PRE-CLINICAL RESEARCH
Prolonged Targeting of Ischemic/Reperfused Myocardium by Liposomal Adenosine Augments Cardioprotection in Rats
Hiroyuki Takahama, MD*, , ,
Tetsuo Minamino, MD, PhD ,*,
Hiroshi Asanuma, MD, PhD,
Masashi Fujita, MD, PhD,
Tomohiro Asai, PhD¶,
Masakatsu Wakeno, MD, PhD*,,,
Hideyuki Sasaki, MD*,,,
Hiroshi Kikuchi, PhD#,
Kouichi Hashimoto**,
Naoto Oku, PhD¶,
Masanori Asakura, MD, PhD,
Jiyoong Kim, MD,
Seiji Takashima, MD, PhD,
Kazuo Komamura, MD, PhD||,
Masaru Sugimachi, MD, PhD||,
Naoki Mochizuki, MD, PhD*, and
Masafumi Kitakaze, MD, PhD, FACC
* Department of Molecular Imaging in Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
Department of Cardiovascular Medicine, National Cardiovascular Center, Osaka, Japan
Department of Structural Analysis, Research Institute, National Cardiovascular Center, Osaka, Japan
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
|| Department of Cardiovascular Dynamics, Research Institute, National Cardiovascular Center, Osaka, Japan
¶ Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
# Daiichi Pharmaceutical Co., Tokyo, Japan
** Daiichi-Sankyo Pharmaceutical Co., Tokyo, Japan
Manuscript received September 4, 2008;
revised manuscript received October 21, 2008,
accepted November 3, 2008.
* Reprint requests and correspondence: Dr. Tetsuo Minamino, Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan (Email: minamino{at}medone.med.osaka-u.ac.jp).
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Abstract
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Objectives: The purpose of this study was to investigate whether liposomal adenosine has stronger cardioprotective effects and fewer side effects than free adenosine.
Background: Liposomes are nanoparticles that can deliver various agents to target tissues and delay degradation of these agents. Liposomes coated with polyethylene glycol (PEG) prolong the residence time of drugs in the blood. Although adenosine reduces the myocardial infarct (MI) size in clinical trials, it also causes hypotension and bradycardia.
Methods: We prepared PEGylated liposomal adenosine (mean diameter 134 ± 21 nm) by the hydration method. In rats, we evaluated the myocardial accumulation of liposomes and MI size at 3 h after 30 min of ischemia followed by reperfusion.
Results: The electron microscopy and ex vivo bioluminescence imaging showed the specific accumulation of liposomes in ischemic/reperfused myocardium. Investigation of radioisotope-labeled adenosine encapsulated in PEGylated liposomes revealed a prolonged blood residence time. An intravenous infusion of PEGylated liposomal adenosine (450 µg/kg/min) had a weaker effect on blood pressure and heart rate than the corresponding dose of free adenosine. An intravenous infusion of PEGylated liposomal adenosine (450 µg/kg/min) for 10 min from 5 min before the onset of reperfusion significantly reduced MI size (29.5 ± 6.5%) compared with an infusion of saline (53.2 ± 3.5%, p < 0.05). The antagonist of adenosine A1, A2a, A2b, or A3 subtype receptor blocked cardioprotection observed in the PEGylated liposomal adenosine-treated group.
Conclusions: An infusion as PEGylated liposomes augmented the cardioprotective effects of adenosine against ischemia/reperfusion injury and reduced its unfavorable hemodynamic effects. Liposomes are promising for developing new treatments for acute MI.
Key Words: myocardial infarction • liposome • drug delivery system • adenosine
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Abbreviations and Acronyms
| | 8-SPT = 8-(p-sulfophenyl) theophylline | | EM = electron microscopy | | MI = myocardial infarction | | PEG = polyethylene glycol | | RI = radioisotope | | TTC = triphenyltetrazolium chloride |
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Liposomes are now widely used for drug delivery in cancer treatment to target specific organs actively or passively and to prevent the degradation of chemotherapy agents (1). However, the application of liposomes for cardiovascular diseases is still limited. In ischemic/reperfused myocardium, cellular permeability is enhanced and vascular endothelial integrity is disrupted (2,3), suggesting that nanoparticles such as liposomes may be a promising drug delivery system for targeting damaged myocardium with cardioprotective agents. Additionally, coating liposomes with polyethylene glycol (PEG) prolongs their residence time in the circulation (1). Because enhanced microvascular permeability persists for at least 48 h after the occurrence of myocardial infarction (MI) (2), drugs delivered in PEGylated liposomes should be able to display their maximum beneficial effects on myocardial damage after MI.
