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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.
doi:10.1021/cb400698s
PMCID: PMC3944097  PMID: 24266806
Liquid chromatography–mass spectrometry (LC-MS); activatable probe; matrix metalloproteinases (MMPs); metabolite; near-infrared fluorescence imaging
2.  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.
doi:10.1021/ja410438n
PMCID: PMC4004262  PMID: 24401138
3.  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
4.  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.
doi:10.7150/thno.10226
PMCID: PMC4279188  PMID: 25553112
Exendin-4; PEGylation; cardioprotection; Angiogenesis; myocardial infarction.
5.  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.
doi:10.1002/anie.201307952
PMCID: PMC3874880  PMID: 24155243
acetylcholinesterase-catalyzed hydrolysis; pathogen detection; high-sensitivity; gold nanoparticle; clinical samples
6.  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.
doi:10.1002/anie.201308986
PMCID: PMC4058316  PMID: 24318645
Theranostics; Biodegradable block co-polymer; Gold Nanovesicles; Plasmonic Coupling Effect; Photoacoustic Imaging; Photothermal Therapy
7.  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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.2967/jnumed.113.122069
PMCID: PMC4209961  PMID: 24232871
dual-tracer dynamic PET; parametric imaging; 18F-Alfatide II; 18F-FDG; therapy response
8.  Resistin disrupts glycogen synthesis under high insulin and high glucose levels by down-regulating the hepatic levels of GSK3β 
Gene  2013;529(1):50-56.
The effect of mouse resistin on hepatic insulin resistance in vivo and in vitro, and its possible molecular mechanism were examined. Focusing on liver glycogen metabolism and gluconeogenesis, which are important parts of glucose metabolism, in primary cultures of rat hepatocytes we found that glycogen content was significantly lower (P<0.05) after treatment with recombinant murine resistin only in the presence of insulin plus glucose stimulation. Protein levels of factors in the insulin signaling pathway involved in glycogen synthesis were examined by Western blot analysis, with the only significant change observed being the level of phosphorylated (at Ser 9) glycogen synthase kinase-3β (GSK-3β) (P<0.001). No differences in the protein levels for the insulin receptor β (IRβ), insulin receptor substrates (IRS1 and IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) or their phosphorylated forms were observed between control and resistin treated primary rat hepatocytes. In a mouse model with high liver-specific expression of resistin, fasting blood glucose levels and liver glycogen content changed. Fasting blood glucose levels were significantly higher (P<0.001) in the model mice, compared to the control mice, while the glycogen content of the liver tissue was about 60% of that of the control mice (P<0.05). The gluconeogenic response was not altered between the experimental and control mice. The level of phosphorylated GSK-3β in the liver tissue was also decreased (P<0.05) in the model mice, consistent with the results from the primary rat hepatocytes. Our results suggests that resistin reduces the levels of GSK-3β phosphorylated at Ser 9 leading to impaired hepatic insulin action in primary rat hepatocytes and in a mouse model with high liver-specific expression of resistin.
doi:10.1016/j.gene.2013.06.085
PMCID: PMC3897445  PMID: 23860320 CAMSID: cams3833
Resistin; Insulin resistance; Glycogen synthase kinase-3β (GSK-3β); Liver
9.  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.
doi:10.1016/j.biomaterials.2013.05.004
PMCID: PMC4167587  PMID: 23721793
Fluorescence imaging; Subdiffraction imaging; Fluorogenic PLGA nanoparticle; BODIPY NIR dye; pH sensitive; Molecular binding
10.  In Vivo Bioluminescence Tumor Imaging of RGD Peptide-modified Adenoviral Vector Encoding Firefly Luciferase Reporter Gene 
Purpose
The goal of this study is to demonstrate the feasibility of chemically modified human adenovirus (Ad) vectors for tumor retargeting.
Procedures
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.
Results
RGD-PEG-AdLuc abrogated the native CAR tropism and exhibited significantly enhanced transduction efficiency of integrin-positive tumors than AdLuc through intravenous administration.
