Sigrid L. Fossheim

Paramagnetic liposomes as MRI contrast agents: influence of liposomal physicochemical properties on the in vitro relaxivity

The in vitro contrast efficacy of liposome encapsulated gadolinium-[10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1, 4,7-triacetic acid] (GdHPDO3A) has been assessed by relaxometry. The internal concentrations were 150 and 250 mM Gd. Two types of liposome compositions were investigated: a phospholipid blend consisting of both hydrogenated phosphatidylcholine (HPC) and phosphatidylserine (HPS) with a gel-to-liquid crystalline phase transition temperature (Tm) of 50 degrees C, and a mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) with a Tm of 41 degrees C. The investigated liposome size range was 70-400 nm. The T1 and T2 relaxivities (r1 and r2) of liposome encapsulated GdHPDO3A were significantly reduced at 37 degrees C and 0.47 T, compared to those of non-liposomal metal chelate, due to an exchange limitation of the dipolar relaxation process. The highest relaxivity values were obtained for the DPPC/DPPG liposomes, and were attributed to a higher liposome water permeability and to a more efficient water exchange across the membrane. A reduction in liposome size increased the r1, confirming the exchange limited dipolar relaxation. The increased r1 with increasing temperature demonstrated the prerequisite of rapid water exchange between the interior and exterior of the liposome for efficient dipolar relaxation enhancement. Susceptibility effects were present in the liposome systems as the r2/r1 ratio increased with increasing liposome size and internal Gd concentration. In summary, the current work has shown the influence of key physicochemical properties, such as liposome size, membrane composition and permeability, on the in vitro relaxivity of liposome encapsulated GdHPDO3A.

Liposomes as carriers of amphiphilic gadolinium chelates: the effect of membrane composition on incorporation efficacy and in vitro relaxivity

The effects of membrane composition (phospholipid type and amount of cholesterol), liposome size, drug/lipid ratio (loading) and nature of the amphiphilic gadolinium (Gd) chelate on the incorporation efficacy and magnetic resonance (MR) contrast efficacy (longitudinal (T1) relaxivity) were investigated using a fractional factorial design. A highly lipophilic Gd-chelate was required to ensure complete liposome incorporation. High T1-relaxivity was obtained by using liposomes composed of cholesterol and phospholipids with short acyl chain lengths (dimyristoyl phosphatidyl choline (DMPC) and dimyristoyl phosphatidyl glycerol (DMPG). Two key factors, the loading of Gd-chelate and the amount of cholesterol in small-sized DMPC/DMPG liposomes, were studied further in a central composite optimising design. A robust high relaxivity region was identified, comprising high loading of cholesterol and Gd-chelate. However, the highest T1-relaxivity (52 mM(-1) s(-1)) was found in an area containing no cholesterol and low content of Gd-chelate. Nuclear magnetic resonance dispersion (NMRD) profiles were obtained for five of the liposome compositions from the optimising design, and high relaxivity peaks in the 20 MHz region confirmed the presence of Gd-chelates with a long tau(R). A liposome formulation was selected for surface modification with polyethylene glycol (PEG), without having any effect on the T1-relaxivity.

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.

Lipid membrane composition influences drug release from dioleoylphosphatidylethanolamine-based liposomes on exposure to ultrasound

The effect of membrane composition on calcein release from dioleoylphosphatidylethanolamine (DOPE)-based liposomes on exposure to low doses of 1.13 MHz focused ultrasound (US) was investigated by multivariate analysis, with the goal of designing liposomes for US-mediated drug delivery. Regression analysis revealed a strong correlation between sonosensitivity and the non-bilayer forming lipids DOPE and pegylated distearoylphosphatidylethanolamine (DSPE-PEG 2000), with DOPE having the strongest impact. Unlike most of the previously studied distearoylphosphatidylethanolamine (DSPE)-based liposomes, all the current DOPE-based liposome formulations were found stable in 20% serum in terms of drug retention.

Investigation of lanthanide-based starch particles as a model system for liver contrast agents.

Gadolinium and dysprosium diethylenetriamine pentaacetic acid‐labeled starch microparticles (Gd‐DTPA‐SP and Dy‐DTPA‐SP) were investigated as model liver contrast agents. The liver contrast efficacy of particles with low and high metal contents was compared in two imaging models: in vivo rat liver and ex vivo perfused rat liver. The biodistribution of intravenously injected particles was also assessed by ex vivo relaxometry and inductively coupled plasma atomic emission spectrophotometry of tissues. All particles reduced the liver signal intensity on T2‐weighted spin‐echo and gradient‐recalled echo images as a result of susceptibility effects. Because of their higher magnetic susceptibility, the Dy‐DTPA‐SP were more effective negative contrast enhancers than the Gd‐DTPA‐SP. On T1‐weighted spin‐echo images, only the Gd‐DTPA‐SP with low metal content significantly increased the liver signal intensity. In addition, these low‐loading Gd‐DTPA‐SP markedly reduced the blood T1. The two latter observations were not consistent with the anticipated blood circulation time of microparticles, but were a result of the lower stability of these particles in blood compared with Gd‐DTPA‐SP, which has a high metal content. Regardless of stability or imaging conditions, the paramagnetic starch particles investigated showed potential as negative liver contrast enhancers. However, the observed accumulation of particles in the lungs represented a biological limitation for their use as contrast agents.J. Magn. Reson. Imaging 1999; 9:295–303.

