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).

All in vivo experiments were performed under a protocol approved by the University Health Network Institutional Animal Care and Use Committee.
1. Animal Model
<ol>
	<li>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.</li>
	<li>Harvest cells when they are 80% confluent using a 0.05% trypsin-EDTA solution. After 3-5 min neutralize trypsin-EDTA ...

<ol>
	<li>Seo, J. W., Zhang, H., Kukis, D. L., Meares, C. F., Ferrara, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+label+preformed+liposomes+with+64Cu+for+positron+emission+tomography+(PET)+imaging.">A novel method to label preformed liposomes with 64Cu for positron emission tomography (PET) imaging.</a> Bioconjugate chemistry. 19 (12), 2577-2584 (2008).</li><li>Huang, H., Dunne, M., Lo, J., Jaffray, D., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Computed+Tomography-+and+Optical+Image-Based+Assessment+of+Liposome+Distribution.">Comparison of Computed Tomography- and Optical Image-Based Assessment of Liposome Distribution.</a> Molecular Imaging. 12 (3), 148-160 (2013).</li><li>Stapleton, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mathematical+model+of+the+enhanced+permeability+and+retention+effect+for+liposome+transport+in+solid+tumors.">A mathematical model of the enhanced permeability and retention effect for liposome transport in solid tumors.</a> PloS one. 8 (12), e81157 (2013).</li><li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multimodal+nano+agent+for+image-guided+cancer+surgery.">A multimodal nano agent for image-guided cancer surgery.</a> Biomaterials. 67, 160-168 (2015).</li><li>Zheng, J., Liu, J., Dunne, M., Jaffray, D. A., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+performance+of+a+liposomal+vascular+contrast+agent+for+CT+and+MR-based+image+guidance+applications.">In vivo performance of a liposomal vascular contrast agent for CT and MR-based image guidance applications.</a> Pharmaceutical research. 24 (6), 1193-1201 (2007).</li><li>Harrington, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeting+of+solid+tumors+in+patients+with+locally+advanced+cancers+by+radiolabeled+pegylated+liposomes.">Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes.</a> Clinical Cancer Research. 7 (2), 243-254 (2001).</li><li>Stapleton, S., Allen, C., Pintilie, M., Jaffray, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+perfusion+imaging+predicts+the+intra-tumoral+accumulation+of+liposomes.">Tumor perfusion imaging predicts the intra-tumoral accumulation of liposomes.</a> J Control Release. 172 (1), 351-357 (2013).</li><li>Lammers, T., Kiessling, F., Hennink, W. E., Storm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotheranostics+and+image-guided+drug+delivery:+current+concepts+and+future+directions.">Nanotheranostics and image-guided drug delivery: current concepts and future directions.</a> Mol. Pharm. 7, 1899-1912 (2010).</li><li>Stapleton, S., Milosevic, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Targeted Drug Delivery. , 241-272 (2013).</li><li>Blanco, E., Shen, H., Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nanoparticle+design+for+overcoming+biological+barriers+to+drug+delivery.">Principles of nanoparticle design for overcoming biological barriers to drug delivery.</a> Nature biotechnology. 33 (9), 941-951 (2015).</li><li>Heldin, C. H., Rubin, K., Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+interstitial+fluid+pressure+-+an+obstacle+in+cancer+therapy.">High interstitial fluid pressure - an obstacle in cancer therapy.</a> Nat Rev Cancer. 4 (10), 806-813 (2004).</li><li>Chauhan, V. P., Stylianopoulos, T., Boucher, Y., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+molecular+and+nanoscale+medicine+to+tumors:+transport+barriers+and+strategies.">Delivery of molecular and nanoscale medicine to tumors: transport barriers and strategies.</a> Annual review of chemical and biomolecular engineering. 2, 281-298 (2011).</li><li>Bax, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+image-guided+robotic+needle+positioning+system+for+small+animal+interventions.">3D image-guided robotic needle positioning system for small animal interventions.</a> Medical physics. 40 (1), 011909 (2013).</li><li>Stapleton, S., Milosevic, M., Tannock, I. F., Allen, C., Jaffray, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intra-tumoral+relationship+between+microcirculation,+interstitial+fluid+pressure+and+liposome+accumulation.">The intra-tumoral relationship between microcirculation, interstitial fluid pressure and liposome accumulation.</a> Journal of Controlled Release. 211, 163-170 (2015).</li><li>Stapleton, S., Allen, C., Pintilie, M., Jaffray, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+perfusion+imaging+predicts+the+intra-tumoral+accumulation+of+liposomes.">Tumor perfusion imaging predicts the intra-tumoral accumulation of liposomes.</a> J Control Release. 172 (1), 351-357 (2013).</li><li>Brix, G., Zwick, S., Kiessling, F., Griebel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetic+analysis+of+tissue+microcirculation+using+nested+models:+multimodel+inference+and+parameter+identifiability.">Pharmacokinetic analysis of tissue microcirculation using nested models: multimodel inference and parameter identifiability.</a> Medical physics. 36 (7), 2923-2933 (2009).</li><li>Brix, G., Griebel, J., Kiessling, F., Wenz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracer+kinetic+modelling+of+tumour+angiogenesis+based+on+dynamic+contrast-enhanced+CT+and+MRI+measurements.">Tracer kinetic modelling of tumour angiogenesis based on dynamic contrast-enhanced CT and MRI measurements.</a> European journal of nuclear medicine and molecular imaging. 37 (1), 30-51 (2010).</li></ol>

