Opening doors with ultrasound and microbubbles: Beating biological barriers to promote drug delivery.

Apart from its clinical use in imaging, ultrasound has been thoroughly investigated as a tool to enhance drug delivery in a wide variety of applications. Therapeutic ultrasound, as such or combined with cavitating nuclei or microbubbles, has been explored to cross or permeabilize different biological barriers. This ability to access otherwise impermeable tissues in the body makes the combination of ultrasound and therapeutics very appealing to enhance drug delivery in situ. This review gives an overview of the most important biological barriers that can be tackled using ultrasound and aims to provide insight on how ultrasound has shown to improve accessibility as well as the biggest hurdles. In addition, we discuss the clinical applicability of therapeutic ultrasound with respect to the main challenges that must be addressed to enable the further progression of therapeutic ultrasound towards an effective, safe and easy-to-use treatment tailored for drug delivery in patients.

Sonoprinting liposomes on tumor spheroids by microbubbles and ultrasound.

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed "sonoprinting". However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues.

Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia.

BACKGROUND: The role of protein phosphorylation in the Pasteur effect--the phenomenon whereby anaerobic conditions stimulate glycolysis--has not been addressed. The AMP-activated protein kinase (AMPK) is activated when the oxygen supply is restricted. AMPK acts as an energy-state sensor and inhibits key biosynthetic pathways, thus conserving ATP. Here, we studied whether AMPK is involved in the Pasteur effect in the heart by phosphorylating and activating 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. RESULTS: Heart PFK-2 was phosphorylated on Ser466 and activated by AMPK in vitro. In perfused rat hearts, anaerobic conditions or inhibitors of oxidative phosphorylation (oligomycin and antimycin) induced AMPK activation, which correlated with PFK-2 activation and with an increase in fructose 2,6-bisphosphate concentration. Moreover, in cultured cells transfected with heart PFK-2, oligomycin treatment resulted in a parallel activation of endogenous AMPK and PFK-2. In these cells, the activation of PFK-2 was due to the phosphorylation of Ser466. A dominant-negative construct of AMPK abolished the activation of endogenous and cotransfected AMPK, and prevented both the activation and phosphorylation of transfected PFK-2 by oligomycin. CONCLUSIONS: AMPK phosphorylates and activates heart PFK-2 in vitro and in intact cells. AMPK-mediated PFK-2 activation is likely to be involved in the stimulation of heart glycolysis during ischaemia.

Cyclic AMP suppresses the inhibition of glycolysis by alternative oxidizable substrates in the heart.

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.

[Biological tissue strain imaging with freehand elastography]. / Imagerie de la déformation des tissus biologiques par élastographie main-libre.

An imaging technique of deformation under load of a biological soft tissue, from numerical processing of radiofrequency ultrasound images is presented. The 2D locally regularized estimation method determines deformation parameters as the arguments that maximize a similarity criterion between a pre-compression region and its deformed version, compensated for according to these parameters. The technique was assessed with ultrasound data acquired during freehand scanning on two dedicated elastography phantoms as well as ex vivo bovine liver samples, containing artificial lesions made with agar gel. Although the load conditions are complex, elastograms are easy to interpret, exhibiting the inclusions with sharp boundaries.

A parametric imaging approach for the segmentation of ultrasound data.

When an ultrasonic examination is performed, a segmentation tool would often be very useful, either for the detection of pathologies, the early diagnosis of cancer or the follow-up of the lesions. Such a tool must be both reliable and accurate. However, because of the relatively reduced quality of ultrasound images due to the speckled texture, the segmentation of ultrasound data is a difficult task. We have previously proposed to tackle the problem using a multiresolution Bayesian region-based algorithm. For computation time purposes, a multiresolution version has been implemented. In order to improve the quality of the segmentation, we propose to perform the segmentation not only from the envelope image but to combine more information about the properties of the tissues in the segmentation process. Several acoustical parameters have thus been computed, either directly from the images or from the radio-frequency (RF) signal. In a previous study, two parametric images were involved in the segmentation process. The parameter represented the integrated backscatter (IBS) and the mean central frequency (MCF), which is a measurement related to the attenuation of ultrasound waves in the media. In this study, parameters representative of the scattering conditions in the tissue are evaluated in the multiparametric segmentation process. They are extracted from the K-distribution (alpha,b) and the Nakagami distribution (m,Omega) and are related to the local density of scatterers (alpha,m), the size of the scatterers (b) and the backscattering properties of the medium (Omega). The acoustical features are calculated locally on a sliding window. This procedure allows to built parametric mapping representing the particular characteristics of the medium. To test the influence of the acoustical parameters in the segmentation process, a set of numerical phantoms has been computed using the Field software developed by J.A. Jensen. Each phantom consists in two regions with two different acoustical properties: the density of scatterers and the scattering amplitude. From both the simulated RF signals and envelope images, the parameters have been computed; their relevance to represent a particular characteristic of the medium is evaluated. The segmentation has been processed for each phantom. The ability of each parameter to improve the segmentation results is validated. A agar-gel phantom has also been created, in order to test the accuracy of the parameters in conditions closer to the in vivo ultrasound imaging. This phantom contains four inclusions with different concentrations of silica. A B&K ultrasound device provides the RF data. The quantification of the segmentation quality is based on the rate of correctly classified pixels and it has been computed for all the parameters either from the field images or the phantom images. The large improvement in the segmentation results obtained reveals that the multiparametric segmentation scheme proposed in this study can be a reliable tool for the processing of noisy ultrasound data.

Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: benefits over classical vesicles.

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes [SPC:CHOL:PEG-750-DSPE (47:47:6 molar% ratio)] or pH-sensitive stealth liposomes [DOPE:CHEMS:CHOL:PEG(750)-DSPE (43:21:30:6 molar% ratio)] were used to deliver a therapeutic peptide to its nuclear site of action. The benefit of using stealth pH-sensitive liposomes was investigated and formulations were compared to classical liposomes in terms of size, shape, charge, encapsulation efficiency, stability and, most importantly, in terms of cellular uptake. Confocal microscopy and flow cytometry were used to evaluate the intracellular fate of liposomes themselves and of their hydrophilic encapsulated material. Cellular uptake of peptide-loaded liposomes was also investigated in three cell lines: Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was investigated as a function of time. Characterization studies showed that both formulations possess acceptable size, shape and encapsulation efficiency but cellular uptake studies showed the important benefit of the pH-sensitive formulation over the classical one, in spite of liposome PEGylation. Indeed, stealth pH-sensitive liposomes were able to deliver hydrophilic materials strongly to the cytoplasm. Most importantly, when encapsulated in pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells.

Which techniques to improve the early detection and prevention of pressure ulcers?

Pressure ulcers are a serious health problem for people with mobility disorders, like elders in acute care, long-term care, and home care settings. It also concerns paraplegics, tetraplegics or persons with burned injuries. Pressure ulcers result in significant morbidity and mortality. Consequences are a high human suffering, with high cost in terms of treatment. Several risk factors have been identified for the development of pressure ulcers: they are classified into extrinsic and intrinsic factors. Extrinsic factors include interface pressure, shear forces, friction. Intrinsic factors are the nutritional state of the patient, its age, diseases. There is little information about the mechanism of the formation of pressure sores but it is agreed that it is a complex process. The difficulty of the prevention lies in the evaluation of these factors. It is an essential stage to optimize the preventative measures. Actually, no quantifiable parameters exist to predict the formation of a pressure ulcer. This article is aimed to propose new techniques developed for the early detection of pressure ulcers. First, extrinsic parameters as the interface pressure and its consequences on the mobility are investigated. A new actimeter is presented to monitor the movements of the patient. The second part is dedicated to the presentation of a new imaging technique which can help the physician to control tissue elasticity of the patient. The technique is called elastography, it is a 3D strain estimation of soft biological tissues. Finally, the last way of investigation is the combination of extrinsic and intrinsic factors evaluation for a most relevant earlier diagnosis. Before the description of these techniques, it is essential to understand the phenomenology associated to the development of pressure sores. Only in this way, new techniques can be developed.

Excitation trapping and primary charge stabilization in Rhodopseudomonas viridis cells, measured electrically with picosecond resolution.

The transmembrane primary charge separation in the photosynthetic bacterium Rhodopseudomonas viridis was monitored by electric measurements of the light-gradient type [Trissl, H. W. & Kunze, U. (1985) Biochim. Biophys. Acta 806, 136-144]. Excitation of whole cells with 30-ps laser pulses at either 532 nm or 1064 nm gave rise to a biphasic increase of the photovoltage. The fast phase, contributing about 50% of the total, rose with an exponential time constant

[Value of arteriovenous oxygen difference in the therapeutic approach of post-traumatic intracranial hypertension]. / Intérêt de la différence atério-veineuse en O2 dans l'approache thérapeutique de l'hypertension intracranienne post-traumatique.

Elevated intracranial pressure (ICP) is undoubtedly a determinant factor of outcome in severe head injured patient. Until now, medical treatment of elevated ICP in diffuse brain lesions consisted of hyperventilation, CSF drainage, mannitol and barbiturates without accurate selection criteria. In fact, in comatose patients, the cerebral blood flow (CBF) can vary independently of the cerebral metabolic rate of oxygen (CMRO2) which is typically reduced. The venous oxygen saturation (SjO2) measured in the jugular bulb and the arterial-jugular oxygen difference (ajDO2) can be used for more appropriately selecting therapeutic measures. According to the following equation: CMRO2 = CBF x ajDO2, the measurement of the changes in ajDO2 might serve as an indicator of CBF adequacy, out of the presence of cerebral ischemia. A decreased ajDO2 suggests that CBF is excessive for cerebral metabolic requirements and an elevated ajDO2 indicates a decreased CBF. Consequently, treatment of elevated ICP in the presence of a low ajDO2 should be more oriented towards moderate hyperventilation and administration of metabolic depressive agents with maintenance of normal arterial blood pressure. Conversely, in case of high ajDO2, ICP control should aim to increase CBF by maintaining normocapnia, improving hemodynamic status or reducing extravascular volume accordingly.

