Bioconjugate Chem. 2004, 15, 799−806
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A Liposomal System for Contrast-Enhanced Magnetic Resonance Imaging of Molecular Targets Willem J. M. Mulder,*,† Gustav J. Strijkers,† Arjan W. Griffioen,‡ Louis van Bloois,§ Grietje Molema,| Gert Storm,§ Gerben A. Koning,§,⊥ and Klaas Nicolay† Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. Angiogenesis Laboratory, Department of Pathology/ Internal Medicine, Maastricht University & University Hospital, P.O. Box 5800, 6225 EH Maastricht, The Netherlands, Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, UIPS, TB 3508, Utrecht, The Netherlands, Department of Pathology and Laboratory Medicine, Medical Biology Section, Groningen University, P.O. Box 30001, 9700 RB Groningen, The Netherlands, and Department of Radiochemistry, Interfaculty Reactor Institute, Delft University of Technology, Mekelweg 15, 2629 JB Delft, The Netherlands. Received March 3, 2004; Revised Manuscript Received April 29, 2004
Pegylated paramagnetic and fluorescent immunoliposomes were designed to enable the parallel detection of the induced expression of molecular markers on endothelial cells with magnetic resonance imaging (MRI) and fluorescence microscopy. MRI is capable of three-dimensional noninvasive imaging of opaque tissues at near cellular resolution, while fluorescence microscopy can be used to investigate processes at the subcellular level. As a model for the expression of a molecular marker, human umbilical vein endothelial cells (HUVEC) were treated with the pro-inflammatory cytokine tumor necrosis factor R (TNFR) to upregulate the expression of the adhesion molecule E-selectin/CD62E. E-selectinexpressing HUVEC were incubated with pegylated paramagnetic fluorescently labeled liposomes carrying anti-E-selectin monoclonal antibody as a targeting ligand. Both MRI and fluorescence microscopy revealed the specific association of the liposomal MR contrast agent with stimulated HUVEC. This study suggests that this newly developed system may serve as a useful diagnostic tool to investigate pathological processes in vivo with MRI.
INTRODUCTION
A challenge in diagnostics is noninvasive detection of molecular markers of disease in order to identify and visualize pathology in an early stage and to be able to follow the effect of therapy (1). Traditional immunohistochemical techniques can only be applied after invasive procedures, which excludes their use for in vivo detection and thus makes longitudinal studies of disease progression impossible. In recent years, magnetic resonance imaging (MRI) has emerged as one of the leading noninvasive imaging modalities (2). With MRI it is possible to measure the morphology and function of healthy and diseased soft tissues in vivo by exploiting a remarkable range of physical and chemical properties of water protons, including the T1 relaxation time. MRI, however, suffers from limited resolution and specificity, compared to optical and traditional staining techniques. In contrast to nuclear tracer methods such as PET or SPECT, MRI has a relatively low inherent sensitivity (3). A number of approaches can be taken to overcome these limitations. First, specific information on molecular markers can be obtained with the use of targeted contrast agents, which are directed to a molecular entity of interest, e.g. an * To whom correspondence should be addressed. Telephone: (+31)402474853.Fax: (+31)402432598.E-mail:
[email protected]. † Eindhoven University of Technology. ‡ Maastricht University & University Hospital. § Utrecht Institute for Pharmaceutical Sciences. | Groningen University. ⊥ Delft University of Technology.
