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).

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.

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.

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.

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.