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author:("Chen, xiaoyan")
1.  Rational Design of Matrix Metalloproteinase-13 (MMP-13) Activatable Probes for Enhanced Specificity 
ACS chemical biology  2013;9(2):510-516.
Due to the important roles of matrix metalloproteinases (MMPs) play in tumor invasion and metastasis, various activatable optical probes have been developed to visualize MMP activities in vitro and in vivo. Our recently developed MMP-13 activatable probe, L-MMP-P12, has been successfully applied to image the expression and inhibition of MMPs in a xenografted tumor model (Zhu L et al., Theranostics. 2011;1:18–27). In this study, to further optimize the in vivo behavior of the proteinase activatable probe, we tracked and profiled the metabolites by a high resolution LC/MS system. Two major metabolites that contributed to the fluorescence recovery were identified: One was specifically cleaved between Glycine (G4) and Valine (V5) by MMP, while the other one was generated by non-specific cleavage between Glycine (G7) and Lysine (K8). In order to visualize the MMP activity more accurately and specifically, a new probe D-MMP-P12 was designed by replacing the L-lysine with D-lysine in the MMP substrate sequence. The metabolic profile of the new probe, D-MMP-P12, was further characterized by in vitro enzymatic assay and no non-specific metabolite was found by LC/MS. Our in vivo optical imaging also demonstrated that D-MMP-12 had significantly higher tumor-to-background ratio (TBR, 5.55 ± 0.75) compared with L-MMP-P12 (3.73 ± 0.31) at 2 h post-injection. The improved MMP activatable probe may have the potential for drug screening, tumor diagnosis and therapy response monitoring. Moreover, our research strategy can be further extended to study other protease activatable probes.
PMCID: PMC3944097  PMID: 24266806
Liquid chromatography–mass spectrometry (LC-MS); activatable probe; matrix metalloproteinases (MMPs); metabolite; near-infrared fluorescence imaging
2.  Stability Analysis of Glutamic Acid Linked Peptides Coupled to NOTA through Different Chemical Linkages 
Molecular Pharmaceutics  2014;11(11):3867-3874.
Glutamic acid is a commonly used linker to form dimeric peptides with enhanced binding affinity than their corresponding monomeric counterparts. We have previously labeled NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 (NOTA-PRGD2) [1] with [18F]AlF and 68Ga for imaging tumor angiogenesis. The p-SCN-Bn-NOTA was attached to E[c(RGDyK)]2 [2] through a mini-PEG with a thiourea linkage, and the product [1] was stable at radiolabeling condition of 100 °C and pH 4.0 acetate buffer. However, when the same p-SCN-Bn-NOTA was directly attached to the α-amine of E[c(RGDfK)]2 [3], the product NOTA-Bn-NCS-E[c(RGDfK)]2 [4] became unstable under similar conditions and the release of monomeric c(RGDfK) [5] was observed. The purpose of this work was to use HPLC and LC-MS to monitor the decomposition of glutamic acid linked dimeric peptides and their NOTA derivatives. A c(RGDyK) [6] and bombesin (BBN) [7] heterodimer c(RGDyK)-E-BBN [8], and a dimeric bombesin E(BBN)2 [9], both with a glutamic acid as the linker, along with a model compound PhSCN-E[c(RGDfK)] [10] were also studied. All the compounds were dissolved in 0.5 M pH 4.0 acetate buffer at the concentration of 1 mg/mL, and 0.1 mL of each sample was heated at 100 °C for 10 min and the more stable compounds were heated for another 30 min. The samples at both time points were analyzed with analytical HPLC to monitor the decomposition of the heated samples. The samples with decomposition were further analyzed by LC-MS to determine the mass of products from the decomposition for possible structure elucidation. After 10 min heating, the obvious release of c(RGDfK) [5] was observed for NOTA-Bn-NCS-E[c(RGDfK)]2 [4] and Ph-SCN-E[c(RGDfK)] [10]. Little or no release of monomers was observed for the remaining samples at this time point. After further heating, the release of monomers was clearly observed for E[c(RGDyK)]2 [2], E[c(RGDfK)]2 [3], c(RGDyK)-E-BBN [8], and E(BBN)2 [9]. No decomposition or little decomposition was observed for NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 [1], PEG3-E[c(RGDyK)]2 [11], NOTA-E[c(RGDyK)]2 [12], and NOTA-PEG3-E[c(RGDyK)]2 [13]. The glutamic acid linked dimeric peptides with a free α-amine are labile due to the neighboring amine participation in the hydrolysis. The stability of peptides could be increased by converting the free amine into amide. The instability of thiourea derivatives formed from α-amine was caused by participation of thiol group derived from thiourea.
PMCID: PMC4224566  PMID: 24533430
peptide; glutamate linker; thiourea; hydrolysis; Edman degradation
3.  Self-Illuminating 64Cu-Doped CdSe/ZnS Nanocrystals for in Vivo Tumor Imaging 
Construction of self-illuminating semiconducting nanocrystals, also called quantum dots (QDs), has attracted much attention recently due to their potential as highly sensitive optical probes for biological imaging applications. Here we prepared a self-illuminating QD system by doping positron-emitting radionuclide 64Cu into CdSe/ZnS core/shell QDs via a cation-exchange reaction. The 64Cu-doped CdSe/ZnS QDs exhibit efficient Cerenkov resonance energy transfer (CRET). The signal of 64Cu can accurately reflect the biodistribution of the QDs during circulation with no dissociation of 64Cu from the nanoparticles. We also explored this system for in vivo tumor imaging. This nanoprobe showed high tumor-targeting ability in a U87MG glioblastoma xenograft model (12.7% ID/g at 17 h time point) and feasibility for in vivo luminescence imaging of tumor in the absence of excitation light. The availability of these self-illuminating integrated QDs provides an accurate and convenient tool for in vivo tumor imaging and detection.
