In vitro and in vivo evaluation of superparamagnetic iron oxide nanoparticles coated by bisphosphonates: the effects of electrical charge and molecule length.

Physicochemical coating properties are often considered to be determining factors for in vivo characteristics of superparamagnetic iron oxide nanoparticles, used as contrast agent in Magnetic Resonance Imaging (MRI). To investigate the electrical charge (modified by zero, one or two ammonium groups) and the molecule length (3, 5 or 7 methylene chains) effects of bisphosphonate-type coatings, we assessed the complement activation, in vivo plasma and tissue relaxation time alterations of intravenously injected small iron oxide nanoparticles (<25 nm) on male healthy Wistar rats. The presence of ammonium groups induces a weak activation of the complement whatever the size and the concentration of particles, whereas hydroxyethylenebisphosphonate (HEBP)-coated particles are poor complement activators only at the lowest concentration. In vivo, HEBP-coated nanoparticles have the greatest prolonged relaxation time effects, despite their higher negative electrical charge, contrary to two ammonium bearing coatings. No significant differences were observed between mono-ammonium molecular coatings.

A starch-based microparticulate system dedicated to diagnostic and therapeutic nuclear medicine applications.

The aim of this work was to develop a new microparticulate system able to form a complex with radionuclides with a high yield of purity for diagnostic or therapeutic applications. Owing to its properties potato starch was chosen as starting material and modified by oxidization and coupling of a ligand (polyamine) enabling modified starch to chelate radionuclides. The choice of suitable experiments was based on a combination of a Rechtschaffner experimental design and a surface response design to determine the influence of experimental parameters and to optimize the final product. Starch-based microparticle formulations from the experimental plans were compared and characterized through particle size analysis, scanning electron microscopy, elemental analysis and, for the most promising formulations, by in vitro labeling stability studies and determination of free polyamine content or in vivo imaging studies. The mechanism of starch-based microparticle degradation was identified by means of size measurements. The results of the Rechtschaffner design showed the positive qualitative effect of the temperature and the duration of coupling reaction whereas surface response analysis clearly showed that, by increasing the oxidization level and starch concentration, the nitrogen content in the final product is increased. In vitro and in vivo characterization led to identification of the best formulation. With a size around 30 µm, high radiochemical purity (over 95%) and a high signal-to-noise ratio (over 600), the new starch-based microparticulate system could be prepared as ready-to-use kits and sterilized without modification of its characteristics, and thus meet the requirement for in vivo diagnostic and therapeutic applications.

Volumetric assessment of myocardial viability in rats using 3D double contrast enhanced T1 and T2-weighted MRI.

OBJECTIVE: Volumetric evaluation of the myocardial viability post-infarction in rats using 3D in vivo MR imaging at 7 T using injection of an extracellular paramagnetic contrast agent and intravascular superparamagnetic iron oxide nanoparticles in the same imaging session. MATERIALS AND METHODS: Five hours after induction of permanent myocardial infarction in rats (n=6), 3D in vivo T1- and T2-weighted MR Imaging was performed prior to and after Gd-DOTA injection (0.2 mmol/kg) and prior to and after nanoparticle injection (5 mg Fe/kg) to assess infarct size and myocardial viability. RESULTS: 3D MR Imaging using a successive contrast agent injection showed a difference of infarct size after Gd-DOTA injection on T1-weighted images compared to the one measured on T2-weighted images after Gd-DOTA and nanoparticle injection. CONCLUSION: The use of 3D T1- and T2-weighted MR Imaging using a double contrast agents protocol made possible the accurate characterization of myocardial infarction volume and allowed the detection of myocardial viability post-infarction in rats.

Description and technical pitfalls of a hepatoma model and of intra-arterial injection of radiolabelled lipiodol in the rat.

