Ultrasound-mediated destabilization and drug release from liposomes comprising dioleoylphosphatidylethanolamine

European Journal of Pharmaceutical Sciences 42 (2011) 380–386

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Ultrasound-mediated destabilization and drug release from liposomes comprising dioleoylphosphatidylethanolamine Tove J. Evjen a,b , Esben A. Nilssen a,∗ , Sabine Barnert c , Rolf Schubert c , Martin Brandl b,1 , Sigrid L. Fossheim a,2 a b c

Epitarget AS, Oslo, Norway University of Tromsø, Department of Pharmacy, Tromsø, Norway University of Freiburg, Department of Pharmaceutical Technology and Biopharmacy, Freiburg, Germany

a r t i c l e

i n f o

Article history: Received 2 September 2010 Received in revised form 16 November 2010 Accepted 4 January 2011 Available online 14 January 2011 Keywords: Drug release DOPE Doxorubicin Drug delivery Liposome Ultrasound

a b s t r a c t 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 6 min of 40 kHz 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 DSPEbased 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. © 2011 Elsevier B.V. All rights reserved.

1. Introduction A novel approach to increase delivery of anticancer drugs to tumour cells is to apply ultrasound (US) to tumour tissue upon accumulation of sonosensitive drug-containing liposomes. The strategy is based on the ability of US to increase the permeabilization of phospholipid membranes, which may be exploited to both locally induce drug release from liposomes at the tumour site and increase drug uptake into tumour cells (Pitt et al., 2004; Liu et al.,

Abbreviations: US, ultrasound; Chol, cholesterol; DOPE, 1,2 dioleoyl-sn-glycero-3-phosphatidylethanolamine; DSPE, 1,2 distearoylsn-glycero-3-phosphatidylethanolamine; DSPE-PEG 2000, 1,2 distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(methoxy(polyethylene glycol)-2000); DSPC, 1,2 distearoyl-sn-glycero-3-phosphatidylcholine; HSPC, hydrogenated (soy) L-␣-phosphatidylcholine; DXR, doxorubicin; PP, packing parameter; HII , reversed hexagonal phase; DLS, dynamic light scattering; CryoTEM, cryotransmission electron microscopy; PCS, photon correlation spectroscopy. ∗ Corresponding author at: Epitarget AS, PO Box 7159 Majorstuen, 0307 Oslo, Norway. Tel.: +47 23201200; fax: +47 23201249. E-mail address: [email protected] (E.A. Nilssen). 1 Current address: University of Southern Denmark, Department of Chemistry and Physics, Odense, Denmark. 2 Current address: Clavis Pharma ASA, Oslo, Norway. 0928-0987/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.01.002

1998; Sundaram et al., 2003). This in turn, could give a therapeutic benefit in tumour treatment, as already demonstrated in animal studies (Myhr and Moan, 2006; Schroeder et al., 2009a). In order to achieve a satisfactory response to US, liposomes should be designed to exhibit physicochemical properties that impart sonosensitivity. Others and we have shown that different lipid compositions influence on sonosensitivity (Lin and Thomas, 2003, 2004; Schroeder et al., 2009b; Evjen et al., 2010). Lin and Thomas (2003) have demonstrated that inclusion of surfactants and polyethylenglycol (PEG) in standard phosphatidylcholine (PC)based liposomes enhanced sonosensitivity with respect to drug release in vitro. Recently we found that pegylated liposome formulations comprising distearoylphosphatidylethanolamine (DSPE) displayed markedly improved sonosensitivity as evidenced by an increased release extent of doxorubicin (DXR) (Evjen et al., 2010). Here, we speculated that the underlying mechanism was related to the non-bilayer forming characteristics of DSPE. More specifically, DSPE resembles a cone-shaped geometry and tends to form reversed hexagonal structures when dispersed in water (Israelachvili, 1991). This behaviour can be explained by its packing parameter (PP), which is above 1. PP can be defined as the ratio of the geometrical area of the apolar to polar regions of the amphiphile, PP = V/l × Aw, where V is the volume of the molecule,