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 ...

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 &#34;image-able&#34; 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 to reveal the extent of inter-subject and intra-tumoral heterogeneity in nanoparticle accumulation6,7. However, imaging of nanoparticles alone does not identify the biological barriers that have contributed to their poor accumulation and distribution. This knowledge is paramount to the rational development of more efficacious formulations, and strategies to improve intra-tumoral accumulation8. It has been demonstrated that therapeutic strategies can be applied to modulate specific biological barriers resulting in improved nanoparticle transport9. Additionally, nanoparticle formulations have been developed to specifically overcome specific biological transport barrier10. In both scenarios, measurements of biological barriers could be used to guide the use of an appropriate nanoparticle drug delivery strategy.
Tumor microcirculation and elevated IFP are believed to be two key determinants of the intra-tumoral accumulation of nanoparticles, such as liposomes, in solid tumors9,11. However, other barriers that to contribute to poor liposome accumulation include a dense extracellular matrix, impermeable vasculature, and solid tissue pressure12. These barriers are related in a spatio-temporal manner, with abnormal blood flow and elevated interstitial fluid pressure being two important factors driving the initial delivery and extravasation of nanoparticles. As previously discussed, establishing the relationship between the tumor microcirculation, elevated IFP, and the intra-tumoral accumulation of liposomes is imperative for proper interpretation of liposome imaging data. Herein quantitative methods to measure the relationship between the tumor microcirculation, elevated IFP, and nanoparticle accumulation in a solid tumor are presented. This is accomplished by performing co-localized measurements of the intra-tumoral distribution of a CT liposome contrast agent using volumetric CT imaging, tumor microcirculation using dynamic contrast enhanced computed tomography imaging, and tumor IFP using an image-guided robotic needle positioning system, termed the CT-IFP robot13.