Conclusion
This approach provides a robust platform for site-specific gene delivery and noninvasive monitoring of the transgene delivery efficacy and homing.
doi:10.1007/s11307-007-0079-2
PMCID: PMC4165526  PMID: 17297551
Adenovirus; Firefly luciferase; Reporter gene; Integrin αvβ3; RGD; Bioluminescence imaging
11.  Single Continuous Wave Laser Induced Photodynamic/Plasmonic Photothermal Therapy Using Photosensitizer-Functionalized Gold Nanostars 
doi:10.1002/adma.201204623
PMCID: PMC4138877  PMID: 23404693
Gold nanostar; chlorin e6; continuous wave laser; photodynamic therapy; plasmonic photothermal therapy
12.  The Effect of Injection Routes on the Biodistribution, Clearance and Tumor Uptake of Carbon Dots 
ACS nano  2013;7(7):5684-5693.
The emergence of photoluminescent carbon-based nanomaterials has shown exciting potential in the development of benign nanoprobes. However, the in vivo kinetic behaviors of these particles that are necessary for clinical translation are poorly understood to date. In this study, fluorescent carbon dots (C-dots) were synthesized and the effect of three injection routes on their fate in vivo was explored by using both near-infrared fluorescence (NIRF) and positron emission tomography (PET) imaging techniques. We found that C-dots are efficiently and rapidly excreted from the body after all three injection routes. The clearance rate of C-dots is ranked as: intravenous > intramuscular > subcutaneous. The particles had relatively low retention in the reticuloendothelial system (RES) and showed high tumor-to-background contrast. Furthermore, different injection routes also resulted in different blood clearance patterns and tumor uptakes of C-dots. These results satisfy the need for clinical translation and should promote efforts to further investigate the possibility of using carbon-based nanoprobes in a clinical setting. More broadly, we provide a testing blueprint for in vivo behavior of nanoplatforms under various injection routes, an important step forward towards safety and efficacy analysis of nanoparticles.
doi:10.1021/nn401911k
PMCID: PMC3725601  PMID: 23731122
Biodistribution; carbon dots; clearance; injection routes; translation; tumor uptake
13.  Development of a new thiol site-specific prosthetic group and its conjugation with [cys40]-exendin-4 for in vivo targeting of insulinomas 
Bioconjugate chemistry  2013;24(7):1191-1200.
A new tracer, N-5-[18F]fluoropentylmaleimide ([18F]FPenM), for site-specific labeling of free thiol group in proteins and peptides was developed. The tracer was synthesized in three steps (18F displacement of the aliphatic tosylate, di-Boc removal by TFA to expose free amine and incorporation of the free amine into a maleimide). The radiosynthesis was completed in 110 min with 11–17% radiochemical yield (uncorrected), and specific activity of 20–49 GBq/µmol. [18F]FPenM showed comparable labeling efficiency with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Its application was demonstrated by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys40]-exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution and metabolic stability of the radiolabeled [18F]FPenM-[cys40]-exendin-4 were studied using INS-1 tumor cells and INS-1 xenograft model. Positron emission tomography (PET) results showed that the new thiol-specific tracer, [18F]FPenM-[cys40]-exendin-4, had high tumor uptake (20.32 ± 4.36 %ID/g at 60 min post-injection) and rapid liver and kidney clearance, which was comparable to the imaging results with [18F]FBEM-[cys40]-exendin-4 reported by our group.
doi:10.1021/bc400084u
PMCID: PMC3779517  PMID: 23750453
14.  In vivo quantitative photoacoustic microscopy of gold nanostar kinetics in mouse organs 
Biomedical Optics Express  2014;5(8):2679-2685.
We developed a high-resolution photoacoustic microscopy (PAM) system with a near-infrared (NIR) laser to noninvasively monitor the distribution of gold nanostar (GNS) in blood vessels, liver and spleen in mice. Photoacoustic images of organs at deep depths were continuously acquired in vivo every 30 minutes after a single dose of GNS by tail vein injection. The experimental results showed that GNS accumulated significantly in both liver and spleen from blood circulation after administration, which was qualitatively validated by fluorescence imaging. Our studies demonstrate that PAM might be potentially used for noninvasive tracing the kinetics of exogenous nanoparticles in biological system.
doi:10.1364/BOE.5.002679
PMCID: PMC4132997  PMID: 25136493
(180.0180) Microscopy; (110.5125) Photoacoustics; (000.1430) Biology and medicine; (170.3880) Medical and biological imaging
15.  Gold Nanoparticle Based Activatable Probe for Sensing Ultra-Low Levels of Prostate Specific Antigen 
ACS nano  2013;7(6):5568-5576.
