Abstract
Introduction
Nanoparticles may serve as a promising means to deliver novel therapeutics to the myocardium following myocardial infarction. We sought to determine whether lipid-based liposomal nanoparticles can be shown through different imaging modalities to specifically target injured myocardium following intravenous injection in an ischemia–reperfusion murine myocardial infarction model.
Methods
Mice underwent ischemia–reperfusion surgery and then either received tail-vein injection with gadolinium- and fluorescent-labeled liposomes or no injection (control). The hearts were harvested 24 h later and underwent T1 and T2-weighted ex vivo imaging using a 7 Tesla Bruker magnet. The hearts were then sectioned for immunohistochemistry and optical fluorescent imaging.
Results
The mean size of the liposomes was 100 nm. T1-weighted signal intensity was significantly increased in the ischemic vs. the non-ischemic myocardium for mice that received liposomes compared with control. Optical imaging demonstrated significant fluorescence within the infarct area for the liposome group compared with control (163 ± 31% vs. 13 ± 14%, p = 0.001) and fluorescent microscopy confirmed the presence of liposomes within the ischemic myocardium.
Conclusions
Liposomes traffic to the heart and preferentially home to regions of myocardial injury, enabling improved diagnosis of myocardial injury and could serve as a vehicle for drug delivery.
1
Introduction
The past decade has witnessed major improvements in outcomes following myocardial infarction (MI) . These may be attributed to improvements in revascularization with percutaneous coronary intervention, and to more aggressive use of medical therapies including anti-platelet agents, anticoagulation, beta-blockers, inhibitors of the renin-angiotensin-aldosterone system, and statin therapy. However, despite enhanced outcomes, the development of heart failure (HF) following AMI remains high and is associated with significant morbidity and mortality . It therefore seems important to consider additional strategies that could lead to further improvement in long-term outcomes of patients with MI.
The use of liposomes as drug delivery platforms is well established in oncology. An example of this is the utilization of pegylated liposomes for the delivery of chemotherapeutics . Liposomes provide the ability to increase the duration of circulation for agents with short half-lives and may provide a means to enter the infarcted myocardium through “leaky” endothelium . Lipid-based nanoparticles have been utilized to image myocardial infarction and can be modified to target specific molecules in the ischemic or infarcted myocardium . Studies have been performed using liposomes carrying drugs capable of reducing inflammation , stimulating repair , increasing angiogenesis , and augmenting cardiac function . In addition to therapeutic potential, lipid-based nanoparticles have been utilized to image myocardial infarction and can be modified to target specific molecules in the ischemic or infarcted myocardium .
In the present investigation we sought to determine, using different imaging modalities, whether liposomes specifically target injured myocardium following intravenous injection in an ischemia–reperfusion murine myocardial infarction model. It is our belief that if targeting is relatively specific and robust, then such a delivery platform could prove of value for delivery either imaging agents to identify regions of myocardial infarction, or therapeutic agents to reduce the extent of myocardial injury.
2
Methods
2.1
Liposome preparation
Liposomes were made from the phospholipid 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), the fluorescently-labeled phospholipid 1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine- N -7-nitro-2-1,3-benzoxadiazol-4-yl (DPPE-NBD), 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[maleimide-(poly(ethylene glycol))2000] (DSPE-PEG2000-Mal), 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[methoxy(polyethylene glycol)-2000] (DPSE-PEG2000), and the aliphatic Gd complex Gd-diethylenetriamine pentaacetate-bis(stearylamide) (Gd-DTPA-BSA) in a molar ratio of 78:2:1:12:7. The lipid mixture was dissolved in a 1:1 chloroform/methanol solution (5 mL) and evaporated under nitrogen flux yielding a thin film that was then rehydrated. Thereafter, the lipid film was heated and sonicated twice for 15 min at 70 W at 90% duty cycle. The DSPE-PEG2000-Mal can be utilized to attach the resulting liposome to a monoclonal antibody to target specific molecule. Dynamic light scattering was performed on a Malvern instrument (Zetasizer, Nano-S) to determine the hydrodynamic diameter of a suspension of liposomes. The number of gadolinium ions per liposome was determined by inductively coupled plasma mass spectroscopy (Maxxam Analytics, Burnaby, British Columbia, Canada) and this data was used to determine the number of NBD molecules per liposome.