Adenosine has multiple physiological functions that are mediated via the adenosine A1, A2a, A2b, and A3 receptors (4,5). Although large-scale clinical trials suggested the potential value of adenosine therapy for patients with acute MI (6,7), this agent has an extremely short half-life (1 to 2 s) and causes hypotension and bradycardia because of vasodilatory and negative chronotropic effects (4). Because a high dose of adenosine is required to exert cardioprotective effects, it is difficult to use clinically because of the associated hemodynamic consequences. Therefore, we hypothesized that adenosine encapsulated in PEGylated liposomes would cause less hemodynamic disturbance and might also specifically accumulate in ischemic/reperfused myocardium, leading to augmented cardioprotective effects. To test this hypothesis, we created PEGylated liposomal adenosine by the hydration method and investigated: 1) whether liposomal adenosine accumulated in ischemic/reperfused myocardium and prolonged blood residence time; 2) whether liposomal adenosine caused less severe hypotension and bradycardia than free adenosine; and 3) which adenosine receptor subtype was involved in mediating the cardioprotective effects of liposomal adenosine against ischemia/reperfusion injury.
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Methods
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Materials.
The materials for preparing PEGylated liposomes, including hydrogenated soy phosphatidyl choline (HSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000), and cholesterol were obtained from Nissei Oil Co., Ltd. (Tokyo, Japan) and Wako Pure Chemical Co., Ltd. (Osaka, Japan). [3H]-adenosine was purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). Other materials were obtained from Sigma (St. Louis, Missouri), including 8-(p-sulfophenyl)theophylline (8-SPT; a nonselective adenosine receptor antagonist), 1,3-diethyl-8-phenylxanthine (DPCPX; a selective adenosine A1 receptor antagonist), 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH58261; a selective adenosine A2a receptor antagonist), 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1, 3-di(n-propyl)xanthine (MRS1754; a selective adenosine A2b receptor antagonist), and 5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate (MRS1523, a selective adenosine A3 receptor antagonist).
Animals.
Male Wistar rats (9 weeks old and weighing 250 to 310 g, Japan Animals, Osaka, Japan) were used. The animal experiments were approved by the National Cardiovascular Center Research Committee and were performed according to institutional guidelines.
Preparation of PEGylated liposomes.
The PEGylated liposomes were prepared by the hydration method. Briefly, adenosine was added to the lipid solution. After mixture of lipid and adenosine, DSPE-PEG2000 was added and incubated. The final composition of PEGylated liposomes was HSPC:cholesterol:DSPE-PEG2000 = 6.0:4.0:0.3 (molar ratio). After ultracentrifugation several times, the pellet of liposomal adenosine was resuspended in sodium lactate at each required concentration for use in the experimental protocols. Some samples of final liposomal adenosine were disrupted by dilution with 50% methanol (1.5 ml per 30-µl of liposomes). After 10 min of ultracentrifugation, the concentration of adenosine in the supernatant was measured by high-performance liquid chromatography.
To prepare fluorescent-labeled liposomes, 0.5 mol% tetramethylrhodamine isothiocyanate (rhodamine) was added to the lipid mixture. To prepare radioisotope (RI)-labeled adenosine encapsulated in liposomes, [3H]-radiolabeled adenosine (Daiichi Pure Chemicals, Tokyo, Japan) was diluted with free adenosine and was encapsulated in liposomes as described above.
Characterization of PEGylated liposomal adenosine.
The characterization of the liposomes was performed by the dynamic scatter analysis (Zetasizer Nano ZS, Malvern, Worcestershire, United Kingdom). The analyses were performed 10 times per sample, and results represented analyses of 4 independent experiments.