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes.

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100-300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680(+) Phe(-)) and charge stabilization (formation of P-680(+) QA (-)), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced QA was about 30% of that measured with oxidized QA. The recombination time was 0.8-1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.

Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades.

To understand the insulin-induced activation of 6-phosphofructo-2-kinase (PFK-2) of the bifunctional enzyme PFK-2/fructose-2,6-bisphosphatase in heart, the effect of phosphorylation by protein kinases of the insulin signaling pathways on PFK-2 activity was studied. Purified PFK-2/fructose-2, 6-bisphosphatase from bovine heart is a mixture of two isoforms (Mr 58,000 and 54,000 on SDS-polyacrylamide gels). The Mr 54,000 protein is an alternatively spliced form, lacking phosphorylation sites for protein kinases. Recombinant enzymes corresponding to the Mr 58,000 (BH1) and Mr 54,000 (BH3) forms were expressed and used as substrates for phosphorylation. The recombinant BH1 isoform was phosphorylated by p70 ribosomal S6 kinase (p70(s6k)), mitogen-activated protein kinase-activated protein kinase-1, and protein kinase B (PKB), whereas the recombinant BH3 isoform was a poor substrate for these protein kinases. Treatment with all protein kinases activated PFK-2 in the recombinant BH1 preparation. Phosphorylation of the recombinant BH1 isoform correlated with PFK-2 activation and was reversed by treatment with protein phosphatase 2A. All the protein kinases phosphorylated Ser-466 and Ser-483 in the BH1 isoform, but to different extents: p70(s6k) preferentially phosphorylated Ser-466, whereas mitogen-activated protein kinase-activated protein kinase-1 and PKB phosphorylated Ser-466 and Ser-483 to a similar extent. We propose that PKB is part of the insulin signaling cascade for PFK-2 activation in heart.

Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.

Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase.

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC family. In addition, WISK contained no detectable phosphoinositide-dependent protein kinase-1 (PDK1) activity. WISK phosphorylated recombinant heart PFK-2 in a time-dependent manner to the extent of 0.4 mol of phosphate incorporated/mol of enzyme subunit, and increased the V(max) of PFK-2 twofold, without affecting the K(m) for fructose 6-phosphate. WISK phosphorylated Ser-466 to a greater extent than Ser-483 in recombinant heart PFK-2, and both sites were demonstrated to be phosphorylated to the same extent by PKB. Gel filtration and in-gel kinase analysis indicated that WISK was a monomer with a M(r) of 56500. Treatment of WISK with protein phosphatase 2A (PP2A) catalytic subunits reversed the effect of insulin, suggesting the involvement of an upstream activating kinase. Indeed, PDK1 was able to partially reactivate the PP2A-treated WISK and this reactivation was not enhanced by PtdIns(3,4,5)P(3)-containing vesicles. Moreover, a single 57000-M(r) band was labelled on incubation of the dephosphorylated WISK preparation with PDK1 and [gamma-(32)P]ATP. These findings provide evidence for the existence of a new protein kinase in the insulin signalling pathway, probably downstream of PDK1.

Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not protein kinase B.

Previous studies have shown that (i) the insulin-induced activation of heart 6-phosphofructo-2-kinase (PFK-2) is wortmannin-sensitive, but is insensitive to rapamycin, suggesting the involvement of phosphatidylinositol 3-kinase; and (ii) protein kinase B (PKB) activates PFK-2 in vitro by phosphorylating Ser-466 and Ser-483. In this work, we have studied the effects of phosphorylation of these residues on PFK-2 activity by replacing each or both residues with glutamate. Mutation of Ser-466 increased the V(max) of PFK-2, whereas mutation of Ser-483 decreased citrate inhibition. Mutation of both residues was required to decrease the K(m) for fructose 6-phosphate. We also studied the insulin-induced activation of heart PFK-2 in transfection experiments performed in human embryonic kidney 293 cells. Insulin activated transfected PFK-2 by phosphorylating Ser-466 and Ser-483. Kinase-dead (KD) PKB and KD 3-phosphoinositide-dependent kinase-1 (PDK-1) cotransfectants acted as dominant negatives because both prevented the insulin-induced activation of PKB as well as the inactivation of glycogen-synthase kinase-3, an established substrate of PKB. However, the insulin-induced activation of PFK-2 was prevented only by KD PDK-1, but not by KD PKB. These results indicate that the insulin-induced activation of heart PFK-2 is mediated by a PDK-1-activated protein kinase other than PKB.