Esben A. Nilssen

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.

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.

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.

In vivo monitoring of liposomal release in tumours following ultrasound stimulation

Dioeleoylphosphatidylethanolamine (DOPE)-based liposomes were recently reported as a new class of liposomes for ultrasound (US)-mediated drug delivery. The liposomes showed both high stability and in vitro US-mediated drug release (sonosensitivity). In the current study, in vivo proof-of-principle of US triggered release in tumoured mice was demonstrated using optical imaging. Confocal non-thermal US was used to deliver cavitation to tumours in a well-controlled manner. To detect in vivo release, the near infrared fluorochrome Al (III) Phthalocyanine Chloride Tetrasulphonic acid (AlPcS₄) was encapsulated into both DOPE-based liposomes and control liposomes based on hydrogenated soy phosphatidylcholine (HSPC). Encapsulation causes concentration dependent quenching of fluorescence that is recovered upon AlPcS₄ release from the liposomes. Exposure of tumours to US resulted in a significant increase in fluorescence in mice administered with DOPE-based liposomes, but no change in the mice treated with HSPC-based liposomes. Thus, DOPE-based liposomes showed superior sonosensitivity compared to HSPC-based liposomes in vivo.

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.

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.

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.

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

Abstract 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.

Stable inertial cavitation with a confocal ultrasonic device for drug release from nongaseous sonosensitive liposomes

Encapsulating chemotherapeutic agents in liposomes improves targeting and efficacy of treatments against some tumors. The present work aims at evaluating if sonosensitive liposomes combined with cavitation for drug delivery enhance efficacy and reduce toxicity. Two focused beams were combined for stabilizing the cavitation cloud and an imaging probe used for guidance. Each 1MHz focused transducer had a 5cm diameter and focal length. Exposure conditions were 10.8kW/cm2 Isppa, 250Hz PRF and 1% duty cycle. Phosphatidylcholine-based nongaseous liposomes were loaded with Doxorubicin. To control for mechanical tissue damage, AT2 Dunning tumors on rats were first exposed to ultrasound only. Treatment induced temperature rose below 0.5° C. The tumor growth was not significantly slowed down by ultrasound, but histological examination of tumors evidenced large areas of necrosis which resorbed one week after ultrasound. The new liposomes were compared with conventional HSPC-based liposomes in terms of efficacy and t...