Therapeutic efficacy of the combination of doxorubicin-loaded liposomes with inertial cavitation generated by confocal ultrasound in AT2 Dunning rat tumour model.

The combination of liposomal doxorubicin (DXR) and confocal ultrasound (US) was investigated for the enhancement of drug delivery in a rat tumour model. The liposomes, based on the unsaturated phospholipid dierucoylphosphocholine, were designed to be stable during blood circulation in order to maximize accumulation in tumour tissue and to release drug content upon US stimulation. A confocal US setup was developed for delivering inertial cavitation to tumours in a well-controlled and reproducible manner. In vitro studies confirm drug release from liposomes as a function of inertial cavitation dose, while in vivo pharmacokinetic studies show long blood circulation times and peak tumour accumulation at 24-48 h post intravenous administration. Animals injected 6 mg kg(-1) liposomal DXR exposed to US treatment 48 h after administration show significant tumour growth delay compared to control groups. A liposomal DXR dose of 3 mg kg(-1), however, did not induce any significant therapeutic response. This study demonstrates that inertial cavitation can be generated in such a fashion as to disrupt drug carrying liposomes which have accumulated in the tumour, and thereby increase therapeutic effect with a minimum direct effect on the tissue. Such an approach is an important step towards a therapeutic application of cavitation-induced drug delivery and reduced chemotherapy toxicity.

Non-invasive magnetic resonance imaging follow-up of sono-sensitive liposome tumor delivery and controlled release after high-intensity focused ultrasound.

This work examines the use of lanthanide-based contrast agents and magnetic resonance imaging in monitoring liposomal behavior in vivo. Dysprosium (Dy) and gadolinium (Gd) chelates, Dy-diethylenetriaminepentaacetic acid bismethylamide (Dy-DTPA-BMA) and Gd-DTPA-BMA, were encapsulated in pegylated distearoylphosphatidylethanolamine-based (saturated) liposomes, and then intravenously injected into Copenhagen rats with subcutaneous Dunning AT2 xenografts. Liposome-encapsulated Dy chelate shortens transverse relaxation times (T(2) and T(2)*) of tissue; thus, liposomal accumulation in the tumor can be monitored by observing the decrease in T(2)* relaxation time over time. The tumor was treated at the time of maximum liposomal accumulation (48 h) with confocal, cavitating high-intensity focused ultrasound to induce liposomal payload release. Using liposome-encapsulated Gd chelate at high enough concentrations and saturated liposomal phospholipids induces an exchange-limited longitudinal (T(1)) relaxation when the liposomes are intact; when the liposomes are released, exchange limitation is relieved, thus allowing in vivo observation of payload release as a decrease in tumor T(1).

Physicochemical characterization of liposomes after ultrasound exposure - mechanisms of drug release.

Ultrasound is investigated as a novel drug delivery tool within cancer therapy. Non-thermal ultrasound treatment of solid tumours post i.v.-injection of drug-carrying liposomes may induce local drug release from the carrier followed by enhanced intracellular drug uptake. Recently, ultrasound-mediated drug release of liposomes (sonosensitivity) was shown to strongly depend on liposome membrane composition. In the current study the ultrasound-mediated drug release mechanism of liposomes was investigated. The results showed that differences in ultrasound drug release kinetics obtained for different liposomal compositions were caused by distinctive release mechanisms of the carriers. Two types of liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and hydrogenated soy L-α-phosphatidylcholine (HSPC) as main lipids, respectively, were recently shown to vary in sonosensitivity. Here, these liposomes were analyzed prior to and after a given ultrasound-exposure for their mean size, size distribution and morphology. Cryo-transmission electron microscopy, dynamic light scattering and asymmetric flow field-flow fractionation in combination with multi-angle light scattering revealed a significant change in mean size, size distribution and morphology of DOPE-based liposomes after ultrasound, pointing to an irreversible disruption of the vesicles and concomitant drug release. In contrast, the HSPC-based liposomes remained unchanged in size and structure after ultrasound application, indicating pore-mediated release mechanisms. The results show that the release mechanisms and interactions between ultrasound and liposomes depend on the liposome membrane-composition, explaining their sonosensitive properties.