endothelial cell surface receptor that is overexpressed as a consequence of a disease process. Second, the low inherent sensitivity of MRI requires that the targeted contrast agent is very effective in producing a local MRI signal change. This can be achieved by the use of colloidal particles that can accommodate large amounts of Gdchelates (4-6), which are widely used for contrastenhanced T1-weighted MRI. Additional information can be obtained by labeling such particles with markers for other imaging modalities, such as fluorescence imaging (7), for multimodality characterization of (patho)physiological processes. In this paper, we report on the development and application of targeted liposomes that carry a high payload of MRI contrast agent. Liposomes have been studied extensively during the last two decades as drug carrier vehicles (8-11). For that purpose, liposomes have previously been optimized in terms of their stability, circulation time in vivo, and membrane composition (11). Liposomes are biocompatible, they are composed of either natural or synthetic amphiphilic lipids, they can be sized to a defined diameter in the range of 50 to 500 nm, and they can be coated with polymers to increase stability and to prolong circulation half-life in vivo (12). A wide range of amphiphilic and hydrophobic molecules can be incorporated in the bilayer of the liposomes. The aqueous interior can be loaded with water soluble drugs, proteins, or DNA for therapeutic applications (13). Another potential application is the use of liposomes as a (targetable) contrast agent for MRI (4, 5, 14-16). The goal of this study was to develop paramagnetic pegylated liposomes with a targeting ligand coupled to
10.1021/bc049949r CCC: $27.50 © 2004 American Chemical Society Published on Web 06/15/2004
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Figure 1. Schematic representation of a pegylated paramagnetic liposome that is composed of Gd-DTPA-bis(stearylamide) (GdBSA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(poly(ethylene glycol))-2000] (PEG2000-DSPE) and has antibodies coupled to the distal end of the PEG-chains.
the distal end of the PEG-chains for contrast-enhanced MR imaging of molecular markers (Figure 1). Gd-DTPAbis(sterylamide) (Gd-BSA) was incorporated in the bilayer of the liposomes as a paramagnetic MRI contrast agent. We have used the antibody-conjugated liposomal system to detect E-selectin expression on HUVEC in vitro. This is an attractive model for receptor expression on endothelial cells, since E-selectin levels can be upregulated approximately 10-fold by TNFR (17-19). Eselectin can be considered a model system for a variety of endothelial cell surface receptors that are of pathophysiological and therapeutic importance, including Rvβ3integrins (20, 21) and vascular endothelial growth factor receptor (22, 23). We show that the association to TNFR stimulated HUVEC was specific for liposomes carrying E-selectin antibodies. The antibody-dependent liposomal association was accompanied by a pronounced shortening of the T1 relaxation time of HUVEC suspensions as evidenced by MRI. MATERIALS AND METHODS
Materials. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(poly(ethylene glycol))2000] (PEG2000-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(poly(ethylene glycol))2000] (Mal-PEG2000-DSPE), and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) were obtained from Avanti Polar Lipids (Albaster, AL). Gd-DTPA-bis(stearylamide) (GdBSA) was purchased from Gateway Chemical Technology (St. Louis, MO). N-Succinimidyl S-acetylthioacetate (SATA) and calcein were obtained from Sigma Chemical Co. (St. Louis, MO), and HEPES was obtained from Merck (Darmstadt, Germany). All other chemicals were of analytic grade or the best grade available. Polycarbonate filters for liposome extrusion were from Costar (Cambridge, MA). H18/7 monoclonal antibody against human E-selectin, kindly donated by Dr. M. A. Gimbrone (Boston, MA), was isolated from hybridoma culture supernatant using protein A affinity chromatography according to standard procedures. Preparation of Paramagnetic PEG Liposomes. Liposomes were prepared by lipid film hydration. A
