Hongguang Liu

Molecular optical imaging with radioactive probes.

Background Optical imaging (OI) techniques such as bioluminescence and fluorescence imaging have been widely used to track diseases in a non-invasive manner within living subjects. These techniques generally require bioluminescent and fluorescent probes. Here we demonstrate the feasibility of using radioactive probes for in vivo molecular OI. Methodology/Principal Findings By taking the advantages of low energy window of light (1.2–3.1 eV, 400–1000 nm) resulting from radiation, radionuclides that emit charged particles such as β+ and β− can be successfully imaged with an OI instrument. In vivo optical images can be obtained for several radioactive probes including 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), Na18F, Na131I, 90YCl3 and a 90Y labeled peptide that specifically target tumors. Conclusions/Significance These studies demonstrate generalizability of radioactive OI technique. It provides a new molecular imaging strategy and will likely have significant impact on both small animal and clinical imaging.

Construction and Validation of Nano Gold Tripods for Molecular Imaging of Living Subjects

Anisotropic colloidal hybrid nanoparticles exhibit superior optical and physical properties compared to their counterparts with regular architectures. We herein developed a controlled, stepwise strategy to build novel, anisotropic, branched, gold nanoarchitectures (Au-tripods) with predetermined composition and morphology for bioimaging. The resultant Au-tripods with size less than 20 nm showed great promise as contrast agents for in vivo photoacoustic imaging (PAI). We further identified Au-tripods with two possible configurations as high-absorbance nanomaterials from various gold multipods using a numerical simulation analysis. The PAI signals were linearly correlated with their concentrations after subcutaneous injection. The in vivo biodistribution of Au-tripods favorable for molecular imaging was confirmed using small animal positron emission tomography (PET). Intravenous administration of cyclic Arg-Gly-Asp-d-Phe-Cys (RGDfC) peptide conjugated Au-tripods (RGD-Au-tripods) to U87MG tumor-bearing mice showed PAI contrasts in tumors almost 3-fold higher than for the blocking group. PAI results correlated well with the corresponding PET images. Quantitative biodistribution data revealed that 7.9% ID/g of RGD-Au-tripods had accumulated in the U87MG tumor after 24 h post-injection. A pilot mouse toxicology study confirmed that no evidence of significant acute or systemic toxicity was observed in histopathological examination. Our study suggests that Au-tripods can be reliably synthesized through stringently controlled chemical synthesis and could serve as a new generation of platform with high selectivity and sensitivity for multimodality molecular imaging.

Radiation‐Luminescence‐Excited Quantum Dots for in vivo Multiplexed Optical Imaging

Recently,QDshavedrawnmuchattentionfortheirpotentialbiomedicalapplications and have been widely explored as effectivefluorescent sensors for real-time detection of biomolecules,for the staining ofbiological tissues and formolecular imagingof biological pathways or disease progression in vitro and invivo.

Harnessing the Power of Radionuclides for Optical Imaging: Cerenkov Luminescence Imaging

Over the past several years, nuclear imaging modalities such as PET and SPECT have received much attention because they have been instrumental not only in preclinical cancer research but also in nuclear medicine. Yet nuclear imaging is limited by high instrumentation cost and subsequently low availability to basic researchers. Cerenkov radiation, a relativistic physical phenomenon that was discovered 70 years ago, has recently become an intriguing subject of study in molecular imaging because of its potential in augmenting nuclear imaging, particularly in preclinical small-animal studies. The intrinsic capability of radionuclides emitting luminescent light from decay is promising because of the possibility of bridging nuclear imaging with optical imaging—a modality that is much less expensive, is easier to use, and has higher throughput than its nuclear counterpart. Thus, with the maturation of this novel imaging technology using Cerenkov radiation, which is termed Cerenkov luminescence imaging, it is foreseeable that advances in both nuclear imaging and preclinical research involving radioisotopes will be significantly accelerated in the near future.

