Eric Allémann

In vitro sonothrombolysis of human blood clots with BR38 microbubbles.

Microbubble-mediated sonothrombolysis is a promising approach for ischemic stroke treatment. The aim of this in vitro study was to evaluate a new microbubble (MB) formulation (BR38) for sonothrombolysis and to investigate the involved mechanisms. Human whole-blood clots were exposed to different combinations of recombinant tissue plasminogen activator (rtPA), ultrasound (US) and MB. Ultrasound at 1.6 MHz was used at 150, 300, 600 and 1000 kPa (peak-negative pressure). Thrombolysis efficacy was assessed by measuring clot diameter changes during 60-min US exposure. The rate of clot diameter loss (RDL) in μm/min was determined and clot lysis profiles were analyzed. The most efficient clot lysis (5.9 μm/min) was obtained at acoustic pressures of 600 and 1000 kPa in combination with MB and a low concentration of rtPA (0.3 μg/mL). This is comparable with the rate obtained with rtPA at 3 μg/mL alone (6.6 μm/min, p > 0.05). Clot lysis profiles were shown to be related to US beam profiles and microbubble cavitation.

Sonothrombolysis: the contribution of stable and inertial cavitation to clot lysis.

Microbubble-mediated sonothrombolysis (STL) is a remarkable approach to vascular occlusion therapy. However, STL remains a complex process with multiple interactions between clot, ultrasound (US), microbubbles (MB) and thrombolytic drug. The aim of this study was to evaluate the ability of combining US and MB to degrade fibrin and, more specifically, to assess the roles of both stable (SC) and inertial (IC) cavitation. Human blood clots containing radiolabeled fibrin were exposed to different combinations of recombinant tissue plasminogen activator (rtPA), US (1 MHz) and phospholipid MB. Three acoustic pressures were tested: 200, 350 and 1,300 kPa (peak-negative pressure). Clot lysis was assessed by diameter loss and release of radioactive fibrin degradation products. The combination rtPA + US + MB clearly revealed that IC (1,300 kPa) was able to enhance fibrin degradation significantly (66.3 ± 1.8%) compared with rtPA alone (51.7 ± 2.0%, p < 0.001). However, SC failed to enhance fibrin degradation at an acoustic pressure of 200 kPa. At 350 kPa, a synergistic effect between rtPA and US + MB was observed with an absolute increase of 6% compared to rtPA alone (p < 0.001). Conversely, without rtPA, the combination of US + MB was unable to degrade the fibrin network (0.3 ± 0.1%, p > 0.05 vs. control), but induced a distinct loss of red blood cells throughout the entire thickness of the clot, implying that MB were able to penetrate and cavitate inside the clot.

Gd-nanoparticles functionalization with specific peptides for ß-amyloid plaques targeting.

BackgroundAmyloidoses are characterized by the extracellular deposition of insoluble fibrillar proteinaceous aggregates highly organized into cross-β structure and referred to as amyloid fibrils. Nowadays, the diagnosis of these diseases remains tedious and involves multiple examinations while an early and accurate protein typing is crucial for the patients’ treatment. Routinely used neuroimaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) using Pittsburgh compound B, [11C]PIB, provide structural information and allow to assess the amyloid burden, respectively, but cannot discriminate between different amyloid deposits. Therefore, the availability of efficient multimodal imaging nanoparticles targeting specific amyloid fibrils would provide a minimally-invasive imaging tool useful for amyloidoses typing and early diagnosis. In the present study, we have functionalized gadolinium-based MRI nanoparticles (AGuIX) with peptides highly specific for Aβ amyloid fibrils, LPFFD and KLVFF. The capacity of such nanoparticles grafted with peptide to discriminate among different amyloid proteins, was tested with Aβ(1–42) fibrils and with mutated-(V30M) transthyretin (TTR) fibrils.ResultsThe results of surface plasmon resonance studies showed that both functionalized nanoparticles interact with Aβ(1–42) fibrils with equilibrium dissociation constant (Kd) values of 403 and 350xa0µM respectively, whilst they did not interact with V30M-TTR fibrils. Similar experiments, performed with PIB, displayed an interaction both with Aβ(1–42) fibrils and V30M-TTR fibrils, with Kd values of 6 and 10xa0µM respectively, confirming this agent as a general amyloid fibril marker. Thereafter, the ability of functionalized nanoparticle to target and bind selectively Aβ aggregates was further investigated by immunohistochemistry on AD like-neuropathology brain tissue. Pictures clearly indicated that KLVFF-grafted or LPFFD-grafted to AGuIX nanoparticle recognized and bound the Aβ amyloid plaque localized in the mouse hippocampus.ConclusionThese results constitute a first step for considering these functionalized nanoparticles as a valuable multimodal imaging tool to selectively discriminate and diagnose amyloidoses.