mixture of the appropriate amounts of lipids (typically: 120 µmol of total lipid) was dissolved in chloroform/ methanol 1:1 (v/v) and evaporated to dryness by rotary evaporation at 40 °C. For the paramagnetic PEG-liposomes, Gd-BSA, DSPC, cholesterol, and PEG2000-DSPE were used at a molar ratio of 0.75/1.10/1/0.15. For PEGliposomes that were used to attach antibodies to the distal end of the PEG-chains, Gd-BSA, DSPC, cholesterol, PEG2000-DSPE, and Mal-PEG2000-DSPE were mixed at a molar ratio of 0.75/1.10/1/0.075/0.075. For fluorescence microscopy, 0.1 mol % of rhodamine-PE was added to these preparations. The lipid film was subsequently hydrated in 3 mL of HEPES-buffered saline (HBS), containing 20 mM HEPES, 135 mM NaCl (pH 6.5). The resulting lipid dispersion was extruded sequentially six times through polycarbonate membrane filters with a pore diameter of 400 nm and subsequently 10 times through filters with a pore diameter of 100 nm using a Lipofast Extruder (Avestin, Canada). The temperature during extrusion was 60 °C. Coupling of the Antibodies to Liposomes. The monoclonal antibody H18/7 against human E-selectin (AbEsel) was coupled to liposomes containing MalPEG2000-DSPE by a sulfhydryl-maleimide coupling method as described previously (18, 24). In short, H18/7 monoclonal antibody (1 mg/mL) was modified with N-succinimidyl S-acetylthioacetate (8:1 SATA:antibody mole:mole ratio) and incubated for 45 min at a roller-bench at room temperature. Free SATA was separated from the antibody/ SATA solution by centrifugation on Vivaspin concentrator with a 50 kD MW cut-off filter followed by thorough washing four times with HBS. The SATA-derivatized antibody was deacetylated by incubation with a hydroxylamine solution for 1 h at room temperature. The activated antibody was added to the Mal-PEG2000-DSPEcontaining liposomes in a 50 µg/µmol protein/lipid ratio. This preparation was stored at 4 °C under N2 overnight. Uncoupled antibody was separated from immunoliposomes by centrifugating two times at 65 000 rpm for 45 min. The supernatant was removed and an appropriate amount of buffer, typically 1.5 mL, was added to the pellet to obtain a lipid suspension with a lipid concentration of approximately 20 mM. The final liposomal suspension was stored at 4 °C under N2 and used within 7 days.
A Liposomal System for MR Imaging of Molecular Targets
Liposome Characterization. Size, Lipid/Protein Ratio. The size and size distribution of the bare liposomes and the antibody-coupled liposomes were determined by dynamic light scattering (DLS) at 25 °C with a Malvern 4700 system using an argon-ion laser (488 nm) operating at 10.4 mW (Uniphase) and PCS (photon correlation spectrometry) software for Windows version 1.34 (Malvern, U.K.). For data analysis, the viscosity and refractive index of water was used. The system was calibrated with a polystyrene dispersion containing particles of 100 nm. The phospholipid content of the liposome preparations was determined by phosphate analysis according to Rouser (25) after destruction with perchloric acid, and the protein content was determined according to Petterson (26). Efficiency of Gd-BSA Incorporation. The efficiency of Gd-BSA incorporation in the liposomal bilayer was determined as a function of the Gd-BSA fraction in the lipid mixture. Lipid films of Gd-BSA/DSPC/Chol/MalPEG2000-DSPE with 10, 20, 30, and 50 mol % Gd-BSA were prepared by dissolving the lipids in chloroform/ methanol (1:1), followed by evaporation to dryness. The combined Gd-BSA and DSPC content was kept constant at 62 mol %, while the molar ratios of cholesterol and Mal-PEG2000-DSPE were fixed at 33 and 5 mol %, respectively. The lipid film was subsequently hydrated in 3 mL of HBS and the phosphate and gadolinium content were determined. The resulting lipid dispersion was extruded as described above, after which the phosphate and gadolinium content were again determined. The extruded liposomes were centrifuged at 65 000 rpm for 45 min, and the phosphate and gadolinium content of the pellet and the supernatant were once more determined. Phosphate determinations were done according to Rouser (25) and gadolinium content was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Membrane Stability of the Liposomes. Calcein has been used as a fluorescent marker to investigate membrane permeability (27) and its release profile from the liposomal lumen reflects membrane stability. Calcein containing DSPC/Chol/Mal-PEG2000-DSPE liposomes and calcein containing Gd-BSA/DSPC/Chol/Mal-PEG2000-DSPE (25 mol % Gd-BSA) liposomes were prepared as described above by hydration with 50 mM calcein/150 mM sucrose/ 10 mM Hepes buffer (pH 7.4). The prepared liposomes were dialyzed at 4 °C for 4 days with phosphate-buffered saline (PBS, pH 7.4) to remove the untrapped calcein. The size and the lipid/calcein ratio were determined. Following dialysis both formulations were tested for the release of calcein upon