Synthesis and Radioluminescence of PEGylated Eu3+-doped Nanophosphors as Bioimaging Probes

Lanthanide-doped nanophosphors have received significant attention for use in biological sensing and imaging due to their unique optical properties. Much like semiconductor quantum dots (QDs), these luminescent nanocrystals offer several advantages over conventional organic fluorophores, including high photochemical stability, large Stokes shift, and tunable fluorescence emission. [1] Up-conversion nanophosphors, which are capable of absorbing two or more low-energy photons to emit a higher-energy photon, also exhibit favorable characteristics such as long fluorescence lifetimes, no photoblinking, and reduced autofluorescence. [2] The recent development of lanthanide-doped nanophosphors that function in the near-infrared (NIR) spectral range optimal for optical transmission through biological tissues (650–900 nm) has attracted great interest towards in vivo bioim-aging probes. [3–5] Alternatively, high-energy radiation, currently employed in medical imaging modalities, such as X-ray computed tomography (CT) or positron emission tomo graphy (PET), may also be used to excite NIR-emitting radioluminescent nanophosphors (RLNPs) for bioimaging.

Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging

Development of reporter genes for multimodality molecular imaging is highly important. In contrast to the conventional strategies which have focused on fusing several reporter genes together to serve as multimodal reporters, human tyrosinase (TYR) – the key enzyme in melanin production – was evaluated in this study as a stand-alone reporter gene for in vitro and in vivo photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Human breast cancer cells MCF-7 transfected with a plasmid that encodes TYR (named as MCF-7-TYR) and non-transfected MCF-7 cells were used as positive and negative controls, respectively. Melanin targeted N-(2-(diethylamino)ethyl)-18F-5-fluoropicolinamide was used as a PET reporter probe. In vivo PAI/MRI/PET imaging studies showed that MCF-7-TYR tumors achieved significant higher signals and tumor-to-background contrasts than those of MCF-7 tumor. Our study demonstrates that TYR gene can be utilized as a multifunctional reporter gene for PAI/MRI/PET both in vitro and in vivo.

Affibody modified and radiolabeled gold-iron oxide hetero-nanostructures for tumor PET, optical and MR imaging.

A highly monodispersed hetero-nanostructure with two different functional nanomaterials (gold (Au) and iron oxide (Fe(3)O(4,) IO)) within one structure was successfully developed as Affibody based trimodality nanoprobe (positron emission tomography, PET; optical imaging; and magnetic resonance imaging, MRI) for imaging of epidermal growth factor receptor (EGFR) positive tumors. Unlike other regular nanostructures with a single component, the Au-IO hetero-nanostructures (Au-IONPs) with unique chemical and physical properties have capability to combine several imaging modalities together to provide complementary information. The IO component within hetero-nanostructures serve as a T(2) reporter for MRI; and gold component serve as both optical and PET reporters. Moreover, such hetero-nanoprobes could provide a robust nano-platform for surface-specific modification with both targeting molecules (anti-EGFR Affibody protein) and PET imaging reporters (radiometal (64)Cu chelators) in highly efficient and reliable manner. In vitro and in vivo study showed that the resultant nanoprobe provided high specificity, sensitivity, and excellent tumor contrast for both PET and MRI imaging in the human EGFR-expressing cells and tumors. Our study data also highlighted the EGFR targeting efficiency of hetero-nanoparticles and the feasibility for their further theranostic applications.

Intraoperative Imaging of Tumors Using Cerenkov Luminescence Endoscopy: A Feasibility Experimental Study

Cerenkov luminescence imaging (CLI) is an emerging new molecular imaging modality that is relatively inexpensive, easy to use, and has high throughput. CLI can image clinically available PET and SPECT probes using optical instrumentation. Cerenkov luminescence endoscopy (CLE) is one of the most intriguing applications that promise potential clinical translation. We developed a prototype customized fiberscopic Cerenkov imaging system to investigate the potential in guiding minimally invasive surgical resection. Methods: All experiments were performed in a dark chamber. Cerenkov luminescence from 18F-FDG samples containing decaying radioactivity was transmitted through an optical fiber bundle and imaged by an intensified charge-coupled device camera. Phantoms filled with 18F-FDG were used to assess the imaging spatial resolution. Finally, mice bearing subcutaneous C6 glioma cells were injected intravenously with 18F-FDG to determine the feasibility of in vivo imaging. The tumor tissues were exposed, and CLI was performed on the mouse before and after surgical removal of the tumor using the fiber-based imaging system and compared with a commercial optical imaging system. Results: The sensitivity of this particular setup was approximately 45 kBq (1.21 μCi)/300 μL. The 3 smallest sets of cylindric holes in a commercial SPECT phantom were identifiable via this system, demonstrating that the system has a resolution better than 1.2 mm. Finally, the in vivo tumor imaging study demonstrated the feasibility of using CLI to guide the resection of tumor tissues. Conclusion: This proof-of-concept study explored the feasibility of using fiber-based CLE for the detection of tumor tissue in vivo for guided surgery. With further improvements of the imaging sensitivity and spatial resolution of the current system, CLE may have a significant application in the clinical setting in the near future.

