Name: A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines (Video) | JoVE Journal
Uploaded: 2017-03-04T14:00:00.000+00:00
Duration: 7 min 59 s
Description: Learn a comprehensive procedure to evaluate cancer nanomedicine performance in tumor-bearing mice using dual-labeling and liposome platforms. This method enables quantitative assessment from systemic to subcellular levels.

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The authors would like to thank Drs. Helene Salmon and Miriam Merad from Icahn School of Medicine at Mount Sinai for providing the B16-F10-YFP cells and for their expert advice on melanoma mouse models. The authors further thank the Animal Imaging Core Facility, the Radiochemistry and Molecular Imaging Probes Core Facility, and the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center (MSK) for their support. This work was supported by National Institutes of Health grants NIH 1 R01 HL125703 (W.J.M.M.), R01CA155432 (W.J.M.M.), K25 EB016673 (T.R.) and P30 CA008748 (MSK Center Grant). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) at MSK for their financial support (T.R.).

The procedure consists of the dual radioactive and fluorescent labeling of nanoparticles, in vivo PET-CT imaging, ex vivo biodistribution measurements, and ex vivo immunostaining and flow cytometry analyses. All animal experiments were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center.
1. Preparation of Dual-labeled Liposomes
NOTE: Syngeneic B16 melanoma tumors can be induced by injecting 300,000 B16-F10 cells into the back flanks of C57BL/6 mice under anesthesia from inhaling 2%-isoflurane-containing oxygen. Use sterile reagents and tools in a sterile workspace during surgery. After surgery, carefully observe the animals until their recovery from anesthesia. Put the animals back in their cages only when they regain full consciousness and mobility. House them in sterile rooms for animals with xenografted tumors. Tumors with a size of 300 mm 3 are ideal for nanoparticle characterization due to their pronounced enhanced permeability and retention effects (EPR), which can increase nanoparticle accumulation. The detailed protocols are provided below.
<ol>
	<li>In a round-bottom flask, dissolve a mixture of 20 mg of lipids in 2 mL of chloroform containing 1,2-dipalmitoyl- sn-glycero-3-phophocholine (DPPC), cholesterol, 1,2-disearoyl- sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)2000 (DSPE-PEG2000), DSPE-deferoxamine (DSPE-DFO) 8, and 1,1-diododecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disulfonic acid (DiIC12[5]-DS) at molar ratios of 61.3%, 33.4%, 5, 0.2%, and 0.1%, respectively. 
	NOTE: This lipid composition is very similar to the liposomal formulation of doxorubicin currently used in the clinic 2. The resulting liposomes from our procedure will closely mimic the in vivo performance of the clinical nanomedicine.</li>
	<li>Use a rotary evaporator to remove the organic solvent and form a thin lipid film at room temperature. Add 20 mL of PBS and sonicate for 30 min to generate liposomal nanoparticles by using a 3.8-mm sonication tip and an amplitude of 30 W, with sufficient cooling on ice.</li>
	<li>Centrifuge the liposome solution at 4,000 x g for 10 min to remove the aggregates and wash the nanoparticles with PBS using centrifugal filtration (molecular weight cut-off (MWCO: 100 kDa) to remove free lipids and residual organic solvent. Concentrate the liposomes, which will remain in the top chamber, and transfer them to a new tube. The liposomes can be stored in a fridge in PBS for up to 1 week before the subsequent steps.</li>
	<li>For quality control, characterize the liposomes by dynamic light scattering (DLS). Specifically, mix 50 &#181;L of liposomes with 950 &#181;L of PBS, and then transfer the mixture into the sizing cuvette. Place the cuvette in the analyzer and measure the particle sizes and their size distribution. The particle sizes and their size distribution (polydispersity index (PDI)) are expected to be 90 - 120 nm and 0.1 - 0.2, respectively.</li>
	<li>For radiolabeling, mix the purified liposomes, equivalent to 2 mg of lipids, in PBS at a pH between 6.9 and 7.1 with 1 mCi 89Zr-oxalate at 37 &#176;C for 2 h in a 1.5-mL tube, (total volume: ~ 100 &#181;L). Remove the free, unreacted 89Zr by centrifugal filtration (MWCO = 100 kDa), similar to step 1.3. Wash the retentate with sterile PBS and dilute it to the desired volume. The radiochemical yield should be 80 - 95%. 
	CAUTION! Working with radioactive materials is highly regulated. Institutions must be certificated and provide the necessary facilities, personnel training, and strict monitoring of radioactivity use. Researchers need to receive adequate training and wear personal protective equipment and dosimetry rings/badges when handling radioactivity.</li>
	<li>Determine the radiochemical purity of the radiolabeled liposomes by size exclusion radio-HPLC, which typically should be over 98% 10. Radiochemical purities lower than 95% are indicative of insufficient washing on step 1.5.</li>
	<li>After radiolabeling, determine the size of the nanoparticles by dynamic light scattering. The size and PDI are expected to be within the same range as the measurements in step 1.4 10. 