incubation in 10% calf serum at 37 °C for 24 h. Calcein leakage from the aqueous interior of the liposomes was determined fluorometrically at excitation and emission wavelengths of 485 and 512 nm, respectively, using a fluorescence spectrophotometer. After incubation liposome size was determined again. T1 Relaxation Time Measurements. An important quality criterion for MRI contrast agents is their molar relaxivity r1 (in units of mM-1 s-1). A higher r1 will lower the detection threshold of local contrast agent deposition with T1-weighted MRI. MR spectroscopic measurements of the T1 relaxation times of 0.5 mL of buffered liposome suspensions (leading to final concentrations of 7.1, 3.6, 1.8, and 0.9 mM gadolinium) and Gd-DTPA solutions (5.0, 2.5, 1.3, and 0.6 mM gadolinium) were performed using a 7 T MR spectrometer (Varian, Palo Alto, CA). T1 relaxation times were obtained by the inversion recovery method, using 13 different inversion times, ranging from 10 to 3100 ms in an exponential fashion, and a repetition
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time of 10 s. To determine the temperature dependence of T1, the temperature was varied between 25 and 80 °C, using steps of 5 °C and an equilibration time of 10 min. For comparison the T1 relaxation times of 2 mL of buffered liposome suspensions (7.1, 3.6, and 0.9 mM gadolinium) were also determined at 1.5 T, a field strength frequently used in clinical MRI, using a Philips S15/ACS whole-body MR scanner. The molar relaxivity r1 was obtained from the slope of the linear fit of the inverse of the measured T1-values as a function of the gadolinium concentration (28). In Vitro Targeting Experiments. Human umbilical vein-derived endothelial cells (HUVEC) were cultured on gelatin (0.2% in PBS)-coated tissue culture flasks (Costar, Cambridge, MA) in culture medium, RPMI-1640 (Life Technologies, Breda, The Netherlands), 20% human serum (HS; University Hospital Maastricht, The Netherlands), 2 mM glutamine (Life Technologies), and 100 U/mL penicillin and 0.1 mg/mL streptomycin (ICN Biomedicals, Aurora, OH). For all incubations, 106 cells of passage 2-3 were used for experiments. Association of paramagnetic AbEsel-immunoliposomes (25 mol % Gd-BSA) to endothelial cells was assessed using fluorescence microscopy and magnetic resonance imaging. For this, HUVEC were grown to 70-80% of confluence in tissue culture flasks and stimulated for 3 h with 4 ng/mL human recombinant TNFR (rhTNFR; Boehringer, Germany). The effect of the stimulation was verified by FACS analysis of E-selectin expression (17, 29) and by visual verification of the change in cell shape. Control cells were left untreated. Subsequently, paramagnetic AbEsel-immunoliposomes and bare paramagnetic liposomes were added to control and TNFR-treated cells (1000 nmol lipid/mL medium). After 3.5 h of incubation with liposomes, the cultures were washed two times with PBS, and cells were harvested by incubation in EDTA and a mild trypsinization procedure. Cells and medium were collected in tubes and were washed twice in PBS. A small amount of cells (104) was taken to assess the association of liposomes using fluorescence microscopy. The obtained pellets, 106 ( 105 cells, were put in small eppendorf-cups, and 40 µL of 4% paraformaldehyde solution was added to a final volume of approximately 50 µL. Next, the samples were used for MR imaging. Magnetic Resonance Imaging of Cell Pellets. MRI was performed on pellets of HUVEC, using a 6.3 T horizontal 9.5 cm bore magnet. A 3 cm send and receive birdcage coil was used. In all imaging experiments the samples described above were placed in a custom-made sample holder, capable of carrying four eppendorf-cups. A T1-weighted spin-echo sequence (TR 1200 ms, TE 9 ms) was used for imaging. For absolute quantification of T1, an inversion recovery spin-echo sequence was used with 13 different inversion times, ranging from 10 to 3100 ms in an exponential fashion. For quantification of T2 a spin-echo sequence was used with different echo times, ranging from 9 to 64 ms. In all experiments the FOV was 3 × 3 cm2, matrix size was 256 × 128, and the slice thickness was 1 mm. From the images, a T1-map or a T2-map was calculated using Mathematica (Wolfram Research, Inc.). T1 and T2 of the different cell pellets were determined in regions-of-interest and averaged for three independent experiments. Statistical Analysis. Values for T1, obtained from the pellets of HUVEC as described above were analyzed using multiple t-tests (p < 0.05) with correction for multiple comparisons (30).