2.2
Ischemia–reperfusion surgery and liposome injection
Animal care and procedures were carried out in accordance with the guide for the care and use of laboratory animals and our protocol was approved by the MedStar Health Research Institute Institutional Animal Care and Use Committee. All animal procedures were carried out at the Medstar Heart and Vascular Institute. Adult chow-fed, male CD1 mice at 8 to 10 weeks of age were anesthetized with pentobarbital sodium at 70 mg/kg via intraperitoneal injection with additional doses at 10 mg/kg as needed. The mice underwent endotracheal intubation and were ventilated with room air at a rate of 170 to 220 breaths per minute with a tidal volume 0.27 mL using a rodent ventilator (Harvard Apparatus). Left lateral thoracotomy was performed under sterile fashion and the left anterior descending (LAD) coronary artery was temporarily ligated just below the left atrial appendage with a 7-0 silk suture (Ethicon) using a loose knot tied over a small piece of Intramedic polyethylene PE 50 tubing (Becton, Dickinson and Company) to temporarily occlude blood flow and cause obvious blanching of the anterior left ventricular myocardium. Following 45 min of ischemia, the tubing was removed to re-establish blood flow and reperfusion was verified by reactive hyperemia. The suture was left in place in the myocardium, the ribs and skin were closed with a 5-0 Nylon suture (Ethicon), the animal recovered from anesthesia, and received standard post-operative pain control. The following day, animals either received a tail-vein injection of 200 μL of liposomes or no injection. Approximately 24 h later, mice received a lethal dose of pentobarbital, blood was aspirated from the right ventricle, and the heart and organs were harvested from each animal. The hearts were placed on a plastic rod to maintain orientation and placed in Fomblin solution (perfluoropolyether; Solvay Solexis) for ex vivo imaging.
2.3
Magnetic resonance imaging
Ex vivo magnetic resonance (MR) imaging was performed using a 7 Tesla Bruker scanner (Billerica, Mass) with 29 G/cm gradients. A T1-weighted 3D gradient echo imaging sequence (TR/TE, 100/3.2 ms; flip angle, 60°; 3 averages) was used. The field of view was generally 2.5 × 1 × 1 cm, and the matrix was 128 × 64 × 64, to yield a voxel size of 195 × 156 × 156 μm. T2-weighted spin echo images (TR/TE, 1500/12 ms; flip angle, 180°) were obtained for each heart through a representative section of the infarct and corresponding T1-weighted images were obtained to perform T1 signal intensity analysis. An independent cardiologist (ROE) blinded to treatment allocation (liposomes vs. control) selected regions of interest (ROI) within the infarct/ischemic region supplied by the LAD artery and a corresponding ROI from the inferoseptal wall (non-ischemic region) to generate average pixel intensity from T1-weighted images for each heart using ImageJ software (NIH, Bethesda, Maryland).
2.4
Immunohistochemistry
Following MR imaging, the hearts were placed in OCT embedding medium for frozen tissue specimens (Tissue-Tek) to preserve the orientation of the LAD territory. Five micron thick sagittal or axial sections of the heart were then cut to keep a short-axis orientation using a Thermo Shandon Cryotome (Pittsburg, PA). Following creating slides for frozen sectioning, a 1.5 mm thick short-axis section was cut using 3 slots of the 0.5 mm Zivic heart slicer (Zivic Instruments) to be used for optical imaging. Slides were then fixed and permeablized in acetone at − 20 °C, washed in phosphate buffered saline (PBS), incubated in 10% BSA in PBS for 1 h, washed in PBS, incubated with R -phycoerythrin (PE)-labeled anti-mouse monoclonal antibody against CD11b (BioLegend, San Diego, CA) or PE-labeled anti-mouse monoclonal antibody targeting CD45 (BioLegend, San Diego, CA) at 1:100 in 1% BSA in PBS for 1 h at room temperature, washed in PBS, and treated with DAPI mounting media. Fluorescent microscopy was performed using a Zeiss Axioimager microscope and multichannel black and white camera equipped with fluorescence filters (Carl Zeiss, Oberkochen, Germany).