<table border="0" cellpadding="0" cellspacing="0" width="1520">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">MDA-MB-231 metastatic breast adenocarcinoma tumor cells&#160;</td>
			<td style="width:471px;">ATCC</td>
			<td style="width:119px;">HTB-26</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td style="width:471px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:471px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone Penicillin-Streptomycin 100x Solution</td>
			<td>GE Healthcare Life Sciences</td>
			<td style="width:119px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td>Avanti Lipids Inc., USA</td>
			<td>850355P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol (CH)</td>
			<td>Avanti Lipids Inc., USA</td>
			<td style="width:119px;">700000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol) 2000 (DSPE-PEG2000)</td>
			<td>Avanti Lipids Inc., USA</td>
			<td style="width:119px;">880128P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnipaque (Iohexol) 300 mg of iodine/mL&#160;</td>
			<td>GE Healthcare, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">80 nm pore size Track-Etch polycarbonate membranes</td>
			<td style="width:471px;">Whatman Inc., USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 nm pore size Track-Etch polycarbonate membranes</td>
			<td style="width:471px;">Whatman Inc., USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL Lipex Extruder&#160;</td>
			<td>Nothern Lipids Inc, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Dialysis Bag Molecular Weight Cut Off (MWCO) of 8 kDa</td>
			<td>Spectrum Labs, USA&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">750,000 Nomical Molecular Weight Cut Off (NMWC) Tangential flow column&#160;</td>
			<td>MidGee ultrafiltration cartridge, GE Healthcare, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic pump&#160;</td>
			<td>Watson Marlow Inc., USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV spectrometer</td>
			<td>Helios &#947;, Spectronic Unicam,&#160; USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">90Plus particle size analyzer&#160;</td>
			<td>Brookhaven, Holtsville, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eXplore Locus Ultra micro-CT system&#160;</td>
			<td>GE Healthcare, CA</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Manipulated using CT-Console Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">AxRecon GPU-based Reconstruction</td>
			<td>&#160;Acceleware Corp. CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">27G Catheter SURFLO Winged Infusion Set</td>
			<td>Terumo Medical Products, USA</td>
			<td style="width:119px;">SV*27EL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">PE20 polyethylyne tubing</td>
			<td>Becton Dickinson, USA</td>
			<td style="width:119px;">427406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen tip 25G &#215; 3.5&#8242;&#8242; Whitacre spinal needle&#160;</td>
			<td>Becton Dickinson, USA</td>
			<td style="width:119px;">405140</td>
			<td>IFP needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P23XL&#160; pressure transducer&#160;</td>
			<td>Harvard Apparatus, CA</td>
			<td style="width:119px;">P23XL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">PowerLab 4/35, Bridge Amp, with LabChart Pro 7.0</td>
			<td>ADInstruments Pty Ltd., USA</td>
			<td style="width:119px;">PL3504, FE221</td>
			<td>IFP acquisition system and acquisition software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:632px;">CT-Sabre Small Animall Intervention system (CT-IFP Robot)</td>
			<td style="width:471px;">Parallax Innovations, CA</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Manipulated using CT-IFP robot Control Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">CT-IFP robot alignment software</td>
			<td style="width:471px;"></td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Custom Matlab software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">DCE-CT Analysis Software</td>
			<td style="width:471px;"></td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Custom Matlab software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Matlab 2013b</td>
			<td style="width:471px;">Mathworks, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatial measurements of perfusion, interstitial fluid pressure and liposomes accumulation in solid tumors

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.

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 &#177; 0.3 nm and -45.5 &#177; 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 ...

Watch this Scientific Journal Video about Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors at JoVE.com

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

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.

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and ...