Small-Animal PET Imaging of Human Epidermal Growth Factor Receptor Positive Tumor with a 64Cu Labeled Affibody Protein

Epidermal growth factor receptor (EGFR) has become an attractive target for cancer molecular imaging and therapy. Affibody proteins against EGFR have been reported, and thus, we were interested in evaluating their potential for positron emission tomography (PET) imaging of EGFR positive cancer. An Affibody analogue (Ac-Cys-Z(EGFR:1907)) binding to EGFR was made through conventional solid phase peptide synthesis. The purified protein was site-specifically coupled with the 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to produce the bioconjugate, DOTA-Z(EGFR:1907). (64)Cu labeled probe (64)Cu-DOTA-Z(EGFR:1907) displayed a moderate specific activity (5-8 MBq/nmol, 22-35 microCi/microg). Cell uptake assays by pre-incubating without or with 300 times excess unlabeled Ac-Cys-Z(EGFR:1907) showed high EGFR-specific uptake (20% applied activity at 0.5 h) in A431 epidermoid carcinoma cancer cells. The affinity (K(D)) of (64)Cu-DOTA-Z(EGFR:1907) as tested by cell saturation analysis was 20 nM. The serum stability test showed excellent stability of the probe with >95% intact after 4 h of incubation in mouse serum. In vivo small-animal PET imaging showed fast tumor targeting, high tumor accumulation (approximately 10% ID/g at 1 h p.i.), and good tumor-to-normal tissue contrast of (64)Cu-DOTA-Z(EGFR:1907) spiked with a wide dose range of Ac-Cys-Z(EGFR:1907). Bio-distribution studies further demonstrated that the probe had high tumor, blood, liver, and kidney uptakes, while blood radioactivity concentration dropped dramatically at increased spiking doses. Co-injection of the probe with 500 microg of Ac-Cys-Z(EGFR:1907) for blocking significantly reduced the tumor uptake. Thus, (64)Cu-DOTA-Z(EGFR:1907) showed potential as a high tumor contrast EGFR PET imaging reagent. The probe spiked with 50 microg of Ac-Cys-Z(EGFR:1907) improved tumor imaging contrast which may have important clinical applications.

Proof-of-Concept Study of Monitoring Cancer Drug Therapy with Cerenkov Luminescence Imaging

Cerenkov luminescence imaging (CLI) has emerged as a less expensive, easier-to-use, and higher-throughput alternative to other nuclear imaging modalities such as PET. It is expected that CLI will find many applications in biomedical research such as cancer detection, probe development, drug screening, and therapy monitoring. In this study, we explored the possibility of using CLI to monitor drug efficacy by comparisons against PET. To assess the performance of both modalities in therapy monitoring, 2 murine tumor models (large cell lung cancer cell line H460 and prostate cancer cell line PC3) were given bevacizumab versus vehicle treatments. Two common radiotracers, 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) and 18F-FDG, were used to monitor bevacizumab treatment efficacy. Methods: One group of mice (n = 6) was implanted with H460 xenografts bilaterally in the shoulder region, divided into treatment and control groups (n = 3 each), injected with 18F-FLT, and imaged with PET immediately followed by CLI. The other group of mice (n = 6) was implanted with PC3 xenografts in the same locations, divided into treatment and control groups (n = 3 each), injected with 18F-FDG, and imaged by the same modalities. Bevacizumab treatment was performed by 2 injections of 20 mg/kg at days 0 and 2. Results: On 18F-FLT scans, both CLI and PET revealed significantly decreased signals from H460 xenografts in treated mice from pretreatment to day 3. Moderately increased to unchanged signals were observed in untreated mice. On 18F-FDG scans, both CLI and PET showed relatively unchanged signals from PC3 tumors in both treated and control groups. Quantifications of tumor signals of Cerenkov luminescence and PET images showed that the 2 modalities had excellent correlations (R2 > 0.88 across all study groups). Conclusion: CLI and PET exhibit excellent correlations across different tumor xenografts and radiotracers. This is the first study, to our knowledge, demonstrating the use of CLI for monitoring cancer treatment. The findings warrant further exploration and optimization of CLI as an alternative to PET in preclinical therapeutic monitoring and drug screening.