2.5
Optical imaging
Ex vivo optical fluorescent imaging of the 1.5 mm thick axial or short-axis heart sections was performed with a Xenogen IVIS 100 in vivo imaging system (Caliper Life Sciences) using the GFP channel (excitation 445–490 nm/emission 515–575 nm) and comparison between groups. Fluorescent imaging of all heart sections was performed at the same time by obtaining a single fluorescent image of all heart sections in close proximity to enable uniform measurement of fluorescence, minimize variability, and enable quantitative fluorescent comparison using the same settings. ROIs were again selected of the whole heart section, from the LAD territory, and from the non-ischemic region to obtain the mean level of fluorescence for each ROI.
2.6
Statistics
Mean signal intensity values obtained from T1-weighted MR images and fluorescence imaging for the control and liposome groups were compared using Student’s T test (Prism 6 software, San Diego, CA). Statistical significance was defined as a p-value < 0.05.
2
Methods
2.1
Liposome preparation
Liposomes were made from the phospholipid 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), the fluorescently-labeled phospholipid 1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine- N -7-nitro-2-1,3-benzoxadiazol-4-yl (DPPE-NBD), 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[maleimide-(poly(ethylene glycol))2000] (DSPE-PEG2000-Mal), 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[methoxy(polyethylene glycol)-2000] (DPSE-PEG2000), and the aliphatic Gd complex Gd-diethylenetriamine pentaacetate-bis(stearylamide) (Gd-DTPA-BSA) in a molar ratio of 78:2:1:12:7. The lipid mixture was dissolved in a 1:1 chloroform/methanol solution (5 mL) and evaporated under nitrogen flux yielding a thin film that was then rehydrated. Thereafter, the lipid film was heated and sonicated twice for 15 min at 70 W at 90% duty cycle. The DSPE-PEG2000-Mal can be utilized to attach the resulting liposome to a monoclonal antibody to target specific molecule. Dynamic light scattering was performed on a Malvern instrument (Zetasizer, Nano-S) to determine the hydrodynamic diameter of a suspension of liposomes. The number of gadolinium ions per liposome was determined by inductively coupled plasma mass spectroscopy (Maxxam Analytics, Burnaby, British Columbia, Canada) and this data was used to determine the number of NBD molecules per liposome.
2.2
Ischemia–reperfusion surgery and liposome injection
Animal care and procedures were carried out in accordance with the guide for the care and use of laboratory animals and our protocol was approved by the MedStar Health Research Institute Institutional Animal Care and Use Committee. All animal procedures were carried out at the Medstar Heart and Vascular Institute. Adult chow-fed, male CD1 mice at 8 to 10 weeks of age were anesthetized with pentobarbital sodium at 70 mg/kg via intraperitoneal injection with additional doses at 10 mg/kg as needed. The mice underwent endotracheal intubation and were ventilated with room air at a rate of 170 to 220 breaths per minute with a tidal volume 0.27 mL using a rodent ventilator (Harvard Apparatus). Left lateral thoracotomy was performed under sterile fashion and the left anterior descending (LAD) coronary artery was temporarily ligated just below the left atrial appendage with a 7-0 silk suture (Ethicon) using a loose knot tied over a small piece of Intramedic polyethylene PE 50 tubing (Becton, Dickinson and Company) to temporarily occlude blood flow and cause obvious blanching of the anterior left ventricular myocardium. Following 45 min of ischemia, the tubing was removed to re-establish blood flow and reperfusion was verified by reactive hyperemia. The suture was left in place in the myocardium, the ribs and skin were closed with a 5-0 Nylon suture (Ethicon), the animal recovered from anesthesia, and received standard post-operative pain control. The following day, animals either received a tail-vein injection of 200 μL of liposomes or no injection. Approximately 24 h later, mice received a lethal dose of pentobarbital, blood was aspirated from the right ventricle, and the heart and organs were harvested from each animal. The hearts were placed on a plastic rod to maintain orientation and placed in Fomblin solution (perfluoropolyether; Solvay Solexis) for ex vivo imaging.