PMCID: PMC4004262  PMID: 24401138
4.  The genotype-dependent influence of functionalized multiwalled carbon nanotubes on fetal development 
Biomaterials  2014;35(2):856-865.
In many cases cancer is caused by gene deficiency that is being passed along from generation to generation. Soluble carbon nanotubes (CNTs) have shown promising applications in the diagnosis and therapy of cancer, however, the potential relationship between cancer-prone individuals and response to CNT exposure as a prerequisite for development of personalized nanomedicine, is still poorly understood. Here we report that intravenous injections of multi-walled carbon nanotubes into p53 (a well-known cancer susceptible gene) heterozygous pregnant mice can induce p53- dependent responses in fetal development. Larger sized multi-walled carbon nanotubes moved across the blood-placenta barrier (BPB), restricted the development of fetuses, and induced brain deformity, whereas single-walled and smaller sized multi-walled carbon nanotubes showed no or less fetotoxicity. A molecular mechanism study found that multi-walled carbon nanotubes directly triggered p53-dependent apoptosis and cell cycle arrest in response to DNA damage. Based on the molecular mechanism, we also incorporated N-acetylcysteine (NAC), a FDA approved antioxidant, to prevent CNTs induced nuclear DNA damage and reduce brain development abnormalities. Our findings suggest that CNTs might have genetic background-dependent toxic effect on the normal development of the embryo, and provide new insights into protection against nanoparticle-induced toxicity in potential clinical applications.
PMCID: PMC4091802  PMID: 24344357
Carbon nanotubes; nanotoxicity; genetic background; blood-placenta barrier; fetal development
5.  PEGylated Exendin-4, a Modified GLP-1 Analog Exhibits More Potent Cardioprotection than Its Unmodified Parent Molecule on a Dose to Dose Basis in a Murine Model of Myocardial Infarction 
Theranostics  2015;5(3):240-250.
A Site-specifically PEGylated exendin-4 (denoted as PEG-Ex4) is an exendin-4 (denoted as Ex4) analog we developed by site-specific PEGylation of exendin-4 with a high molecular weight trimeric poly(ethylene glycol) (tPEG). It has been shown to possess prolonged half-life in vivo with similar receptor binding affinity compared to unmodified exendin-4 by our previous work. This study is sought to test whether PEG-Ex4 is suitable for treating myocardial infarction (MI). In the MI model, PEG-Ex4 was administered every 3 days while equivalent amount of Ex4 was administered every 3 days or twice daily. Animal survival rate, heart function, remodeling and neoangiogenesis were evaluated and compared. Tube formation was examined in endothelial cells. In addition, Western blotting and histology were performed to determine the markers of cardiac hypertrophy and angiogenesis and to explore the possible molecular mechanism involved. PEG-Ex4 and Ex4 showed comparable binding affinity to GLP-1 receptor. In MI mice, PEG-Ex4 given at 3 days interval achieved similar extent of protection as Ex4 given twice daily, while Ex4 given at 3 days interval failed to produce protection. PEG-Ex4 elevated endothelial tube formation in vitro and capillary density in the border area of MI. PEG-Ex4 increased Akt activity and VEGF production in a GLP-1R dependent manner in endothelial cells and antagonism of GLP-1R, Akt or VEGF abolished the protection of PEG-Ex4 in the MI model. PEG-Ex4 is a potent long-acting GLP-1 receptor agonist for the treatment of chronic heart disease. Its protection might be attributed to enhanced angiogenesis mediated by the activation of Akt and VEGF.
PMCID: PMC4279188  PMID: 25553112
Exendin-4; PEGylation; cardioprotection; Angiogenesis; myocardial infarction.
6.  Kinetic Analysis of Dynamic 11C-Acetate PET/CT Imaging as a Potential Method for Differentiation of Hepatocellular Carcinoma and Benign Liver Lesions 
Theranostics  2015;5(4):371-377.
Objective: The kinetic analysis of 11C-acetate PET provides more information than routine one time-point static imaging. This study aims to investigate the potential of dynamic 11C-acetate hepatic PET imaging to improve the diagnosis of hepatocellular carcinoma (HCC) and benign liver lesions by using compartmental kinetic modeling and discriminant analysis.
Methods: Twenty-two patients were enrolled in this study, 6 cases were with well-differentiated HCCs, 7 with poorly-differentiated HCCs and 9 with benign pathologies. Following the CT scan, all patients underwent 11C-acetate dynamic PET imaging. A three-compartment irreversible dual-input model was applied to the lesion time activity curves (TACs) to estimate the kinetic rate constants K1-k3, vascular fraction (VB) and the coefficient α representing the relative hepatic artery (HA) contribution to the hepatic blood supply on lesions and non-lesion liver tissue. The parameter Ki (=K1×k3/(k2 + k3)) was calculated to evaluate the local hepatic metabolic rate of acetate (LHMAct). The lesions were further classified by discriminant analysis with all the above parameters.