T.J. Evjen et al. / European Journal of Pharmaceutical Sciences 42 (2011) 380–386

l is the length of the acyl chains, and Aw is the area of the polar head group (Israelachvili, 1991). It is likely that inclusion of nonbilayer forming lipids like DSPE into liposomes induce membrane perturbations or even phase transitions in the liposome membrane during US treatment, followed by drug release. In the current study we wanted to further explore this theory by investigating the influence of the unsaturated analogue of DSPE; dioleoylphosphatidylethanolamine (DOPE) on sonosensitivity of liposomes. DOPE, due to its unsaturated acyl chains, has a more pronounced inverted conical shape than DSPE, as given by a higher PP value. Under physiological conditions (pH 7.4) DOPE tends to form reversed hexagonal (HII) structures. Liposome bilayers can be formed, however, when DOPE is mixed with other lipids such as pegylated phospholipids (Holland et al., 1996; Johnsson and Edwards, 2001). This study focuses on designing a drug carrier system based on DOPE that is stabilised when combined with PEGylated DSPE (DSPE-PEG 2000), but which is selectively destabilized upon exposure to ultrasound, releasing its drug payload. First, sonosensitivity of DOPE-based liposomes was tested in comparison to DSPE-based liposomes and reference PC-based liposomes in buffered sucrose solution and 20% serum, respectively. DXR release was monitored as a function of US duration time at a frequency of 40 kHz and 20% amplitude. Dynamic light scattering (DLS) and cryo-transmission electron microscopy (CryoTEM) were performed on DOPE-based liposomes before and after US treatment in order to study the size and morphology of the liposomes. Finally, the stability of the formulation was investigated as a function of pH and temperature in 20% serum, respectively. 2. Materials and methods

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Canada) through Nucleopore polycarbonate filters with decreasing pore size from 800 to 80 nm (Nuclepore, West Chester, PA, USA). The lipid hydration, liposome extrusion and thawing process were performed at 25 ◦ C for DOPE-based liposomes and at 75 ◦ C for DSPE-based liposomes, which is above the gel-to-liquid crystalline phase transition temperature of the respective phospholipid blends. 2.3. Liposome entrapment of doxorubicin An ammonium sulphate transmembrane gradient was obtained by extensive dialysis of DOPE-based liposomes against an isotonic sucrose solution containing 10 mM HEPES (pH 7.4), herein referred to as sucrose/HEPES buffer. Unbuffered isotonic sucrose solution was used for dialysis of DSPE-based liposomes as previously described (Evjen et al., 2010). The dialysis was performed by placing disposable dialysis bags (MW cut off 100 000 D), (Float-A-Lyzer® , Spectra/Por® , Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) containing the liposome dispersions in a magnetically stirred dialysis solution for approximately 2–3 days (volume ratio liposome dispersion:dialysis solution, 1:100) with intermediate exchanges of the dialysis solution. Doxorubicin hydrochloride solution was added to the liposome dispersion to give a final ratio of drug-to-lipid of 1:16 (w/w). The resulting lipid concentration was 16 mg/ml. To provide optimal loading efficiency, the DOPE- and DSPE-based liposome dispersions were, after DXR addition, further incubated under stirring for 60 min at 35 ◦ C and for 30 min at 75 ◦ C, respectively. Any remaining non-encapsulated DXR was removed by liposome dialysis against sucrose/HEPES buffer containing 0.01% (w/v) sodium azide, as described previously.