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RESULTS
Characterization of the Paramagnetic Liposomes. The mean size of a typical batch of liposomes before coupling of the AbEsel-antibody was 120 nm with a polydispersity index of 0.05. After the antibody was coupled, the mean size of the liposomes increased to 150 nm and the polydispersity index increased to 0.2. The initial coupling started with 50 µg protein/µmol lipid. The final protein-to-lipid ratio was 25 µg protein/µmol lipid, corresponding to a 50% efficiency. This corresponds to an estimated number of 30 antibody molecules per liposome. First, we established the maximal amount of Gd-BSA that could be incorporated in the liposomal bilayer. From a standard DSPC/Chol/PEG-DSPE 1.85/1/0.15 formulation an increasing amount of DSPC was replaced by GdBSA. When DSPC was replaced by 10, 20, or 30 mol % Gd-BSA (percentage of the total amount of lipid), all GdBSA did incorporate into the bilayer, and the phosphate/ Gd ratio before and after extrusion of the liposomal dispersion was identical. Higher percentages of Gd-BSA resulted in a decrease of the incorporation efficiency: at 50 mol % Gd-BSA, the Gd-to-total lipid ratio in the liposomal suspension after extrusion was decreased from an expected value of 50 mol % to a value of 40 mol %. Incorporation of a large amount of Gd-DTPA-bis(stearylamide) (Gd-BSA) might affect the stability of the liposomes. Therefore, the stability of liposomes with a Gd-BSA/DSPC/Chol/PEG (25 mol % Gd-BSA) formulation was compared with a DSPC/Chol/PEG formulation by enclosing calcein in the aqueous lumen and monitoring the release of calcein upon incubation in serum. Both formulations did not show a measurable release of calcein upon incubation at 4 °C in buffer for 24 h. Upon incubation of the liposomes in 10% calf serum buffer at 37 °C for 24 h, both formulations showed a calcein release of 4-6%. The size of both liposome preparations remained the same after the 24 h incubation in serum, as determined from dynamic light scattering data. Based on the above data, it was decided to use a molar percentage of 25% Gd-BSA in the E-selectin targeting studies on HUVEC cells. Since the Gd-BSA-containing liposomes are to be used in contrast-enhanced T1-weighted MRI, it is critical to determine the efficiency with which the paramagnetic particles shorten the T1 relaxation time of water. This efficiency is conveniently expressed as the molar relaxivity, r1, in units mM-1 s-1, which is determined from the dependence of the measured T1 relaxation rate on the contrast agent concentration. The longitudinal relaxivity r1 of a buffered liposome suspension was determined at 7 T as a function of temperature and was compared with the r1 of free Gd-DTPA that is in clinical use (Figure 2). At 37 °C, the r1 of Gd-BSA contained in the liposomal membrane was 1.5 times that of the solution with free Gd-DTPA. The r1 of Gd-DTPA decreased with increasing temperature. For the liposomal Gd-BSA suspension, r1 increased up to 55 °C, while decreasing above 55 °C. The r1 of the liposome suspension as measured at 1.5 T was 5.5 mM-1 s-1 at room temperature, which is similar to the r1 at 7 T at room temperature (5.2 mM-1 s-1). In Vitro Targeting Experiments. Treatment of endothelial cells with TNFR leads to activation and overexpression of adhesion molecules involved in leukocyte interactions. When HUVEC were treated with 4 ng/ mL TNFR, next to a morphological change, E-selectin protein expression increased 10-fold as deduced from FACS analysis (data not shown). Fluorescence micros-
Figure 2. The molar relaxivities r1 of free Gd-DTPA (O) and Gd-BSA containing liposomes (9) were determined as function of the temperature at a field strength of 7 T. The Gd-DTPA concentration was varied between 0.6 and 5.0 mM. The Gd-BSA content of the liposomes was fixed at 25 mol %, while the effective Gd-BSA concentration in the medium was varied from 0.9 to 7.1 mM Gd-BSA to determine r1. The lines connecting the data points are aimed to guide the eye.