	NOTE: If radioactive samples are not allowed in the flow cytometer or fluorescence microscope, non-radioactive natZr-oxalate can be used to label liposomes in step 1.5. These non-radioactive liposomes have identical characteristics to their radioactive analogs. A depiction of this procedure is provided in Figure 2.</li>
</ol>
2. In Vivo PET-CT Imaging and Biodistribution of Dual-labeled Liposomes
<ol>
	<li>Inject about 100 &#181;L of dual-labeled liposomes with about 300 &#181;Ci of radioactivity into restrained melanoma mice (C57BL/6, female, 10 - 16 weeks old) through their tail veins, at about 10 mCi 89Zr/kg body weight.</li>
	<li>To determine the half-life, draw blood from the tail vein from anesthetized animals under 2% isoflurane-containing oxygen using 28-G insulin syringes. Collect the blood (10 - 20 &#181;L) in pre-weighed tubes at 2 min, 15 min, 1 h, 4 h, 8 h, 24 h, and 48 h after injection. Weigh the blood by difference, determine radioactivity content using a gamma counter, and calculate radioactivity concentration as percentage of injected dose per gram (%ID/g). 
	NOTE: Perform the procedure in a room equipped with an anesthesia system, recovery cages, heating, and a biohazard hood. Make sure that the animals fully recover and gain normal mobility after the procedure before transferring them from recovery cages to holding cages. House the tumor-bearing animals in rooms designated for animals administered with radioactive materials.</li>
	<li>24 or 48 h after the injection of the liposomes, image the mice in a small-animal MicroPET-CT scanner 10. Place the anesthetized animal horizontally on its belly and keep it anesthetized. Administer 2% isoflurane-oxygen through a nose cone. Apply ophthalmic ointment to its eyes before the scan to prevent them from drying.</li>
	<li>Perform a whole-body PET static scan recording at least 50 million coincident events, with an approximate duration of 15 min. Set the energy and coincidence timing window at 350-700 keV and 6 ns, respectively. Afterwards, perform a 5-min computed tomography (CT) scan. Set the X-ray tube to a voltage of 80 kV and a current of 500 &#181;A; the CT scan uses 120 rotational steps for a total of 220 degrees, with approximately 145 ms per frame 10.</li>
	<li>After the PET-CT scan, immediately sacrifice the mice by carbon dioxide asphyxiation and confirm the deaths by pinching the paws of the animals. Once confirmed, perform cervical dislocations.</li>
	<li>Remove the blood in mouse tissues by first cutting open the right atrium and then injecting 20 mL PBS into the left ventricle, and subsequently collect the relevant tissues such as tumor, liver, spleen, lung, kidney, muscle, and others 16.</li>
	<li>Weigh the tissues, measure their radioactivity content by gamma counting 8 , 9, and calculate the tissue radioactivity accumulation as a percentage of the injected dose per g (%ID/g).</li>
</ol>
3. Ex Vivo Flow Cytometry and Immunostaining
NOTE: Inject about 100 &#181;L of fluorescent liposomes into melanoma mice through their tail vein at a dose of 0.5 mg of dye per kg of body weight. If radioactive samples are not allowed in the flow cytometer and fluorescence microscope, non-radioactive liposomes must be used.
<ol>
	<li>Sacrifice the mice 24 h post injection, as in step 2.5, and collect the tumors, which must be stored in cold PBS.</li>
	<li>For flow cytometry, follow these steps 17:
	<ol>
		<li>Prepare an enzyme cocktail consisting of a mixture of purified collagenase I and II (4 U/mL), hyaluronidase (60 U/mL), and DNase I (60 U/mL) in flow cytometry buffer (PBS with 0.5% BSA and 1 mM EDTA).</li>
		<li>Mix 100 mg of tumor tissue with 200 &#181;L of enzyme cocktail buffer in a 1.5-mL tube and dice the tissue to small pieces with fine scissors. Add another 1.3 mL of enzyme cocktail into the tube and transfer its contents to a 6-well plate.</li>
		<li>Shake the 6-well plate on a horizontal shaker placed inside a 37 &#176;C oven for 60 min at 60 rpm.</li>
		<li>Wash the tissue homogenate with flow cytometry buffer 3 times to obtain a single cell suspension.</li>
		<li>Stain the cells with a cocktail of antibodies specific to biomarkers including CD45, CD11b, Ly6C, CD11c, CD64, CD3, MHCII, and CD31 (1:200 dilution for all antibodies; clone information is provided in the materials spreadsheet). Identify relevant cell types and determine their mean fluorescence intensity (MFI) of DiIC12(5)-DS in each cell population with an appropriate flow cytometry analysis software.</li>
	</ol>
	</li>
	<li>For immunostaining, follow these steps:
	<ol>
		<li>Place a tumor into a cassette filled with optimal cutting medium (OCT) and freeze it on dry ice.</li>
		<li>Make 6-&#181;m frozen sections from tumor tissue and place them on histology glass slides; the sections should cover the largest cross-sections of the tumor, which provide the most representative information on the tissue.</li>
		<li>Stain the biomarkers ( e.g., CD31 for endothelial cells) of interest with the corresponding antibodies ( e.g., anti-CD31, clone MEC13.3). Fix the sections with paraformaldehyde; block them with serum matching the species origin of the secondary antibodies; incubate with primary antibodies for 12 h at 4 &#176;C and then with secondary antibodies for 2 h; and finally, wash with PBS and cover with cover slips. Image the stained slides with a Leica upright confocal SP5 microscope 13 , 16.</li>
	</ol>
	</li>
</ol>