Feasibility study of cavitation-induced liposomal doxorubicin release in an AT2 Dunning rat tumor model.

BACKGROUND: Targeted and triggered release of liposomal drug using heat or ultrasound represents a promising treatment modality able to increase the therapeutic-totoxicity ratio of encapsulated drugs. PURPOSE: To study the ability for high-intensity focused ultrasound to induce liposomal drug release mainly by focused inertial cavitation in vitro and in an animal model. METHODS: A 1 MHz ultrasound setup has been developed for in vitro and in vivo drug release from a specific liposomal doxorubicin formulation at a target cavitation dose. RESULTS: Controlled cavitation at 1 MHz was applied within the tumors 48 hours after liposome injection according to preliminary pharmacokinetic study. A small non-significant therapeutic effect of US-liposomal treatment was observed compared to liposomes alone suggesting no beneficial effect of ultrasound in the current setup. CONCLUSION: The in vitro study provided a suitable ultrasound setup for delivering a cavitation dose appropriate for safe liposomal drug release. However, when converting to an in vivo model, no therapeutic benefit was observed. This may be due to a number of reasons, one of which may be the difficulty in converting in vitro findings to an in vivo model. In light of these findings, we discuss important design features for future studies.

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.

Ultrasound enhanced antitumor activity of liposomal doxorubicin in mice.

Liposomal encapsulation of doxorubicin (DXR) improves tumor accumulation and reduces adverse effects. One possible strategy for further optimization of this delivery technology would be to design the liposome carrier to release its content within the tumor tissue in response to specific stimuli such as ultrasound (US). In this study, the tumor uptake properties and therapeutic efficacy of 1,2 distearoyl-sn-glycero-3-phosphatidylethanolamine-based liposomes containing DXR were investigated in nude mice bearing tumor xenografts. The liposomal DXR formulation alone showed no inhibitory effect on tumor growth. However, upon exposure to low frequency US in situ inhibition of tumor growth was demonstrated.

Compressibility study of quaternary phospholipid blend monolayers.

The mechanical properties of liposome membranes are strongly dependent on type and ratio of lipid compounds, which can have important role in drug targeting and release processes when liposome is used as drug carrier. In this work we have used Brewster's angle microscopy to monitor the lateral compression process of lipid monolayers containing as helper lipids either distearoyl phosphatidylethanolamine (DSPE) or dioleoyl phophatidylethanolamine (DOPE) molecules on the Langmuir trough. The compressibility coefficient was determined for lipid blend monolayers containing the helper lipids above, cholesterol, distearoyl phosphatidylcholine (DSPC) and pegylated-DSPE at room temperature. Two variables, the cholesterol fraction and the ratio ρ between the helper lipid (either DSPE or DOPE) and the reference lipid DSPC, were studied by multivariate analysis to evaluate their impact on the compressibility coefficient of the monolayers. The cholesterol level was found to be the most significant variable for DSPE blends while the ratio ρ was the most significant one for DOPE blend monolayers. It was also found that these two variables can exhibit positive interaction and the same compressibility value can be obtained with different blend compositions.

Ultrasound-mediated destabilization and drug release from liposomes comprising dioleoylphosphatidylethanolamine.

Novel sonosensitive doxorubicin-containing liposomes comprising dioleoylphosphatidylethanolamine (DOPE) as the main lipid constituent were developed and characterized in terms of ultrasound-mediated drug release in vitro. The liposome formulation showed high sonosensitivity; where approximately 95% doxorubicin was released from liposomes after 6min of 40kHz US exposure in buffered sucrose solution. This represented a 30% increase in release extent in absolute terms compared to liposomes comprising the saturated lipid analogue distearoylphosphatidylethanolamine (DSPE), and a 9-fold improvement in release extent when compared to standard pegylated liposomal doxorubicin, respectively. Ultrasound release experiments in the presence of serum showed a significantly reduction in sonosensitivity of DSPE-based liposomes, whilst the release properties of DOPE-based liposomes were essentially maintained. Dynamic light scattering measurements and cryo-transmission electron microscopy of DOPE-based liposomes after ultrasound treatment indicated liposome disruption and formation of various lipid structures, corroborating the high release extent. The results point to the potential of DOPE-based liposomes as a new class of drug carriers for ultrasound-mediated drug delivery.