Results: K1 and lesion to non-lesion standardized uptake value (SUV) ratio (T/L) were found to be the parameters best characterizing the differences among well-differentiated HCC, poorly-differentiated HCC and benign lesions in stepwise discriminant analysis. With discriminant functions consisting of these two parameters, the accuracy of lesion prediction was 87.5% for well-differentiated HCC, 50% for poorly-differentiated HCC and 66.7% for benign lesions. The classification was much better than that with SUV and T/L, where the corresponding classification accuracy of the three kinds of lesions was 57.1%, 33.3% and 44.4%.
Conclusion: 11C-acetate kinetic parameter K1 could improve the identification of HCC from benign lesions in combination with T/L in discriminant analysis. The discriminant analysis using static and kinetic parameters appears to be a very helpful method for clinical liver masses diagnosis and staging.
PMCID: PMC4329501
11C-Acetate, dynamic PET; hepatocellular carcinoma; kinetic modeling; discriminant analysis
7.  Acetylcholinesterase-Catalyzed Hydrolysis Allows Ultrasensitive Detection of Pathogens with the Naked Eye** 
Angewandte Chemie (International ed. in English)  2013;52(52):10.1002/anie.201307952.
This paper describes a rapid diagnostic platform for pathogen detection based on the acetylcholinesterase-catalyzed hydrolysis reaction. Owing to the signal amplification strategies, the sensitivity of this assay is comparable to that of PCR. In addition, the readout of this assay is based on the color change of solutions, which can be easily observed by the naked eye alone.
PMCID: PMC3874880  PMID: 24155243
acetylcholinesterase-catalyzed hydrolysis; pathogen detection; high-sensitivity; gold nanoparticle; clinical samples
8.  Biodegradable Gold Nanovesicles with Ultra-Strong Plasmonic Coupling Effect for Photoacoustic Imaging and Photothermal Therapy** 
Hierarchical assembling of gold nanoparticles (GNPs) allows one to engineer the localized surface plasmon resonance (LSPR) peaks to the near-infrared (NIR) region for enhanced photothermal Therapy (PTT). Herein we report a novel theranostic platform based on biodegradable plasmonic gold nanovesicles for photoacoustic (PA) Imaging and PTT. The disulfide bond (S-S) termed PEG-b-PCL block copolymer graft allows dense packing of GNPs during the assembly process and induces ultra-strong plasmonic coupling effect between adjacent GNPs. The strong NIR absorption induced by plasmon coupling and very high photothermal conversion efficiency (η= 37 %) enable simultaneous thermal/PA imaging and enhanced PTT efficacy with improved clearance of the dissociated particles after the completion of PTT. These vesicle-architectures assembling of various nanocrystals with tailored optical, magnetic, and electronic properties opens new possibilities for constructing multifunctional biodegradable platforms for biomedical applications, particularly in cancer theranotics.
PMCID: PMC4058316  PMID: 24318645
Theranostics; Biodegradable block co-polymer; Gold Nanovesicles; Plasmonic Coupling Effect; Photoacoustic Imaging; Photothermal Therapy
9.  Color Tunable Gd-Zn-Cu-In-S/ZnS Quantum Dots for Dual Modality Magnetic Resonance and Fluorescence Imaging 
Nano research  2014;7(11):1581-1591.
Inorganic nanoparticles have been introduced into biological systems as useful probes for in vitro diagnosis and in vivo imaging, due to their relatively small size and exceptional physical and chemical properties. A new kind of color tunable Gd-Zn-Cu-In-S/ZnS (GZCIS/ZnS) quantum dots (QDs) with stable crystal structure was successfully synthesized and utilized for magnetic resonance (MR) and fluorescence dual modality imaging. This strategy allows successful fabrication of GZCIS/ZnS QDs by incorporating Gd into ZCIS/ZnS QDs to achieve great MR enhancement without compromising the fluorescence properties of the initial ZCIS/ZnS QDs. The as-prepared GZCIS/ZnS QDs show high T1 MR contrast as well as “color-tunable” photoluminescence (PL) in the range of 550–725 nm by adjusting the Zn/Cu feeding ratio with high PL quantum yield (QY). The GZCIS/ZnS QDs were transferred into water via a bovine serum albumin (BSA) coating strategy. The resulting Cd-free GZCIS/ZnS QDs reveal negligible cytotoxicity on both HeLa and A549 cells. Both fluorescence and MR imaging studies were successfully performed in vitro and in vivo. The results demonstrated that GZCIS/ZnS QDs could be a dual-modal contrast agent to simultaneously produce strong MR contrast enhancement as well as fluorescence emission for in vivo imaging.
PMCID: PMC4254824  PMID: 25485043
CuInS2 quantum dot; magnetic resonance imaging; photoluminescence; multimodality imaging; gadolinium doped
10.  18F-Alfatide II and 18F-FDG Dual Tracer Dynamic PET for Parametric, Early Prediction of Tumor Response to Therapy 
A single dynamic PET acquisition using multiple tracers administered closely in time could provide valuable complementary information about a tumor’s status under quasi-constant conditions. This study aims to investigate the utility of dual-tracer dynamic PET imaging with 18F-Alfatide II (18F-AlF-NOTA-E[PEG4-c(RGDfk)]2) and 18F-FDG for parametric monitoring of tumor responses to therapy.
