The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.
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Method Article
Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors
In This Article
Summary
Abstract
The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.
Introduction
Measuring the intra-tumoral accumulation of nanoparticle drug delivery systems may provide an important tool to determine if an adequate concentration of cytotoxic drug has been achieved within the tumor. The development of "image-able" liposomal systems allows for non-invasive and quantitative in vivo detection of the drug delivery vehicle using imaging modalities such as positron emission tomography (PET)1, optical fluorescence2, and computed tomography (CT)3,4 and magnetic resonance imaging (MRI)5. Imaging has been used to determine the pharmacokinetics and biodistribution of liposome delivery systems and....
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Protocol
All in vivo experiments were performed under a protocol approved by the University Health Network Institutional Animal Care and Use Committee.
1. Animal Model
- Culture between 5 to 7 x 106 MDA-MB-231 breast adenocarcinoma tumor cells in DMEM together with 10% Fetal Bovine Serum (FBS) and 100x dilution of penicillin-streptomycin.
- Harvest cells when they are 80% confluent using a 0.05% trypsin-EDTA solution. After 3-5 min neutralize trypsin-EDTA with a 3x volume of DMEM. Take a 15 µl aliquot of cells and count using a hemocytometer. Centrifuge cells into a pellet for 5 min at 200 x g, and....
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Results
The aforementioned protocol should yield CT-liposomes with an encapsulated concentration of iohexol, mean liposome diameter, and zeta potential of 55 mg ml-1, 91.8 ± 0.3 nm and -45.5 ± 2.5 mV, respectively. Figure 1a includes representative DCE-CT imaging results, yielding a time series of volumetric data that show the temporal changes in intra-tumoral accumulation of iohexol. Selecting a ROI within the tumor yields a TIC that can be quantified using tracer kin.......
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Discussion
The methods for image-based measurement presented herein enable determination of the spatial distribution of tumor microcirculation properties, IFP, and CT-liposome accumulation. Previous attempts to relate these properties have relied on performing bulk measurements across multiple tumor-bearing animals and therefore lack the sensitivity to elucidate mechanisms responsible for heterogeneity in intra-tumoral accumulation that has commonly been observed for nano-sized drug delivery systems15. DCE-CT provides a .......
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Disclosures
The authors have nothing to disclose
Acknowledgements
The authors would like to thank Dr. Javed Mahmood for assistance with culturing MDA-MB-231 cells and implanting the MDA-MB-231 xenografts, Linyu Fan for preparing the CT-liposomes. Shawn Stapleton is grateful for funding from the Natural Sciences and Engineering Research Postgraduate Scholarships Program and the Terry Fox Foundation Strategic Initiative for Excellence in Radiation Research for the 21st Century (EIRR21) at CIHR. This study was supported by grants from the Terry Fox New Frontiers Program (020005) and the Canadian Institutes of Health Research (102569).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| MDA-MB-231 metastatic breast adenocarcinoma tumor cells | ATCC | HTB-26 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 11965-092 | |
| Fetal Bovine Serum (FBS) | Sigma-Aldrich | F1051 | |
| HyClone Penicillin-Streptomycin 100x Solution | GE Healthcare Life Sciences | SV30010 | |
| Trypsin-EDTA (0.05%), phenol red | ThermoFisher Scientific | 25300-054 | |
| 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Avanti Lipids Inc., USA | 850355P | |
| Cholesterol (CH) | Avanti Lipids Inc., USA | 700000P | |
| 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol) 2000 (DSPE-PEG2000) | Avanti Lipids Inc., USA | 880128P | |
| Omnipaque (Iohexol) 300 mg of iodine/ml | GE Healthcare, CA | ||
| 80 nm pore size Track-Etch polycarbonate membranes | Whatman Inc., USA | ||
| 200 nm pore size Track-Etch polycarbonate membranes | Whatman Inc., USA | ||
| 10 m Lipex Extruder | Nothern Lipids Inc, CA | ||
| Dialysis Bag Molecular Weight Cut Off (MWCO) of 8 kDa | Spectrum Labs, USA | ||
| 750,000 Nomical Molecular Weight Cut Off (NMWC) Tangential flow column | MidGee ultrafiltration cartridge, GE Healthcare, CA | ||
| Peristaltic pump | Watson Marlow Inc., USA | ||
| UV spectrometer | Helios γ, Spectronic Unicam, USA | ||
| 90Plus particle size analyzer | Brookhaven, Holtsville, USA | ||
| eXplore Locus Ultra micro-CT system | GE Healthcare, CA | Manipulated using CT-Console Software | |
| AxRecon GPU-based Reconstruction | Acceleware Corp. CA | ||
| 27 G Catheter SURFLO Winged Infusion Set | Terumo Medical Products, USA | SV*27EL | |
| PE20 polyethylyne tubing | Becton Dickinson, USA | 427406 | |
| Pen tip 25 G × 3.5′′ Whitacre spinal needle | Becton Dickinson, USA | 405140 | IFP needle |
| P23XL pressure transducer | Harvard Apparatus, CA | P23XL | |
| PowerLab 4/35, Bridge Amp, with LabChart Pro 7.0 | ADInstruments Pty Ltd., USA | PL3504, FE221 | IFP acquisition system and acquisition software |
| CT-Sabre Small Animall Intervention system (CT-IFP Robot) | Parallax Innovations, CA | Manipulated using CT-IFP robot Control Software | |
| CT-IFP robot alignment software | Custom Matlab software | ||
| DCE-CT Analysis Software | Custom Matlab software | ||
| Matlab 2013b | Mathworks, USA |
References
- Seo, J. W., Zhang, H., Kukis, D. L., Meares, C. F., Ferrara, K. W. A novel method to label preformed liposomes with 64Cu for positron emission tomography (PET) imaging. Bioconjugate chemistry. 19 (12), 2577-2584 (2008).
- Huang, H., Dunne, M., Lo, J., Jaffray, D., Allen, C.
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