It is still in high demand to develop extremely sensitive and accurate clinical tools for biomarkers of interest for early diagnosis and monitoring of diseases. In this report, we present a highly sensitive and compatible gold nanoparticle (AuNP)-based fluorescence activatable probe for sensing ultra-low levels of prostate-specific antigen (PSA) in patient serum samples. The limit of detection of the newly-developed probe for PSA was pushed down to 0.032 pg/mL, which is more than two orders of magnitude lower than that of the conventional fluorescence probe. The ultrahigh sensitivity of this probe was attributed to the high loading efficiency of the dyes on AuNP surfaces and high fluorescence quenching unquenching abilities of the dye-AuNP pairs. The efficiency and robustness of this probe was investigated in patient serum samples, demonstrating the great potential of this probe in real-world applications.
doi:10.1021/nn401837q
PMCID: PMC3696512  PMID: 23683064
fluorescence activatable probe; gold nanoparticle; prostate specific antigen (PSA); Rhodamine B isothiocyanate (RBITC)
16.  Photosensitizer-Loaded Gold Vesicles with Strong Plasmonic Coupling Effect for Imaging-Guided Photothermal/Photodynamic Therapy 
ACS nano  2013;7(6):5320-5329.
A multifunctional theranostic platform based on photosensitizer-loaded plasmonic vesicular assemblies of gold nanoparticles (GNPs) is developed for effective cancer imaging and treatment. The gold vesicles (GVs) composed of a monolayer of assembled GNPs show strong absorbance in the near-infrared (NIR) range of 650–800 nm, as a result of the plasmonic coupling effect between neighboring GNPs in the vesicular membranes. The strong NIR absorption and the capability of encapsulating photosensitizer Ce6 in gold vesicles (GVs) enable tri-modality NIR fluorescence/thermal/photoacoustic imaging-guided synergistic photothermal/photodynamic therapy (PTT/PDT) with improved efficacy. The Ce6-loaded GVs (GV-Ce6) have the following characteristics: i) high Ce6 loading efficiency (up to ~18.4 wt%; ii) enhanced cellular uptake efficiency of Ce6; iii) simultaneous tri-modality NIR fluorescence/thermal/photoacoustic imaging; iv) synergistic PTT/PDT treatment with improved efficacy using single wavelength continuous wave laser irradiation.
doi:10.1021/nn4011686
PMCID: PMC3709863  PMID: 23721576
Gold vesicles; plasmonic coupling effect; photoacoustic imaging; photothermal therapy; photodynamic therapy; synergistic therapy
17.  Photosensitizer-Conjugated Silica-Coated Gold Nanoclusters for Fluorescence Imaging-Guided Photodynamic Therapy 
Biomaterials  2013;34(19):4643-4654.
Multifunctional theranostics have recently been intensively explored to optimize the efficacy and safety of therapeutic regimens. In this work, a photo-theranostic agent based on chlorin e6 (Ce6) photosensitizer-conjugated silica-coated gold nanoclusters (AuNCs@SiO2-Ce6) is strategically designed and prepared for fluorescence imaging-guided photodynamic therapy (PDT). The AuNCs@SiO2-Ce6 shows the following features: i) high Ce6 photosensitizer loading; ii) no non-specific release of Ce6 during its circulation; iii) significantly enhanced cellular uptake efficiency of Ce6, offering a remarkably improved photodynamic therapeutic efficacy compared to free Ce6; iv) subcellular characterization of the nanoformula via both the fluorescence of Ce6 and plasmon luminescence of AuNCs; v) fluorescence imaging-guided photodynamic therapy (PDT). This photo-theranostics owns good stability, high water dispersibility and solubility, non-cytotoxicity, and good biocompatibility, thus facilitating its biomedical applications, particularly for multi-modal optical, CT and photoacoustic (PA) imaging guided PDT or sonodynamic therapy.
doi:10.1016/j.biomaterials.2013.02.063
PMCID: PMC3645870  PMID: 23523428
Theranostics; silica; gold nanocluster; chlorin e6 (Ce6); fluorescence imaging; photodynamic therapy
18.  Differential expressed genes of change in glucose metabolism in the liver of GCK knockout mice 
Gene  2013;516(2):248-254.