Intra-arterial metabolic radiotherapy (using lipiodol labelled with iodine-131 or rhenium-188) is a therapeutic approach that can be used for the treatment of hepatocellular carcinomas (HCC). We propose a detailed description of the tumoral model using the N1-S1 cell line as well as a technique for intra-arterial injection of radiolabelled lipiodol in order to undertake preclinical studies necessary for the evaluation of a new molecule. We also report the principal technical pitfalls that were faced. The speed of injection of the tumoral cells is a key factor in the tumoral induction since slow injections lead to a tumoral induction rate of 36.3% compared with 76.6% (P<0.01) when using very slow injections. This parameter should thus be controlled carefully during the subcapsular injection of the tumoral cells. In addition, when injecting radiolabelled lipiodol, anaesthesia should not be performed with isoflurane since this leads to a reduction in tumoral uptake. Indeed, we found a 'tumour/healthy liver' uptake ratio of only 2.1+/-0.7 with isoflurane as against 4.4+/-2.6 (P<0.05) when anaesthesia was carried out by intraperitoneal injection of ketamine. Lastly, we show that the tumour size has an influence on the tumoral uptake of radiolabelled lipiodol; therefore, this parameter must also be carefully controlled.

New thermoluminescent dosimeters (TLD): optimization and characterization of TLD threads sterilizable by autoclave.

To improve the performance of mono-extruded TLD threads as a dosimetric thermoluminescent tool (French Patent 9903729), a new process was developed by co-extrusion methodology leading to threads of 600 microm diameter with a 50 microm homogeneous polypropylene sheath. In this optimization work, study of parameters such as LiF:Mg,Cu,P powder granulometry, load rate and proportion of components led to an increased sensitivity of around 40%. Moreover, the co-extrusion technique allowed the threads to be sterilized by humid steam (134 degrees C/18 min) without significant variation of the linearity response between 0 and 30 Gy after gamma irradiation (60Co).

High field magnetic resonance imaging evaluation of superparamagnetic iron oxide nanoparticles in a permanent rat myocardial infarction.

RATIONALE AND OBJECTIVES: The purpose of this study was to evaluate superparamagnetic iron oxide (SPIO) nanoparticles to discriminate infarcted from normal tissue after myocardial infarction using high field MR imaging (7 tesla). MATERIALS AND METHODS: Permanent myocardial infarction was induced in rats. SPIO nanoparticles (1 mg Fe/kg) were assessed with T1-weighted gradient echo sequence to visualize the myocardial infarction 48 hours after ligature (n = 6). Furthermore, MR Imaging was performed using a T2-weighted RARE sequence and nanoparticles were injected (5 or 10 mg Fe/kg) on 36 rats 5, 24 or 48 hours after infarction. RESULTS: No changes in contrast between normal and infarcted myocardium was observed after nanoparticle injection on T1-weighted images. However, nanoparticles induced a significant contrast increase between normal and infarcted myocardium on T2-weighted images whatever the delay between infarction and imaging (2.99 +/- 1.66 preinjection vs. 7.82 +/- 1.96 after SPIO injection at a dose of 5 mg Fe/kg 5 hours postinfarction, P = 0.0001). CONCLUSIONS: Nanoparticle injection made it possible to discriminate normal from infarcted myocardium on T2-weighted images. However, the high magnetic field prevented the visualization of the T1 effect of SPIO nanoparticles.

Biodistribution of dual radiolabeled lipidic nanocapsules in the rat using scintigraphy and gamma counting.

The aim of the present work was to study the biodistribution of a radiolabeled lipidic nanocapsule formulation after intravenous administration in rat by scintigraphy and gamma counting. This formulation is expected to be used as anticancer agent delivery devices and as transfection complexes. For this purpose, 99mTc-oxine was incorporated in the lipidic core, while 125I labeled tensioactive shell of the nanocapsule. First, in vitro stability of radiolabeled nanocapsules was evaluated by dialysis against distilled water and size measurements. Second, the nanocapsule biodistribution was followed after intravenous administration for 3 h by dynamic scintigraphic acquisition and up to 24 h by determining the gamma activity in blood and tissues. Radiolabeling was efficient and stable in vitro. After intravenous injection blood radioactivity decreased with an early half disappearance time of about 45 min for both radioisotopes. Liver and intestine radioactivities raised up to 24 h. The relatively long remanence in blood of the tracers which is probably due to the presence of PEG at the nanocarrier surface seems promising for the use of these solvent free lipidic nanocapsules as carrier of lipophilic drugs.

