pH-sensitive paramagnetic liposomes as MRI contrast agents: in vitro feasibility studies

Share Embed

Descrição do Produto

Magnetic Resonance Imaging 19 (2001) 731–738

pH-sensitive paramagnetic liposomes as MRI contrast agents: in vitro feasibility studies Knut-Egil Løklinga,*, Sigrid L. Fossheimb, Roald Skurtveitb, Atle Bjørnerudb, Jo Klavenessa a

Department of Medicinal Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway b Nycomed Imaging AS, Oslo, Norway Received 2 January 2001; accepted 7 March 2001

Abstract A novel type of pH-sensitive paramagnetic contrast agent is introduced; a low molecular weight gadolinium (Gd) chelate (GdDTPABMA) 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. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Paramagnetic liposomes; pH-sensitivity; Tumour pH; Phase transitions; Exchange limited relaxation

1. Introduction It is well known that in the presence of pathology, physiological parameters may be altered [1,2]. For instance, pH values in solid human tumours are reported to be approximately 0.4 units lower than those encountered in healthy subcutaneous and muscle tissue [3]. This phenomenon has been attributed to the extensive production of lactic acid within tumour cells and its extrusion to the interstitial fluid where the acid is accumulated due to an impaired clearance. The reduced interstitial pH in tumour tissue has formed basis for several studies on selective and/or enhanced delivery of cytostatic drugs with ionisable functions [4 – 6], and pH activation of prodrugs [7,8]. In vivo measurements of extracellular pH have traditionally been obtained by application of microelectrodes. This invasive technique is presumed to reflect predominantly the pH of the interstitial fluid, with an unknown component * Corresponding author. Tel.: ⫹47-23-18-5605; fax: ⫹47-23-18-6014. E-mail address: [email protected] (K.-E. Løkling).

from damaged cells and ruptured blood capillaries [3]. Microelectrodes are normally large compared to cells, and are not suited to study pH variations over small distances, which could be of great interest for tumour characterisation. The extracellular pH has also been studied by magnetic resonance spectroscopy (MRS) using exogenous shift reagents with extracellular distribution [9 –11]. In general, MRS suffers from low spatial resolution, and together with poor availability of clinical spectroscopy systems, the diagnostic utilisation of this method has so far been limited. In diagnostic imaging, the research interest in novel paramagnetic contrast agents has changed focus since the early launch of low molecular weight (LMW) gadolinium (Gd) chelates with extracellular distribution and, later, tissue-specific agents such as manganese dipyridoxyl diphosphate [12,13]. Contrast materials that can “visualise” pathology based on alterations in physiological parameters are presently investigated. For example, paramagnetic contrast agents whose in vitro contrast efficacy is modulated by pH have been reported [14,15]. Recently, the potential of a pH-sensitive Gd-labeled polymer as a tumour agent has been demonstrated [16]. A common feature of these novel

0730-725X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 0 - 7 2 5 X ( 0 1 ) 0 0 3 8 0 - 0


K.-E. Løkling et al. / Magnetic Resonance Imaging 19 (2001) 731–738

contrast materials is their ability to function as “off-on” switches; e.g. the MR efficacy being markedly enhanced at a given pH value. pH-sensitive drug delivery systems are gaining increasing interest in the field of pharmaceutics [17–19]. More specifically, pH-sensitive liposomes are currently evaluated as a delivery vehicle in several therapeutic concepts, such as gene therapy and intracellular targeting [20,21]. Such liposomes become unstable and leaky under weakly acidic conditions. This concept of pH-mediated drug release could be exploited in MRI of tumours. Paramagnetic pH-sensitive liposomes accumulated in the acidic environment within a tumour could undergo a structural change and release the contrast agent. If properly designed, these liposomes would exhibit a markedly increased contrast effect and, hence, function as “off-on” switches. Consequently, the tumour region may be visualised as a hyperintense area compared to surrounding healthy tissue. It is well established that long-circulating liposomes preferentially accumulate in the interstitial space of tumours, due to the “enhanced permeability and retention effect” (EPRE) [22]. Given this suitable pharmacokinetic feature, and the low interstitial pH within tumours, the potential of pH-responsive MR liposomes as an in vivo pH-meter/tumour marker was investigated. In the present work, the pH-dependent contrast properties of GdDTPABMA encapsulated within different types of pH-sensitive liposomes were assessed in various in vitro models by both relaxometry and MR imaging.