2.1. Materials 2.4. Determination of entrapment efficiency 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2 distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2 distearoyl-sn-glycero-3-phosphatidylethanolamine-N(methoxy(polyethylene glycol)-2000) (DSPE-PEG 2000) were purchased from Genzyme Pharmaceuticals, Liestal, Switzerland. Cholesterol (Chol), calcein, 2-(N-morpholino)ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), ammonium sulphate, cholesterol (Chol), sodium azide, Triton® X-100 solution and sucrose were obtained from Sigma Aldrich, Oslo, Norway. Serum of fetal bovine origin (Autonorm) was obtained from Sero, Billingstad, Norway. Doxorubicin hydrochloride (DXR) was purchased from Nycomed, Asker, Norway. DXR-containing liposomes, Caelyx® (herein termed reference PC-based liposomes) comprising hydrogenated soy phosphatidylcholine, DSPE-PEG 2000 and Chol (57:5:38 mol%) was supplied from the pharmacy at the Norwegian Radium Hospital, Oslo, Norway (European distributor Schering-Plough). 2.2. Liposome preparation Small-sized liposomes with the two lipid compositions DOPE:DSPC:DSPE-PEG 2000:Chol and DSPE:DSPC:DSPE-PEG 2000:Chol (both 62:10:8:20 mol%) were prepared by the thin film hydration and sequential extrusion method (Lasic, 1993). Lipids were dissolved in a choloroform/methanol mixture (9/1, v/v) at 60 ◦ C, and rotary evaporated to dryness under vacuum for 6–8 h. The dry lipid film was hydrated with 300 mM ammonium sulphate solution giving a nominal lipid concentration of 20 mg/ml. After three freeze–thaw cycles in a dry-ice/aceton/methanol mixture and water, respectively, the liposomes were downsized by stepwise extrusion (Lipex extruder, Biomembrane Inc., Vancouver B.C.,

To estimate the percentage of drug entrapment aliquots of both the dialyzed and the non-dialyzed liposome sample were diluted 1:500 (v/v) with sucrose/HEPES buffer and further dissolved with Triton X-100 surfactant solution in a 50:1 (v/v) ratio. The entrapment efficiency (%) was calculated according to: Fen − Fb × 100% Ftot − Fb

(1)

where Fen is the fluorescence intensity in the dialyzed and surfactant treated liposome sample, Fb is the initial background signal of the dispersion medium (sucrose/HEPES buffer), and Ftot is the fluorescence intensity in the non-dialyzed and surfactant treated liposome sample. Fluorescence intensity measurements were performed using a fluorescence spectrometer from Ocean Optics (model QE65000, Duiven, Netherlands). The excitation and emission wavelength of DXR were 488 and 595 nm, respectively. Triplicate samples were measured. 2.5. Liposome size determination The mean intensity-weighted hydrodynamic liposome diameter was determined by photon correlation spectroscopy (PCS) (Nanosizer, Malvern Instruments, Malvern, UK) prior and after ultrasound treatment. The measurements were performed at 23 ◦ C and at a scattering angle of 90◦ on liposome dispersions diluted with 0.22 ␮m-filtered sucrose/HEPES buffer (dilution factor liposome dispersion/buffer: 1:500, v/v). The width of the particle size distribution was expressed by the polydispersity index (PI). Triplicate measurements were performed.

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2.6. Ultrasound-mediated release methodology US release was conducted using a 40 kHz US transducer (VC 750, Sonic and Materials, Inc, Newtown, CT, USA) connected to a custom-built sample chamber as previously described by Evjen et al. (2010). The temperature in the sample chamber was kept constant at 25 ◦ C by circulating water. The liposome dispersions were diluted in a 1:500 (v/v) ratio with sucrose/HEPES buffer and/or sucrose/HEPES buffer containing 20% (v/v) serum, just prior to the US experiments. The diluted liposome dispersions were exposed in a continuous mode (100% duty cycle) to 40 kHz US at an amplitude of 20%. At this amplitude, acoustic pressure measurements in the sample chamber gave ∼240 kPa (pk–pk). Pressure measurements were taken with a hydrophone (Bruel and Kjaer type 8103, Denmark). The DXR release could be monitored due to the relief of DXR-mediated fluorescence self-quenching in the external liposomal phase, and concomitant increase in fluorescence intensity (Düzgünes et al., 2003; Zhigaltsev, 2002). An optical fiber probe (Ocean Optics, type QP600-2-VIS/BX, Florida, USA) connected to a fluorescence spectrometer (Ocean Optics QE65000, Duiven, the Netherlands) and a 470 nm LED lamp (LS-LED Ocean Optics, Duiven, the Netherlands) was used to measure the increase in 595 nm fluorescence intensity as a result of DXR release, using the following equation: % Drug release =

