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 ....

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 too....

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