Experimental protocols.
Protocol 1: Effects of PEGylated Liposomal Adenosine on Hemodynamics in Rats
Rats were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg). Catheters were advanced into a femoral artery and vein for the measurement of systemic blood pressure and infusion of drugs, respectively. Both blood pressure and heart rate were monitored continuously during the study using a Power Lab (AD Instruments, Castle Hill, Australia). After hemodynamics became stable, we intravenously administered empty PEGylated liposomes (n = 8), free adenosine (n = 8), or PEGylated liposomal adenosine (n = 8) for 10 min. Either PEGylated liposomal or free adenosine was infused at an initial dose of 225 µg/kg/min (0.1 ml/min) for 10 min. After a 5-min interval, either PEGylated liposomal adenosine or free adenosine was infused at 450 µg/kg/min (0.1 ml/min) for 10 min. In the same manner, PEGylated liposomal adenosine or free adenosine was then infused at 900 µg/kg/min (0.1 ml/min).
Protocol 2: Effects of PEGylated Liposomal Adenosine on Infarct Size in Rats
The MI was induced by transient ligation of the left coronary artery as described previously (8). In the first series of experiments, to examine the dose-dependent effects of liposomal adenosine on MI size, PEGylated liposomal adenosine was infused intravenously at 50, 150, or 450 µg/kg/min for a 10-min period starting from 5 min before the onset of reperfusion. In the second series of experiments, to determine the adenosine receptor subtype involved in cardioprotective effects by the liposomal adenosine, the antagonist of adenosine subtype receptor was intravenously injected as a bolus followed by the infusion of liposomal adenosine for 10 min. The MI size was evaluated at 3 h after the start of reperfusion. The doses of adenosine receptor subtype antagonists were determined according to the previous reports (9–11).
Measurement of infarct size.
At 3 h after the onset of reperfusion, the area at risk and the infarcted area were determined by Evans blue and triphenyltetrazolium chloride (TTC) staining, respectively, as previously described (8). Infarct size was calculated as [infarcted area/area at risk] x 100(%) in a blind manner. The area at risk was composed of border (TTC staining) and infarcted (TTC nonstaining) areas.
Electron microscopy (EM).
Myocardial samples for EM were obtained from the central and peripheral areas in ischemic/reperfused myocardium, which roughly corresponded to the infarcted and border areas, respectively, after the left coronary artery was occluded for 30 min of ischemia followed by 3 h of reperfusion. Samples were prepared as previously reported (12). Liposomes, whose major membrane component is unsaturated phospholipids, were visualized as homogenous dark dots with a diameter of 100 to 150 nm (13).
Accumulation of fluorescent-labeled PEGylated liposomes in ischemic/reperfused myocardium.
Unlabeled or fluorescent-labeled PEGylated liposomes were infused intravenously at a dose of 0.1 ml/min as liposomal adenosine was infused in protocol 2. At 3 h after reperfusion, hearts were quickly removed and cut into 4 sections parallel to the axis from base to apex. Then ex vivo bioluminescence imaging was performed with an Olympus OV 100 imaging system (Olympus, Tokyo, Japan) and signals were quantified using WASABI quantitative software (Hamamatsu Photonics K.K., Shizuoka, Japan). Fluorescent intensity in the region of interest was measured as previously reported (14). Control intensity indicated the fluorescent intensity in the nonischemic area of the individual rat.
Time-course changes of free and PEGylated liposomal RI-labeled adenosine in plasma and myocardium.
Free or PEGylated liposomal [3H]-adenosine (83 kBq per rat) was infused intravenously at a dose of 0.1 ml/min as liposomal adenosine was infused in protocol 2. At the time indicated, rat hearts were harvested for counting of radioactivity (LSC-3100, Aloka Co., Tokyo, Japan). Results are expressed as a percentage of the injected dose per 1 ml of blood or 1 g of wet tissue weight.
Statistical analysis.