We administered doxorubicin to one group of athymic nude mice with U87MG tumors and Abraxane to another group of mice with MDA-MB-435 tumors. To monitor therapeutic responses, we performed dual-tracer dynamic imaging, in sessions that lasted 90 min, starting by injecting the mice via tail vein catheters with 18F-Alfatide II, followed 40 minutes later by 18F-FDG. To achieve signal separation of the two tracers, we fit a three-compartment reversible model to the time activity curve (TAC) of 18F-Alfatide II for the 40 min prior to 18F-FDG injection, and then extrapolated to 90 min. The 18F-FDG tumor TAC was isolated from the 90 min dual tracer tumor TAC by subtracting the fitted 18F-Alfatide II tumor TAC. With separated tumor TACs, the 18F-Alfatide II binding potential (Bp=k3/k4) and volume of distribution (VD), and 18F-FDG influx rate ((K1×k3)/(k2 + k3)) based on the Patlak method were calculated to validate the signal recovery in a comparison with 60-min single tracer imaging and to monitor therapeutic response.
The transport and binding rate parameters K1-k3 of 18F-Alfatide II, calculated from the first 40 min of dual tracer dynamic scan, as well as Bp and VD, correlated well with the parameters from the 60 min single tracer scan (R2 > 0.95). Compared with the results of single tracer PET imaging, FDG tumor uptake and influx were recovered well from dual tracer imaging. Upon doxorubicin treatment, while no significant changes in static tracer uptake values of 18F-Alfatide II or 18F-FDG were observed, both 18F-Alfatide II Bp and 18F-FDG influx from kinetic analysis in tumors showed significant decreases. For Abraxane therapy of MDA-MB-435 tumors, significant decrease was only observed with 18F-Alfatide II Bp value from kinetic analysis but not 18F-FDG influx.
The parameters fitted with compartmental modeling from the dual tracer dynamic imaging are consistent with those from single tracer imaging, substantiating the feasibility of this methodology. Even though no significant differences in tumor size were found until 5 days after doxorubicin treatment started, at day 3 there were already substantial differences in 18F-Alfatide II Bp and 18F-FDG influx rate. Dual tracer imaging can measure 18F-Alfatide II Bp value and 18F-FDG influx simultaneously to evaluate tumor angiogenesis and metabolism. Such changes are known to precede anatomical changes, and thus parametric imaging may offer the promise of early prediction of therapy response.
PMCID: PMC4209961  PMID: 24232871
dual-tracer dynamic PET; parametric imaging; 18F-Alfatide II; 18F-FDG; therapy response
11.  microPET of Tumor Integrin αvβ3 Expression Using 18F-Labeled PEGylated Tetrameric RGD Peptide (18F-FPRGD4) 
In vivo imaging of αvβ3 expression has important diagnostic and therapeutic applications. Multimeric cyclic RGD peptides are capable of improving the integrin αvβ3–binding affinity due to the polyvalency effect. Here we report an example of 18F-labeled tetrameric RGD peptide for PET of αvβ3 expression in both xenograft and spontaneous tumor models.
The tetrameric RGD peptide E{E[c(RGDyK)]2}2 was derived with amino-3,6,9-trioxaundecanoic acid (mini-PEG; PEG is poly(ethylene glycol)) linker through the glutamate α-amino group. NH2-mini-PEG-E{E[c(RGDyK)]2}2 (PRGD4) was labeled with 18F via the N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) prosthetic group. The receptor-binding characteristics of the tetrameric RGD peptide tracer 18F-FPRGD4 were evaluated in vitro by a cell-binding assay and in vivo by quantitative microPET imaging studies.
The decay-corrected radiochemical yield for 18F-FPRGD4 was about 15%, with a total reaction time of 180 min starting from 18F-F−. The PEGylation had minimal effect on integrin-binding affinity of the RGD peptide. 18F-FPRGD4 has significantly higher tumor uptake compared with monomeric and dimeric RGD peptide tracer analogs. The receptor specificity of 18F-FPRGD4 in vivo was confirmed by effective blocking of the uptake in both tumors and normal organs or tissues with excess c(RGDyK).
The tetrameric RGD peptide tracer 18F-FPRGD4 possessing high integrin-binding affinity and favorable biokinetics is a promising tracer for PET of integrin αvβ3 expression in cancer and other angiogenesis related diseases.
PMCID: PMC4183663  PMID: 17704249
microPET; integrin αvβ3; tetrameric RGD peptide; PEGylation; 18F
12.  Click Chemistry for 18F-Labeling of RGD Peptides and microPET Imaging of Tumor Integrin αvβ3 Expression 
Bioconjugate chemistry  2007;18(6):1987-1994.