2. Materials and methods 2.1. Materials Gd diethylene pentaacetic acid bismethylamide (GdDTPA-BMA) was obtained from Nycomed Imaging AS, Oslo, Norway. Dipalmitoyl phosphatidyl ethanolamine (DPPE) was purchased from Sygena Ltd., Liestal, Switzerland. Dioleoyl phosphatidyl ethanolamine (DOPE) was supplied from Avanti Polar Lipids Inc., Alabaster, AL, USA. Oleic acid (OA) and glucose monohydrate were obtained from Fluka Chemie AG, Buchs, Switzerland. Palmitic acid (PA) was purchased from Merck, Hohenbrunn, Germany. Tris(hydroxymethyl)aminomethane (tris) and Triton X-100 were supplied from Sigma Chemical Co., St. Louis, MO, USA. Citric acid was supplied from Acros Organics, Geel, Belgium. All materials were used without further purification. 2.2. Preparation of liposomal GdDTPA-BMA Blends of the saturated lipids DPPE and PA and the unsaturated lipids DOPE and OA were employed for the liposome preparation, both in a 4/1 mole ratio between

phospholipid and fatty acid. The liposomes were prepared by the thin film hydration method [23]. Briefly, a 10/1 (v/v) chloroform/methanol solution of the lipids was rotary evaporated to dryness and the resulting film was further dried under vacuum over night. The lipids were hydrated with an aqueous solution containing 0.250 M GdDTPA-BMA and 0.05 M tris/hydrochloric acid (HCl) buffer (pH 8.4), giving a total lipid concentration of 25 mg/ml. The lipid dispersions were allowed to swell for two hours and were then subjected to three freeze-thaw cycles in methanol/dry ice and water, respectively. The liposomes were sized down by sequential extrusion (Lipex Extruder威, Lipex Biomembranes Inc., Vancouver, B.C., Canada) through polycarbonate filters (Millipore, Cork, Ireland) with pore sizes ranging from 2000 to 200 nm. The lipid hydration, thawing, swelling and extrusion procedures were performed at approximately 75°C for the saturated lipid mixture and at approximately 25°C for the unsaturated lipid mixture, well above the gel-to-liquid crystalline phase transition temperature (Tm) of the respective lipid membranes. Untrapped metal chelate was removed by dialysis (Spectra/Por威 membrane tubing MW cutoff 50000, Spectrum Medical Industries Inc., Houston, TX, USA) against isoosmotic and isoprotic glucose solution. 2.3. Physicochemical characterisation The characterisation was performed on dialysed liposomes (pH 8.4). The osmolality was determined by vapour phase osmometry (Wescor 5500 XR Vapor Pressure Osmometer, Wescor Inc., Logan, UT, USA). For the size measurements, liposomes were diluted with isoosmotic and isoprotic glucose solution. The intensity-weighted hydrodynamic liposome diameter was determined by photon correlation spectroscopy at a scattering angle of 90° and 25°C (Malvern PS/MW 4700, Malvern Instruments Ltd., Malvern, England). The width of the particle size distribution was expressed by the polydispersity index. The Tm of the liposomal membrane was measured by differential scanning calorimetry (DSC 7, Perkin Elmer Inc., Norwalk, CT, USA) at pH 8.4. The effective Gd concentration (Ceff) in the liposome preparations, defined as the Gd concentration in the total sample volume, was determined by inductively coupled plasma atomic emission spectrophotometry (ICPAES, Perkin Elmer Optima 3300 DV, Perkin Elmer Inc., Norwalk, CT, USA). For the determination of Ceff, liposomes were diluted with a 0.2% (v/v) solution of Triton X-100. Yttrium (III) was added to samples and standard solutions to perform simultaneous internal standardisation. The Gd concentration in the samples was determined using a multipoint standard calibration curve. pH measurements were performed with a calomel reference pH electrode (Cole-Parmer International, Vernon Hills, IL, USA) attached to a pH meter (Orion 250A, Orion Research Inc., Beverly, MA, USA).

