pH-responsive polymeric micelle carriers for siRNA drugs.

The ability of small interfering RNA (siRNA) to efficiently silence the expression of specific genes provides the basis for exciting new therapies based on RNA interference (RNAi). The efficient intracellular delivery of siRNA from cell uptake through the endosomal trafficking pathways into the cytoplasm remains a significant challenge. Previously we described the synthesis of a new family of diblock copolymer siRNA carriers using controlled reversible addition-fragmentation chain transfer (RAFT) polymerization. The carriers were composed of a positively charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA binding and a second pH-responsive endosome releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios and butyl methacylate (BMA). Here we describe the development of a new generation of siRNA delivery polymers based on this design that exhibit enhanced transfection efficiency and low cytotoxicity. This design incorporates a longer endosomolytic block with increased hydrophobic content to induce micelle formation. These polymers spontaneously form spherical micelles in the size range of 40 nm with CMC (critical micelle concentration) values of approximately 2 µg/mL based on dynamic light scattering (DLS), (1)H NMR, electron microscopy, and selective partitioning of the small molecule pyrene into the hydrophobic micelle core. The siRNA binding to the cationic shell block did not perturb micelle stability or significantly increase particle size. The self-assembly of the diblock copolymers into particles was shown to provide a significant enhancement in mRNA knockdown at siRNA concentrations as low as 12.5 nM. Under these conditions, the micelle-based systems showed an 89% reduction in GAPDH mRNA levels as compared to only 23% (10 nM siRNA) for the nonmicelle system. The reduction in mRNA levels becomes nearly quantitative as the siRNA concentration is increased to 25 nM and higher. Flow cytometry analysis of fluorescent-labeled siRNA showed uptake in 90% of cells and a 3-fold increase in siRNA per cell compared to a standard lipid transfection agent. These results demonstrate the potential utility of this carrier design for siRNA drug delivery.

Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins.

The aggregation of integral membrane proteins (IMPs) in aqueous media is a significant concern for mechanistic investigations and pharmaceutical applications of this important class of proteins. Complexation of IMPs with amphiphiles, either detergents or short amphiphilic polymers known as amphipols (APols), renders IMPs water-soluble. It is common knowledge that IMP-detergent complexes are labile, while IMP-APol complexes are exceptionally stable and do not dissociate even under conditions of extreme dilution. To understand the thermodynamic origin of this difference in stability and to guide the design of new APols, we have studied by isothermal titration calorimetry (ITC) the heat exchanges during two reciprocal processes, the "trapping" of detergent-solubilized IMPs in APols and the "stripping" of IMP-APol complexes by detergents, using two IMPs (the transmembrane domain of porin OmpA from Escherichia coli and bacteriorhodopsin from Halobium salinarium), two APols [an anionic polymer derived from acrylic acid (A8-35) and a cationic phosphorylcholine-based polymer (C22-43)], and two neutral detergents [n-octyl thioglucoside (OTG) and n-octyltetraethylene glycol (C(8)E(4))]. In the presence of detergent, free APols and IMP-APol complexes form mixed particles, APol-detergent and IMP-APol-detergent, respectively, according to the regular mixing model. Diluting IMP-APol-detergent complexes below the critical micellar concentration (CMC) of the detergent triggers the dispersion of detergent molecules as monomers, a process characterized by an enthalpy of demicellization. The enthalpy of APol <--> detergent exchange on the hydrophobic surface of IMPs is negligibly small, an indication of the similarity of the molecular interactions of IMPs with the two types of amphiphiles. The enhanced stability against dilution of IMP-APol complexes, compared to IMP-detergent ones, originates from the difference in entropy gain achieved upon release in water of a few APol molecules (in the case of IMP-APol complexes) or several hundred detergent molecules (in the case of IMP-detergent complexes). The data account both for the stability of IMP-APols complexes in the absence of detergent and for the ease with which detergents displace APols from the surface of proteins.

Complexation of integral membrane proteins by phosphorylcholine-based amphipols.

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b(6)f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols ( approximately 22 kDa) of low dispersity in length, containing approximately 30 mol% octyl side groups, approximately 35 mol% PC-groups, and approximately 35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH> or =5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.

Enthalpy of interaction and binding isotherms of non-ionic surfactants onto micellar amphiphilic polymers (amphipols).

The interactions in water between short amphiphilic macromomolecules, known as amphipols, and three neutral surfactants (detergents), dodecylmaltoside (DM), n-octylthioglucoside (OTG), and n-octyltetraethyleneoxide (C8E4), have been assessed by static and dynamic light-scattering (SLS and DLS), capillary electrophoresis (CE), and isothermal titration calorimetry (ITC). The amphipols selected are random copolymers of the hydrophobic n-octylacrylamide (25-30 mol %), a charged hydrophilic monomer, either acrylic acid ( approximately 35 mol %) or a phosphorylcholine-modified acrylamide (40-70 mol %), and, optionally, N-isopropylacrylamide (30-40 mol %). In water, the copolymers form micelles of small size (hydrodynamic radius: approximately 5 nm). Neutral surfactants, below their critical micellar concentration (cmc), form mixed micelles with the amphipols irrespective of the chemical structure of the detergent or the polymer. The fraction of detergent in the surfactant/polymer complexes increases significantly (cooperatively) as the surfactant concentration nears the cmc. The ITC data, together with data gathered by CE, were fitted via a regular mixing model, which allowed us to predict the detergent concentration in equilibrium with complexes and the heat evolved upon transfer of detergent from water into a mixed surfactant/polymer complex. The enthalpy of transfer was found to be almost equal to the enthalpy of micellization, and the regular mixing model points to a near-ideal mixing behavior for all systems. Amphipols are promising tools in biochemistry where they are used, together with neutral surfactants, for the stabilization and handling of proteins. This study provides guidelines for the optimization of current protein purification protocols and for the formulations of surfactant/polymer systems used in pharmaceutics, cosmetics, and foodstuffs.