Production of new thermoluminescent mini-dosimeters.

A method of producing CaSO4:Dy thermoluminescent mini-dosimeters was reported in 1986 by B W Wessels for determination of the in vivo absorbed dose in radioimmunotherapy, a field in which absorbed dose gradients are important. These dosimeters, which undergo dissolution when used in a liquid environment, showed a sensitivity loss of up to 30% after 4 days of immersion in our tests. Moreover, several studies have shown that biocompatibility problems can occur during in vivo studies in animals. This paper describes the production and testing of a new type of thermoluminescent mini-dosimeter obtained by microextrusion of a mixture of LiF:Mg,Cu,P polypropylene and plastic adjuvants. These dosimeters, in the form of long 400 microm diameter filaments, can be cut to the desired length. The production process allows an LiF:Mg,Cu,P load of up to 50%. Results obtained in external irradiation indicate that these new miniature LiF:Mg,Cu,P dosimeters have good sensitivity (about 1.6 times that of CaSO4:Dy mini-TLDs), homogeneous response within a production batch (mean +/-4%), response stability in water (0.7% of variation in sensitivity after 2 weeks of immersion) and stability in aqueous solutions at different pH. LiF:Mg,Cu,P mini-dosimeters appear to be highly promising for internal dosimetry, and evaluation is in progress in animals.

Thallium-201 right lung/heart ratio during exercise in patients with coronary artery disease: relation to thallium-201 myocardial single-photon emission tomography, rest and exercise left ventricular function and coronary angiography.

The aim of this study was to correlate lung thallium-201 uptake on exercise with 201Tl single-photon emission tomography (SPET) myocardial perfusion imaging, rest and exercise equilibrium radionuclide angiographic and coronary angiographic findings in patients with coronary artery disease (CAD) using a simple, reproducible lung/heart (L/H) ratio that would be easy to use in clinical practice. L/H ratio was defined on the anterior planar image obtained during exercise 201Tl SPET acquisition as the mean counts per pixel in an entire right lung field region of interest divided by the mean counts per pixel in the hottest myocardial wall region of interest. We studied 103 patients. Fifty-nine patients (group I) with <5% likelihood of CAD were used as a reference group. In 44 CAD patients (group II), L/H ratio was compared with 201Tl SPET, radionuclide angiographic and coronary angiographic variables. The group I L/H ratio of 0.35+/-0.05 (mean +/-1 SD) was significantly lower (P<0.001) than the group II L/H ratio of 0. 45+/-0.10. An L/H ratio >0.45 (mean + 2 SD in group I) was considered abnormal. In group II, L/H ratio showed a significant correlation with stress and rest 201Tl perfusion defect size (r = 0. 39 and r = 0.42, P<0.01, respectively), but not with extent of ischaemic myocardium. The mean L/H ratio was 0.41+/-0.10 in patients with one-vessel disease (n = 15), 0.46+/-0.08 in those with two-vessel disease (n = 17) and 0.47+/-0.12 in those with three-vessel disease (n = 12), but no significant difference was found between the three subgroups. L/H ratio showed a significant inverse relation with rest and exercise left ventricular ejection fraction (r = -0.37 and r = -0.50, P<0.05 and P<0.001, respectively). Using stepwise multiple regression analysis, exercise left ventricular ejection fraction and previous history of hypertension were the sole two variables independently predictive of the L/H ratio. In conclusion, although lung thallium uptake is usually found to correlate with extent and severity of CAD, increased L/H ratio should primarily be considered as a marker of exercise-induced left ventricular systolic and perhaps diastolic dysfunction, probably independent of the underlying cardiac disease.