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.

Distearoylphosphatidylethanolamine-based liposomes for ultrasound-mediated drug delivery.

The ability of ultrasound (US) to permeabilize phospholipid membranes has opened the potential of using US as a means to enhance delivery of anti-cancer drugs to tumour cells via liposomes. In this study, novel US sensitive or sonosensitive doxorubicin-containing liposomes based on 1,2 distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) as the main lipid component are reported. A variety of lipid bilayer compositions was studied with respect to in vitro US triggered release of drug as well as serum stability in terms of drug retention, using experimental design. The multivariate data analysis indicated a strong correlation between DSPE content and sonosensitivity, both alone and in interplay with cholesterol. The most optimal formulation showed approximately 70% release of doxorubicin after 6min of US exposure. This represented a 7-fold increase in release extent when compared to standard pegylated liposomal doxorubicin. The significant enhancement in sonosensitivity of the liposomes shows the potential of engineering liposomal lipid composition for US-mediated drug delivery.

Dynamic and structural aspects of PEGylated liposomes monitored by NMR.

Proton-detected NMR diffusion and (31)P NMR chemical shifts/bandwidths measurements were used to investigate a series of liposomal formulations where size and PEGylation extent need to be controlled for ultrasound mediated drug release. The width of the (31)P line is sensitive to aggregate size and shape and self-diffusion (1)H NMR conveys information about diffusional motion, size, and PEGylation extent. Measurements were performed on the formulations at their original pH, osmolality, and lipid concentration. These contained variable amounts of PEGylated phospholipid (herein referred to as PEG-lipid) and cholesterol. At high levels of PEG-lipid (11.5 and 15 mol%) the self-diffusion (1)H NMR revealed the coexistence of two entities with distinct diffusion coefficients: micelles (1.3 to 3x10(-11) m(2)/s) and liposomes (approximately 5x10(-12) m(2)/s). The (31)P spectra showed a broad liposome signal and two distinct narrow lines that were unaffected by temperature. The narrow lines arise from mixed micelles comprising both PEG-lipids and phospholipids. The echo decay in the diffusion experiments could be described as a sum of exponentials revealing that the exchange of PEG-lipid between liposomes and micellar aggregates is slower than the experimental observation time. For low amounts of PEG-lipid (1 and 4.5 mol%) the (31)P spectra consisted of a broad signal typically obtained for liposomes and the diffusion data were best described by a single exponential decay attributed solely to liposomes. For intermediate amounts of PEG-lipid (8 mol%), micellization started to occur and the diffusion data could no longer be fitted to a single or bi-exponential decay. Instead, the data were best described by a log-normal distribution of diffusion coefficients. The most efficient PEG-lipid incorporation in liposomes (about 8 mol%) was achieved for lower molecular weight PEG (2000 Da vs 5000 Da) and when the PEG-lipid acyl chain length matched the acyl chain length of the liposomal core phospholipid. Simultaneously to the PEGylation extent, self-diffusion (1)H NMR provides information about the size of micelles and liposomes. The size of the micellar aggregates decreased as the PEG-lipid content was increased while the liposome size remained invariant.

Local delivery of magnetic resonance (MR) contrast agent in kidney using thermosensitive liposomes and MR imaging-guided local hyperthermia: a feasibility study in vivo.

PURPOSE: To investigate the feasibility of local delivery of a magnetic resonance (MR) contrast agent in vivo using paramagnetic thermosensitive liposomes and infrared (IR) laser-induced local hyperthermia under real-time MR thermometry on rabbit kidney. MATERIALS AND METHODS: Respiratory gated, radio frequency (RF)-spoiled gradient-echo sequences were used for precise MR temperature mapping (SD = 1 degrees C). In vivo heating experiments confirmed local release of MR contrast agent from liposomes. RESULTS: T1 decreased from 800 msec to about 500 msec, as measured after tissue cooling, in those locations where the renal parenchyma was heated above the phase transition temperature of the liposome membrane. CONCLUSION: The release of MR contrast agent has been demonstrated in rabbit kidney in vivo. This may be used as a reporter for simultaneous release of therapeutic agents.