K.-E. Løkling et al. / Magnetic Resonance Imaging 19 (2001) 731–738

2.4. Cryogenic transmission electron microscopy (Cryo-TEM) The DPPE/PA liposomal dispersion was diluted 3/1 and 1/1 (v/v) with isoosmotic buffer solutions (0.05 M citrate— phosphate buffer and 0.05 M tris—HCl buffer) at pH 4.2 and 8.4, respectively. The liposomal dispersion buffered at pH 4.2 was incubated at 37°C for 1 hour prior to the Cryo-TEM procedure, while the corresponding sample at pH 8.4 was assessed directly after dilution. The Cryo-TEM preparation procedure consisted, in short, of the following: The liposome dispersions were equilibrated at 25°C and 98 –99% relative humidity within a climate chamber, which comprised an improved version of a system described elsewhere [24]. A droplet (⬃2 ␮l) of sample dispersion was deposited on glow discharge treated 300 mesh copper grids (Agar Scientific Ltd, Stansted Essex, England) coated with a perforated carbon film. Excess liquid was blotted away with filter paper, followed by immediate plunging of the grid into liquid ethane held just above its freezing point. The vitrified sample was then transferred under protection against atmospheric conditions to a Zeiss EM 902A electron microscope (LEO Electron Microscopy, Oberkochen, Germany). The temperature of the specimen was kept below ⫺165°C during the whole process and all observations were made in zero loss brightfield mode and at an accelerating voltage of 80 kV. A minimum/micro-dose focussing system (MDF) was used to minimise radiation damage. The images were acquired by employing a cooled slow-scan CCD camera (Proscan GmbH, Scheuring, Germany), and analySIS software (Soft Imaging System GmbH, Mu¨nster, Germany). 2.5. In vitro relaxometry 2.5.1. pH-relaxivity profile The relaxation measurements were performed at 0.24 Tesla (T) (Minispec PC-110b, Bruker GmbH, Rheinstetten, Germany). The effect of pH on the T1 relaxivity (r1) was studied at 37°C for DPPE/PA liposomal GdDTPA-BMA, and at both 23 and 37°C for DOPE/OA liposomal GdDTPA-BMA. The T1 relaxation times were measured in different isoosmotic buffer solutions (0.05 M citrate—phosphate buffer and 0.05 M tris—HCl buffer) containing liposomes. The Ceff in the buffered liposomal dispersions was approximately 1 mM Gd. The investigated pH range was 4.3– 8.5. The buffered liposome samples were incubated at 23 or 37°C for 20 minutes prior to the relaxation measurements. Triplicates at each pH value were analysed. The T1 relaxation times were obtained by the inversion recovery (IR) method. The r1 at any pH value was obtained from the following relationship1: 1 A two-point relaxivity could be calculated due to the linear dependence of Robs on Ceff in the concentration range 0.3–2.5 mM Gd (data not 1 shown).

r1 ⫽



⫺R C eff

obs 1

m 1


⫺1 where Robs and Rm ) at a 1 1 are the T1 relaxation rates (s given pH value of the buffer-diluted liposomal sample and matrix (isoosmotic buffer-solution), respectively, and Ceff is the effective Gd concentration (mM). For comparative purposes, the pH-r1 dependency of non-liposomal GdDTPABMA was investigated.

2.5.2. Kinetics The time-dependent change in r1 of DPPE/PA liposomal GdDTPA-BMA was studied at three different pH values (4.7, 6.5 and 7.3) at 37°C. The buffer solutions and the liposome dispersions were preheated to 37°C before mixing. The T1 relaxation times were measured immediately and frequently during the first hour after mixing of buffer solution and liposome dispersion. The T1 relaxation times were then monitored less frequently for several weeks. The samples were kept at 37°C during the study. Duplicates at each pH value were analysed. 2.6. MR imaging The in vitro MR imaging efficacy of DPPE/PA liposomal GdDTPA-BMA was investigated in a gel phantom at 1.5 T on a Gyroscan ACS-NT system (Philips Medical Systems, Best, The Netherlands). The phantom was composed of thirteen glass vials (10 mm inner diameter) placed in a circular glass container. The latter was filled with an agar gel (2% w/v) doped with GdDTPA-BMA to give a T1 of about 900 ms at 1.5 T. The glass vials were filled with isoosmotic buffer solutions with pH values ranging from 4.8 to 8.2. The temperature of the phantom was essentially held constant at 37°C by circulating heated water through the shell of the reactor with a water pump. Liposomes were added successively to each vial (ascending pH) with a time interval of 1 minute. The imaging was initiated 45 minutes after addition of liposomes to the first vial. The phantom was placed in a quadratune knee coil and T1-weighted (T1-w) images were acquired using an IR-TSE pulse sequence (TR/TE/TI ⫽ 800/15/500 ms). Other scan parameters were: slice thickness ⫽ 7 mm, FoV ⫽ 230 ⫻ 230 mm2 and matrix ⫽ 256 ⫻ 256.