The parameters of the liposomes were expressed as the average ± SD, whereas other data were expressed as the average ± SEM. Comparison of time-course changes in hemodynamic parameters between groups was performed by 2-way repeated-measures analysis of variance (ANOVA) followed by a post-hoc Bonferroni test. For comparison of RI activity between groups, statistical analysis was done with the Mann-Whitney U test. To address the differences in infarct size among groups, we performed a nonparametric (Kruskal-Wallis) test followed by evaluation with the Mann-Whitney U test. Resulting p values were corrected according to the Bonferroni method. To compare parameters of liposomes, an unpaired t test was performed. In all analyses, p < 0.05 was considered to indicate statistical significance.
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Results
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Characterization of liposomes by dynamic light scatter analysis.
The dynamic light scatter analysis showed no significant difference in mean diameter, polydispersity index, or zeta-potential distribution between empty and adenosine-loaded PEGylated liposomes (Table 1).
Liposomes in ischemic/reperfused myocardium.
The EM revealed the intact vascular endothelial cells and cardiomyocytes in the nonischemic myocardium (Figs. 1A and 1B). There were no homogenous dark dots indicating liposomes in the nonischemic myocardium of rats that received either saline (Fig. 1A) or liposomes (Fig. 1B). In the border area, many homogenous dark dots indicating liposomes were accumulated in rats that received liposomes, but not saline (Figs. 1C and 1D). In this area, significant structural damage was not observed in endothelium, but slight swelling of mitochondria was often observed. In the infarcted area, numerous liposomes were detected in rats that received liposomes, but not saline (Figs. 1E and 1F). In this area, the disrupted endothelial integrity and marked swelling of mitochondria were often observed.
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Figure 1 Liposomes in Ischemic/Reperfused Myocardium
(A, B) Representative electron micrographs of the nonischemic area in rats that received saline (A) or liposomes (Lp) (B). (C, D) Representative electron micrographs of border area at 3 h after myocardial infarction (MI). Many dark dots accumulated in this area in the rat that received liposomes but not saline. (E, F) Representative electron micrographs of infarcted areas at 3 h after MI. Numerous dark dots accumulated in this area in the rat that received liposomes but not saline. Scale bars represent 1 µm.
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Fluorescent-labeled PEGylated liposomes in ischemic/reperfused myocardium.
Quantitative analysis by bioluminescence ex vivo bioluminescene imaging revealed that the target to control fluorescent intensity ratio was higher in the border (noninfarcted area at risk) as well as infarcted areas compared with a nonischemic one, suggesting that fluorescent-labeled liposomes were accumulated in the border as well as infarcted areas. Since there was no high-intensity area when unlabeled liposomes were infused, it was suggested that this was not a nonspecific phenomenon to MI by the ex vivo bioluminescence imaging system (Fig. 2). The Evans blue staining was unrelated to the fluorescence intensity (data not shown).
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Figure 2 Detection of Fluorescence-Labeled PEGylated Liposomes in Ischemic/Reperfused Myocardium
Quantitative analysis of target-to-control fluorescent intensity ratio for each area in rats (n = 3 each group) that received nonfluorescent (A) or fluorescent (B) liposomes. The values of bioluminescence signals in the border and infarcted areas were expressed as the fold to that of the each nonischemic area. Values are expressed as the mean ± SEM (error bars). *p < 0.05 versus nonischemic areas. #p < 0.05 versus border areas.
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Plasma radioactivity of RI-labeled adenosine was markedly higher in the PEGylated liposomal adenosine group at 10 min and 3 h after the intravenous infusion than in the free adenosine group (Fig. 3A). Encapsulation within PEGylated liposomes also augmented the accumulation of adenosine in ischemic/reperfused myocardium compared with that of free adenosine (Fig. 3B).
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Figure 3 Radioisotope-Labeled Adenosine in Plasma and Ischemic/Reperfused Myocardium
(A) Changes in plasma radioactivity after infusion of radioisotope-labeled adenosine. Solid and open bars indicate the PEGylated liposomal adenosine and free adenosine groups, respectively (n = 4 each). In the PEGylated liposomal adenosine group, plasma radioactivity was markedly higher than in the free adenosine group. (B) Changes in radioactivity in ischemic/reperfused myocardium. Solid and open bars indicate the PEGylated liposomal adenosine and free adenosine groups, respectively (n = 4 each). In the PEGylated liposomal adenosine group, myocardial radioactivity was markedly higher than in the free adenosine group. Values are expressed as the mean ± SEM (error bars). *p < 0.05 versus the free adenosine group at the corresponding time.