Ft − F0 × 100 Fmax − F0

(2)

where Ft is the fluorescence intensity in the liposome sample after a given duration (t) of US, F0 is the initial background fluorescence of the diluted liposome sample prior to US, and Fmax is the fluorescence intensity after liposome solubilisation with surfactant (Triton-X 100). The diluted liposome samples were solubilised with Triton-X 100 solution at a 50:1 (v/v) ratio. The release test was performed on triplicate samples. 2.7. Serum stability Liposome stability was studied using a well-established serum-induced leakage assay mimicking biological conditions as previously described by Evjen et al. (2010). Liposome dispersions, diluted 1:125 (v/v) with sucrose/HEPES buffer containing 20% serum, were incubated up to 24 h at 37 ◦ C. Time-dependent leakage of liposomal DXR was quantified by fluorescence measurements of serum samples further diluted 1:4 (v/v) with sucrose/HEPES buffer, according to Eq. (2). Triplicate samples of each liposome batch were measured.

Temperature-mediated drug release from liposomes was tested upon incubation in sucrose/HEPES buffer (dilution ratio of 1:125 (v/v) liposome dispersion/sucrose/HEPES buffer) up to 2 h at 60 ◦ C. In both experiments, the extent of DXR leakage was quantified using fluorescence measurements according to Eq. (2). 2.9. Cryo-transmission electron microscopy (Cryo-TEM) Cryo-TEM micrographs were obtained prior to and after exposure of the liposome dispersions to 6 and 12 min US. Prior to US the liposomal dispersions were diluted in the ratio of 1:1 (v/v) with sucrose/HEPES buffer. The untreated liposomes were not diluted prior to imaging. Cryo-TEM investigations were performed as previously described by Rank et al. (2009), using a LEO 912 OMEGA electron microscope (Zeiss, Oberkochen, Germany) operating at 120 kV. A drop of the sample was placed onto a copper grid (Quantifoil® S7/2 Cu 400 mesh, holey carbon films Quantifoil Micro Tools GmbH, Jena, Germany). Excess solution was removed by a filter paper, leaving a thin liquid film of 100–500 nm. The sample was then immediately shock-frozen by plunging it into liquid ethane (Dubochet et al., 1988). The vitrified sample was stored at 90 K in liquid nitrogen until it was loaded into a cryogenic sample holder (D626,Gatan Inc., Pleasanton, USA). The specimens were examined at −174 ◦ C. Digital images with a magnification of 6300× or 12,500× were recorded with a slow-scan CCD camera system (Proscan HSC 2). Minimal under-focus of the microscope objective lens was provided to obtain sufficient phase contrast (Talmon, 1996). 2.10. Statistical analysis All results are reported as mean values with standard deviation. Test of significance was performed using a student t-test with p < 0.01. 3. Results 3.1. Liposome characterization The liposome batches produced were comparable in size showing mean diameters in the range of 84–88 nm with PI below 0.14, indicating a narrow size-distribution. The DXR encapsulation efficiencies of the liposome batches were above 94%. 3.2. In vitro sonosensitivity