Experimental application of thermosensitive paramagnetic liposomes for monitoring magnetic resonance imaging guided thermal ablation.

The use of a liposomal paramagnetic agent with a T(1)-relaxivity that increases markedly at temperatures above the phase transition temperature (T(m)) of the liposomal membrane was evaluated during magnetic resonance imaging (MRI) guided hyperthermia ablation. A neodymium-yttrium aluminum garnet (Nd-YAG) laser unit and a radiofrequency ablation system were used for tissue ablation in eight rabbit livers in vivo. One ablation was made in each animal prior to administration of the liposomal agent. Liposomes with a T(m) of 57 degrees C containing gadodiamide (GdDTPA-BMA) were injected iv, and two additional ablations were performed. T(1)-weighted scans were performed in heated tissue, after tissue temperature had normalized, and 15-20 min after normalization of tissue temperature. Increase in signal intensity (DeltaSI) for ablations prior to injection of the agent was 13.0% (SD = 5.7) for the laser group and 9.1% (SD = 7.9) for the radiofrequency group. Signal intensity after administration of the agent unrelated to heating was not statistically significant (DeltaSI = 1.4%, P = 0.35). For ablations made after injection of the agent, a significant increase was found in the laser (DeltaSI = 34.5%, SD = 11.9) and radiofrequency group (DeltaSI = 21.6%, SD = 22.7). The persistent signal enhancement found in areas exposed to a temperature above the threshold temperature above T(m) allows thermal monitoring of MRI guided thermal ablation.

Biodistribution of pH-responsive liposomes for MRI and a novel approach to improve the pH-responsiveness.

The potential of pH-sensitive paramagnetic liposomes as a probe for monitoring acidic pH in tumours with magnetic resonance imaging has recently been demonstrated. If the blood retention time is prolonged, such liposomes can accumulate in tumour interstitium due to increased vascular permeability and interstitial retention. In the present study, biodistribution studies in healthy rats showed rapid clearance of the pH-sensitive system dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylglycerosuccinate (DPSG) liposomal GdDTPA-BMA from the blood circulation with most of the Gd dose in the liver at 15 min post intravenous injection. Incorporation of 1.5 mol% polyethylene glycol (PEG) grafted DPPE (DPPE-PEG) in the above-mentioned formulation resulted in a significantly prolonged blood circulation time. However, the relaxometric pH-response of the DPPE/DPSG/DPPE-PEG system decreased as a function of mol% DPPE-PEG. Therefore, a compromise would be necessary between long blood residence time and a suitable pH-sensitivity of the liposomes. A possible approach to compensate for the reduced pH-sensitivity was investigated. Gadofosveset, a low-molecular weight Gd-chelate with high affinity for albumin, was encapsulated within DPPE/DPSG liposomes. This promising system showed in blood a markedly higher relaxometric response than the corresponding system with GdDTPA-BMA, due to release of gadofosveset at low pH and subsequent binding to albumin.

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.

Tuning the MR properties of blood-stable pH-responsive paramagnetic liposomes.

Paramagnetic pH-responsive liposomes have recently been suggested as a promising approach for monitoring by magnetic resonance imaging (MRI) pH changes in tumours. In the present study, the effects of variations in bilayer composition on the relaxometric properties of diacylphosphatidylethanolamine (PE)/dipalmitoylglycerosuccinate (DPSG) liposomal GdDTPA-BMA were investigated both in buffer and blood. A factorial experimental design was used with the variables PE chain length and mol% DPSG. All the relaxometric profiles displayed a semi-sigmoidal shape with a minimum plateau at high pH (r1(min)) and a maximum at low pH (r1(max,E)). Relevant sigmoidal curve fit parameters were evaluated by partial least squares regression. Systematic variations in the relaxometric response (r1(max,E)-r1(min)) were shown for the liposomal systems both in buffer and blood. The pH value at which the r1 was 20% of r1(max,E) relative to r1(min), i.e. pH20, decreased significantly both in buffer and blood as a function of the mol% DPSG. This phenomenon could be understood by the increased surface charge density with increasing mol% DPSG and, hence, higher barrier against liposome aggregation with consequent leakage of contrast agent. Furthermore, the pH relaxometric profiles in blood were shifted laterally to higher, and likely more clinically relevant pH values than the corresponding profiles in buffer. The liposome formulations displayed minimal leakage of contrast agent after prolonged incubation in blood at physiological pH and retained their pH sensitivity after pre-incubation in blood.