2.3
Magnetic resonance imaging
Ex vivo magnetic resonance (MR) imaging was performed using a 7 Tesla Bruker scanner (Billerica, Mass) with 29 G/cm gradients. A T1-weighted 3D gradient echo imaging sequence (TR/TE, 100/3.2 ms; flip angle, 60°; 3 averages) was used. The field of view was generally 2.5 × 1 × 1 cm, and the matrix was 128 × 64 × 64, to yield a voxel size of 195 × 156 × 156 μm. T2-weighted spin echo images (TR/TE, 1500/12 ms; flip angle, 180°) were obtained for each heart through a representative section of the infarct and corresponding T1-weighted images were obtained to perform T1 signal intensity analysis. An independent cardiologist (ROE) blinded to treatment allocation (liposomes vs. control) selected regions of interest (ROI) within the infarct/ischemic region supplied by the LAD artery and a corresponding ROI from the inferoseptal wall (non-ischemic region) to generate average pixel intensity from T1-weighted images for each heart using ImageJ software (NIH, Bethesda, Maryland).
2.4
Immunohistochemistry
Following MR imaging, the hearts were placed in OCT embedding medium for frozen tissue specimens (Tissue-Tek) to preserve the orientation of the LAD territory. Five micron thick sagittal or axial sections of the heart were then cut to keep a short-axis orientation using a Thermo Shandon Cryotome (Pittsburg, PA). Following creating slides for frozen sectioning, a 1.5 mm thick short-axis section was cut using 3 slots of the 0.5 mm Zivic heart slicer (Zivic Instruments) to be used for optical imaging. Slides were then fixed and permeablized in acetone at − 20 °C, washed in phosphate buffered saline (PBS), incubated in 10% BSA in PBS for 1 h, washed in PBS, incubated with R -phycoerythrin (PE)-labeled anti-mouse monoclonal antibody against CD11b (BioLegend, San Diego, CA) or PE-labeled anti-mouse monoclonal antibody targeting CD45 (BioLegend, San Diego, CA) at 1:100 in 1% BSA in PBS for 1 h at room temperature, washed in PBS, and treated with DAPI mounting media. Fluorescent microscopy was performed using a Zeiss Axioimager microscope and multichannel black and white camera equipped with fluorescence filters (Carl Zeiss, Oberkochen, Germany).
2.5
Optical imaging
Ex vivo optical fluorescent imaging of the 1.5 mm thick axial or short-axis heart sections was performed with a Xenogen IVIS 100 in vivo imaging system (Caliper Life Sciences) using the GFP channel (excitation 445–490 nm/emission 515–575 nm) and comparison between groups. Fluorescent imaging of all heart sections was performed at the same time by obtaining a single fluorescent image of all heart sections in close proximity to enable uniform measurement of fluorescence, minimize variability, and enable quantitative fluorescent comparison using the same settings. ROIs were again selected of the whole heart section, from the LAD territory, and from the non-ischemic region to obtain the mean level of fluorescence for each ROI.
2.6
Statistics
Mean signal intensity values obtained from T1-weighted MR images and fluorescence imaging for the control and liposome groups were compared using Student’s T test (Prism 6 software, San Diego, CA). Statistical significance was defined as a p-value < 0.05.
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