Emily B. Ehlerding

In Vivo Tumor Vasculature Targeting of CuS@MSN Based Theranostic Nanomedicine

Actively targeted theranostic nanomedicine may be the key for future personalized cancer management. Although numerous types of theranostic nanoparticles have been developed in the past decade for cancer treatment, challenges still exist in the engineering of biocompatible theranostic nanoparticles with highly specific in vivo tumor targeting capabilities. Here, we report the design, synthesis, surface engineering, and in vivo active vasculature targeting of a new category of theranostic nanoparticle for future cancer management. Water-soluble photothermally sensitive copper sulfide nanoparticles were encapsulated in biocompatible mesoporous silica shells, followed by multistep surface engineering to form the final theranostic nanoparticles. Systematic in vitro targeting, an in vivo long-term toxicity study, photothermal ablation evaluation, in vivo vasculature targeted imaging, biodistribution and histology studies were performed to fully explore the potential of as-developed new theranostic nanoparticles.

Biodegradable and Renal Clearable Inorganic Nanoparticles.

Personalized treatment plans for cancer therapy have been at the forefront of oncology research for many years. With the advent of many novel nanoplatforms, this goal is closer to realization today than ever before. Inorganic nanoparticles hold immense potential in the field of nano‐oncology, but have considerable toxicity concerns that have limited their translation to date. In this review, an overview of emerging biologically safe inorganic nanoplatforms is provided, along with considerations of the challenges that need to be overcome for cancer theranostics with inorganic nanoparticles to become a reality. The clinical and preclinical studies of both biodegradable and renal clearable inorganic nanoparticles are discussed, along with their implications.

Surface engineering of graphene-based nanomaterials for biomedical applications.

Graphene-based nanomaterials have attracted tremendous interest over the past decade due to their unique electronic, optical, mechanical, and chemical properties. However, the biomedical applications of these intriguing nanomaterials are still limited due to their suboptimal solubility/biocompatibility, potential toxicity, and difficulties in achieving active tumor targeting, just to name a few. In this Topical Review, we will discuss in detail the important role of surface engineering (i.e., bioconjugation) in improving the in vitro/in vivo stability and enriching the functionality of graphene-based nanomaterials, which can enable single/multimodality imaging (e.g., optical imaging, positron emission tomography, magnetic resonance imaging) and therapy (e.g., photothermal therapy, photodynamic therapy, and drug/gene delivery) of cancer. Current challenges and future research directions are also discussed and we believe that graphene-based nanomaterials are attractive nanoplatforms for a broad array of future biomedical applications.

Intrinsically Radiolabeled Nanoparticles: An Emerging Paradigm

Although chelator-based radiolabeling techniques have been used for decades, concerns about the complexity of coordination chemistry, possible altering of pharmacokinetics of carriers, and potential detachment of radioisotopes during imaging have driven the need for developing a simple yet better technique for future radiolabeling. Here, the emerging concept of intrinsically radiolabeled nanoparticles, which could be synthesized using methods such as hot-plus-cold precursors, specific trapping, cation exchange, and proton beam activation, is introduced. Representative examples of using these multifunctional nanoparticles for multimodality molecular imaging are highlighted together with current challenges and future research directions. Although still in the early stages, design and synthesis of intrinsically radiolabeled nanoparticles has shown attractive potential to offer easier, faster, and more specific radiolabeling possibilities for the next generation of molecular imaging.

NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.

Preclinical Pharmacokinetics and Biodistribution Studies of 89Zr-labeled Pembrolizumab