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The authors have no disclosure to make.

Critical Steps within the Protocol:
The high quality of dual-labeled liposomes is the key to producing consistent results over a long period of time. Free fluorescent dyes or 89Zr ions can generate totally different targeting patterns and must be completely removed during the purification step. In addition, if the immune system significantly affects experimental cancer nanomedicine performance, the use of immunocompetent mouse models should be preferable, such as the B16-F10 melanoma m...

Nanomedicine is shifting the paradigm of cancer treatment development1. Inspired by the tremendous clinical impact of previous cancer nanomedicines, such as liposome- and albumin-based nanotherapies2,3, many novel formulations have been produced in the past decade. However, recent analyses of the clinical translation success of these cancer nanomedicines indicate that only a few of them have been approved for clinical use4,5. One major obstacle to the clinical translation of new cancer nanomedicines is their limited improvement of the therapeutic index compared with the direct administration of the free therapeutic compounds6. As such, accurate evaluation of the in vivo performance of nanomedicines at systemic, tissue, and cellular levels in preclinical animal models is essential to identify those with optimal therapeutic indices for future clinical translation.
Nanomaterials can be radiolabeled for quantitative characterization in living animals with positron emission tomography (PET) imaging, which has superb sensitivity and reproducibility among all clinical imaging modalities7. For example, 89Zr-labeled long-circulating nanomedicines have been characterized in mouse models for cancer8,9,10, as well as in other disease models11. In addition, the blood half-life and biodistribution of the nanomedicines can be extensively evaluated by using ex vivo radioactivity measurements in individual tissues8. Therefore, radiolabeling allows for the quantitative evaluation of nanomedicines at systemic and tissue levels.
Importantly, radiolabeled nanomedicines generally cannot be analyzed at the single-cell or subcellular levels due to the limited spatial resolution of the radioactive signal. Therefore, fluorescent labeling proves to be a complementary modality for the evaluation of nanoparticles with optical imaging techniques such as flow cytometry and fluorescence microscopy12. To this end, nanoparticles labeled with radioisotopes and fluorescent tags can be quantitatively evaluated in vivo by nuclear imaging and ex vivo by radioactivity counting, and they can also be extensively characterized at the cellular level by optical imaging.
Previously, we have developed modular procedures to incorporate radioactive and fluorescent labels into various nanoparticles, including high-density lipoprotein (HDL)11, liposomes9,10, polymeric nanoparticles, antibody fragments, and nanoemulsions10,13. These labeled nanoparticles have allowed for quantitative characterization in relevant animal models at different levels, which guided the optimization of these nanomaterials for their specific applications. In the current study, the aim is to use liposomal nanoparticles&#8212;the most established nanomedicine platform14&#8212;as an example to demonstrate comprehensive procedures to generate a dual-labeled nanoparticle and to thoroughly characterize it in a classic syngeneic melanoma B16-F10 mouse model15. From the results, we are confident this nanoparticle characterization approach can be adapted to evaluate other cancer nanomedicines in relevant mouse models.

