-glycerol lipids (mPEG-(MeO-PIVE])-DOG) showed higher transfection efficiency compared to that of polyethylenimine (PEI), a positive control, on HEK 293 and COS-7 cells. In addition, lower cytotoxicity of PEG-PIVE-lipid:DOPE liposomes/PS/DNA was observed in comparison to PEI. These results suggest that PEG-PIVE-lipid:DOPE liposomes can be considered as nonviral vehicles for drug and gene delivery applications.

Intracellular trafficking and gene expression of pH-sensitive, artificially enveloped adenoviruses in vitro and in vivo.

Recombinant adenovirus (Ad) has shown great promise in gene therapy. Artificial envelopment of adenovirus within lipid bilayers has previously been shown to decrease the immunogenicity and hepatic affinity of naked Ad in vivo. Unfortunately, this also resulted in a significant reduction of gene expression, which we attributed to poor endosomal release of the Ad from its artificial lipid envelope. In this work, we explored the artificial envelopment of Ad within pH-sensitive DOPE:CHEMS bilayers and characterized this vector by TEM, AFM, dot blot, dynamic light scattering and zeta potential measurements. The artificially enveloped viral vectors exhibited good stability at physiological pH but immediately collapsed and released naked Ad virions at pH 5.5. Intracellular trafficking using confocal laser scanning microscopy (CLSM) revealed that Cy3-labelled Ad enveloped in DOPE:CHEMS bilayers exhibited the characteristic Ad distribution within the cytoplasm that led to virion accumulation around the nuclear membrane, indicating endosomal release of Ad. We obtained equivalent levels of gene expression as those of naked Ad in a series of CAR-positive (CAR+) and CAR-negative (CAR-) cell lines. This suggested that the mechanism of infection for the artificially enveloped Ad remained dependent on the presence of CAR receptors. Finally, the pH-sensitive enveloped Ad were injected intratumorally in human cervical carcinoma xenograft-bearing nude mice, also illustrating their capacity for efficient in vivo marker gene expression. This study is a step forward toward the engineering of functional, artificially enveloped adenovirus vectors for gene transfer applications.

Efficient receptor-independent intracellular translocation of aptamers mediated by conjugation to carbon nanotubes.

We have covalently grafted aptamers onto carboxylated carbon nanotubes to design a novel vector system that can easily translocate into the cytosol of different cell types independent of receptor-mediated uptake. We propose the use of carbon nanotubes for the efficient intracellular delivery of biologically active aptamers for potential therapeutic applications.

Folate-mediated intracellular drug delivery increases the anticancer efficacy of nanoparticulate formulation of arsenic trioxide.

Arsenic trioxide (As(2)O(3)) is a frontline drug for treatment of acute promyelocytic leukemia and is in clinical trials for treatment of other malignancies, including multiple myeloma; however, efforts to expand clinical utility to solid tumors have been limited by toxicity. Nanoparticulate forms of As(2)O(3) encapsulated in 100-nm-scale, folate-targeted liposomes have been developed to lower systematic toxicity and provide a platform for targeting this agent. The resultant arsenic "nanobins" are stable under physiologic conditions but undergo triggered drug release when the pH is lowered to endosomal/lysosomal levels. Cellular uptake and antitumor efficacy of these arsenic liposomes have been evaluated in folate receptor (FR)-positive human nasopharyngeal (KB) and cervix (HeLa) cells, as well as FR-negative human breast (MCF-7) tumor cells through confocal microscopy, inductively coupled plasma mass spectroscopy, and cytotoxicity studies. Uptake of folate-targeted liposomal arsenic by KB cells was three to six times higher than that of free As(2)O(3) or nontargeted liposomal arsenic; the enhanced uptake occurs through folate-mediated endocytosis, leading to a 28-fold increase in cytotoxicity. In contrast, tumor cells with lower FR density on the surface (HeLa and MCF-7) showed much less uptake of the folate-targeted drug and lower efficacy. In cocultures of KB and MCF-7 cells, the folate-targeted arsenic liposomes were exclusively internalized by KB cells, showing high targeting specificity. Our studies further indicate that folate-targeted delivery of As(2)O(3) with coencapsulated nickel(II) ions (as a nontoxic adjuvant) potentiates the As(2)O(3) efficacy in relatively insensitive solid tumor-derived cells and holds the promise of improving drug therapeutic index.

Improved plasmalogen synthesis using organobarium intermediates.

An improved synthesis of plasmalogen type lipids is described. Transmetalation of lithioalkoxy allyl intermediates with BaI(2) and subsequent alkylation with 1-iodoalkanes enables the stereoselective formation of O-(Z)-alkenyl ether as precursors for the synthesis of plasmenyl- and bisplasmenylcholines. This method provides a simple and adaptable approach for the stereocontrolled synthesis of plasmenyl derivatives with variations at the sn-1, sn-2, and sn-3 positions of the glycerol backbone.

Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells.

This paper reports the creation of Au nanoparticles (AuNP) that are soluble in aqueous solution over a broad range of pH and ionic strength values and that are capable of selective uptake by folate receptor positive (FR+) cancer cells. A novel poly(ethylene glycol) (PEG) construct with thioctic acid and folic acid coupled on opposite ends of the polymer chain was synthesized for targeting the AuNP to FR+ tumor cells via receptor-mediated endocytosis. These folic acid-PEG-thioctic acid conjugates were grafted onto 10-nm-diameter Au particles in aqueous solution. The resulting folate-PEG-coated nanoparticles do not aggregate over a pH range of from 2 to 12 and at electrolyte concentrations of up to 0.5 M NaCl with particle concentrations as high as 1.5 x 10(13) particles/mL. Transmission electron microscopy was used to document the performance of these coated nanoparticles in cell culture. Selective uptake of folate-PEG grafted AuNPs by KB cells, a FR+ cell line that overexpress the folate receptor, was observed. AuNP uptake was minimal in cells that (1) do not overexpress the folate receptor, (2) were exposed to AuNP lacking the folate-PEG conjugate, or (3) were co-incubated with free folic acid in large excess relative to the folate-PEG grafted AuNP. Understanding this process is an important step in the development of methods that use targeted metal nanoparticles for tumor imaging and ablation.