The cell adhesion molecule integrin αvβ3 plays a key role in tumor angiogenesis and metastasis. A series of 18F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine reactive prosthetic groups. In this study, we report the use of the Cu(I)-catalyzed Huisgen cycloaddition, also known as a click reaction, to label RGD peptides with 18F by forming 1,2,3-triazoles. Nucleophilic fluorination of a toluenesulfonic alkyne provided 18F-alkyne in high yield (nondecay-corrected yield: 65.0 ± 1.9%, starting from the azeotropically dried 18F-fluoride), which was then reacted with an RGD azide (nondecay-corrected yield: 52.0 ± 8.3% within 45 min including HPLC purification). The 18F-labeled peptide was subjected to microPET studies in murine xenograft models. Murine microPET experiments showed good tumor uptake (2.1 ± 0.4%ID/g at 1 h postinjection (p.i.)) with rapid renal and hepatic clearance of 18F-fluoro-PEG-triazoles-RGD2 (18F-FPTA-RGD2) in a subcutaneous U87MG glioblastoma xenograft model (kidney 2.7 ± 0.8%ID/g; liver 1.9 ± 0.4%ID/g at 1 h p.i.). Metabolic stability of the newly synthesized tracer was also analyzed (intact tracer ranging from 75% to 99% at 1 h p.i.). In brief, the new tracer 18F-FPTA-RGD2 was synthesized with high radiochemical yield and high specific activity. This tracer exhibited good tumor-targeting efficacy and relatively good metabolic stability, as well as favorable in vivo pharmacokinetics. This new 18F labeling method based on click reaction may also be useful for radiolabeling of other biomolecules with azide groups in high yield.
PMCID: PMC4183694  PMID: 18030991
14.  Dual-factor triggered fluorogenic nanoprobe for ultrahigh contrast and subdiffraction fluorescence imaging 
Biomaterials  2013;34(26):6194-6201.
Ultrahigh contrast fluorescence molecular imaging has long been pursued over the past few decades from basic sciences to clinics. Although new classes of near-infrared (NIR) molecular probes are emerging, the requirement of fluorophores with high quantum yield, high signal to noise (S/N) ratio, and being activatable to microenvironment changes can hardly be fulfilled. In this study, a new NIR dye embedded fluorogenic nanoprobe (fg-nanoprobe) was developed for ultrahigh contrast in vitro and in vivo imaging with negligible background interference. The achieved S/N ratio was found to be attributed to the synergistic effects of the cellular compartmental triggered fluorogenicity and pH tunable fluorescence on/off character. In addition, this constructed fluorogenic nanoprobe could be coupled with image processing method for super-resolution subdiffraction imaging. The developed fg-nanoprobe integrated photophysical merits of the synthesized NIR fluorophore and advantages of engineered nanoparticle for enhanced fluorescence molecular imaging. This probe may open another avenue for ultrahigh contrast fluorescence molecular imaging in the future.
PMCID: PMC4167587  PMID: 23721793
Fluorescence imaging; Subdiffraction imaging; Fluorogenic PLGA nanoparticle; BODIPY NIR dye; pH sensitive; Molecular binding
15.  18F-labeled mini-PEG spacered RGD dimer (18F-FPRGD2): synthesis and microPET imaging of αvβ3 integrin expression 
We have previously reported that 18F-FB-E[c(RGDyK)]2 (18F-FRGD2) allows quantitative PET imaging of integrin αvβ3 expression. However, the potential clinical translation was hampered by the relatively low radiochemical yield. The goal of this study was to improve the radiolabeling yield, without compromising the tumor targeting efficiency and in vivo kinetics, by incorporating a hydrophilic bifunctional mini-PEG spacer.
18F-FB-mini-PEG-E[c(RGDyK)]2 (18F-FPRGD2) was synthesized by coupling N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) with NH2-mini-PEG-E[c(RGDyK)]2 (denoted as PRGD2). In vitro receptor binding affinity, metabolic stability, and integrin αvβ3 specificity of the new tracer 18F-FPRGD2 were assessed. The diagnostic value of 18F-FPRGD2 was evaluated in subcutaneous U87MG glioblastoma xenografted mice and in c-neu transgenic mice by quantitative microPET imaging studies.
The decay-corrected radiochemical yield based on 18F-SFB was more than 60% with radiochemical purity of >99%. 18F-FPRGD2 had high receptor binding affinity, metabolic stability, and integrin αvβ3-specific tumor uptake in the U87MG glioma xenograft model comparable to those of 18F-FRGD2. The kidney uptake was appreciably lower for 18F-FPRGD2 compared with 18F-FRGD2 [2.0±0.2%ID/ g for 18F-FPRGD2 vs 3.0±0.2%ID/g for 18F-FRGD2 at 1 h post injection (p.i.)]. The uptake in all the other organs except the urinary bladder was at background level. 18F-FPRGD2 also exhibited excellent tumor uptake in c-neu oncomice (3.6±0.1%ID/g at 30 min p.i.).
Incorporation of a mini-PEG spacer significantly improved the overall radiolabeling yield of 18F-FPRGD2. 18F-FPRGD2 also had reduced renal uptake and similar tumor targeting efficacy as compared with 18F-FRGD2. Further testing and clinical translation of 18F-FRGD2 are warranted.
PMCID: PMC4167588  PMID: 17492285
Integrin αvβ3; Dimeric RGD peptide; Mini-PEG spacer; MicroPET; Fluorine-18
16.  In Vivo Bioluminescence Tumor Imaging of RGD Peptide-modified Adenoviral Vector Encoding Firefly Luciferase Reporter Gene 
The goal of this study is to demonstrate the feasibility of chemically modified human adenovirus (Ad) vectors for tumor retargeting.
