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

Summary

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.

Abstract

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.

Introduction

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 nanotherapies^2,3, 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 use^4,5. 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 cancer^8,9,10, 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, liposomes^9,10, polymeric nanoparticles, antibody fragments, and nanoemulsions^10,13. 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—the most established nanomedicine platform¹⁴—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.

Protocol

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.

1. 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.
2. 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.
3. 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.
4. For quality control, characterize the liposomes by dynamic light scattering (DLS). Specifically, mix 50 µL of liposomes with 950 µL 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.
5. 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 °C for 2 h in a 1.5-mL tube, (total volume: ~ 100 µL). 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.
6. 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.
7. 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.

2. In Vivo PET-CT Imaging and Biodistribution of Dual-labeled Liposomes

1. Inject about 100 µL of dual-labeled liposomes with about 300 µCi 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.
2. 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 µL) 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.
3. 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.
4. 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 µA; the CT scan uses 120 rotational steps for a total of 220 degrees, with approximately 145 ms per frame ¹⁰.
5. 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.
6. 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.
7. 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).

3. Ex Vivo Flow Cytometry and Immunostaining

NOTE: Inject about 100 µL 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.

1. Sacrifice the mice 24 h post injection, as in step 2.5, and collect the tumors, which must be stored in cold PBS.
2. For flow cytometry, follow these steps ¹⁷:
   1. 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).
   2. Mix 100 mg of tumor tissue with 200 µL 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.
   3. Shake the 6-well plate on a horizontal shaker placed inside a 37 °C oven for 60 min at 60 rpm.
   4. Wash the tissue homogenate with flow cytometry buffer 3 times to obtain a single cell suspension.
   5. 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.
3. For immunostaining, follow these steps:
   1. Place a tumor into a cassette filled with optimal cutting medium (OCT) and freeze it on dry ice.
   2. Make 6-µm 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.
   3. 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 °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}.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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