Pembrolizumab is a humanized monoclonal antibody targeting programmed cell death protein 1 (PD-1) found on T and pro-B cells. Pembrolizumab prevents PD-1 ligation by both PD-L1 and PD-L2, preventing the immune dysregulation that otherwise occurs when T-cells encounter cells expressing these ligands. Clinically, PD-1 blockade elicits potent antitumor immune responses, and antibodies blocking PD-1 ligation, including pembrolizumab, have recently received Food and Drug Administration approval for the treatment of advanced melanoma, renal cell cancer, and non–small cell lung cancer. Methods: In this study, we evaluated the pharmacokinetics, biodistribution, and dosimetry of pembrolizumab in vivo, accomplished through radiolabeling with the positron emitter 89Zr. PET imaging was used to evaluate the whole-body distribution of 89Zr-deferoxamine (Df)-pembrolizumab in two rodent models (mice and rats). Data obtained from PET scans and biodistribution studies were extrapolated to humans to estimate the dosimetry of the tracer. As a proof of concept, the biodistribution of 89Zr-Df-pembrolizumab was further investigated in a humanized murine model. Results: The tracer remained stable in blood circulation throughout the study and accumulated the greatest in liver and spleen tissues. Both mice and rats showed similar biodistribution and pharmacokinetics of 89Zr-Df-pembrolizumab. In the humanized mouse model, T-cell infiltration into the salivary and lacrimal glands could be successfully visualized. Conclusion: These data will augment our understanding of the pharmacokinetics and biodistribution of radiolabeled pembrolizumab in vivo, while providing detailed dosimetry data that may lead to better dosing strategies in the future. These findings further demonstrate the utility of noninvasive in vivo PET imaging to dynamically track T-cell checkpoint receptor expression and localization in a humanized mouse model.

Molecular Imaging of Immunotherapy Targets in Cancer

Immunotherapy has emerged as a promising alternative in the arsenal against cancer by harnessing the power of the immune system to specifically target malignant tissues. As the field of immunotherapy continues to expand, researchers will require newer methods for studying the interactions between the immune system, tumor cells, and immunotherapy agents. Recently, several noninvasive imaging strategies have been used to map the biodistribution of immune checkpoint molecules, monitor the efficacy and potential toxicities of the treatments, and identify patients who are likely to benefit from immunotherapies. In this review, we outline the current applications of noninvasive techniques for the preclinical imaging of immunotherapy targets and suggest future pathways for molecular imaging to contribute to this developing field.

CD146-targeted immunoPET and NIRF Imaging of Hepatocellular Carcinoma with a Dual-Labeled Monoclonal Antibody.

Overexpression of CD146 has been correlated with aggressiveness, recurrence rate, and poor overall survival in hepatocellular carcinoma (HCC) patients. In this study, we set out to develop a CD146-targeting probe for high-contrast noninvasive in vivo positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of HCCs. YY146, an anti-CD146 monoclonal antibody, was employed as a targeting molecule to which we conjugated the zwitterionic near-infrared fluorescence (NIRF) dye ZW800-1 and the chelator deferoxamine (Df). This enabled labeling of Df-YY146-ZW800 with 89Zr and its subsequent detection using PET and NIRF imaging, all without compromising antibody binding properties. Two HCC cell lines expressing high (HepG2) and low (Huh7) levels of CD146 were employed to generate subcutaneous (s.c.) and orthotopic xenografts in athymic nude mice. Sequential PET and NIRF imaging performed after intravenous injection of 89Zr-Df-YY146-ZW800 into tumor-bearing mice unveiled prominent and persistent uptake of the tracer in HepG2 tumors that peaked at 31.65 ± 7.15 percentage of injected dose per gram (%ID/g; n=4) 72 h post-injection. Owing to such marked accumulation, tumor delineation was successful by both PET and NIRF, which facilitated the fluorescence image-guided resection of orthotopic HepG2 tumors, despite the relatively high liver background. CD146-negative Huh7 and CD146-blocked HepG2 tumors exhibited significantly lower 89Zr-Df-YY146-ZW800 accretion (6.1 ± 0.5 and 8.1 ± 1.0 %ID/g at 72 h p.i., respectively; n=4), demonstrating the CD146-specificity of the tracer in vivo. Ex vivo biodistribution and immunofluorescent staining corroborated the accuracy of the imaging data and correlated tracer uptake with in situ CD146 expression. Overall, 89Zr-Df-YY146-ZW800 showed excellent properties as a PET/NIRF imaging agent, including high in vivo affinity and specificity for CD146-expressing HCC. CD146-targeted molecular imaging using dual-labeled YY146 has great potential for early detection, prognostication, and image-guided surgical resection of liver malignancies.