E1- and E3-deleted Ad vectors carrying firefly luciferase reporter gene under cytomegalovirus promoter (AdLuc) was surface-modified with cyclic arginine–glycine–aspartic acid (RGD) peptides through a bifunctional poly(ethyleneglycol) linker (RGD-PEG-AdLuc) for integrin αvβ3 specific delivery. The Coxsackie and adenovirus viral receptor (CAR) and integrin αvβ3 expression in various tumor cell lines was determined by reverse transcriptase PCR and fluorescence-activated cell sorting. Bioluminescence imaging was performed in vitro and in vivo to evaluate RGD-modified AdLuc infectivity.
RGD-PEG-AdLuc abrogated the native CAR tropism and exhibited significantly enhanced transduction efficiency of integrin-positive tumors than AdLuc through intravenous administration.
This approach provides a robust platform for site-specific gene delivery and noninvasive monitoring of the transgene delivery efficacy and homing.
PMCID: PMC4165526  PMID: 17297551
Adenovirus; Firefly luciferase; Reporter gene; Integrin αvβ3; RGD; Bioluminescence imaging
17.  Trafficking Mesenchymal Stem Cell Engraftment and Differentiation in Tumor-Bearing Mice by Bioluminescence Imaging 
Stem cells (Dayton, Ohio)  2009;27(7):1548-1558.
The objective of the study was to track the distribution and differentiation of mesenchymal stem cells (MSCs) in tumor-bearing mice. The 4T1 murine breast cancer cells were labeled with renilla luciferase-monomeric red fluorescence protein (rLuc-mRFP) reporter gene. The MSCs labeled with firefly luciferase-enhanced green fluorescence protein (fLuc-eGFP) reporter gene (MSCs-R) were isolated from L2G85 transgenic mice that constitutively express fLuc-eGFP reporter gene. To study the tumor tropism of MSCs, we established both subcutaneous and lung metastasis models. In lung metastasis tumor mice, we injected MSCs-R intravenously either on the same day or 4 days after 4T1 tumor cell injection. In subcutaneous tumor mice, we injected MSCs-R intravenously 7 days after subcutaneous 4T1 tumor inoculation. The tumor growth was monitored by rLuc bioluminescence imaging (BLI). The fate of MSCs-R was monitored by fLuc BLI. The localization of MSCs-R in tumors was examined histologically. The osteogenic and adipogenic differentiation of MSCs-R was investigated by alizarin red S and oil red O staining, respectively. The mechanism of the dissimilar differentiation potential of MSCs-R under different tumor microenvironments was investigated. We found that the 4T1 cells were successfully labeled with rLuc-mRFP. The MSCs-R isolated from L2G85 transgenic mice constitutively express fLuc-eGFP reporter gene. When injected intravenously, MSCs-R survived, proliferated, and differentiated in tumor sites but not elsewhere. The localization of GFP+ MSCs-R in tumor lesions was confirmed ex vivo. In conclusion, the MSCs-R can selectively localize, survive, and proliferate in both subcutaneous tumor and lung metastasis as evidenced by noninvasive bioluminescence imaging and ex vivo validation. The MSCs-R migrated to lung tumor differentiated into osteoblasts, whereas the MSCs-R targeting subcutaneous tumor differentiated into adipocytes.
PMCID: PMC4161123  PMID: 19544460
Mesenchymal stem cell; Multipotent differentiation; Adipogenesis; Osteoblastogenesis; Bioluminescence imaging; Molecular imaging
18.  Role of Albumin in the Formation and Stabilization of Nanoparticle Aggregates in Serum Studied by Continuous Photon Correlation Spectroscopy and Multiscale Computer Simulations 
Recently, small (<5 nm diameter) nanoparticles (NPs) have shown improved in vivo biocompatibility compared to that of larger (>10 nm) NPs. However, the fate of small NPs under physiological conditions is poorly understood and remains unexplored. Here, the long-term aggregation behavior of gold nanoparticles (AuNPs) exposed to serum proteins in a near-physiological setup is studied using continuous photon correlation spectroscopy and computer simulations. It is found that the medium, temperature, and NP concentration affect the aggregation of AuNPs, but the observed aggregates are much smaller than previously reported. Simulations show that a single layer of albumin is deposited on the NP surface, but the properties of the aggregates (size, shape, and internal structure) depend critically on the charge distribution on the proteins, which changes with the conditions of the solution. These results explain the seemingly conflicting data reported in the literature regarding the size of aggregates and the morphology of the albumin corona. The simulations suggest that controlling the concentration of NPs as well as the pH and ionic strength of the solution prior to intravenous administration may help to preserve properties of the functionalized NPs in the bloodstream.
PMCID: PMC4159775  PMID: 25221633
19.  Quantitative PET Imaging of Tumor Integrin αvβ3 Expression with 18F-FRGD2 
The development of noninvasive methods to visualize and quantify integrin αvβ3 expression in vivo appears to be crucial for the success of antiangiogenic therapy based on integrin antagonism. Precise documentation of integrin receptor levels will allow appropriate selection of patients who will most likely benefit from an antiintegrin treatment regimen. Imaging can also be used to provide an optimal dosage and time course for treatment based on receptor occupancy studies. In addition, imaging integrin expression will be important to evaluate antiintegrin treatment efficacy and to develop new therapeutic drugs with favorable tumor targeting and in vivo kinetics. We labeled the dimeric RGD peptide E[c(RGDyK)]2 with 18F and evaluated its tumor-targeting efficacy and pharmacokinetics of 18F-FB–E[c(RGDyK)]2 (18F-FRGD2).
