Roald Skurtveit

pH-sensitive paramagnetic liposomes as MRI contrast agents: in vitro feasibility studies

A novel type of pH-sensitive paramagnetic contrast agent is introduced; a low molecular weight gadolinium (Gd) chelate (GdDTPA-BMA) encapsulated within pH-sensitive liposomes. The in vitro relaxometric properties of the liposomal Gd chelate were shown to be a function of the pH in the liposomal dispersion and the membrane composition. Only a minor pH-dependency of the T1 relaxivity (r1) was observed for liposomal GdDTPA-BMA composed of the unsaturated lipids dioleoyl phosphatidyl ethanolamine (DOPE) and oleic acid (OA). On the other hand, the r1 of GdDTPA-BMA encapsulated within saturated dipalmitoyl phosphatidyl ethanolamine/palmitic acid (DPPE/PA) liposomes demonstrated a strong pH-dependency. At physiological pH and above, the r1 of this system was significantly lowered compared to that of non-liposomal Gd chelate, which was explained by an exchange limited relaxation process. Lowering the pH below physiological value, however, gave a sharp and 6-7 fold increase in r1, due to liposome destabilisation and subsequent leakage of entrapped GdDTPA-BMA. The pH-sensitivity of the DPPE/PA liposome system was confirmed in an in vitro magnetic resonance imaging (MRI) phantom study.

Novel pH-sensitive paramagnetic liposomes with improved MR properties.

The use of paramagnetic pH‐sensitive liposomes was recently suggested as a new approach for monitoring pathologic changes in pH by MRI. Such liposomes must be stable in blood and selectively release the encapsulated paramagnetic agent when exposed to lower pH in the target tissue. In the present study, different liposomal systems were formulated and characterized by relaxometry, cryo‐transmission electron microscopy (cryo‐TEM), and MRI. The pH‐sensitive system dipalmitoylphosphatidylethanolamine/palmitic acid (DPPE/PA) liposomal GdDTPA‐BMA, which was previously shown to be unstable in blood, was modified to improve its stability. The incorporation of cholesterol into the DPPE/PA liposomes significantly increased their stability in blood, but the pH sensitivity was diminished. Polyethylene glycol (PEG)‐modified DPPE/PA liposomes were pH‐insensitive in buffer, and unstable in blood. However, exchanging PA with the double‐chained amphiphile dipalmitoylglycerosuccinate (DPSG) yielded liposomes with improved properties. DPPE/DPSG liposomal GdDTPA‐BMA was stable in blood at physiological pH, and displayed a marked pH sensitivity. The pH sensitivity was not diminished after preincubation in blood, contrary to what has been reported for analogues containing unsaturated lipids. The potential of this system for monitoring pH was demonstrated in an in vitro MRI phantom study. Magn Reson Med 51:688–696, 2004.

Air-filled polymeric microcapsules from emulsions containing different organic phases

Air-filled polymeric microcapsules for use as a contrast agent in ultrasonography have been prepared by the freeze-drying of different oil-in-water emulsions. The water phases consisted of a block copolymer in water. The organic phases consisted of a biodegradable polyester dissolved in (-)-camphene, cyclooctane, cyclohexane or tricyclene, which were relatively poor solvents for the polyester. A polymeric wall was, therefore, precipitated at the droplet surface early in the process, i.e. during freezing. Removing the solvent during freeze-drying, resulted in air-filled microcapsules. The microcapsules were suspended in saline after freeze-drying. All the suspensions contained echogenic microcapsules with a volume mean diameter of 5-7mum. Microscopic investigations showed that the microcapsules were spherical and hollow. Tricyclene and, to some degree, (-)-camphene were found unsuitable for industrial production due to melting points above 30°C. Cyclooctane and cyclohexane were investigated as replacements for the initially chosen (-)camphene, since they are liquids over a wider temperature range. These solvents gave improved yields, measured both as particle volume concentration per amount of polymer in suspension and acoustic attenuation at 3.5MHz per amount of polymer in suspension, although the freeze-drying cycle was not optimized for these systems.Air-filled polymeric microcapsules for use as a contrast agent in ultrasonography have been prepared by the freeze-drying of different oil-in-water emulsions. The water phases consisted of a block copolymer in water. The organic phases consisted of a biodegradable polyester dissolved in (-)-camphene, cyclooctane, cyclohexane or tricyclene, which were relatively poor solvents for the polyester. A polymeric wall was, therefore, precipitated at the droplet surface early in the process, i.e. during freezing. Removing the solvent during freeze-drying, resulted in air-filled microcapsules. The microcapsules were suspended in saline after freeze-drying. All the suspensions contained echogenic microcapsules with a volume mean diameter of approximately 5-7 microm. Microscopic investigations showed that the microcapsules were spherical and hollow. Tricyclene and, to some degree, (-)-camphene were found unsuitable for industrial production due to melting points above 30 degrees C. Cyclooctane and cyclohexane were investigated as replacements for the initially chosen (-)-camphene, since they are liquids over a wider temperature range. These solvents gave improved yields, measured both as particle volume concentration per amount of polymer in suspension and acoustic attenuation at 3.5 MHz per amount of polymer in suspension, although the freeze-drying cycle was not optimized for these systems.

Acoustic Cluster Therapy (ACT) – A novel concept for ultrasound mediated, targeted drug delivery

A novel approach for ultrasound (US) mediated drug delivery - Acoustic Cluster Therapy (ACT) - is proposed, and basic characteristics of the ACT formulation are elucidated. The concept comprises administration of free flowing clusters of negatively charged microbubbles and positively charged microdroplets. The clusters are activated within the target pathology by diagnostic US, undergo an ensuing liquid-to-gas phase shift and transiently deposit 20-30 μm large bubbles in the microvasculature, occluding blood flow for ∼5-10 min. Further application of US will induce biomechanical effects that increases the vascular permeability, leading to a locally enhanced extravasation of components from the vascular compartment (e.g. released or co-administered drugs). Methodologies are detailed for determination of vital in-vitro characteristics of the ACT compound; cluster concentration and size distribution. It is shown how these attributes can be engineered through various formulation parameters, and their significance as predictors of biological behaviour, such as deposit characteristics, is demonstrated by US imaging in a dog model. Furthermore, in-vivo properties of the activated ACT bubbles are studied by intravital microscopy in a rat model, confirming the postulated behaviour of the concept.