2.8. pH- and thermo sensitivity studies DOPE has traditionally been used as the main lipid constituent together with protonable lipids in pH-sensitive liposomes to induce drug release under mildly acidification in the tumour tissue (Slepushkin et al., 2004; De Oliveira et al., 1998; Løkling et al., 2004). At low pH the aminogroup of the DOPE molecule is protonated. This in turn, affects the size of the headgroup, promoting adoption of the molecule into a HII phase, followed by drug leakage. For the DOPE-based liposomal formulation studied here, inclusion of DSPE-PEG 2000 should hinder pH-induced destabilization by preventing head-group interactions between the DOPE-molecules in the bilayer. However, to exclude the possibility that potential drug leakage from liposomes in tumour tissue is due to the acidic tumour environment and not US, liposomal drug leakage was studied as a function of pH. In detail, the liposome dispersions were diluted at a 1:125 (v/v) ratio with 10 mM MES buffered isotonic sucrose solution at pH 4.0, 6.0, 7.4 and 8.5, followed by 2 h incubation at 37 ◦ C.

Fig. 1 shows the US-mediated release profiles of DXR from liposomes in sucrose/HEPES buffer as a function of US duration time. DOPE-based liposomes showed an exponential release kinetic profile where approximately 70 and 95% DXR was released after 2 and 6 min US exposure, respectively. DSPEbased liposomes displayed a significantly lower release extent; 60% DXR being released after 6 min US. Both the PE-based liposome formulations displayed a several-fold increase in drug release extent as compared to reference PC-based liposomes (Fig. 1). 3.3. Sonosensitivity in serum US-mediated dXR release from liposomes was studied in 20% serum. As seen in Fig. 2 the sonosensitivity of DOPE-based liposomes was approximately maintained in the presence of serum components. By contrast, the DSPE-based liposomes revealed a drastic reduction in sonosensitivity in 20% serum as compared to

T.J. Evjen et al. / European Journal of Pharmaceutical Sciences 42 (2011) 380–386

Table 1 Mean size and size distribution of DXR-containing DOPE-based liposomes (DOPE:DSPC:DSPE-PEG 2000:Chol; 62:10:8:20 mol%) before and after 6 and 12 min US treatment. Mean and SD of triplicate measurements are given.

% released DXR from liposomes

100 90 80

Liposome formulation

70 60 50

DOPEliposomes

40

a

30 20 10 0 0

1

2

3

4

5

6

US duration, min Fig. 1. US-mediated release profiles of DXR from (䊉) DOPE-based liposomes (DOPE:DSPC:DSPE-PEG 2000:Chol; 62:10:8:20 mol%) in sucrose/HEPES buffer (pH 7.4). () DSPE-based liposomes (DSPE:DSPC:DSPE-PEG 2000:Chol; 62:10:8:20 mol%) and () reference PC-based liposomes (HSPC:DSPE-PEG 2000:Chol; 57:5:38 mol%) are included for comparison. The mean and standard deviation of triplicate measurements of duplicate batches are given.

sucrose/HEPES buffer; with only 5% DXR released after 6 min US. The sonosensitivity of reference PC-based liposomes was also significantly reduced in 20% serum (Fig. 2). 3.4. Effect of US on liposome size and morphology To investigate the mechanism of sonosensitivity of DOPE-based liposomes, liposome size and morphology were investigated before and after US exposure by PCS measurements and Cryo-TEM (see Sections 2.5 and 2.9 for experimental details). Table 1 summarises the mean liposome size and size-distribution before and after US treatment. The UStreated liposomes showed a significant change towards a larger mean size and a broader size-distribution as evidenced by an increased PI. After 12 min US the lipo100

% released DXR from liposomes

383

80

60

40

20

Mean size (nm)

PI

Untreated 6 min US

12 min US

Untreated

86 ± 2

a

0.14 ± 0.03 0.45 ± 0.07a

150 ± 27

6 min US

12 min US

The liposome sample was too heterogeneous in size for valid PCS measurements.