3. Results 3.1. Physicochemical characterisation The physicochemical properties of the dialysed liposome dispersions are summarised in Table 1. The Tm of DOPE and various DOPE-OA mixtures in buffer (pH 8.5) is reported to be ⫺11°C [25]. More specifically, no change in Tm was observed on addition of OA up to a mole ratio of 5/2.


K.-E. Løkling et al. / Magnetic Resonance Imaging 19 (2001) 731–738

Table 1 Physicochemical properties of liposomal GdDTPA-BMA Liposome composition

Experiment type

Liposome diameter (nm) Polydispersity Index

Transition temperature(°C)

Effective Gd concentration(mM)

Osmolality (mosmol/kg)


In vitro Relaxometry In vitro MR Imaging In vitro Relaxometry

121 ⫾ 1




124 ⫾ 2




155 ⫾ 2





63.4 ⫺11‡

From reference 25.

3.2. Cryogenic transmission electron microscopy (Cryo-TEM) Fig. 1A and B show Cryo-TEM micrographs of the DPPE/PA system at pH 8.4 and 4.2, respectively. Different dilution factors (1/1 and 3/1, respectively) were used at the two pH values in order to obtain micrographs with similar material content. A 1/1 diluted sample at pH 4.2 (micrograph not shown) gave a too high material content. Mainly

unilamellar liposomes were observed at pH 8.4, but other structures like deformed liposomes and disks were also present (Fig. 1A). Aggregated structures were evident at pH 4.2, along with bilayer-fragments and large bilayers (Fig. 1B). Fig. 1A is presumed to reflect a kinetically stable system, since the liposomal dispersion (pH 8.4) was diluted with isoprotic buffer solution. At pH 4.2 (Fig. 1B), however, the system was examined after 1 hour incubation at 37°C, and equilibrium may not have been fully reached. The white spots in the micrographs are irradiation-damaged areas. 3.3. In vitro relaxometry 3.3.1. pH-relaxivity profile The r1 of non-liposomal GdDTPA-BMA was unaffected by pH in the investigated pH interval with a mean value of 4.6 mM⫺1 s⫺1. Fig. 2 shows the pH-dependence of the r1 for DOPE/OA and DPPE/PA liposomal GdDTPA-BMA, given

Fig. 1. Cryo-electron micrographs of DPPE/PA liposomal GdDTPA-BMA at pH 8.4 (A) and after 60 minutes incubation at pH 4.2 (B). Note that a different dilution factor has been used in (A) and (B). Bar represents 200 nm.

Fig. 2. pH dependence of the r1 of DPPE/PA liposomal GdDTPA-BMA at 37°C (■), and DOPE/OA liposomal GdDTPA-BMA at 23°C (F) and 37°C (Œ) (0.24 T, 20 minutes incubation). Pooled standard deviation: 0.05, 0.07 and 0.13, respectively. The lines are for guidance only.

K.-E. Løkling et al. / Magnetic Resonance Imaging 19 (2001) 731–738


Fig. 3. Time evolution of the r1 of DPPE/PA liposomal GdDTPA-BMA at pH 4.7 (■), 6.5 (F) and 7.3 (Œ) (0.24 T, 37°C). Pooled standard deviation: 0.13, 0.05 and 0.07, respectively. The lines are for guidance only.

as the mean of triplicates. For the DOPE/OA system the r1 was relatively constant at 37°C (⬇4.6 mM⫺1 s⫺1) at pH values above 6.7. With decreasing pH, the r1 increased slightly before levelling off at about 5.2 mM⫺1 s⫺1 in the pH range 4.3 to 6.0. At 23°C the r1 of this system was also relatively invariable above pH 6.7 (⬇4.3 mM⫺1 s⫺1). When lowering the pH the r1 increased slightly and reached a plateau at about 5.7 mM⫺1 s⫺1 in the pH range 4.3– 6.0. The r1 of DPPE/PA liposomal GdDTPA-BMA was significantly reduced compared to that of non-liposomal GdDTPA-BMA at pH values above 7.3, and relatively constant (⬇0.6 mM⫺1 s⫺1). The r1 increased significantly by lowering the pH from 7.0 to 6.0, and reached a plateau at about 3.9 mM⫺1 s⫺1 at pH values below 5.5. The change in buffer type at pH 7.3 did not seem to affect significantly the pH-dependence of the r1 for DOPE/OA and DPPE/PA liposomal GdDTPABMA.

