Weibo Cai

In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice.

Single-walled carbon nanotubes (SWNTs) exhibit unique size, shape and physical properties1,2,3 that make them promising candidates for biological applications. Here, we investigate the biodistribution of radio-labelled SWNTs in mice by in vivo positron emission tomography (PET), ex vivo biodistribution and Raman spectroscopy. It is found that SWNTs that are functionalized with phospholipids bearing polyethylene-glycol (PEG) are surprisingly stable in vivo. The effect of PEG chain length on the biodistribution and circulation of the SWNTs is studied. Effectively PEGylated SWNTs exhibit relatively long blood circulation times and low uptake by the reticuloendothelial system (RES). Efficient targeting of integrin positive tumour in mice is achieved with SWNTs coated with PEG chains linked to an arginine–glycine–aspartic acid (RGD) peptide. A high tumour accumulation is attributed to the multivalent effect of the SWNTs. The Raman signatures of SWNTs are used to directly probe the presence of nanotubes in mice tissues and confirm the radio-label-based results.

Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy

Carbon nanotubes are promising new materials for molecular delivery in biological systems. The long-term fate of nanotubes intravenously injected into animals in vivo is currently unknown, an issue critical to potential clinical applications of these materials. Here, using the intrinsic Raman spectroscopic signatures of single-walled carbon nanotubes (SWNTs), we measured the blood circulation of intravenously injected SWNTs and detect SWNTs in various organs and tissues of mice ex vivo over a period of three months. Functionalization of SWNTs by branched polyethylene-glycol (PEG) chains was developed, enabling thus far the longest SWNT blood circulation up to 1 day, relatively low uptake in the reticuloendothelial system (RES), and near-complete clearance from the main organs in ≈2 months. Raman spectroscopy detected SWNT in the intestine, feces, kidney, and bladder of mice, suggesting excretion and clearance of SWNTs from mice via the biliary and renal pathways. No toxic side effect of SWNTs to mice was observed in necropsy, histology, and blood chemistry measurements. These findings pave the way to future biomedical applications of carbon nanotubes.

Multimodality Molecular Imaging of Tumor Angiogenesis

Molecular imaging is a key component of 21st-century cancer management. The vascular endothelial growth factor (VEGF)/VEGF receptor signaling pathway and integrin αvβ3, a cell adhesion molecule, play pivotal roles in regulating tumor angiogenesis, the growth of new blood vessels. This review summarizes the current status of tumor angiogenesis imaging with SPECT, PET, molecular MRI, targeted ultrasound, and optical techniques. For integrin αvβ3 imaging, only nanoparticle-based probes, which truly target the tumor vasculature rather than tumor cells because of poor extravasation, are discussed. Once improvements in the in vivo stability, tumor-targeting efficacy, and pharmacokinetics of tumor angiogenesis imaging probes are made, translation to clinical applications will be critical for the maximum benefit of these novel agents. The future of tumor angiogenesis imaging lies in multimodality and nanoparticle-based approaches, imaging of protein–protein interactions, and quantitative molecular imaging. Combinations of multiple modalities can yield complementary information and offer synergistic advantages over any modality alone. Nanoparticles, possessing multifunctionality and enormous flexibility, can allow for the integration of therapeutic components, targeting ligands, and multimodality imaging labels into one entity, termed “nanomedicine,” for which the ideal target is tumor neovasculature. Quantitative imaging of tumor angiogenesis and protein–protein interactions that modulate angiogenesis will lead to more robust and effective monitoring of personalized molecular cancer therapy. Multidisciplinary approaches and cooperative efforts from many individuals, institutions, industries, and organizations are needed to quickly translate multimodality tumor angiogenesis imaging into multiple facets of cancer management. Not limited to cancer, these novel agents can also have broad applications for many other angiogenesis-related diseases.