Paramagnetic Liposomes as Magnetic Resonance Imaging Contrast Agents: Assessment of Contrast Efficacy in Various Liver Models

RATIONALE AND OBJECTIVES Liposomal gadolinium (Gd)-HP-DO3A has been evaluated as a contrast agent for liver magnetic resonance imaging. The influence of various liposomal physicochemical properties on the liver uptake and contrast efficacy was investigated in various ex vivo and in vivo liver models. METHODS Liposomes of different size and membrane properties were prepared. The liposome size ranged from 74 to 304 nm. Two types of phospholipid compositions were studied; a mixture of hydrogenated phosphatidylcholine (HPC) and hydrogenated phosphatidylserine (HPS) with a phase transition temperature (Tm) of 51 degrees C and, a blend composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) displaying a Tm of 41 degrees C. Ex vivo tissue relaxometry and in vivo liver imaging were used to study the influence of liposome composition on the liver uptake and contrast efficacy of intravenously injected liposomes. The influence of liposome size and composition on the kinetics of liver uptake and imaging effect was assessed ex vivo in the perfused rat liver. RESULTS The HPC/HPS preparations showed generally a higher and faster liver uptake than the DPPC/DPPG preparations due to a higher stability in blood/perfusate (high Tm) and to the HPS component. The liposome size modulated the extent and kinetics of liver uptake; the larger the size, the faster and more extensive was the liver uptake. Both types of liposome preparations were shown to be efficient liver susceptibility agents both ex vivo and in vivo due to their uptake by the Kupffer cells of liver. The lack of full correlation between the extent of liver uptake and degree of contrast enhancement might be attributed to different regimes of susceptibility-based relaxation. CONCLUSIONS The present study has demonstrated the influence of key liposomal physicochemical properties on the liver uptake and contrast efficacy of liposome-encapsulated Gd chelates, exemplified by Gd-HP-DO3A.

Synthesis and physicochemical characterisation of new amphiphilic gadolinium DO3A complexes as contrast agents for MRI

Two approaches were employed in the syntheses of four 1,4,7-tris(carboxymethyl)-10-(2-hydroxyalkyl)-1,4,7,10-tetraazacyclododecanes (4) with alkyl chain lengths from 4 to 16 carbons. Physicochemical properties, such as critical micelle concentration (CMC), micelle size, partition coefficient (P) between water and octan-1-ol and T1 relaxivity (r1), were investigated for the corresponding gadolinium (Gd) complexes. The Gd complexes containing the shortest alkyl chains (5a and 5b) showed properties typical of water-soluble Gd complexes. On the other hand, the long-chained chelates (5c and 5d) were found to possess amphiphilic properties and to form micelles. The relaxivities of these amphiphilic complexes were found to be concentration dependent, consistent with the formation of micelles. An unexpectedly high relaxivity was measured for compound 5d below its CMC. This feature is probably caused by cluster formation due to low solubility in water.

Low-molecular weight lanthanide contrast agents: Evaluation of susceptibility and dipolar effects in red blood cell suspensions

Red blood cell (RBC) suspensions, containing low-molecular weight (LMW) dysprosium (Dy) and gadolinium (Gd) chelates, were selected as a two-compartment system for the evaluation of the magnetic dipolar and susceptibility contributions to the transverse (T2) relaxation of solvent water protons. The influence of RBC geometry and degree of metal chelate compartmentalization on T2 was investigated by variation of the osmolality and hematocrit (HC), respectively. The T2-relaxation ability of Dy-chelates was markedly improved in RBC suspensions, in comparison to aqueous solutions, due to the presence of susceptibility effects that more than compensated for the low dipolar relaxation efficacy. Despite a smaller susceptibility effect, the Gd-chelates were still the most efficacious in shortening T2 due to their comparatively larger dipolar relaxation contribution. The results obtained with the Dy-chelates allowed the evaluation of the relative contributions of susceptibility and dipolar mediated relaxation for the Gd-chelates. The RBC geometry and degree of compartmentalization influenced strongly the T2 relaxation efficacy of Dy-chelates, as opposed to the Gd-chelates. Hemolysis eliminated the susceptibility effect, essentially removing the T2 relaxation ability of Dy-chelates. The T2 relaxation efficacy of Gd-chelates was improved by hemolysis due to enhancement of the dipolar relaxation. As a conclusion, RBC suspensions have clearly been shown to be a suitable ex vivo model with which to distinguish the different contrast mechanisms of LMW Dy- and Gd-based MRI contrast agents.

Sonosensitive dioleoylphosphatidylethanolamine-containing liposomes with prolonged blood circulation time of doxorubicin

Ultrasound sensitive (sonosensitive) liposomes represent a drug delivery system designed for releasing a drug load upon exposure to ultrasound (US). Inclusion of dioleoylphosphatidylethanolamine (DOPE) in liposome membranes was previously shown to induce sonosensitivity. Long blood circulation time of the liposomal drug is required for high tumour uptake and efficient US-mediated drug delivery. In this study, blood pharmacokinetics of DOPE-based liposomal doxorubicin (DXR) were evaluated in non-tumoured mice. A markedly faster blood clearance of DXR was observed for DOPE-rich liposomes compared to Caelyx® (standard liposomal DXR). Subsequently, liposome membrane composition was altered to improve drug retention in the bloodstream, whilst maintaining sonosensitivity. Formulations with reduced blood clearance of DXR were obtained by reducing the content of DOPE from 62 to 32 or 25 mol%. These formulations showed long blood circulation time, as approximately 20% of the administered DXR dose was present in the bloodstream 24 h after intravenous injection. The reduction in liposomal DOPE content did not significantly reduce US-mediated DXR release in vitro, indicating that DOPE is a potent modulator of sonosensitivity. The novel liposome formulations, containing moderate amounts of DOPE, displayed similar blood pharmacokinetic profiles as standard liposomal DXR, but a markedly improved sonosensitivity.