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Hemodynamic effects of PEGylated liposomal adenosine.
Baseline hemodynamic parameters did not differ among the groups. An intravenous infusion of free adenosine at doses of 225, 450, and 900 µg/kg/min decreased the mean blood pressure by 14.8%, 25.4%, and 33.7%, respectively, compared with the effect of empty PEGylated liposomes. In contrast, the intravenous infusion of PEGylated liposomal adenosine at a dose of either 225 or 450 µg/kg/min did not significantly alter mean blood pressure (Fig. 4). Changes of the heart rate after infusion of PEGylated liposomal adenosine or free adenosine were similar to those observed for mean blood pressure (Fig. 4).
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Figure 4 Hemodynamic Effects of PEGylated Liposomal Adenosine
Changes in the mean blood pressure (A) and heart rate (B) after intravenous infusion of various doses of empty PEGylated liposomes (triangles), PEGylated liposomal adenosine (circles), or free adenosine (squares) (n = 8 each). Values are expressed as the mean ± SEM. *p < 0.05 versus baseline at the corresponding group. #p < 0.05 versus PEGylated liposomes.
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Effects of PEGylated liposomal adenosine on MI size.
Baseline hemodynamic parameters were similar among all of the groups (Table 2). Intravenous infusion of free adenosine for 10 min reduced both the blood pressure and the heart rate, although these parameters returned to baseline within 5 min of ceasing infusion (Table 2). In contrast, hemodynamic parameters of the other groups were not altered (Table 2). The area at risk in the control group (61 ± 3%) did not differ compared with those of other groups that received liposomal adenosine. Intravenous infusion of PEGylated liposomal adenosine caused a dose-dependent decrease of MI size compared with that in the control group, whereas intravenous infusion of empty PEGylated liposomes or free adenosine did not (Fig. 5B).
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Figure 5 Dose-Dependent Effects of PEGylated Liposomal Adenosine on Myocardial Infarct Size
(A) Protocol showing the timing of infusion of PEGylated liposomal adenosine and of infarct size measurement. (B) Myocardial infarct size. Values are expressed as mean ± SEM (error bars). *p < 0.05 versus the control group. Free-Ado = free adenosine; Lp-Ado = PEGylated liposomal adenosine; Vehicle = PEGylated liposomes.
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The bolus injection of adenosine receptor antagonist did not alter the hemodynamic parameters (Table 3). The area at risk in the liposomal adenosine group (58 ± 3%) did not differ compared with those of other groups that received adenosine receptor antagonist. Infusion of 8-SPT, a nonspecific adenosine receptor antagonist, blunted the cardioprotective effect of liposomal adenosine (Fig. 6B). Furthermore, the infusion of the adenosine A1, A2a, A2b, or A3 receptor antagonist also blunted cardioprotective effects of liposomal adenosine (Fig. 6B). Infusion of 8-SPT alone did not significantly affect myocardial infarct size compared with the control (52 ± 5%, n = 4).
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Figure 6 Effects of Adenosine Receptor Antagonists on Myocardial Infarct Size
(A) Protocol showing the timing of infusion of PEGylated liposomal adenosine and bolus injection adenosine receptor antagonists. (B) Myocardial infarct size. Values are expressed as the mean ± SEM (error bars). *p < 0.05 versus the control group. The abbreviations for adenosine antagonists were described in the text. Abbreviations as in Figure 5.
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Discussion
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In the present study, EM, bioluminescence ex vivo imaging, and fluorescent analysis revealed the accumulation of liposomes in the border (noninfarcted areas at risk) as well as infarcted ones, but not nonischemic myocardium, at 3 h after MI. These findings suggested that liposomes could specifically accumulate in ischemic/reperfused myocardium. Interestingly, EM revealed the existence of liposomes at sites where endothelial integrity was still morphologically maintained. Endothelial dysfunction such as enhanced permeability is induced by ischemic insult without morphological endothelial disruption (3,15). Enhanced permeability might lead to the accumulation of liposomes in the border as well as infarcted area, which will contribute to salvage the ischemic/reperfused myocardium. However, further investigation will be needed to determine the precise mechanism by which liposomes accumulate in ischemic/reperfused myocardium.