copy showed a pronounced association of the AbEselimmunoliposomes to TNFR-treated HUVEC (Figure 3B). The association of the AbEsel-immunoliposomes by the HUVEC was partly due to internalization of the liposomes into the cytosol (insert Figure 3B). Nontargeted liposomes did not associate with either resting (data not shown) or TNFR-activated HUVEC (Figure 3C). For MR imaging, HUVEC treated with TNFR and incubated with AbEsel-immunoliposomes were compared with three controls. Figure 4 shows a schematic representation of these experiments. A T1-weighted image, a T1-map, and a T2-map of a typical experiment are depicted in Figure 5. The average T1 and T2 of different systems are listed in Table 1. In the T1-weighted image (Figure 5A) the region corresponding with the test incubation, TNFR-treated HUVEC incubated with AbEsel-immunoliposomes, was much brighter than the three control incubations, indicating that there is a distinct difference in T1 of the test incubation compared to the controls. This difference was quantified by generating a T1-map of the setup (Figure 5B) with an inversion recovery experiment and averaging the T1s found in three independent experiments (Table 1). The T1 of the pellet of stimulated HUVEC incubated with AbEsel-immunoliposomes was 457 ( 62 ms, which is almost a factor 5 lower than the T1 of the nonstimulated cells incubated with AbEsel-immunoliposomes (T1 of 2156 ( 130 ms). The stimulated cells that were incubated with nontargeted liposomes had a T1, which was comparable with the T1 of nonstimulated cells incubated with AbEselimmunoliposomes: both T1s were approximately 2150 ms. The pellet of control HUVEC had a T1 of 2412 ( 122 ms, which was significantly longer than all other three systems. The same trend was observed for the T2 values (Figure 5D, Table 1). The association of AbEsel-immunoliposomes with TNFR-activated HUVEC led to a decrease in the T2 relaxation time from 103 ( 12 to 16 ( 1 ms (Table 1). The other incubations of paramagnetic liposomes with HUVEC did not cause significant alterations in the T2 of water.
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Figure 3. Fluorescence microscopy of liposomal targeting to HUVEC. (A) Fluorescence microscopy image of nonstimulated HUVEC that were incubated for 3.5 h with paramagnetic AbEsel-immunoliposomes showed very little fluorescence. (B) TNFR-treated HUVEC showed massive association of paramagnetic AbEsel-immunoliposomes. Insert in B demonstrates that the fluorescence partly originates from the cell interior (magnification 630 ×). (C) TNFR-treated HUVEC incubated with nontargeted liposomes. (D) Control HUVEC that were not incubated with liposomes.