To investigate the role of liver-specific expression of GCK in the pathogenesis of hyperglycemia and identify candidate genes involved in the mechanisms for onset and progression of MODY2, we examined the differentially expression of genes in the liver of liver-specific glucokinase (GCK) knockout mice (gckw/−) model at 2 and 26 weeks by suppression subtractive hybridization (SSH). The expression levels of the ACAT2 and PEPCK genes identified by SSH, glycogen Synthase (GS) and glycogen phosphorylase (GP) genes were further examined at different ages by Real time PCR. In addition, we also characterized the expression of mouse GCK mRNA at different ages as well as the fasting blood glucose, serum insulin, GCK activity, total cholesterol (TC), triglyceride(TG) and glycogen content. The results show that, except for 2-week-old gckw/− mice, the fasting blood glucose levels are significantly higher for gckw/− mice (P<0.01)and the GCK activity of gckw/− mice lower about 50% than for GCK wild type(gckw/w )mice(P<0.05). The glycogen content of 4-week old and 40-week old gckw/− mice was lower than that of 4-week old and 40-week old gckw/w mice. The GP mRNA levels decrease at 40-week old gckw/− mice compared to age-matched gckw/w mice. PEPCK mRNA decreases at 2-week old gckw/− mice, but increases at 4-week old gckw/− mice(P<0.05). Changes in the expression of PEPCK genes, delayed development of GCK and impaired hepatic glycogen synthesis in liver potentially lead to onset and progression of maturity onset diabetes of the young, type 2 (MODY-2).
doi:10.1016/j.gene.2012.12.036
PMCID: PMC3642073  PMID: 23291412 CAMSID: cams2937
glucokinase; MODY2; differentially expressed genes
19.  Assessment of Iron Deposition and White Matter Maturation in Infant Brains by Using Enhanced T2 Star Weighted Angiography (ESWAN): R2* versus Phase Values 
PLoS ONE  2014;9(2):e89888.
Background and Purpose
Iron deposition and white matter (WM) maturation are very important for brain development in infants. It has been reported that the R2* and phase values originating from the gradient-echo sequence could both reflect the iron and myelination. The aim of this study was to investigate age-related changes of R2* and phase value, and compare their performances for monitoring iron deposition and WM maturation in infant brains.
Methods
56 infants were examined by enhanced T2 star weighted angiography (ESWAN) and diffusion tensor imaging in the 1.5T MRI system. The R2* and phase values were measured from the deep gray nuclei and WM. Fractional anisotropy (FA) values were measured only in the WM regions. Correlation analyses were performed to explore the relation among the two parameters (R2* and phase values) and postmenstrual age (PMA), previously published iron concentrations as well as FA values.
Results
We found significantly positive correlations between the R2* values and PMA in both of the gray nuclei and WM. Moreover, R2* values had a positive correlation with the iron reference concentrations in the deep gray nuclei and the FA in the WM. However, phase values only had the positive correlation with PMA and FA in the internal capsule, and no significant correlation with PMA and iron content in the deep gray nuclei.
Conclusions
Compared with the phase values, R2* may be a preferable method to estimate the iron deposition and WM maturation in infant brains.
doi:10.1371/journal.pone.0089888
PMCID: PMC3934963  PMID: 24587101
20.  Positron Emission Tomography Imaging of Vascular Endothelial Growth Factor Receptor Expression with 61Cu-Labeled Lysine-Tagged VEGF121 
Molecular pharmaceutics  2012;9(12):3586-3594.