Perivascular sustained release of atorvastatin from a hydrogel-microparticle delivery system decreases intimal hyperplasia

Intimal hyperplasia (IH) is the major cause of grafted vessel occlusion and occurs frequently after bypass intervention. No pharmaceutical formulation is currently available to prevent this pathology. Local perivascular delivery of an appropriate active compound released in a time-dependent manner (from day one up to 4weeks) is necessary for an efficient single-administration preventive therapy. To this aim, we propose the combination of gel and microparticles delivery system containing atorvastatin (ATV). The incorporation of ATV in a cross-linked hyaluronic acid gel, provided in vitro a fast release over 3days, while ATV-loaded poly-lactic-co-glycolic acid (PLGA) microparticles dispersed in the gel gave a sustained release over 4weeks. In vivo, ATV formulations were applied perivascularly in mice undergoing carotid artery ligation. IH was significantly reduced (-68%) in presence of ATV incorporated in hyaluronic acid gel and encapsulated in microparticles compared to control. No significant IH alteration was observed when ATV was incorporated only in the gel (fast release) or only in the microparticles (slow release) demonstrating that a biphasic release of ATV is essential to interfere with the development of IH. ATV was detected in adjacent tissues 28days after the intervention, showing the sustained presence of the drug in vivo. After four weeks ATV was not detected in remote tissues, except at a very low concentration (0.044ng/mg) in the liver, suggesting a very low risk of systemic toxicity of locally delivered ATV. Additionally, the ex vivo data showed that ATV in solution permeates through isolated human saphenous veins and thus is a good candidate for perivascular delivery. Our data demonstrate that a local biphasic ATV release on the mice ligated carotid efficiently prevents the development of IH without apparent toxicity.

Effect of particle size on the biodistribution of nano- and microparticles following intra-articular injection in mice

Intra-articular (IA) injection of extended drug release forms based on biodegradable microparticles holds promise for the treatment of joint diseases. However, the fate of microparticles following intra-articular injection is controversial and has not been thoroughly investigated. The aim of this work was therefore to evaluate the biodistribution of fluorescent poly(lactic acid) particles of different sizes after IA injection in arthritic or healthy mice. Regardless of the inflammatory status of the joint, 300 nm-nanoparticles leaked from the joint. Due to inflammation and related increase of vascular permeability, 3 μm-microparticles that were retained in the non-inflamed synovial membrane leaked from the inflamed joint. Complete retention of 10 μm-microparticles was observed independently of the joint inflammatory status. Embedding particles in a hyaluronic acid gel prolonged the retention of the formulations only in inflamed joints. Depending on particles size, formulations were preferentially eliminated by blood vessels or lymphatic pathways. Poly(lactic acid) particles of 3 μm were biocompatible and retained in knee joints at least for 6 weeks. This work highlights the need to deliver hyaluronic acid-embedded particles of at least 3 μm to guarantee their retention in inflamed joints. These results will contribute to the rational design of long-lasting formulations to treat acute and chronic joint diseases.

Tunable phosphatase-sensitive stable prodrugs of 5-aminolevulinic acid for tumor fluorescence photodetection.

5-Aminolevulinic acid (5-ALA) has been at the forefront of small molecule based fluorescence-guided tumor resection and photodynamic therapy. 5-ALA and two of its esters received marketing authorization but suffer from several major limitations, namely low stability and poor pharmacokinetic profile. Here, we present a new class of 5-ALA derivatives aiming at the stabilization of 5-ALA by incorporating a phosphatase sensitive group, with or without self-cleavable linker. Compared to 5-ALA hexyl ester (5-ALA-Hex), these compounds display an excellent stability under acidic, basic and physiological conditions. The activation and conversion into the 5-ALA is controlled and can be structure-tailored. The prodrugs display reduced acute toxicity compared to 5-ALA-Hex with superior dose response profiles of protoporphyrin IX synthesis and fluorescence intensity in human glioblastoma cells in vitro. Clinically relevant fluorescence kinetics in vivo shown in U87MG glioblastoma spheroid tumor model in chick embryos provide a solid basis for their further development and translation to clinical fluorescence guided tumor resection and photodynamic therapy.

