Quantifying the average number of nucleic acid therapeutics per nanocarrier by single particle tracking microscopy

Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies

Dendritic cell vaccination as postremission treatment to prevent or delay relapse in acute myeloid leukemia

Relapse is a major problem in acute myeloid leukemia (AML) and adversely impacts survival. In this phase II study, we investigated the effect of vaccination with dendritic cells (DCs) electroporated with Wilms’ tumor 1 (WT1) mRNA as post-remission treatment in 30 AML patients at very high risk of relapse. There was a demonstrable anti-leukemic response in 13 patients. Nine patients achieved molecular remission as demonstrated by normalization of WT1 transcript levels, 5 of which are sustained after a median follow-up of 109.4 months. Disease stabilization was achieved in 4 other patients. Five-year overall survival (OS) was higher in responders than in non-responders (53.8% vs. 25.0%; P=0.01). In patients receiving DCs in first complete remission (CR1), there was a vaccine-induced relapse reduction rate of 25% and the 5-year relapse-free survival was higher in responders than in non-responders (50% vs. 7.7%; P65 years who received DCs in CR1, 5-year OS was 69.2% and 30.8% respectively, as compared to 51.7% and 18% in the Swedish Acute Leukemia Registry (SALR). Long-term clinical response was correlated with increased circulating frequencies of poly-epitope WT1-specific CD8+ T-cells. Long-term OS was correlated with interferon-γ+ and tumor necrosis factor-α+ WT1-specific responses in delayed type hypersensitivity-infiltrating CD8+ T-lymphocytes. In conclusion, vaccination of AML patients with WT1 mRNA-electroporated DCs can be an effective strategy to prevent or delay relapse after standard chemotherapy, translating into improved OS rates, which are correlated with the induction of WT1-specific CD8+ T-cell response. This trial was registered at www.clinicaltrials.gov as #NCT00965224

Assessment of hypovolaemia in the critically ill

Assessment of the intravascular volume status of patients is one of the most challenging tasks for the intensive care clinician. It is also one of the most important skills in intensive care management as both hypervolaemia and hypovolaemia lead to increased morbidity and mortality. The assessment of hypovolaemic patients is aided by several clinical signs, laboratory investigations, and a multitude of haemodynamic monitoring systems. This review aims to outline the definitions, pathophysiology, and various assessment techniques (both old and new) employed by intensivists on the critically ill patient

Biogenic silver for disinfection of water contaminated with viruses

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag-0. The antiviral action of biogenic Ag-0 was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter(-1) biogenic Ag-0 caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg(0)) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter(-1) ionic Ag+ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag-0 were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag-0 was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag-0 m(-2) on the filter (135 mg biogenic Ag-0 kg(-1) filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag-0 and its promising opportunities for continuous water disinfection

The black box revelation: monitoring gastrointestinal function

The gastrointestinal tract comprises diverse functions. Despite recent developments in technology and science,there is no single and universal tool to monitor GI function in intensive care unit (ICU) patients. Clinical evaluationis complex and has a low sensitivity to diagnose pathological processes in the abdomen. We performed a MEDLINEand Pubmed search connecting abdominal assessment and critical care. Based on these findings we defined the followingmajor categories of monitoring and diagnostic measures: clinical investigation; assessment of motility anddigestive function; microbiome monitoring; perfusion monitoring; laboratory biomarkers and hormonal function;intra-abdominal pressure measurement; and imaging techniques. Only a few of these monitoring and assessmenttools have found their way into clinical practice, as most of them have one or more significant objections preventingbroad implementation in daily clinical practice. Further research should be directed to reaffirm and define the useof current techniques to ascertain their validity and usefulness to monitor gastrointestinal function in ICU patients