some sample was too polydisperse to be measured by PCS. The significant change towards a more polydisperse size distribution of US-treated DOPE-based liposomes was confirmed by the CryoTEM data. Prior to US treatment the micrographs revealed mainly small, circular and unilamellar vesicles (Fig. 3a). The rod-like structures spanning the aqueous core indicated DXR precipitate. After 6 min US, a mixture of larger empty vesicles as well as smaller drug loaded liposomes were observed (Fig. 3b). This was even more evident after 12 min US, where a mixture of small and larger empty vesicles as well as non-lamellar structures were observed (Fig. 3c). Note that all of the US-treated liposome samples were diluted 1:1 (v/v) with sucrose/HEPES buffer. 3.5. Serum stability Stability of the DOPE-based liposome formulation in terms of DXR leakage was studied by incubation in sucrose/HEPES buffer containing 20% serum for 24 h at 37 ◦ C. The data showed negligible leakage of DXR (4.0 ± 2.0%). Similar serum stability was observed for reference PC-based liposomes (4 ± 0% DXR leakage after 24 h incubation at 37 ◦ C). 3.6. Effect of pH and temperature on DOPE-based liposomes pH and temperature-mediated leakage from liposomes was studied according to Section 2.8. The data showed no detectable leakage of DXR from the liposomes within the tested pH range of 4.0–8.5, indicating pH-resilient liposomes. No temperature induced leakage of DXR (0.4 ± 0%) occurred after 2 h incubation at 60 ◦ C, a temperature that is well above both the gel-to-liquid crystalline phase transition temperature and the HII phase transition temperature of DOPE (Gruner et al., 1985). The sample temperature reached during an US run never exceeded 30 ◦ C, indicating that the observed in vitro drug release was not temperature mediated. The results also indicated that potential drug release during US exposure in an in vivo setting might mainly be attributed to the non-thermal effects of the delivered acoustic energy. 4. Discussion

0 0

1

2

3

4

5

6

US duration time, min Fig. 2. US-mediated release profiles of DXR from (䊉) DOPE-based liposomes (DOPE:DSPC:DSPE-PEG 2000:Chol; 62:10:8:20 mol%) in sucrose/HEPES buffer containing 20% serum. () DSPE-based liposomes (DSPE:DSPC:DSPE-PEG 2000:Chol; 62:10:8:20 mol%) and () reference PC-based liposomes (HSPC:DSPEPEG 2000:Chol; 57:5:38 mol%) are included for comparison. The mean and standard deviation of triplicate measurements of duplicate batches are given.

This study presents novel sonosensitive liposomes containing DOPE as the main lipid constituent. The DOPE-based liposomes displayed a fast release kinetic profile showing approximately 70 and 95% DXR release in buffered sucrose solution after 2 and 6 min US exposure, respectively. This represented a 9-fold increase in release extent compared to reference PC-based liposomes. DOPE showed to be a more potent lipid modulator to sonosensitivity than its saturated analogue DSPE, indicating that lipid polymorphism plays a key role to liposomal sonosensitivity.

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Fig. 3. Cryo-TEM micrographs showing lipid structures before and after US treatment of DOPE-based liposomes (DOPE:DSPC:DSPE-PEG 2000:Chol; 62:10:8:20 mol%). (a) Liposomes before US treatment, (b) liposomes exposed to 6 min US, (c) liposomes exposed to 12 min US.

Interestingly, DSPE-based liposomes experienced a total loss of sonosensitivity in 20% serum, whilst sonosensitivity of DOPE-based liposomes was maintained (Fig. 2). The reason for this behaviour remains unclear at present. One explanation could be interactions between serum proteins and the DSPE-based liposome membrane affecting the lipid packing of the bilayer. Alternatively, reduced cavitation extent in 20% serum due to higher viscosity may be below the threshold necessary for release from DSPE-based liposomes, due to threshold. Irrespective of mechanism, the high sonosensitivity of the DOPE-based liposomes in 20% serum shows promise for in vivo US-mediated drug delivery to tumour tissue.