Dual-Function Probe for PET and Near-Infrared Fluorescence Imaging of Tumor Vasculature

To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function PET/near-infrared fluorescence (NIRF) probe can allow for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs. Methods: A QD with an amine-functionalized surface was modified with RGD peptides and 1,4,7,10-tetraazacyclodocecane-N,N′,N″,N‴-tetraacetic acid (DOTA) chelators for integrin αvβ3–targeted PET/NIRF imaging. A cell-binding assay and fluorescence cell staining were performed with U87MG human glioblastoma cells (integrin αvβ3–positive). PET/NIRF imaging, tissue homogenate fluorescence measurement, and immunofluorescence staining were performed with U87MG tumor–bearing mice to quantify the probe uptake in the tumor and major organs. Results: There are about 90 RGD peptides per QD particle, and DOTA–QD–RGD exhibited integrin αvβ3–specific binding in cell cultures. The U87MG tumor uptake of 64Cu-labeled DOTA–QD was less than 1 percentage injected dose per gram (%ID/g), significantly lower than that of 64Cu-labeled DOTA–QD–RGD (2.2 ± 0.3 [mean ± SD] and 4.0 ± 1.0 %ID/g at 5 and 18 h after injection, respectively; n = 3). Taking into account all measurements, the liver-, spleen-, and kidney-to-muscle ratios for 64Cu-labeled DOTA–QD–RGD were about 100:1, 40:1, and 1:1, respectively. On the basis of the PET results, the U87MG tumor-to-muscle ratios for DOTA–QD–RGD and DOTA–QD were about 4:1 and 1:1, respectively. Excellent linear correlation was obtained between the results measured by in vivo PET imaging and those measured by ex vivo NIRF imaging and tissue homogenate fluorescence (r2 = 0.93). Histologic examination revealed that DOTA–QD–RGD targets primarily the tumor vasculature through an RGD–integrin αvβ3 interaction, with little extravasation. Conclusion: We quantitatively evaluated the tumor-targeting efficacy of a dual-function QD-based probe with PET and NIRF imaging. This dual-function probe has significantly reduced potential toxicity and overcomes the tissue penetration limitation of optical imaging, allowing for quantitative targeted imaging in deep tissue.

Graphene: a versatile nanoplatform for biomedical applications

Graphene, with its excellent physical, chemical, and mechanical properties, holds tremendous potential for a wide variety of biomedical applications. As research on graphene-based nanomaterials is still at a nascent stage due to the short time span since its initial report in 2004, a focused review on this topic is timely and necessary. In this feature review, we first summarize the results from toxicity studies of graphene and its derivatives. Although literature reports have mixed findings, we emphasize that the key question is not how toxic graphene itself is, but how to modify and functionalize it and its derivatives so that they do not exhibit acute/chronic toxicity, can be cleared from the body over time, and thereby can be best used for biomedical applications. We then discuss in detail the exploration of graphene-based nanomaterials for tissue engineering, molecular imaging, and drug/gene delivery applications. The future of graphene-based nanomaterials in biomedicine looks brighter than ever, and it is expected that they will find a wide range of biomedical applications with future research effort and interdisciplinary collaboration.

Iron Oxide Decorated MoS2 Nanosheets with Double PEGylation for Chelator-Free Radiolabeling and Multimodal Imaging Guided Photothermal Therapy

Theranostics for in vivo cancer diagnosis and treatment generally requires well-designed nanoscale platforms with multiple integrated functionalities. In this study, we uncover that functionalized iron oxide nanoparticles (IONPs) could be self-assembled on the surface of two-dimensional MoS2 nanosheets via sulfur chemistry, forming MoS2-IO nanocomposites, which are then modified with two types of polyethylene glycol (PEG) to acquire enhanced stability in physiological environments. Interestingly, (64)Cu, a commonly used positron-emitting radioisotope, could be firmly adsorbed on the surface of MoS2 without the need of chelating molecules, to enable in vivo positron emission tomography (PET) imaging. On the other hand, the strong near-infrared (NIR) and superparamagnetism of MoS2-IO-PEG could also be utilized for photoacoustic tomography (PAT) and magnetic resonance (MR) imaging, respectively. Under the guidance by such triple-modal imaging, which uncovers efficient tumor retention of MoS2-IO-(d)PEG upon intravenous injection, in vivo photothermal therapy is finally conducted, achieving effective tumor ablation in an animal tumor model. Our study highlights the promise of constructing multifunctional theranostic nanocomposites based on 2D transitional metal dichalcogenides for multimodal imaging-guided cancer therapy.