Fig. 4. T1-w image of phantom containing inserts with DPPE/PA liposomal GdDTPA-BMA 45 minutes after start of incubation (1.5 T, 37°C). The pH values within the liposomal dispersions are shown above each insert. The hyperintense spot to the left of the insert at pH 6.4 is due to the thermometer.

3.4. MR Imaging Fig. 4 shows the T1-w image of the phantom containing inserts of DPPE/PA liposomal GdDTPA-BMA 45 minutes after addition of liposomes to the first vial. The signal intensity (SI) within the liposome inserts was low at pH values between 7.1 and 8.2. The SI increased gradually by lowering the pH from 7.1 to 6.3, before levelling off at pH values below 6.3. The difference in SI at pH values above 7.1 could be explained by a minor temperature inhomogeneity in this region of the phantom. Images acquired at later timepoints (up to 60 minutes after liposome addition to the first vial) showed no significant difference in SI within the liposome inserts (images not shown).

3.3.2. Kinetics

4. Discussion

Fig. 3 shows the time-evolution of the r1 of DPPE/PA liposomal GdDTPA-BMA at three different pH values (4.7, 6.5 and 7.3) and 37°C, given as the mean of duplicates. At pH 4.7 the r1 increased sharply during the first hours and then levelled off, reaching the maximum relaxivity (⬇5.3 mM⫺1 s⫺1) after approximately 24 hours. At pH 6.5 the r1 also increased markedly during the first hours, although not to the same extent as the observed increase at pH 4.7. The r1 at pH 6.5 was still increasing after 25 days of incubation. At pH 7.3 the r1 increased slowly during the investigated time period. Only a minor increase in r1 was observed during the first hours of incubation.

pH-sensitive MR contrast agents could be designed by using the technology of pH-sensitive liposomes, the latter being well described in the literature [21]. The main constituent of these liposomes is unsaturated phosphatidylethanolamine (PE), which under normal physiological conditions has a high propensity to form the inverted hexagonal phase (HII) as shown in Fig. 5A [26]. Lamellar phase stabilisation (Fig. 5B), which is necessary for liposome formation, can for instance be achieved under these conditions by incorporation of a negatively charged, acidic amphiphile [21]. This charge prevents the HII phase formation, due to increased headgroup repulsion in the membrane and re-


K.-E. Løkling et al. / Magnetic Resonance Imaging 19 (2001) 731–738

Fig. 5. Schematic presentation of the inverted hexagonal HII (A) and the lamellar (B) phases. The lipid molecules are presented as a circle and two lines, representing the hydrophilic headgroup and the hydrophobic hydrocarbon chains, respectively.

duced interbilayer contact. Neutralisation of the acidic headgroups, caused by a reduction in pH, destabilises the liposomes as the PE converts to the HII phase, concomitant with release of encapsulated material to the surroundings. Some important criteria should be fulfilled if such pHsensitive liposomes are to be employed as “off-on” switches for MRI purposes. At physiological pH, the liposomal MR contrast agent must exhibit a low r1 due to slow water exchange between the liposome interior and exterior. Such an exchange limited dipolar relaxivity could be achieved if the water permeability of the membrane is low and the internal contrast agent concentration is high [27]. Upon conversion to the HII phase at lower pH, contrast material is released, increasing the relaxivity markedly. Under optimal conditions, the release must be immediate and quantitative, resulting in a r1 that is analogous to that of the agent in aqueous solution. PE based pH-sensitive liposomes reported in the literature are generally based on unsaturated or partly unsaturated PE, in combination with acidic amphiphiles. One of the best-described systems is composed of DOPE and OA [28]. The relaxometric properties of GdDTPA-BMA encapsulated within this unsaturated liposome system were first investigated at 37°C, well above the Tm of the liposomes. The r1 was expected to be at its maximum (i.e. fast water exchange) at all the measured pH values, due to the anticipated high fluidity and thereby high water permeability of