The underlying mechanisms behind US-mediated drug release from liposomes have not been fully investigated in the literature. This might partly be due to methodological limitations in determining reversible alterations in phospholipid bilayers occurring only during US treatment. Previous reports have suggested that US-sensitization of PC-based liposomes induces reversible porelike defects in the membrane allowing drug to release (Lin and Thomas, 2003; Schroeder et al., 2007). Schroeder et al. (2007) demonstrated that no significant changes in size or structure of PC-based liposomes occurred after exposure to low unfocused ultrasound.

T.J. Evjen et al. / European Journal of Pharmaceutical Sciences 42 (2011) 380–386

In contrast, the present DOPE-based liposome formulation showed a significant change both in size distribution and liposome structure after US exposure. The liposome size change towards a broader size distribution after US was in agreement with previous observations for DSPE-based liposomes (Evjen et al., 2010). The differences in size distribution, however, were more pronounced for the DOPE-based liposomes, which might explain the higher sonosensitivity for this particular formulation. In the absence of US, the vast majority of the vesicles showed small round unilamellar structures slightly below 100 nm with rod-like structures spanning the aqueous core, indicating DXR precipitate (Fig. 3a). After US exposure, various lipid aggregates with a polydisperse size distribution were evident (Fig. 3b and c). The heterogeneity in liposome size increased with US exposure time and after 12 min US the liposome dispersions were too polydisperse to be measured by PCS. These data suggest that the high sonosensitivity of DOPE-based liposomes is due to destabilization of the liposome-bilayer into various lipid aggregates upon US exposure. As already mentioned, inclusion of PEGylated phospholipids stabilizes DOPE into a lamellar phase (Holland et al., 1996; Johnsson and Edwards, 2001). Hence, potential US-induced removal of DSPE-PEG 2000 from the current liposomes might destabilize the DOPE-bilayer. US or pressure-driven exchange of pegylated lipids with the external aqueous phase is possible as pegylated lipids have significantly greater water solubility than their parent lipids (Marsh, 1990). This said, the exact mechanism behind the US-induced disruption of the DOPE-based liposomes remains to be clarified. However, the fact that less acoustic energy is required to obtain drug release from DOPE-based liposomes compared to PC-based reference liposomes could be of major clinical importance, because the damage to surrounding healthy cells and tissues will be minimized during treatment. It should be noted, however, that for clinical applications a focused US transducer generating higher US frequencies is needed to improve focusing ability. A limitation of liposomes based on unsaturated lipids with low phase transition temperatures has been fast leakage of entrapped drug in blood circulation (Gabizon et al., 1993; Charrois and Allen, 2004). In order to obtain sufficient drug delivery to the tumour site, it is crucial that the liposomes are able to retain their drug content in the blood circulation. The investigated DOPE-based liposomes showed no leakage of drug during 24 h incubation in 20% serum at 37 ◦ C. This could be explained by the steric stabilization effect of DSPE-PEG 2000, which is known to maintain carrier stability by reducing headgroup interactions between bilayer-lipids as well as interactions between the bilayer and serum proteins (Woodle and Lasic, 1992; Woodle, 1998). It should be emphasized, however, that the liposome formulation described here was designed primarily to illustrate the effect of DOPE per se on sonosensitivity of liposomes, and was not optimized as a drug carrier for US-mediated drug delivery in vivo. However, the satisfactory in vitro stability of the DOPE-based formulation as well as the high sonosensitivity in 20% serum are encouraging with regard to future therapeutic efficacy studies in animal models. In conclusion, this study has shown that sonosensitivity of liposomes is significantly enhanced by incorporating the non-bilayer forming lipid DOPE into the liposome membrane. For the first time it is shown that US induces disruption of DOPE-based liposomes into various lipid structures, which might explain the improved drug release properties when compared to reference PC-based liposomes. We suggest that the high sonosensitivity of DOPE-based liposomes is related to the lipid geometry and non-bilayer forming characteristics of DOPE, which induce a fast and marked destabilization of the bilayer upon US exposure. The liposomes were stable drug carriers in terms of in vitro serum stability. The cur-

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