Figure 4. Schematic representation of the setup used for MRI analysis of liposomal targeting to HUVEC. The left side of the figure schematically depicts the setup of the experiment, in which B is the test incubation (TNFR-treated HUVEC incubated with AbEselimmunoliposomes). A (non-TNFR-treated HUVEC incubated with AbEsel-immunoliposomes), C (TNFR-treated HUVEC incubated with bare liposomes), and D (HUVEC) are controls. The right side of the figure gives the setup of the pellets of HUVEC used for MR imaging. DISCUSSION
The in vivo upregulation of receptors expressed on vascular endothelial cells at diseased sites represents an attractive target for early detection of a range of important disease processes (31). In inflamed or angiogenic tissues, such as tumors, atherosclerotic plaques, or rheumatic joints, blood vessels express high and upregulated levels of activation-related receptors. E-selectin, a molecule involved in leukocyte vessel wall interactions, is profoundly upregulated in tissues exposed to inflammatory cytokines (32). Detection of such receptors can be of prime importance in diagnosis of these diseases at an early stage. An established in vitro system for endot-
helial cells, which allows the induction of the typical inflammation related adhesion molecule E-selectin has been described previously (18, 19, 33, 34). In these studies endothelial cells (HUVEC) were stimulated by cytokines such as IL-1β or TNFR, which resulted in a strong upregulation of E-selectin. Particles carrying antibodies against E-selectin are capable of specific binding to HUVEC-expressing E-selectin and can be internalized by these cells. Previous studies have shown an efficient association of AbEsel-immunoliposomes targeted to Eselectin, using the H18/7 antibody as a targeting ligand, by activated endothelial cells in vitro (18, 19). The use of an H18/7 Fab-coupled iron oxide particle to target
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Figure 5. MR image of a typical experiment shows a slice through four pellets of HUVEC of the setup schematically depicted in Figure 4. The test incubation (B) showed enhanced signal intensity in the T1-weighted image (TR 1200 ms, TE 9 ms). The T1-map and T2-map, generated as described in Materials and Methods, revealed a dramatic shortening of both T1 and T2 of the stimulated cells incubated with paramagnetic AbEsel-immunoliposomes, while the T1 and T2 of nonstimulated cells and the stimulated cells that were incubated with liposomes, that did not contain an antibody, only slightly differed from the control HUVEC. Table 1. Quantification of T1 and T2 of HUVECa batch (n ) 3)
T1 (ms)
T2 (ms)
A HUVEC, - TNFR, AbEsel-L B HUVEC, + TNFR, AbEsel-L C HUVEC, + TNFR, Bare-L D HUVEC
2156 ( 130 457 ( 62 2135 ( 84 2412 ( 122
90 ( 11 16 ( 1 106 ( 12 103 ( 12
a MRI-based quantification of the water T and T (mean ( SD) 1 2 in pellets of 106 ( 105 HUVEC of the different incubations (n ) 3). A-D refer to the systems described in Figure 4. The T1s of the different incubations have been analyzed using multiple t-tests (p < 0.05) with correction for multiple comparisons. The T1 of incubation B is significantly different from incubations A, C, and D. The difference between the T1 of A and C is not significant, whereas the T1 of D is significantly higher than that of other incubations.
E-selectin on endothelial cells in vitro showed the capability of detecting E-selectin expression with MRI (33). The contrast generated by iron oxide particles can be ascribed to their superparamagnetic properties. This results in a dramatic shortening of T2 and causes dark spots in T2- and T2*-weighted images, which is termed negative contrast (35, 35, 36). In this study we tested the possibility to MR image the uptake of Gd-DTPA-based paramagnetic AbEselimmunoliposomes, which is accompanied by signal enhancement (i.e., positive contrast) in T1-weighted imaging, by activated endothelial cells. AbEsel (H18/7) antibodies were covalently coupled to the distal end of the PEG-chains of the paramagnetic liposomes, which resulted in an efficient conjugation of 25 µg protein/umol lipid without compromising the liposomal size distribution. The average diameter of the AbEsel-immunoliposomes was 150 nm diameter, with a polydispersity index of 0.2. Sipkins et al. (4) and Lanza et al. (6) have described paramagnetic particles of approximately 250 nm in size, without a PEG-coating, to which antibody was coupled via a noncovalent biotin-avidin interaction. When comparing these two paramagnetic particles, the immunoliposomal system described in this study has several advantages: it has a high flexibility in size (ranging from ca. 80 to 500 nm), it may be expected to have long circulation times because of the PEG-coating (11), and it has the antibody covalently coupled. The applied covalent coupling of targeting ligands is advantageous as it is more efficient (37) and does not involve a large protein complex like in the biotin-avidin-biotin method, which is known to be strongly immunogenic (38). The coupling of the antibody at the terminal end of the PEG chain is known to result in liposomes that retain