<table><tbody><tr><td>DPPC</td><td>Avantilipids</td><td>850355</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C8667</td><td></td></tr><tr><td>DSPE-PEG2000</td><td>Avantilipids</td><td>880120P</td><td></td></tr><tr><td>DSPE-DFO</td><td>Home made</td><td>110634</td><td>Perez-Medina &lt;em&gt;et al.&lt;/em&gt;, JNM, 2014</td></tr><tr><td>DiIC12[5]-DS</td><td>AAT Bioquest</td><td>22051</td><td></td></tr><tr><td>Centrifugal filter</td><td>Vivaproducts</td><td>VS2061</td><td></td></tr><tr><td>Rotary evaporator</td><td>Buchi</td><td>R-100</td><td></td></tr><tr><td>Radio-HPLC</td><td>Shimadzu HPLC with 2 LC-10AT pumps</td><td>N/A</td><td></td></tr><tr><td>&lt;sup&gt;89&lt;/sup&gt;Zr-oxalate</td><td>MSKCC</td><td>Synthesized in house</td><td>TR19/9 variable beam cyclotron (Ebco Industries Inc.)</td></tr><tr><td>Micro PET-CT</td><td>Siemens</td><td>Inveon Micro-PET/CT</td><td></td></tr><tr><td>Gamma counter</td><td>PerkinElmer</td><td>2470-0150</td><td></td></tr><tr><td>Flow cytometry</td><td>BD Biosciences</td><td>Fortessa</td><td>Any multi-parametric flow cytometry analyzers would suffice</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Laboratories</td><td></td><td></td></tr><tr><td>B16-YFP melanoma cells</td><td>Home made</td><td>N/A</td><td>Salmon &lt;em&gt;et al.&lt;/em&gt;, Immunity, 2016</td></tr><tr><td>Ly6C (clone HK1.4)--APC-Cy7</td><td>128025</td><td>Biolegend</td><td></td></tr><tr><td>MHCII (M5/114/152)--APC</td><td>107613</td><td>Biolegend</td><td></td></tr><tr><td>CD45 (30-F11)--BV510</td><td>103137</td><td>Biolegend</td><td></td></tr><tr><td>CD64 (X54-5/7.1)--PE-Cy7</td><td>139313</td><td>Biolegend</td><td></td></tr><tr><td>CD11b (M1/70)--BV605</td><td>101237</td><td>Biolegend</td><td></td></tr><tr><td>CD3 (17A2)--BV711</td><td>100241</td><td>Biolegend</td><td></td></tr><tr><td>CD31 (13.3)--PE</td><td>561073</td><td>Biolegend</td><td></td></tr><tr><td>CD11c (M418)--PerCP-Cy5.5</td><td>117327</td><td>BD Biosciences</td><td></td></tr><tr><td>CD31 (13.3) no fluorophore</td><td>550274</td><td>BD Biosciences</td><td></td></tr></tbody></table>

a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

Figure 1 shows an overview of the procedure. Figure 2 presents the schematic synthesis procedure of the dual-labeled liposomes described in step 110. Figure 3 displays a representative PET-CT image (Figure 3a), radioactivity quantification from PET imaging (Figure 3b), blood half-life (Figure 3c), and biodistribution (Figure 3d) of radioactive nano...

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

The overall goal of this procedure is to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single cell, and subcellular levels in tumor-bearing, immunocompetent mice. This method can help answer the key question in the cancer nanomedicine field. How do nanomedicines accumulate quantitatively in different tissues of living animals over time?The main advantage of this technique is that this dual-labeling approach allows for in vivo nanomedicine characterization from the macroscopic to the microscopic level. We choose liposomes as example to demostrate our procedure because liposomes are currently in clinical use, and they're also a common platform for experimental nanomedicines. To begin the procedure, dissolve 20 milligrams of a lipid mixture, which include 0.1%by mole of a lipophilic cationic fluorescent dye and two milliliters of chloroform.Then, dry the lipids with a rotary evaporator. Add 20 milliliters of phosphate-buffered saline to the dried lipids. Sonicate the mixture on ice with a 3.8 millimeter probe sonicator at 30 watts for 30 minutes, keeping the mixture between four and 20 degrees Celsius throughout.Centrifuge the resulting liposomal nanoparti

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JoVE Journal

Cancer Research

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Viral Nanoparticles for In vivo Tumor Imaging

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol presents techniques and methodology necessary for the accurate delivery of magnetic nanoparticle hyperthermia using a sophisticated delivery and monitoring system.

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1α in a syngeneic mouse model of HNSCC.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Learn a comprehensive procedure to evaluate cancer nanomedicine performance in tumor-bearing mice using dual-labeling and liposome platforms. This method enables quantitative assessment from systemic to subcellular levels.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

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