Analysis using RI-labeled adenosine encapsulated in liposomes revealed that plasma radioactivity was markedly higher in the PEGylated liposomal adenosine group compared with the free adenosine group. This indicates that encapsulation of adenosine by PEGylated liposomes considerably prolonged its residence time in the circulation and delayed its degradation. Consistent with the histological data, RI-labeled adenosine also showed preferential accumulation in ischemic/reperfused myocardium.
Furthermore, this study showed that PEGylated liposomal adenosine had a weaker effect on the blood pressure and heart rate than free adenosine. Thus, encapsulating adenosine in PEGylated liposomes attenuated its vasodilatory and negative chronotropic effects, presumably by reducing the amount of circulating free adenosine. However, the changes of hemodynamic parameters in this in vivo model suggested that significant release of adenosine from PEGylated liposomes would still occur if a large dose of liposomal adenosine (e.g., 900 µg/kg/min) were administered. Thus, further investigation of the in vivo pharmacodynamics of PEGylated liposomal adenosine is needed.
An intravenous infusion of PEGylated liposomal adenosine at the maximum dose that did not disturb hemodynamic parameters for 10 min before reperfusion reduced MI size in a dose-dependent manner, and this improvement was blocked by 8-SPT, a nonselective adenosine receptor antagonist. These findings suggest that adenosine released from liposomes acts via an adenosine receptor-dependent pathway. One possible mechanism by which PEGylated liposomes could augment cardioprotective effects of liposomal adenosine with minimum effects on hemodynamic parameters is the enhanced accumulation of PEGylated liposomal adenosine in ischemic/reperfused myocardium, which could augment various beneficial actions such as preventing calcium overload in the myocardium (5). The prolonged persistence of PEGylated liposomal adenosine would also increase its beneficial effect on ischemic/reperfused myocardium. Although continuous high-dose, long-term infusion of free adenosine was reported to reduce infarct size in rats (16), the present study did not confirm such a cardioprotective effect, probably because the total dose of free adenosine that we used was not high enough.
We found that myocardial infarct size in the group that received PEGylated liposomal adenosine with the antagonist of adenosine A1, A2a, A2b, or A3 subtype receptor was no different from the control group, indicating that every adenosine subtype receptor could possibly play a role in mediating cardioprotection by liposomal adenosine and that it was difficult to identify one particular subtype in the present study. Numerous studies reported that A1, A2a, A2b, and A3 receptors have been involved in cardioprotection against ischemia/reperfusion injury, and it remains controversial which adenosine subtype receptor is most responsible for cardioprotection (17–20). Furthermore, because the adenosine receptor antagonists used in the present study had some nonspecific effects, future investigation will be needed to examine the precise role of each adenosine receptor subtype using genetically engineered mice.
Because liposomal adenosine infused during reperfusion could reduce MI size, this agent could be a candidate for the adjunctive therapy of patients with acute MI. Importantly, adenosine is currently used for the diagnosis of ischemic heart disease and PEGylated liposomes are used to deliver anticancer agents (21). Thus, it should not be difficult to introduce PEGylated liposomal adenosine into clinical practice. Finally, PEGylated liposomes may provide a useful drug delivery system for targeting ischemic/reperfused myocardium with other agents.
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Acknowledgments
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The authors thank Akiko Ogai and Yoko Nakano for their excellent technical assistance; Motohide Takahama, Hiroyuki Hao, and Hatsue Ishibashi-Ueda for advice about the electron microscopy figure; and Syunichi Kuroda and Takashi Matsuzaki for assistance with bioluminescence imaging.
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Footnotes
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Supported by a grant for Scientific Research and a grant for the Advancement of Medical Equipment from the Japanese Ministry of Health, Labor, and Welfare, as well as a grant from the Japan Cardiovascular Research Foundation.
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References
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Abstract
Methods