the liposomal membrane at elevated pH, and the quantitative release of Gd-chelate at low pH. A minor difference in r1 was however observed between low and high pH (Fig. 2). This finding could be due to a slight exchange limited dipolar relaxation enhancement, as indicated by the positive temperature dependence of r1 at high pH (Fig. 2) [27]. At low pH values, the higher r1 of the DOPE/OA system compared to that of non-liposomal GdDTPA-BMA (5.2 vs. 4.6 mM⫺1 s⫺1 at 37°C) could be explained by a higher microviscosity of the bulk. Furthermore, the negative temperature dependence of r1 at low pH values confirmed the release of GdDTPA-BMA to the medium. Regardless, this particular DOPE/OA system was found to be unsuitable for MRI purposes, due to the poor relaxometric pH response. Liposomes composed of the saturated lipids DPPE and PA were formulated to increase the membrane rigidity, thereby potentially obtaining a more pronounced “off-on” relaxivity switch. The DSC measurement of this system indicated that the liposomal membrane was in the lamellar gel state (L␤) at 37°C and pH 8.4. The membrane was rigid enough as to significantly reduce the r1 compared to that of non-liposomal Gd-chelate (Fig. 2). This low relaxivity is most likely due to an exchange limited relaxation enhancement. The r1 increased markedly when the pH was lowered below physiological pH, and reached a plateau of approximately 3.9 mM⫺1 s⫺1 at pH 5.1 after 20 minutes incubation at 37°C. The kinetic studies (Fig. 3) showed that the r1 increased further above this plateau level by prolonged incubation of the liposomal dispersion at low pH (4.7), and reached the maximum level (5.3 mM⫺1 s⫺1) after approximately 24 hours. At pH 6.5, the kinetics of the r1 increase was slower and the maximum r1 was not reached during the investigated time period. The liposome dispersion seemed to be quite stable at pH 7.3 and 37°C with respect to leakage. At lower temperature (25°C), preliminary results suggest slower kinetics of the relaxivity increase when the pH is lowered below physiological level. The mechanism(s) behind the pH-dependent relaxometric response of DPPE/PA liposomal GdDTPA-BMA is less clear. At pH values below physiological level the liposomal dispersions became unstable and massive aggregation was observed in the glass vials. After some time the associated structures precipitated. Relaxometry of the resulting supernatant confirmed that leakage of Gd-chelate had occurred during this process. The phase behaviour of a mixture of DPPE and PA is unknown, although the phase behaviour of pure DPPE is well characterised [26]. The phase behaviour of saturated and unsaturated PE is very different, exemplified by DPPE and DOPE, respectively. DPPE has a Tm of 63°C and a liquid crystalline (L␣) to HII phase transition temperature (Th) of 118°C, while the corresponding phase transition temperatures for DOPE are ⫺11°C and 10°C. In other words; at 37°C and physiological pH DOPE has a high propensity to form the HII phase, while DPPE spontaneously forms the L␤ phase. Therefore, it seems unlikely that

K.-E. Løkling et al. / Magnetic Resonance Imaging 19 (2001) 731–738

the postulated mechanism of the acid induced destabilisation of unsaturated pH-sensitive liposomes is applicable for the DPPE/PA system. The Cryo-TEM micrograph actually confirms the existence of lamellar phase after incubation in buffer at pH 4.2 (Fig. 1B). The micrograph shows that acid-induced aggregation had occurred, but also formation of bilayer-fragments and large bilayers. One or a combination of these processes resulted in leakage of contrast agent. A possible mechanism could be a collapse of the aggregated liposomes to bilayers, concomitant with release of encapsulated GdDTPA-BMA. The relatively slow conversion from aggregated liposomes to the lamellar sheets might explain the time-dependent increase in r1 at sub-physiological pH values, and that the kinetics of the relaxivity increase are faster at lower pH values. Another factor that can contribute to this time dependency is the intra-liposomal buffer capacity of tris/HCl (0.05 M). This buffer could slow down the equalisation of pH across the liposomal membrane, and affect the phase behaviour of the system. The in vitro imaging study confirmed the potential of pH-sensitive paramagnetic liposomes for pH monitoring (Fig. 4). A good correlation was observed between the relaxometric and T1-w imaging results. However, the utilisation of such a liposomal system in vivo is much more complex. First, the pH-sensitivity of the system could be altered and even diminished in vivo, a phenomenon that has previously been reported for DOPE/OA liposomes [29]. Secondly, the liposomal approach would only succeed if the liposomal agent accumulates in the tumour region at a sufficiently high enough concentration. Sterically stabilised or/and small liposomes should therefore be considered. Such liposomes display minimised uptake by the mononuclear phagocyte system and prolonged circulation time in the blood stream, which in turn increases the probability of tumour extravasation. In fact, polyethylene glycol (PEG) grafted liposomes have been successfully applied for selective delivery of cytostatics to tumours [30]. Sterical stabilisation of the DPPE/PA system could influence the pHsensitivity of the liposomes, especially if an aggregation step is involved in the process. This aspect remains to be investigated. Regardless, even if both the pH-relaxometric response of such liposome systems is retained in vivo and the pharmacokinetics are suitable, their application as a pH-meter may be challenging. For example, it is evident that during a tumour extravasation process the liposomes would be subjected to the acidic environment for different time periods. And, since the relaxometric pH-response of the DPPE/PA system is both pH- and time-dependent, a quantitative in vivo assessment of pH seems unlikely. However, the DPPE/PA system still has a potential as a marker of low pH in tumours (“off-on” switch). On a T1-w image, the tumour area would appear as a hyperintense region compared to healthy tissue, due to the accumulation of liposomes and subsequent acid-induced leakage of contrast agent. Finally, compared to the previously mentioned “off-on”