their favorable pharmacokinetic behavior (39, 40) and likely have prolonged circulation times compared to the existing paramagnetic systems. These properties may result in a more efficient targeting of our liposomal contrast agent in vivo. Liposomes composed of DSPC/ Chol/PEG were stable upon incubation in serum containing buffer. The incorporation of 20-30 mol % of Gd-BSA did not significantly alter the stability properties of the liposomes. The stability data suggest that the Gd-BSAcontaining liposomes may also be used as a drug carrier vehicle, carrying a payload of water soluble drugs in the aqueous interior or carrying hydrophobic drugs in the liposomal bilayer. The system could thus be a tool for combining molecular marker imaging with local therapy. The relaxivity properties of the paramagnetic liposomes differed from the relaxivity properties of free GdDTPA. At room temperature the r1 of the paramagnetic liposomes and Gd-DTPA were approximately the same. The longer rotational correlation time of the liposomes was expected to result in a larger relaxivity. With increasing temperature the r1 of Gd-DTPA decreased, whereas the r1 of the liposomes increased up to a temperature of 55 °C (Figure 2). From 55 °C the r1 of the liposomes decreased again but remained higher than the r1 of Gd-DTPA. The peak of the liposomal r1 at 55 °C might be explained by the increasing water exchange across the liposome membrane at higher temperatures allowing Gd-chelates present on the inner side of the bilayer to contribute more to the overall relaxivity. The transition temperature of this liposomal formulation is ca. 55 °C (41), implying that the liposomal bilayer is in the gel phase below 55 °C and in the fluid phase above 55 °C. A bilayer in the fluid phase is more permeable to water (41). These results suggest that at room temperature only part of the gadolinium contributes to the overall relaxivity. Therefore, a liposomal formulation with a transition temperature below the physiological temperature may be more suitable as an in vivo contrast agent in terms of relaxation properties. On the other hand, the altered composition might result in impaired stability and/or decreased Gd-BSA loading capacity. Strongly enhanced interaction of AbEsel-immunoliposomes with TNFR-treated cells was observed by fluorescence microscopy compared to control incubations. The fluorescence pattern represents a combination of cell membrane binding of fluorescent liposomes as well as intracellularly present fluorescence representing internalized liposomes, which is in agreement with previous studies (18, 19). Internalization of the liposomes might
A Liposomal System for MR Imaging of Molecular Targets
be important for in vivo detection since the low inherent sensitivity of MRI requires large amounts of Gd-DTPA to be present at the targeted site. Binding of the liposomes to the receptors expressed at the endothelium alone might not generate sufficient contrast in MR imaging to localize the liposomes with this technique. MRI experiments on pellets of 106 HUVEC were in excellent agreement with fluorescence microscopy. The T1-weighted image of the pellet containing TNFR-treated HUVEC incubated with AbEsel-immunoliposomes (Figure 5A) had a much higher signal intensity than that of the three control systems. This can be explained by the large shortening of the T1 in the former system (Figure 5B). The much less pronounced shortening of T1 for the nonactivated HUVEC, incubated with AbEsel-immunoliposomes, as well as the mild shortening of T1 for the HUVEC that were stimulated and incubated with liposomes that did not contain the anti-E-Selectin antibody can be explained by a low affinity, nonspecific cellular uptake of liposomes. In vivo, this anti-E-selectin MR contrast agent can be useful for the detection of atherosclerosis, angiogenesis, and inflammation. The described technique is also applicable for detection of other internalizing cell surface receptors as markers of the activated and/or angiogenic endothelial phenotype. CONCLUSIONS
We described the preparation, characterization, and successful in vitro targeting of paramagnetic liposomes for use in combination with MRI. The liposomes have a PEG-coat, and they do not leak upon incubation in serum. An anti-E-selectin antibody was coupled to the distal end of the PEG-chains of the liposomes. Other targeting ligands, like peptides (42) can also be coupled. The system both carries a MR-marker and a fluorescent marker, and we have demonstrated the E-selectin-dependent association of the liposomes to stimulated HUVEC by MRI and fluorescence microscopy. These results imply that this novel MR contrast agent may potentially serve as a useful diagnostic tool to investigate disease processes in vivo and to follow therapeutic efficacy. ACKNOWLEDGMENT
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