E[c(RGDyK)]2 was labeled with 18F by conjugation coupling with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under a slightly basic condition. The in vivo metabolic stability of 18F-FRGD2 was determined. The diagnostic value after injection of 18F-FRGD2 was evaluated in various xenograft models by dynamic microPET followed by ex vivo quantification of tumor integrin level.
Starting with 18F− Kryptofix 2.2.2./K2CO3 solution, the total reaction time for 18F-FRGD2, including final high-performance liquid chromatography purification, is about 200 ± 20 min. Typical decay-corrected radiochemical yield is 23% ± 2% (n = 20). 18F-FRGD2 is metabolically stable. The binding potential extrapolated from graphical analysis of PET data and Logan plot correlates well with the receptor density measured by sodium dodecyl sulfate polyacrylamide electrophoresis and autoradiography in various xenograft models. The tumor-to-background ratio at 1 h after injection of 18F-FRGD2 also gives a good linear relationship with the tumor tissue integrin level.
The dimeric RGD peptide tracer 18F-FRGD2, with high integrin specificity and favorable excretion profile, may be translated into the clinic for imaging integrin αvβ3 expression. The binding potential calculated from simplified tracer kinetic modeling such as the Logan plot appears to be an excellent indicator of tumor integrin density.
PMCID: PMC4160026  PMID: 16391195
molecular imaging; integrin αvβ3; dimeric RGD peptide; dynamic microPET; Logan plot
20.  Near-Infrared Fluorescent RGD Peptides for Optical Imaging of Integrin αvβ3 Expression in Living Mice 
Bioconjugate chemistry  2005;16(6):1433-1441.
Near-infrared fluorescence optical imaging is a powerful technique for studying diseases at the molecular level in preclinical models. We recently reported that monomeric RGD peptide c(RGDyK) conjugated to the NIR fluorescent dye specifically targets integrin receptor both in cell culture and in living subjects. In this report, Cy5.5-conjugated mono-, di-, and tetrameric RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model in order to investigate the effect of multimerization of RGD peptide on integrin avidity and tumor targeting efficacy. The binding affinities of Cy5.5-conjugated RGD monomer, dimer, and tetramer for αvβ3 integrin expressed on U87MG cell surface were determined to be 42.9 ± 1.2, 27.5 ± 1.2, and 12.1 ± 1.3 nmol/L, respectively. All three peptide–dye conjugates had integrin specific uptake both in vitro and in vivo. The subcutaneous U87MG tumor can be clearly visualized with each of these three fluorescent probes. Among them, tetramer displayed highest tumor uptake and tumor-to-normal tissue ratio from 0.5 to 4 h postinjection. Tumor-to-normal tissue ratio for Cy5.5-conjugated RGD monomer, dimer, and tetramer were found to be 3.18 ± 0.16, 2.98 ± 0.05, and 3.63 ± 0.09, respectively, at 4 h postinjection. These results suggest that Cy5.5-conjugated monomeric, dimeric, and tetrameric RGD peptides are all suitable for integrin expression imaging. The multmerization of RGD peptide results in moderate improvement of imaging characteristics of the tetramer, compared to that of the monomer and dimeric counterparts.
PMCID: PMC4160083  PMID: 16287239
21.  Monitoring of the Biological Response to Murine Hindlimb Ischemia With 64Cu-Labeled Vascular Endothelial Growth Factor-121 Positron Emission Tomography 
Circulation  2008;117(7):915-922.
Vascular endothelial growth factor-121 (VEGF121), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF121 positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training.
Methods and Results
64Cu-labeled VEGF121 and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF121 positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem γ-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF121 showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF121 uptake in ischemic hindlimbs increased significantly (P<0.001) from a control level of 0.61 ±0.17% ID/g (percentage of injected dose per gram) to 1.62±0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59±0.14% ID/g at day 29), and correlated with γ-counting (R2=0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P≤0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2=0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice.
64Cu-VEGF121 positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.
PMCID: PMC4157592  PMID: 18250264
imaging; arteriosclerosis; exercise; angiogenesis; tomography; peripheral vascular disease; growth substances
22.  US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice1 
Radiology  2008;246(2):508-518.
To prospectively evaluate contrast material–enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models.
Materials and Methods
Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MBV), control microbubbles (MBC), and nonlabeled microbubbles (MBN) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MBV and MBC was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance.
In cell culture, adherence of MBV on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01– 0.10 microbubble per SVR cell; P < .001) and significantly more MBV attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MBV compared with MBC for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types.
US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.
PMCID: PMC4157631  PMID: 18180339
23.  Gadolinium embedded iron oxide nanoclusters as T1-T2 dual-modal MRI-visible vectors for safe and efficient siRNA delivery 
Nanoscale  2013;5(17):8098-8104.
This report illustrates a new strategy in designing a T1-T2 dual-modal magnetic resonance imaging (MRI)-visible vector for siRNA delivery and MRI. Hydrophobic gadolinium embedded iron oxide (GdIO) nanocrystals are self-assembled into nanoclusters in water phase with the help of stearic acid modified low molecular weight polyethylenimine (stPEI). The resulting water-dispersible GdIO-stPEI nanoclusters possess good stability, monodispersity with narrow size distribution and competitive T1-T2 dual-modal MR imaging properties. The nanocomposite system is capable of binding and delivering siR-NA for knockdown of a gene of interest while maintaining magnetic properties and biocompatibility. This new gadolinium embedded iron oxide nanocluster provides an important platform for safe and efficient gene delivery with non-invasive T1-T2 dual-modal MRI monitoring capability.