switches currently evaluated, the liposome approach may be more advantageous. For instance, a relaxivity increase from 0.6 to about 4 mM⫺1 s⫺1, as obtained in this study, should be better than an increase from about 3 to 8 mM⫺1 s⫺1 [15]. More importantly, the ability to target tumour should be higher for liposomes compared to a LMW Gd chelate that undergoes fast renal elimination.

6. Conclusion In the present work, the pH-dependence of the r1 for DOPE/OA and DPPE/PA liposomal GdDTPA-BMA was investigated in vitro in various models. The membrane fluidity was important for the pH-dependent relaxometric effect of these liposomes. The paramagnetic DOPE/OA system had a poor pH-r1 response, most likely due to the high water permeability of the liquid crystalline membrane at pH values above physiological level. This system was therefore found to be unsuitable for MRI purposes. The use of a gel-phase DPPE/PA membrane resulted in a strong pH-dependent relaxometric response. At high pH, the r1 was low due to exchange limitations but increased substantially when lowering the pH below physiological level. The CryoTEM results of the DPPE/PA system at low pH indicated a different phase behaviour than that of the well-known DOPE/OA system. The MR imaging study of DPPE/PA liposomal GdDTPA-BMA showed its ability as a probe for in vitro pH mapping. However, the current use of this liposome formulation requires further investigations to fully evaluate its potential as a tumour agent.

Acknowledgments The authors thank Åse J. Korsmo and Lars Johansson (both Nycomed Imaging AS, Oslo, Norway) for providing technical assistance and Go¨ran Karlsson (Department of Physical Chemistry, University of Uppsala, Sweden) for performing the Cry-TEM.

References [1] Stefanidis C, Dimantopoulos L, Vlachopoulos C, Tsiamis E, Dernellis J, Toutouzas K, Stefanadi E, Toutouzas P. Thermal heterogeneity within human atherosclerotic coronary arteries detected in vivo. A new method of detection by application of a special thermography catheter. Circulation 1999;99:1965–71. [2] Bates SE. Clinical applications of serum tumor markers. Ann Intern Med 1991;115:623–38. [3] Wike-Hooley JL, Haveman J, Reinhold HS. The relevance of tumour pH to the treatment of malignant disease. Radiother Oncol 1984;2: 343– 66. [4] Gerveck LE. Tumor pH: implications for treatment and novel drug design. Semin Radiat Oncol 1998;8:176 – 82. [5] Tannock IF, Rotin D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res 1989;49:4373– 84.