Early detection of liposome brain localization in rat experimental allergic encephalomyelitis.

Blood-brain barrier (BBB) permeability increases prior to the development of clinical signs in early-stage multiple sclerosis (MS). Detection of subtle changes would thus be helpful for diagnostic purposes and rapid therapeutic decisions before new episodes. Since multiple sclerosis and experimental allergic encephalomyelitis (EAE) have numerous common features, in particular BBB-permeability characteristics, and since we have previously shown that BBB localization is disturbed by tumors, embolism, and mannitol injection, we investigated BBB-liposome permeability in an EAE rat model. Twenty young male Lewis rats received a single intradermal inoculation of guinea-pig spinal cord. The effect of the Freund's adjuvant and spinal cord alone on brain permeability were also assessed. In order to compare solution permeability and liposome localization, radioactive liposomes and, 1 h later, 99mTc-DTPA were injected intravenously. Scintigraphic acquisitions were obtained to follow the biodistribution of radioactivity in the whole body. Each rat was subjected to a first examination before inoculation and then every two days until completion and may be considered as its own control. EAE induced a previously unreported increase in global-body permeability, probably due to inflammation. Liposome brain localization and brain/heart ratio were significantly different between normal animals and those with early-stage EAE (before appearance of clinical signs) and distinguished between different disease stages in clinically patent EAE. The index of disease progression was modified earlier than with 99mTc-DTPA injection. One explanation may be particle pick-up by circulating macrophages, which cross the BBB during this pathology. For clinical applications, experiments must be confirmed on models more reliable for human multiple sclerosis.

Synthesis of Iron Oxide Nanoparticles Used as MRI Contrast Agents: A Parametric Study.

Colloidal iron oxides play an important role as magnetic resonance imaging (MRI) contrast agents. The superparamagnetic particles actually used are constituted by solid cores (diameter of 5-15 nm), generally coated by a thick polysaccharidic layer (hydrodynamic radii of 30-100 nm), and formulated by direct coprecipitation of iron salts in the presence of polymeric material. To better control the synthesis, we attempted to formulate new stable uncoated superparamagnetic nanoparticles. Colloids were generated by coprecipitation of an aqueous solution of iron salts and tetramethylammonium hydroxide (TMAOH) solution. The influence of parameters such as media composition, iron media, injection fluxes, Fe and TMAOH concentrations, temperature, and oxygen on size, magnetic and magnetic resonance relaxometric properties, and colloidal stability of particles were evaluated. We have determined the relative importance of these parameters as well as the optimal conditions for obtaining uncoated stable particles with an average size of 5 nm and interesting relaxivities. The interpretation of the observed limits takes into account diffusibilities of reactants and product, feeding rates of reactants, and surface properties of nanoparticles. A model of synthesis, related to spontaneous emulsification of suspensions, is proposed. Copyright 1999 Academic Press.

Phosphorylcholine Coating of Iron Oxide Nanoparticles.

In order to develop thin-walled superparamagnetic nanoparticle suspensions as a contrast agent for magnetic resonance imaging, phosphorylcholine PC was used to coat iron oxide cores of 5 nm. Weak stable positively charged suspensions can be obtained at concentration greater than 3 mmol.l-1 (corresponding to about 3.2 molecules per nm2), while the addition of phosphorylglycerol PG decreases the electrophoretic mobility. Raising the pH over 6 leads to flocculation: the binding of PC on iron oxides as a function of pH appears to be reversible. By Langmuir analysis, two adsorption domains may be observed with a maximal density of 3.48 and 6.55 mol.nm-2, interpreted as a multilayer formation. Copyright 1999 Academic Press.

NGF release from poly(D,L-lactide-co-glycolide) microspheres. Effect of some formulation parameters on encapsulated NGF stability.