[What can be expected from off-pump coronary artery surgery?]. / Que faut-il attendre de la chirurgie coronaire sans circulation extracorporelle?

Coronary artery bypass surgery with beating heart (off-pump) has become more common in the last ten years allowing seven randomized studies with at least 60 patients, comparing off-pump and on-pump coronary bypass. Anaesthesia, monitoring and haemodynamic complications are described. Randomized studies concluded to less elevation of biochemical markers of myocardial and renal injury, less hydric inflation, less cerebral microemboli, reduction of homologous blood transfusions, of hospital stay and global costs. However in low risk patients no reduction in myocardial infarction, atrial fibrillation, stroke, acute renal failure, early reoperation, surgical site infection and mortality were observed. Non-randomized studies suggest a benefit in stroke and mortality in elderly patients but the possibility of incomplete revascularization remains.

[Severe digestive complications after heart surgery using extracorporeal circulation]. / Complications digestives sévères après chirurgie cardiaque sous circulation extracorporelle.

PURPOSE: To determine the incidence, circumstances of occurrence and evolution of gastrointestinal complications after cardiac surgery with extracorporeal circulation (ECC). METHODS: Retrospective chart study of gastrointestinal complications in 6.281 patients undergoing ECC between january 1994 and December 1997. RESULTS: Sixty patients developed 68 gastrointestinal complications (1%). Complications included: upper gastrointestinal bleeding (n = 23), intestinal ischemia (n = 19), cholecystitis (n = 7), pancreatitis (n = 6), and paralytic ileus (n = 16). The incidence of these complications was low after coronary artery (0.4%) or valvular surgery (0.8%) and high after cardiac transplantation (6%) and after surgery for acute aortic dissection (9%). Compared with a control population, patients with gastrointestinal complication had a higher Parsonnet score (29 +/- 15 vs 13 +/- 12 points; P = 0.002), were more frequently operated upon as an emergency (40/60, 66% vs 1120/6221, 18%; P = 0.01), underwent ECC of longer duration (114 +/- 66 vs 74 +/- 42 min; P = 0.01), and presented more frequently with low cardiac output after surgery (45/60, 75% vs 435/6221, 7%; P = 0.001). The mortality rate after gastrointestinal complications was 52%. The major factor associated with mortality was the occurrence of sepsis (OR = 38.7). Other factors were: renal failure (OR = 7.9), age > 75 yr (OR = 3.5), mechanical ventilation for more than seven days (OR = 2.7), associated cerebral damage (OR = 3.9). CONCLUSION: Gastrointestinal complications after ECC occur in high risk surgical patients. These complications are frequently associated with other complications leading to a high mortality rate.

[Reversible graft dysfunction after cardiac transplantation]. / Dysfonction réversible du greffon après transplantation cardiaque.

The authors report 3 cases of major graft dysfunction after cardiac transplantation which recovered completely with biventricular mechanical assistance in 4 to 8 days. All three cases were primary biventricular graft failures in patients with normal preoperative pulmonary resistances. These early dysfunctions (with no signs of myocardial infarction on electro- or echocardiography and in the absence of abnormal increased peri-operative enzyme levels) associated with total functional recovery conforming to the definition of the phenomenon of myocardial stunning. These results argue in favour of aggressive management of primary graft dysfunction.

[Use of the internal mammary artery as a free graft with reimplantation of the proximal end onto the ascending aorta. Apropos of 39 cases]. / Utilisation de l'artère mammaire interne comme greffon libre avec réimplantation de l'extrémité proximale sur l'aorte ascendante. A propos de trente-neuf cas.

From January 1987 to January 1991, 104 patients received bilateral internal mammary artery grafts and 39 of them had coronary bypass with a free graft implanted on the ascending aorta. There were 35 men, with a mean age of 57.35 years (range 41 to 70 years). 87% of them had stable angina, and 23 had preoperative myocardial infarction. The left ventricular function was good in 77 per cent of cases. 23 patients had three vessel disease (61.5%), 13 two vessel diseases (30.7%), 2 one vessel disease (5.12%) and one a left main coronary stenosis. Left internal mammary artery was used in two patients on the left descending artery and the right internal mammary artery was used in 37 patients: 15 on the circumflex, 15 on the right coronary, 4 on the LDA and 3 on the diagonal artery. Five patients had one graft, 32 two grafts and one three grafts (bilateral mammary and saphenous vein). Hospital mortality was 2.56% (1 patient) and there were 2 late deaths (5.12%). There were 5 perioperative myocardial infractions (12.8%) and no sternal infections. The mean follow up is 21 months (range 3 to 46 months). At follow-up, 34 patients (87.2%) were asymptomatic, and there were no myocardial infarctions. Postoperative angiography in 8 patients (mean postoperative time 2.5 months) showed that all the grafts were patent. This analysis demonstrates that free IMA graft has a low operative risk and provides excellent long term functional improvement and survival.