The overall goal of this experiment is to relate the intratumoral accumulation of nanotherapeutics to the properties of the tumor microenvironment, including the tumor microcirculation and the elevated interstitial fluid pressure, or IFP. This method lets us answer important questions about nanomedicines. Questions like what drives heterogeneous uptake of nanoparticles inside a tumor?The main advantage of this technique is that allows for co-localized spatial mapping of the properties of the tumor microenvironment and the intratumoral distribution of nanoparticles. After confirming the appropriate level of anesthesia by topenge, apply ointment to the mouse's eyes and tape the animal's limbs in the prone position on a thin plastic board. Next, insert a custom 27-gauge catheter connected to a 20-centimeter piece of PE10 tubing into the lateral tail vein and secure the tubing with several pieces of tape.Now, fill one one milliliter syringe with at least 200 microliters of computed tomography, or CT liposomes and one one milliliter syringe with at least 150 microliters of a 9-1 ratio by volume of free iohexol mixed with saline. Place the CT liposome syringe in a syringe pump and attach the catheter to the syringe, setting a pump rate of 600 microliters per minute, equivalent to 10 microliters per second. Then place the mouse on the micro CT scanner bed and use the laser positioning system to adjust the tumor so that it will be approximately the same orientation for each scan.Using the CT scanner console software for each imaging protocol of interest, select bright dark from the drop-down menu and click the scan button to initiate the calibration and to initialize the system. To obtain a volumetric anatomical micro CT of the tumor before any contrast agent is injected, first check the CT scanner console software indicator to confirm that the CT scanner safety interlocks have been cleared. Then select the scan that uses an x-ray energy of 80 kilovolts, a tube current of 70 milliamps and captures 1, 000 image projections.Then, initiate the scan. When the scan is finished, set the pump to inject approximately 150 microliters of the liposome solution and press the start button to inject the bolus of CT liposomes at a 55 milligram of iodine per milliliter of solution concentration. Manually flush the catheter with 50 microliters of saline to ensure the entire amount of liposome agent was injected and that the catheter has been cleared.After 10 minutes, perform a second anatomical scan of the tumor, as just demonstrated. To perform a DCE-CT, place a syringe of free iohexol solution into the syringe pump and set the pump to inject 100 microliters of the iohexol at the same injection rate. These injection volumes are higher than the standard of 200 microliters, but the animals are monitored during recovery and no adverse events have been observed.Then, on the CT scanner console, select a five minute dynamic scan using an x-ray energy of 80 kilovolts and a tube current of 90 milliamps, as just demonstrated, that captures 416 image projections every one second for the first 30 seconds followed by 416 image projections every 10 seconds. Capture five seconds of DCE-CT data, then start the injection pump. At the end of the scan, perform a third volumetric anatomical micro CT scan.48-70 hours later, capture anatomical CT images of the liposomes using the same volumetric settings as just demonstrated. To measure the IFP, tape the animal on the CT IFP robot platform such that the tumor is immobilized and accessible to the CT IFP robot system. Obtain an anatomic micro CT scan as just demonstrated.Then load the pre-needle insertion data into the CT IFP robot alignment software and adjust the window and level to visualize the tumor. Click on the rim of the tumor in any image followed by the selection of a second rim location on the adjacent side of the tumor. The software will calculate a series of positions along a linear line between the two points.Then, select the X, Y and Z coordinates for a series of five to eight evenly-spaced positions from the list. Next, flush the IFP system needle with a saline heparin solution and enter the first predetermined needle positions into the X, Y, Z coordinate windows of the CT IFP robot control software. Press the go button to move the robot to the desired location.Then, for each needle position in turn, click the insert needle button to insert the needle into the tissue. Pinch and release the PE20 tubing to confirm good fluid communication between the IFP needle and the tissue and observe that the IFP measurement increases and returns to the pre-pinching value on the IFP acquisition software. Finally, acquire an anatomic CT scan with the needle inserted, clicking the retract needle button at the end of the scan to remove the needle from the tissue.Selecting a region of interest within the tumor yields a time/intensity curve that can be used to yield quantitative estimates of select hemodynamic parameters in the tumor. Segmenting the tumor volume into equally-sized multiple regions of interest allows the quantification of the spatial distribution of these parameters within the tumor volume. The biodistribution of the CT liposomes at 48 hours post-injection can also be observed.As indicated, the agent is still circulating in the vascular system with a substantial uptake observed in the spleen and liver. The intratumoral accumulation of these CT liposomes is heterogeneous with a predominantly peripheral accumulation compared to the center of the tumor tissue. The needle can clearly be identified by high resolution micro CT, allowing the spatial localization of the IFP measurements within the tumor volume.Spatially co-localized measurement of the profusion and the plasma volume fraction demonstrates a significant correlation with the intratumoral accumulation of CT liposomes in subcutaneous tumors. Further, the radial distribution of the IFP correlates with the other hemodynamic measurements, suggesting a complex spatial/temporal relationship between the tumor microcirculation, the IFP and the intratumoral accumulation of the liposomes. While attempting this procedure, it's important to secure the animal on the scanner bed, ensuring minimal tumor motion between IFP measurements.The implications of this work extend to the development of new therapies. The transport of nanoparticles is a key first step in building efficacious therapeutics based on nanomedicine. After watching this video, you should have a good idea of how to spatially map tumor interstitial fluid pressure, the tumor microcirculation, and nanoparticle distribution.

spatial-measurements-perfusion-interstitial-fluid-pressure-liposomes

Research

JoVE Journal

Medicine

7.6K vistas.  University of Toronto. The overall goal of this experiment is to relate the intratumoral accumulation of nanotherapeutics to the properties of the tumor microenvironment, including the tumor microcirculation and the elevated interstitial fluid pressure, or IFP. This method lets us answer important questions about nanomedicines. Questions like what drives heterogeneous uptake of nanoparticles inside a tumor?The main advantage of this technique is that allows for co-localized spatial mapping of the properties ...