Overexpression of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) indicates poor prognosis for cancer patients in a variety of clinical studies. Our goal is to develop a tracer for positron emission tomography (PET) imaging of VEGFR expression using recombinant human VEGF121 with three lysine residues fused to the N-terminus (denoted as K3-VEGF121), which can facilitate radiolabeling without affecting its VEGFR binding affinity. K3-VEGF121 was conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with 61Cu (t1/2: 3.3 h; 62% β+). The IC50 value of NOTA-K3-VEGF121 for VEGFR-2 was comparable to K3-VEGF121 (1.50 and 0.65 nM, respectively) based on cell binding assay. 61Cu labeling was achieved with good yield (55 ± 10 %) and specific activity (4.2 GBq/mg). Serial PET imaging showed that the 4T1 tumor uptake of 61Cu-NOTA-K3-VEGF121 was 3.4 ± 0.5, 4.9 ± 1.0, 5.2 ± 1.0, and 4.8 ± 0.8 %ID/g (n = 4) at 0.5, 2, 4, and 8 h post-injection respectively, which was consistent with biodistribution data measured by gamma counting. Blocking experiments and ex vivo histology confirmed VEGFR specificity of 61Cu-NOTA-K3-VEGF121. Extrapolated human dosimetry calculation showed that liver was the organ with the highest radiation dose. The use of 61Cu as the radiolabel is desirable for small proteins like K3-VEGF121, which has much higher β+ branching ratio than the commonly used 64Cu (62% vs. 17%) thereby offering stronger signal intensity and lower tracer dose for PET imaging.
doi:10.1021/mp3005269
PMCID: PMC3619014  PMID: 23137334
Vascular endothelial growth factor (VEGF); VEGF receptor (VEGFR); 61Cu; Positron emission tomography (PET); Tumor angiogenesis; Molecular imaging
21.  Differentiation of Reactive and Tumor Metastatic Lymph Nodes with Diffusion-weighted and SPIO Enhanced MRI 
Objectives
Determination of lymphatic metastasis is of great importance for both treatment planning and patient prognosis. We aim to distinguish tumor metastatic lymph nodes (TLNs) and reactive lymph nodes (RLNs) with diffusion-weighted and superparamagnetic iron oxide (SPIO) enhanced magnetic resonance imaging (MRI).
Materials and methods
Ipsilateral popliteal lymph node metastasis or lymphadenitis model was established by hock injection of either luciferase-expressing 4T1 murine breast cancer cells or Complete Freund Adjuvant (CFA) in male Balb/C mice. At different time points after inoculation, bioluminescence imaging, T2-weighted, diffusion-weighted and SPIO enhanced MRI were performed. Imaging findings were confirmed by histopathological staining.
Results
Size enlargement was observed in both TLNs and RLNs. At day 28, TLNs showed strong bioluminescence signal and bigger size than RLNs (p < 0.01). At early stages up to day 21, both TLNs and RLNs appeared homogeneous on diffusion-weighted imaging (DWI). At day 28, TLNs showed heterogeneous apparent diffusion coefficient (ADC) map with significantly higher average ADC value of 0.41 ± 0.03 × 10−3 mm2/s than that of RLNs (0.34 ± 0.02 10−3 mm2/s, p < 0.05). On SPIO enhanced MRI, both TLNs and RLNs showed distinct T2 signal reduction at day 21 after inoculation. At day 28, TLNs demonstrated partial uptake of the iron oxide particles, which was confirmed by Prussian blue staining.
Conclusions
Both diffusion-weighted and SPIO enhanced MRI can distinguish tumor metastatic lymph nodes from reactive lymph nodes. However, neither method is able to detect tumor metastasis to the draining lymph nodes at early stages.
doi:10.1007/s11307-012-0562-2
PMCID: PMC3460060  PMID: 22588595
Lymph node metastasis; diffusion-weighted imaging (DWI); superparamagnetic iron oxide (SPIO); magnetic resonance imaging (MRI); apparent diffusion coefficient (ADC) map
22.  Mesenchymal stem cell-based cell engineering with multifunctional mesoporous silica nanoparticles for tumor delivery 
Biomaterials  2012;34(7):1772-1780.
Stem cell engineering, the manipulation and control of cells, harnesses tremendous potential for diagnosis and therapy of disease; however, it is still challenging to impart multifunctionalization onto stem cells to achieve both. Here we describe a mesenchymal stem cell (MSC)-based multifunctional platform to target orthotopic glioblastoma by integrating the tumor targeted delivery of mesenchymal stem cells and the multimodal imaging advantage of mesoporous silica nanoparticles (MSNs). Rapid cellular uptake, long retention time and stability of particles exemplify the potential that the combination of MSNs and MSCs has as a stem cell-based multifunctional platform. Using such a platform, we verified tumor-targeted delivery of MSCs by in vivo multimodal imaging in an orthotopic U87MG glioblastoma model, displaying higher tumor uptake than particles without MSCs. As a proof-of-concept, this MSC platform opens a new vision for multifunctional applications of cell products by combining the superiority of stem cells and nanoparticles for actively targeted delivery.
