Development of Cationic Lipid LAH4-L1 siRNA Complexes for Focused Ultrasound Enhanced Tumor Uptake.

RNAi has considerable potential as a cancer therapeutic approach, but effective and efficient delivery of short interfering RNA (siRNA) to tumors remains a major hurdle. It remains a challenge to prepare a functional siRNA complex, target enough dose to the tumor, and stimulate its internalization into tumor cells and its release to the cytoplasm. Here, we show how these key barriers to siRNA delivery can be overcome with a complex─comprising siRNA, cationic lipids, and pH-responsive peptides─that is suited to tumor uptake enhancement via focused ultrasound (FUS). The complex provides effective nucleic acid encapsulation, nuclease protection, and endosomal escape such that gene silencing in cells is substantially more effective than that obtained with either equivalent lipoplexes or commercial reagents. In mice bearing MDA-MB-231 breast cancer xenografts, both lipid and ternary, lipid:peptide:siRNA complexes, prepared with near-infrared fluorescently labeled siRNA, accumulate in tumors following FUS treatments. Therefore, combining a well-designed lipid:peptide:siRNA complex with FUS tumor treatments is a promising route to achieve robust in vivo gene delivery.

Image-guided thermosensitive liposomes for focused ultrasound drug delivery: Using NIRF-labelled lipids and topotecan to visualise the effects of hyperthermia in tumours.

Image guided drug delivery using imageable thermosensitive liposomes (iTSLs) and high intensity focused ultrasound (FUS or HIFU) has attracted interest as a novel and non-invasive route to targeted delivery of anti-cancer therapeutics. FUS-induced hyperthermia is used as an externally applied "trigger" for the release of a drug cargo from within thermosensitive drug carriers. It is suggested that sub-ablative hyperthermia significantly modifies the permeability of tumour vasculature and enhances nanoparticle uptake. Here we describe the preparation and use of magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF) labelled thermosensitive liposomes for imaging and tracking of biodistribution and drug release in a murine cancer model. We prepared iTSLs to encapsulate topotecan (Hycamtin®), a chemotherapeutic agent which when released in tumours can be monitored by an increase in its intrinsic drug fluorescence. FUS was applied using feedback via subcutaneously placed fine-wire thermocouples to maintain and monitor hyperthermic temperatures. iTSL accumulation was detected within tumours using NIRF imaging immediately after liposome administration. Mild FUS-induced hyperthermia (3 min at 42 °C, 30 min post i.v. administration) greatly enhanced iTSLs uptake. A co-localised enhancement of topotecan fluorescence emission was also observed immediately after application of FUS indicating rapid triggered drug release. The phenomena of increased iTSL accumulation and concomitant topotecan release appeared to be amplified by a second mild hyperthermia treatment applied one hour after the first. MRI in vivo also confirmed enhanced iTSLs uptake due to the FUS treatments. Our imaging results indicate the effects of hyperthermia on the uptake of carriers and drug. FUS-induced hyperthermia combined with real time imaging could be used as a tool for tumour targeted drug delivery.

Focused ultrasound induced hyperthermia accelerates and increases the uptake of anti-HER-2 antibodies in a xenograft model.

Image guided drug delivery has gained significant attention during the last few years. Labelling nanoparticles or macromolecules and monitoring their fate in the body provides information that can be used to modulate their biodistribution and improve their pharmacokinetics. In this study we label antibodies and monitor their distribution in the tumours post intravenous injection. Using Focused Ultrasound (FUS, a non-invasive method of hyperthermia) we increase the tumour temperature to 42°C for a short period of time (3-5min) and we observe an increased accumulation of labelled antibody. Repetition of focused ultrasound induced hyperthermic treatment increased still further the accumulation of the antibodies in the tumour. This treatment also augmented the accumulation of other macromolecules non-specific to the tumour, such as IgG and albumin. These effects may be used to enhance the therapeutic efficiency of antibodies and/or targeted nanoparticles.

Nitric oxide-dependent bradycardia in mutant analbuminemic rats.