Poly(d,l-lactide-co-glycolide) (PLGA 37.5/25 and 25/50) biodegradable microparticles, which allow the locally delivery of a precise amount of a drug by stereotactic injection in the brain, were prepared by a W/O/W emulsion solvent evaporation/extraction method which had been previously optimized. The aim of this work was to study the influence of two formulation parameters (the presence of NaCl in the dispersing phase and the type of PLGA) on the NGF release profiles and NGF stability during microencapsulation. A honey-comb-like structure characterized the internal morphology of the microspheres. The initial burst was attributed to the rapid penetration of the release medium inside the matrix through a network of pores and to the desorption of weakly adsorbed protein from the surface of the internal cavities. The non-release fraction of the encapsulated protein observed after twelve weeks of incubation was accounted for firstly by the adsorption of the released protein on the degrading microparticles and secondly by the entanglement of the encapsulated protein in the polymer chains. The use of sodium chloride in the dispersing phase of the double emulsion markedly reduced the burst effect by making the microparticle morphology more compact. Unfortunately, it induced in parallel a pronounced NGF denaturation. Finally, it appeared that microparticles made from a hydrophilic uncapped PLGA 37.5/25 in the absence of salt, allowed the release of intact NGF at least during the first 24 h as determined by both ELISA and a PC12 cell-based bioassay.

[Renovascular hypertension due to renal artery thrombosis]. / Hypertension artérielle rénovasculaire liée á une thrombose de l'artère rénale.

The authors report the case of a 74 year old woman admitted to hospital for severe hypertension with unilateral renal artery thrombosis. Recanalisation of the renal artery was obtained by transluminal angioplasty leading to rapid control of the hypertension. Dynamic renal scintigraphy with MAG 3 confirmed the viability of the kidney distal to the thrombosis and, secondarily, the functional recovery of the affected kidney.

Investigation of blood-brain barrier permeability to magnetite-dextran nanoparticles (MD3) after osmotic disruption in rats.

The permeability of experimentally disrupted blood-brain barrier (BBB) to superparamagnetic nanoparticles (MD3) was studied in rats. BBB opening was induced by intracarotid injection of mannitol. One hundred eighty rats were used for the study. Rats were examined at two time points, 30 minutes and 12 hours after intracarotid mannitol injection. Different preparations intravenously injected 30 minutes before rat sacrifice were used for characterization of BBB disruption. BBB integrity was determined with 99mTc-diethylenetriamine pentaacetic acid (DTPA) and 99mTc-albumin. Iron oxide-glucose particles (12-nm mean diameter), 99mTc-labeled lecithin-cholesterol liposomes of three different sizes (50, 100, and 200 nm), and polyethylene glycol (PEG)-coated 99mTc liposomes (50 nm) were used for investigations of the dependence of BBB permeability on particle system size or surface. Magnetite-dextran nanoparticles (MD3) were evaluated as superparamagnetic contrast agent to monitor with magnetic resonance imaging (MRI) the BBB breakdown. In vitro T1 and T2 relaxation times of the brain tissue were measured at 40 MHz and 37 degrees C, and T2-weighted MR images were acquired at 0.5 T. After intracarotid mannitol infusion, as expected, the BBB breakdown was immediate and temporary as judged by soluble molecule diffusion. MD3 nanoparticles crossed the BBB 12 hours after intravenous mannitol injection, at a time when brain permeability for molecules or small particles returns to normal. Magnetite crystals were found in cytoplasmic vesicles of glial cells. On MRI, signal intensity decreased after injection of MD3, even 12 hours after mannitol injection. This particularity could be useful in the study of focal pathological lesions accompanied by BBB permeability modifications. In such conditions, superparamagnetic particle contrast agents could be caught by the BBB, allowing the observation of impaired BBB areas without detectable cellular lesions.

Determination of phospholipids from pulmonary surfactant using an on-line coupled silica/reversed-phase high-performance liquid chromatography system.