K.-E. Løkling et al. / Magnetic Resonance Imaging 19 (2001) 731–738

[6] Mikkelsen RB, Asher C, Hicks T. Extracellular pH, transmembrane distribution and cytotoxicity of chlorambucil. Biochem Pharmacol 1985;34:2531– 4. [7] Tietze LF, Neumann M, Mo¨llers T, Fischer R, Glu¨senkamp K-H, Rajewsky MF, Ja¨hde E. Proton-mediated liberation of aldophosphamide from a nontoxic prodrug: a strategy for tumor-selective activation of cytocidal drugs. Cancer Res 1989;49:4179 – 84. [8] Lavie E, Hirschberg DL, Schreiber G, Thor K, Hill L, Hellstrom I, Hellstrom K-E. Monoclonal antibody L6-daunomycin conjugates constructed to release free drug at the lower pH of tumor tissue. Cancer Immunol Immunother 1991;33:223–30. [9] Gillies RJ, Liu Z, Bhujwalla Z. 31P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate. Am J Physiol 1994;267:C195–203. [10] Hunjan S, Mason RP, Mehta VD, Kulkami PV, Aravind S, Arora V, Antich PP. Simultaneous intracellular and extracellular pH measurement in the heart by 19F NMR of 6-fluoropyridoxol. Magn Reson Med 1998;39:551– 6. [11] van Sluis R, Bhujwalla ZM, Raghunand N, Ballesteros P, Alvarez J, Cerda´n S, Galons J-P, Gillies RJ. In vivo imaging of extracellular pH using 1H MRSI. Magn Reson Med 1999;41:743–50. [12] Rocklage SM, Watson AD, Carvlin MJ. Contrast agents in magnetic resonance imaging. In: Stark DD, Bradley WG. Magnetic resonance imaging, Vol. 1. Mosby-Year Book, 1992. p. 372– 437. [13] Ni Y, Marchal G. Enhanced magnetic resonance imaging for tissue characterization of liver abnormalities with hepatobiliary contrast agents: an overview of preclinical animal experiments. Top Magn Reson Imaging 1998;9:183–95. [14] Aime S, Barge A, Botta M, Howard JAK, Kataky R, Lowe MP, Moloney JM, Parker D, de Sousa AS. Dependence of the relaxivity and luminescence of gadolinium and europium amino-acid complexes on hydrogencarbonate and pH. Chem Commun 1999:1047– 8. [15] Mikawa M, Miwa N, Bra¨utigam M, Akaike T, Maruyama A. Gd3⫹loaded polyion complex for pH depiction with magnetic resonance imaging. J Biomed Mater Res 2000;49:390 –5. [16] Mikawa M, Miwa N, Akaike T, Maruyama A. An intelligent MRI contrast agent for tumor sensing. In: proceedings of the 26th International Symposium on Controlled Release of Bioactive Materials. Boston, (MA): CRS, 1999, p. 1158 –9. [17] Kost J, Langer R. Responsive polymer systems for controlled delivery of therapy. Trends Biotechnol 1992;10:127–31.

[18] Serres A, Baudys M, Kim SW. Temperature and pH-sensitive polymers for human calcitonin delivery. Pharm Res 1996;13:196 –201. [19] Nakamura K, Maitani Y, Lowman AM, Takayama K, Peppas NA. Uptake and release of budesonide from mucoadhesive, pH-sensitive copolymers and their application to nasal delivery. J Control Release 1999;61:329 –35. [20] Couvreur P, Fattal E, Malvy C, Dubernet C. pH-sensitive liposomes: an intelligent system for the delivery of antisense oligonucleotides. J Liposome Res 1997;7:1–18. [21] Litzinger DC, Huang L. Phosphatidylethanolamine liposomes: drug delivery, gene transfer and immunodiagnostic applications. Biochim Biophys Acta 1992;1113:201–27. [22] Drummond DC, Meyer O, Hong K, Kirpotin DB, Papahadjopoulos D. Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev 1999;51:691–743. [23] Lasic DD. Preparation of liposomes. In: Lasic, D.D., editor. Liposomes from Physics to Applications. Amsterdam: Elsevier Science Publishers B.V., The Netherlands, 1993. p. 67–73. [24] Bellare JR, Davis HT, Scriven LE, Talmon Y. Controlled environment vitrification system: an improved sample preparation technique. J Electron Microsc Tech 1988;10:87–111. [25] De Oliveira MC, Fattal E, Couvreur P, Lesieur P, Bourgaux C, Ollivon M, Dubernet C. pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in quasi-anhydrous samples. Biochim Biophys Acta 1998;1372:301–10. [26] Koynova R, Caffrey M. Phases and phase transitions of the hydrated phosphatidylethanolamines. Chem Phys Lipids 1994;69:1–34. [27] Fossheim SL, Fahlvik AK, Klaveness J, Muller RN. Paramagnetic liposomes as MRI contrast agents: influence of liposomal physicochemical properties on the in vitro relaxivity. Magn Reson Imaging 1999;17:83–9. [28] Hazemoto N, Harada M, Komatsubara N, Haga M, Kato Y. pHsensitive liposomes composed of phosphatidylethanolamine and fatty acid. Chem Pharm Bull (Tokyo) 1990;38:748 –51. [29] Liu DX, Huang L. Small, but not large, unilamellar liposomes composed of dioleoylphosphatidylethanolamine and oleic acid can be stabilized by human plasma. Biochemistry 1989;28:7700 –7. [30] Gabizon A, Martin F. Polyethylene glycol-coated (pegylated) liposomal doxorubicin. Rationale for use in solid tumours. Drugs 1997;54: 15–21.

Lihat lebih banyak...


Copyright © 2017 DADOSPDF Inc.