Nagase analbuminemic rats (NAR) are natural mutant Sprague-Dawley rats which do not express albumin due to a single splice mutation in the albumin gene. We accidentally discovered that NAR have a significant bradycardia compared with wild type Sprague-Dawley rats, and the present study was carried out to investigate the mechanism of bradycardia in analbuminemic rats. In vitro studies showed that the basal spontaneous beating rate of isolated atria is similar in NAR compared with wild type animals. However, the chronotropic responsiveness of isolated atria to cholinergic stimulation was markedly increased in NAR, an effect which was prevented by incubation with a nitric oxide synthase (NOS) or guanylyl cyclase inhibitor. NAR had a significant increase in plasma nitrite/nitrate concentrations. Administration of a NOS inhibitor for 5 days normalized heart rate in NAR. The level of NOS isoforms, caveolin-1 and caveolin-3 expression in the atria was assessed by real time PCR. There was no significant difference in the expression of NOS isoforms or caveolin-3 in NAR compared with wild type controls. However, NAR exhibited a significant decrease in caveolin-1 expression in the atria. Since caveolin-1 is known to inhibit endothelial NOS activity in cardiomyocytes, we suggest that decreased caveolin-1 levels may have a role in increased nitric oxide production in NAR. Our data suggest that a NOS/cGMP-dependent mechanism might be involved in increased responsiveness to vagal stimulation and bradycardia in analbuminemic condition.

Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo.

The endothelial protein C receptor (EPCR) plays an anticoagulant role by improving protein C activation. Although low levels of activated protein C (APC) constitute a thrombosis risk factor, the relationship between modulating EPCR function and thrombosis has not been addressed so far. Monoclonal antibodies (mAb) against murine EPCR were raised, and their ability to block protein C/APC binding was tested. The ferric chloride carotid artery injury model in mice was chosen to test the effect of anti-EPCR mAb on thrombus formation. The time to total occlusion of the vessel was analysed in three groups, given an isotype control mAb (IC), a blocking (RCR-16) or a non-blocking (RCR-20) anti-EPCR mAb. RCR-16 prevented the interaction between protein C/APC and EPCR as demonstrated by surface plasmon resonance and flow cytometry, and inhibited the activation of protein C on the endothelium. IC and RCR-20 were unable to induce such effects. In vivo , RCR-16 shortened the time to total vessel occlusion with respect to IC [13.4 +/- 1.0 (mean +/- SD) and 17.8 +/- 3.2 minutes, respectively, p<0.001]. Occlusive thrombi lasting for more than one hour were observed in all RCR-16-treated animals, but only in 43% of IC-treated ones. Results with RCR-20 were indistinguishable from those observed with IC. For the first time, a direct relationship between blocking EPCR and thrombosis is demonstrated. Blocking anti-EPCR autoantibodies can predispose to thrombosis episodes and may constitute a new therapeutic target.

Influence of the chitosan nature on the transfection efficacy of DNA-loaded nanoparticles after hydrodynamic administration in mice.

A commercially available chitosan with a degree of deacetylation (DD) of 85% and a molecular weight (Mw) of 400 kDa was modified by acetylation with acetic anhydride to obtain a chitosan with a DD of 75%. Both polysaccharides were used to prepare DNA-chitosan nanoparticles by charge interactions with pDNA (coacervation process). Both resulting nanoparticles showed an almost total DNA loading efficiency (96%) and displayed similar physico-chemical properties with a size of approximately 200 nm and a zeta potential close to +20 mV. In order to study the effect of the DD on the properties of DNA-chitosan nanoparticles as gene delivery systems, the hydrodynamics-based procedure was used. The transgene expression was observed using either the green fluorescent protein (GFP) or the luciferase (Luc) as reporter genes. After the hydrodynamic injection, the DNA-chitosan nanoparticles were accumulated in the liver, where the transgene expression was mostly localized. Interestingly, the decrease of the DD affected the transgene expression, improving the initial burst effect and accelerating the DNA release. Both combined effects led to an increase in the transgene expression levels. In addition, the emitted bioluminescence could be detected over 105 days for all the formulations injected. The calculation of the kinetic parameters (C(max), AUC, Ke, t(1/2) Ke and MET) gave some interesting information regarding the abilities to control the DNA release of the two DNA-chitosan formulations tested and allowed narrower comparisons.

Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: a review.

Hydrogen sulfide is rapidly emerging as an important vasoactive mediator formed in health and disease. Its biological action is centered on its reactivity with heme-proteins and its ability to activate K(ATP) channels. Hydrogen sulfide is a signalling molecule of the inflammatory and nervous systems, and in particular the cardiovascular system where it regulates vascular tone, cardiac work, and exerts cardioprotection. This has led to an explosion of papers in which the role of hydrogen sulfide generated in vitro has been used to stimulate biological responses, and where a variety of methods have been used to measure the concentration of this compound in biological fluids. Understanding the chemistry and the inherent problems in the analytical techniques used to measure hydrogen sulfide concentrations is critical to our expanding knowledge on the biology of hydrogen sulfide. In this brief review we will cover the chemistry of hydrogen sulfide, including sources of hydrogen sulfide, its speciation at physiological pH, the susceptibility of sulfide to aerobic oxidation, and the methods used to measure hydrogen sulfide concentrations in solution, including biological fluids. We also give a brief overview of knockout animals and inhibition of the enzymes involved in the formation of hydrogen sulfide in vivo.

Development of bioadhesive amino-pegylated poly(anhydride) nanoparticles designed for oral DNA delivery.

Amino-pegylated poly(methyl vinyl ether-co-maleic anhydride) nanoparticles were prepared applying a solvent displacement method. The surface charge of the resulting pegylated particles was considerably higher (-2.7 mV) than that of the non-pegylated (-33.5 mV). After oral administration to rats the amino-pegylated nanoparticles exhibited great ability for bioadhesive interactions with the gastrointestinal mucosa. Furthermore, fluorescence microscopy revealed that the amino-pegylated were able to cross the cellular membrane of the absorptive enterocytes. Genomic salmon testes DNA was associated to the amino-pegylated poly(anhydride) particles by applying two procedures: (i) incubation of aqueous DNA solution with the freshly formed amino-pegylated particles; and (ii) initial incubation of DNA and DAP-PEG simultaneously, followed by blending with preformed non-pegylated particles. Gel electrophoresis showed that both methods were safe and DNA integrity was not affected. Based on the results describing their adhesive properties and intracellular transport, the amino-pegylated nanoparticles were considered as a suitable carrier for DNA.

New methodologies to characterize the effectiveness of the gene transfer mediated by DNA-chitosan nanoparticles.

In this work three DNA-chitosan nanoparticle formulations (Np), differing in the molecular weight (MW; 150 kDa, 400 kDa, and 600 kDa) of the polysaccharide, were prepared and administered by two different administration routes: the hydrodynamics-based procedure and the intraduodenal injection. After the hydrodynamic injection, DNA-chitosan nanoparticles were predominantly accumulated in the liver, where the transgene was expressed during at least 105 days. No significant influence of MW was observed on the levels of luciferase expression. The curves of bioluminescence versus time obtained using the charge-coupled device (CCD) camera were described and divided in three phases: (i) the initial phase, (ii) the sustained release step and (iii) the decline phase (promotor inactivation, immunological and physiological processes). From these curves, which describe the transgene expression profile, the behavior of the different formulations as gene delivery systems was characterized. Therefore, the following parameters such as C(max) (maximum level of detected bioluminescence), AUC (area under the bioluminescence-time curve) and MET (mean time of the transgene expression) were calculated. This approach offers the possibility of studying and comparing transgene expression kinetics among a wide variety of gene delivery systems. Finally, the intraduodenal administration of naked DNA permitted the gene transfer in a dose dependent manner quantifiable with the CCD camera within 3 days. Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA. This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.