PMCID: PMC3775844  PMID: 23884164
24.  Small-Animal PET of Melanocortin 1 Receptor Expression Using a 18F-Labeled α-Melanocyte-Stimulating Hormone Analog 
18F-Labeled small synthetic peptides have emerged as attractive probes for imaging various molecular targets with PET. The α-melanocyte-stimulating hormone (α-MSH) receptor (melano-cortin type 1 receptor [MC1R]) is overexpressed in most murine and human melanomas. It is a promising molecular target for diagnosis and therapy of melanomas. However, 18F compounds have not been successfully developed for imaging the MC1R.
In this study, an α-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH2 (NAPamide), was radiolabeled with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB). The resulting radiopeptide was evaluated as a potential molecular probe for small-animal PET of melanoma and MC1R expression in melanoma xenografted mouse models.
The binding affinity of 19F-SFB–conjugated NAPamide, 19F-FB-NAPamide, was determined to be 7.2 ± 1.2 nM (mean ± SD) using B16/F10 cells and 125I-(Tyr2)-[Nle4,D-Phe7]-α-MSH [125I-(Tyr2)-NDP] as a radio-ligand. The biodistribution of 18F-FB-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16/F10 melanoma tumors with high expression of MC1Rs and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Biodistribution experiments showed that tumor uptake values (percentage injected dose per gram of tumor [%ID/g]) of 18F-FB-NAPamide were 1.19 ± 0.11 %ID/g and 0.46 ± 0.11 %ID/g, in B16/F10 and A375M xenografted melanoma at 1 h after injection, respectively. Furthermore, the B16/F10 tumor uptake was significantly inhibited by coinjection with excess α-MSH peptide (P < 0.05), indicating that 18F-FB-NAPamide specifically recognizes the MC1R in living mice. Small-animal PET of 18F-FB-NAPamide in mice bearing B16/F10 and A375M tumors at 1 h after tail vein injection revealed good B16/F10 tumor-to-background contrast and low A375M tumor-to-background ratios.
18F-FB-NAPamide is a promising molecular probe for α-MSH receptor-positive melanoma PET and warrants further study.
PMCID: PMC4154809  PMID: 17504880
melanoma; α-melanocyte-stimulating hormone; PET; imaging; 18F
25.  Imaging Chemically Modified Adenovirus for Targeting Tumors Expressing Integrin αvβ3 in Living Mice with Mutant Herpes Simplex Virus Type 1 Thymidine Kinase PET Reporter Gene 
The aim of this study was to change adenovirus tropism by chemical modification of the fiber knobs with PEGylated RGD peptide for targeting integrin αvβ3 that is uniquely or highly expressed in tumor cells and neovasculature of tumors of various origins.
The first generation Ad (Ad) vector, which expresses the herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) gene under the control of cytomegalovirus (CMV) promoter was conjugated with poly(ethylene glycol) (PEG) or RGD-PEG. The transduction efficiency of Ads (Adtk, PEG-Adtk, and RGD-PEG-Adtk) into different types of cells (293T, MCF7, MDA-MB-435, and U87MG) was analyzed and quantified by thymidine kinase (TK) assay using 8-3H-penciclovir (8-3H-PCV) as substrate. The in vivo infectivity of the Ad vectors after intravenous administration into integrin αvβ3–positive U87MG and MDA-MB-435 tumor-bearing athymic nude mice was measured by both noninvasive microPET using 9-[4-18F-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) as a reporter probe and ex vivo TK assay of the tumor and tissue homogenates.
PEGylation completely abrogated coxsackievirus and adenovirus receptor (CAR)–knob interaction and the infectivity of PEG-Adtk is significantly lower than that of unmodified Adtk in CAR-positive cells. RGD-PEG–modified virus (RGD-PEG-Adtk) had significantly higher infectivity than PEG-Adtk and the extent of increase is related to both CAR and integrin αvβ3 expression levels. 18F-FHBG had minimal nonspecific uptake in the liver and tumors that are void of sr39tk. Mice preinjected intravenously with unmodified Adtk resulted in high hepatic uptake and moderate tumor accumulation of the tracer. In contrast, RGD-PEG-Adtk administration resulted in significantly lower liver uptake without compromising the tumor accumulation of 18F-FHBG. Expression of TK in the liver and tumor homogenates corroborated with the magnitude of 18F-FHBG uptake quantified by noninvasive microPET. Analysis of liver and tumor tissue integrin level confirmed that RGD–integrin interaction is responsible for the enhanced tumor infectivity of RGD-PEG-Adtk.
The results of this study suggest that RGD-PEG conjugation is an effective way to modify Ad vector tropism for improved systemic gene delivery. Noninvasive PET and 18F-FHBG are able to monitor in vivo transfectivity of both Adtk and RGD-PEG-Adtk vectors in the liver and tumors after intravenous injection.
PMCID: PMC4154793  PMID: 16391197
adenovirus; reporter gene; gene therapy; integrin; RGD; PET

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