Heat-activated liposomal MR contrast agent: initial in vivo results in rabbit liver and kidney.

PURPOSE: To evaluate by using in vivo magnetic resonance (MR) imaging the functionality of a liposomal paramagnetic contrast agent with T1 relaxivity that rapidly and markedly increases at temperatures above the gel-to-liquid crystalline phase transition temperature (T(c)) of the liposome membrane. MATERIALS AND METHODS: Liposomal gadolinium diethylenetriaminepentaacetic acid bis(methylamide) was injected intravenously at a dose of 0.4 or 1.2 mL (containing 10 or 30 micromol of gadolinium, respectively) per kilogram of body weight shortly before the application of focused ultrasound in liver (seven rabbits) or kidney (three rabbits). VX2 tumors had been implanted in liver in four of the rabbits. Eighteen locations in liver (13 in normal tissue, five in tumor) and 12 locations in kidney were sonicated. MR thermometry was performed during sonications. Signal intensity enhancement was evaluated on T1-weighted images acquired after the tissue cooled, and enhanced zones were compared with isotherms at T(c) of the liposome membrane (approximately 57 degrees C) by using Bland-Altman analysis. In liver, enhanced zones also were compared with areas of histologically verified thermal damage. The threshold temperature of enhancement at T1-weighted imaging was verified by monitoring the signal intensity increase after 10 sonications at varied powers in two locations in normal liver tissue. RESULTS: Persistent enhancement was observed on T1-weighted images at all sonicated liver locations. In liver, enhanced zones on T1-weighted images were contiguous both with 57 degrees C isotherms (25 measurements; mean difference +/- SD, 0.4 mm +/- 1.2) and with histologically verified areas of necrosis (seven measurements; mean difference +/- SD, 0.1 mm +/- 0.9). The threshold temperature of enhancement at T1-weighted imaging in normal liver was 53 degrees -57 degrees C. In kidney, enhanced zones on T1-weighted images did not match the isotherms. CONCLUSION: The liposomal contrast agent was effective at in vivo MR thermometry in liver but not in kidney.

pH-sensitive paramagnetic liposomes for MRI: assessment of stability in blood.

The pH-dependent stability of dipalmitoyl phosphatidyl ethanolamine/palmitic acid (DPPE/PA) liposomal GdDTPA-BMA was investigated in human blood and after exposure to selected blood components. Relaxometry, visual observations and cryo-transmission electron microscopy (cryo-TEM) were employed for the assessment of stability. The liposomes were stable in buffer at physiological pH and the T(1)-relaxivity (r(1)) of the system was significantly lowered compared to that of non-liposomal GdDTPA-BMA, which could be explained by an exchange limited relaxation process. Lowering the pH, however, gave a marked increase in r(1), due to liposome aggregation and subsequent leakage of GdDTPA-BMA. After a few minutes incubation in human blood the liposomes were destabilised and leaky at both high and low pH, and blood components likely to cause the instability were studied. Physiological level of Na(+) (150 mM) did not affect the relaxometric behavior of the liposomes at pH 7.4, but shifted the pH-r(1) profile laterally to higher pH-values compared to a level of 50 mM Na(+). Increased screening of the surface charges and, concomitantly, a lowering of the energy-barrier against aggregation is a plausible explanation for this phenomenon. In contrast, both Ca(2+) and Mg(2+) (physiological level, both 2 mM) caused massive aggregation of the liposomes and leakage of contents, and were therefore much more detrimental to the stability of the liposomes than a physiological level of Na(+). This could be due to the higher screening ability of divalent cations, but aggregation could also be induced through an inter-liposomal "bridging" effect. Physiological level of both Na(+) and Ca(2+) caused less leakage than for lower Na(+) concentration (50 mM Na(+) and 2 mM Ca(2+)), probably due to competition for the negative surface charges. Albumin also destabilised the liposomes, and it was shown to be due to an interaction between albumin and PA in the liposomal membrane.

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.