Microbubbles and ultrasound-mediated thrombolysis: a review of recent in vitro studies

Current treatment of acute ischemic stroke with recombinant tissue plasminogen activator (rtPA) is limited to a small percentage of patients due to exclusion criteria and side effects. Sonothrombolysis (ultrasound-mediated thrombolysis) is a new treatment strategy shown to be effective for clot lysis, particularly when combined with ultrasound contrast agents (UCAs). The present review surveys recent in vitro studies of sonothrombolysis (STL). Different UCAs and STL approaches are presented and, in particular, the involved mechanisms are discussed. Acoustic cavitations generate microstreaming and microjets at the clot surface leading to direct (clot damage) and/or indirect (enhancement of thrombolytic drug effect) clot destruction. Microbubble-based UCAs have been shown to greatly potentiate cavitation effects and reduce the required acoustic pressure. However, STL remains a complex process with multiple interactions between blood clots, ultrasound, UCAs and thrombolytic drugs. All these parameters have to be optimized with regard to efficacy and safety.

Intra-articular bioactivity of a p38 MAPK inhibitor and development of an extended-release system.

In the treatment of arthritic diseases, oral or systemic administration of anti-inflammatory substances, such as p38 MAPK inhibitors, is hampered by numerous side effects. To overcome them, formulations of rapid and extended drug delivery systems were studied in intra-articular administration. For the first time, VX-745, a highly selective p38 MAPK inhibitor, demonstrated in vivo bioactivity, similar to dexamethasone activity, following intra-articular administration in an antigen-induced arthritic (AIA) mouse model. The in vitro bioactivity of VX-745 was also shown on synoviocytes, reducing the IL-6 concentration. Process and formulation parameters (i.e., polymer concentration, aqueous/organic phase ratio, emulsification speed and process, and evaporation pressure) and particle characterisation (i.e., drug loading, size of particle, and surface aspect) were extensively examined to produce optimised formulations. Indeed, a burst release provides a rapid saturation of intracellular p38 MAPK to relieve patients from pain and inflammation. Then, drug diffusion would be sufficient to maintain an effective dose over 2-3 months. This study confirms the effectiveness of encapsulated p38 MAPK inhibitors in extended drug delivery systems and seems to be a promising strategy for intra-articular treatment.

Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting.

AIMnGadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses.nnnMATERIALS & METHODSnThe ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy.nnnRESULTSnNanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with KD values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues.nnnCONCLUSIONnSuch functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimers disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy.

Activity of phosphatase-sensitive 5-aminolevulinic acid prodrugs in cancer cell lines

5-aminolevulinc acid (5-ALA)-based photodynamic therapy (PDT) and photodiagnosis (PD) present many advantages over treatments with conventional photosensitizers (PS). It offers great tumor specificity, reduced photosensitivity reactions caused by PS accumulation in non-targeted tissues and also inherent PS metabolism into endogenous non-fluorescent heme. However, chemical instability, low bioavailability and poor pharmacokinetic profile limit systemic efficacy of 5-ALA. Here, we present a comprehensive in vitro evaluation of novel phosphatase-sensitive prodrugs of 5-ALA. These prodrugs are designed to be activated by ubiquitously expressed phosphatases with much improved chemical stability and reduced acute toxicity profile. PpIX kinetic measurements and flow cytometry show accumulation of PpIX upon incubation with phosphatase-sensitive prodrugs in PC3 human prostate cell cancer, MCF7 breast adenocarcinoma, U87MG glioblastoma, T24 bladder cancer and A549 lung carcinoma cells. They revealed a different fluorescence kinetics and dose-response curves for the different types of 5-ALA prodrugs. These experiments have allowed us to identify the most promising cancer cell types for phospho- 5-ALA prodrugs. Confocal fluorescence microscopy provided further evidence of fluorescent protoporphyrin IX accumulation and sub-cellular localisation. These findings, together with the low toxicity profile of phosphatase-sensitive prodrugs of 5-ALA and good response to PDT provide solid basis for future translational development in PC3, MCF7 and U87MG cancer types.