In Vivo Targeting and Imaging of Tumor Vasculature with Radiolabeled, Antibody-Conjugated Nanographene

Herein we demonstrate that nanographene can be specifically directed to the tumor neovasculature in vivo through targeting of CD105 (i.e., endoglin), a vascular marker for tumor angiogenesis. The covalently functionalized nanographene oxide (GO) exhibited excellent stability and target specificity. Pharmacokinetics and tumor targeting efficacy of the GO conjugates were investigated with serial noninvasive positron emission tomography imaging and biodistribution studies, which were validated by in vitro, in vivo, and ex vivo experiments. The incorporation of an active targeting ligand (TRC105, a monoclonal antibody that binds to CD105) led to significantly improved tumor uptake of functionalized GO, which was specific for the neovasculature with little extravasation, warranting future investigation of these GO conjugates for cancer-targeted drug delivery and/or photothermal therapy to enhance therapeutic efficacy. Since poor extravasation is a major hurdle for nanomaterial-based tumor targeting in vivo, this study also establishes CD105 as a promising vascular target for future cancer nanomedicine.

64Cu-Labeled Tetrameric and Octameric RGD Peptides for Small-Animal PET of Tumor αvβ3 Integrin Expression

Integrin αvβ3 plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive imaging of αvβ3 expression and targeted radionuclide therapy. In this study, we developed 64Cu-labeled multimeric RGD peptides, E{E[c(RGDyK)]2}2 (RGD tetramer) and E(E{E[c(RGDyK)]2}2)2 (RGD octamer), for PET imaging of tumor integrin αvβ3 expression. Methods: Both RGD tetramer and RGD octamer were synthesized with glutamate as the linker. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), the peptides were labeled with 64Cu for biodistribution and small-animal PET imaging studies (U87MG human glioblastoma xenograft model and c-neu oncomouse model). A cell adhesion assay, a cell-binding assay, receptor blocking experiments, and immunohistochemistry were also performed to evaluate the αvβ3-binding affinity/specificity of the RGD peptide-based conjugates in vitro and in vivo. Results: RGD octamer had significantly higher integrin αvβ3-binding affinity and specificity than RGD tetramer analog (inhibitory concentration of 50% was 10 nM for octamer vs. 35 nM for tetramer). 64Cu-DOTA-RGD octamer had higher tumor uptake and longer tumor retention than 64Cu-DOTA-RGD tetramer in both tumor models tested. The integrin αvβ3 specificity of both tracers was confirmed by successful receptor-blocking experiments. The high uptake and slow clearance of 64Cu-DOTA-RGD octamer in the kidneys was attributed mainly to the integrin positivity of the kidneys, significantly higher integrin αvβ3-binding affinity, and the larger molecular size of the octamer, as compared with the other RGD analogs. Conclusion: Polyvalency has a profound effect on the receptor-binding affinity and in vivo kinetics of radiolabeled RGD multimers. The information obtained here may guide the future development of RGD peptide-based imaging and internal radiotherapeutic agents targeting integrin αvβ3.

Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

PurposeCetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab.MethodsWe conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), labeled with 64Cu, and tested the resulting 64Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated.ResultsMicroPET imaging showed that 64Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R2 = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver.ConclusionThe success of EGFR-positive tumor imaging using 64Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents.

In Vivo Tumor Targeting and Image-Guided Drug Delivery with Antibody-Conjugated, Radiolabeled Mesoporous Silica Nanoparticles

Since the first use of biocompatible mesoporous silica (mSiO2) nanoparticles as drug delivery vehicles, in vivo tumor targeted imaging and enhanced anticancer drug delivery has remained a major challenge. In this work, we describe the development of functionalized mSiO2 nanoparticles for actively targeted positron emission tomography (PET) imaging and drug delivery in 4T1 murine breast tumor-bearing mice. Our structural design involves the synthesis, surface functionalization with thiol groups, PEGylation, TRC105 antibody (specific for CD105/endoglin) conjugation, and (64)Cu-labeling of uniform 80 nm sized mSiO2 nanoparticles. Systematic in vivo tumor targeting studies clearly demonstrated that (64)Cu-NOTA-mSiO2-PEG-TRC105 could accumulate prominently at the 4T1 tumor site via both the enhanced permeability and retention effect and TRC105-mediated binding to tumor vasculature CD105. As a proof-of-concept, we also demonstrated successful enhanced tumor targeted delivery of doxorubicin (DOX) in 4T1 tumor-bearing mice after intravenous injection of DOX-loaded NOTA-mSiO2-PEG-TRC105, which holds great potential for future image-guided drug delivery and targeted cancer therapy.