A basic normal-phase HPLC separation of phospholipids can be improved by introducing a limited contribution of solvophobic retention. For this purpose, the effect of an additional alkylsilica (C18) column of variable length coupled in series with a silica column was investigated. With increasing percentage of reversed phase in this system, the retention of phosphatidylglycerol increased. Phosphatidylinositol and phosphatidylserine were separated into molecular species. The "selective retention" defined in this study permits an evaluation of the solvophobic retention of phospholipids in the coupled system. An alternative column switching procedure is used for specific applications of the biphasic separation on chosen phospholipids. With this system, determination of phosphatidylglycerol and six other phospholipids from pulmonary surfactant could be performed.

A structural study of interfacial phospholipid and lung surfactant layers by transmission electron microscopy after Blodgett sampling: influence of surface pressure and temperature.

Monolayer studies of the lung surfactant extract (LSE), dipalmitoyl phosphatidilcholine (DPPC) and dioleyl phosphatidilcholine (DOPC) have been performed in the dynamic condition at various temperatures. These compounds were also studied by differential scanning calorimetry, and the Langmuir Blodgett films were examined by electron microscopy. The combination of these techniques allowed us to describe precisely the collapse process, which was found to be different above and below the transition temperature of the lipids. However, whereas a phase separation for DPPC/DOPC mixtures occurred at all temperatures studied, this separation was observed for LSE only at temperatures lower than that characteristic of the "rigid state" to "liquid-like state" transition temperature. The ability of LSE to rapidly respread upon decompression appears to be due to the formation of piled amorphous aggregates formed during compression of its monolayers.

[Tension activity of pulmonary surfactant: adsorption of liposomes of model phospholipids]. / Tensioactivié du surfactant pulmonaire: adsorption de liposomes de phospholipides modèles.

One peculiarity of pulmonary surfactant, which is the tensioactive material physiologically present at the surface of alveoli, lies in its very quick localization at in the air-water interface. This being one of the limiting factors of artificial exogenous surfactants for the treatment of patients suffering of respiratory distress syndromes, we have studied the mechanisms which are intervening in the adsorption kinetics of a pure liquid--phase phospholipid, the dioleylphosphatidylcholine (DOPC), and of mixtures of dipalmitoylphosphatidylcholine (DPPC) and phosphatidic acid (PA) in presence of divalent cations (Ca++ and Mg++). The adsorption kinetics of liposomal suspensions of DOPC, which were studied by the Wilhelmy plate method, are determined by the existence of a barrier potential which height depends on the temperature and medium osmolarity, and on the deformability of vesicules. The study of PA-DPPC liposomes was performed with the help of a pulsating bubble surfactometer, a physicochemical instrumentation which mimics the pulmonary alveoli. To obtain performant responses with this model, high concentrations of PA and of divalent cations Ca++ and Mg++ are needed. These results, which are similar to those observed during the study of liposomal fusion, allow to propose a model, according to which adsorption of liposomes at the air-water interface is comparable to liposomal fusion and may be related to the presence of a thin aqueous film.

[Problems posed by the development of exogenous surfactants for the treatment of hyaline membrane disease]. / Les problèmes posés par la mise au point de surfactants exogènes pour le traitement de la maladie des membranes hyalines.

The hyaline membrane disease (HMD) is a respiratory distress occurring at birth of some premature infants, attributed to an endogenous pulmonary surfactant deficiency. The present treatments are iatrogenic and inefficient for the most diseased infants. The exogenous surfactant supplementation intends to give to the baby formulations playing in vivo the role of the natural surfactant. The research developed for the formulation and the diffusion of these exogenous surfactants have to solve physicochemical problems but also of industrial production and of safety use. In spite of their adequate physicochemical and pharmacological properties, surfactants of natural sources, for industrial grounds, are not still distributed at a large scale. The efficiency of artificial surfactants is variable, and the use of non-biodegradable molecules in several preparations let to safety problems. The development of new types of artificial surfactants, more efficient and safe, implies a better knowledge of the physico-chemical mechanisms intervening in the pulmonary surfactant dynamics.