89 Zr-labeled nivolumab for imaging of T-cell infiltration in a humanized murine model of lung cancer

PurposeNivolumab is a human monoclonal antibody specific for programmed cell death-1 (PD-1), a negative regulator of T-cell activation and response. Acting as an immune checkpoint inhibitor, nivolumab binds to PD-1 expressed on the surface of many immune cells and prevents ligation by its natural ligands. Nivolumab is only effective in a subset of patients, and there is limited evidence supporting its use for diagnostic, monitoring, or stratification purposes.Methods89Zr-Df-nivolumab was synthesized to map the biodistribution of PD-1-expressing tumor infiltrating T-cells in vivo using a humanized murine model of lung cancer. The tracer was developed by radiolabeling the antibody with the positron emitter zirconium-89 (89Zr). Imaging results were validated by ex vivo biodistribution studies, and PD-1 expression was validated by immunohistochemistry. Data obtained from PET imaging were used to determine human dosimetry estimations.ResultsThe tracer showed elevated binding to stimulated PD-1 expressing T-cells in vitro and in vivo. PET imaging of 89Zr-Df-nivolumab allowed for clear delineation of subcutaneous tumors through targeting of localized activated T-cells expressing PD-1 in the tumors and salivary glands of humanized A549 tumor-bearing mice. In addition to tumor uptake, salivary and lacrimal gland infiltration of T-cells was noticeably visible and confirmed via histological analysis.ConclusionsThese data support our claim that PD-1-targeted agents allow for tumor imaging in vivo, which may assist in the design and development of new immunotherapies. In the future, noninvasive imaging of immunotherapy biomarkers may assist in disease diagnostics, disease monitoring, and patient stratification.

Radiolabeling Silica-Based Nanoparticles via Coordination Chemistry: Basic Principles, Strategies, and Applications

As one of the most biocompatible and well-tolerated inorganic nanomaterials, silica-based nanoparticles (SiNPs) have received extensive attention over the last several decades. Recently, positron emission tomography (PET) imaging of radiolabeled SiNPs has provided a highly sensitive, noninvasive, and quantitative readout of the organ/tissue distribution, pharmacokinetics, and tumor targeting efficiency in vivo, which can greatly expedite the clinical translation of these promising NPs. Encouraged by the successful PET imaging of patients with metastatic melanoma using 124I-labeled ultrasmall SiNPs (known as Cornell dots or C dots) and their approval as an Investigational New Drug (IND) by the United States Food and Drug Administration, different radioisotopes (64Cu, 89Zr, 18F, 68Ga, 124I, etc.) have been reported to radiolabel a wide variety of SiNPs-based nanostructures, including dense silica (dSiO2), mesoporous silica (MSN), biodegradable mesoporous silica (bMSN), and hollow mesoporous silica nanoparticles (HMSN). With in-depth knowledge of coordination chemistry, abundant silanol groups (-Si-O-) on the silica surface or inside mesoporous channels not only can be directly used for chelator-free radiolabeling but also can be readily modified with the right chelators for chelator-based labeling. However, integrating these labeling strategies for constructing stably radiolabeled SiNPs with high efficiency has proven difficult because of the complexity of the involved key parameters, such as the choice of radioisotopes and chelators, nanostructures, and radiolabeling strategy. In this Account, we present an overview of recent progress in the development of radiolabeled SiNPs for cancer theranostics in the hope of speeding up their biomedical applications and potential translation into the clinic. We first introduce the basic principles and mechanisms for radiolabeling SiNPs via coordination chemistry, including general rules of selecting proper radioisotopes, engineering silica nanoplatforms (e.g., dSiO2, MSN, HMSN) accordingly, and chelation strategies for enhanced labeling efficiency and stability, on which our group has focused over the past decade. Generally, the medical applications guide the choice of specific SiNPs for radiolabeling by considering the inherent functionality of SiNPs. The radioisotopes can then be determined according to the amenability of the particular SiNPs for chelator-based or chelator-free radiolabeling to obtain high labeling stability in vivo, which is a prerequisite for PET to truly reflect the behavior of SiNPs since PET imaging detects the isotopes rather than nanoparticles. Next, we highlight several recent representative biomedical applications of radiolabeled SiNPs including molecular imaging to detect specific lesions, PET-guided drug delivery, SiNP-based theranostic cancer agents, and clinical studies. Finally, the challenges and prospects of radiolabeled SiNPs are briefly discussed toward clinical cancer research. We hope that this Account will clarify the recent progress on the radiolabeling of SiNPs for specific medical applications and generate broad interest in integrating nanotechnology and PET imaging. With several ongoing clinical trials, radiolabeled SiNPs offer great potential for future patient stratification and cancer management in clinical settings.