doi:10.1016/j.biomaterials.2012.11.032
PMCID: PMC3538138  PMID: 23228423
Mesenchymal stem cells (MSCs); mesoporous silica nanoparticles (MSNs); cell engineering; multimodal imaging; targeted delivery
23.  Long term liver specific glucokinase gene defect induced diabetic cardiomyopathy by up regulating NADPH oxidase and down regulating insulin receptor and p-AMPK 
Background
The liver-specific glucokinase knockout (gckw/–) mouse experiences long-term hyperglycemia and insulin resistance. This study was designed to evaluate the functional and structural changes in the myocardium of 60 week-old gckw/– mice, and to investigate the effect of rosiglitazone on the myocardium in this model.
Methods
60 week-old gckw/– mice were randomly divided into 3 groups: gckw/–, gckw/– mice treated with insulin (1 U/kg) and gckw/– mice treated with rosiglitazone (18 mg/kg). Insulin or rosiglitazone treatment was for 4 weeks. Gckw/w litermates were used as controls. Echocardiography, electrocardiogram, biochemical, histopathological, ultrastructural, real time PCR and Western blot studies were performed to examine for structural and functional changes.
Results
Long-term liver-specific gck knockout in mice elicits hyperglycaemia and insulin resistance. Compared to age matched gckw/w mice, 60 week-old gckw/– mice showed decreased LV internal dimension, increased posterior wall thickness, lengthened PR and QRS intervals, up-regulated MLC2 protein expression, decreased SOD activity, increased MDA levels and up-regulated Cyba mRNA. Morphological studies revealed that there was an increase in the amount of PAS and Masson positively stained material, as did the number and proportion of the cell occupied by mitochondria in the gckw/– mice. Western blot analysis revealed that the levels of the insulin receptor, Akt, phosphorylated AMPK beta and phosphorylated ACC were reduced in gckw/– mice. These effects were partly attenuated or ablated by treatment with rosiglitazone.
Conclusions
Our results indicate that changes in the myocardium occur in the liver-specific glucokinase knockout mouse and suggest that reduced glucokinase expression in the liver may induce diabetic cardiomyopathy by up regulating NADPH oxidase and down regulating insulin receptor and p-AMPK protein levels. Rosiglitazone treatment may protect against diabetic cardiomyopathy by altering the levels of a set of proteins involved in cardiac damage.
doi:10.1186/1475-2840-13-24
PMCID: PMC3901845  PMID: 24447392
Liver-specific glucokinase knockout; Diabetic cardiomyopathy; Rosiglitazone; Insulin receptor; AMPK
24.  Multimodality Imaging of Breast Cancer Experimental Lung Metastasis with Bioluminescence and a Monoclonal Antibody Dual-Labeled with 89Zr and IRDye 800CW 
Molecular pharmaceutics  2012;9(8):2339-2349.
Metastatic breast cancer is incurable. The goal of this study was to develop a positron emission tomography (PET)/near-infrared fluorescent (NIRF) probe for imaging of CD105 expression in breast cancer experimental lung metastasis. TRC105, a chimeric anti-CD105 antibody, was dual-labeled with a NIRF dye (IRDye 800CW) and 89Zr to yield 89Zr-Df-TRC105-800CW. Luciferase-transfected 4T1 murine breast cancer cells were injected intravenously into female mice to establish the tumor model. Bioluminescence imaging (BLI) was carried out to non-invasively monitor the lung tumor burden. PET imaging revealed that 4T1 lung tumor uptake of 89Zr-Df-TRC105-800CW was 8.7±1.4, 10.9±0.5, and 9.7±1.1 %ID/g at 4, 24, and 48 h post-injection (n = 4), with excellent tumor contrast. Biodistribution studies, blocking, control studies with 89Zr-Df-cetuximab-800CW, ex vivo BLI/PET/NIRF imaging, and histology all confirmed CD105 specificity of the tracer. Broad clinical potential of TRC105-based agents was shown in many tumor types, which also enabled early detection of small metastasis and intraoperative guidance for tumor removal.
doi:10.1021/mp300277f
PMCID: PMC3500677  PMID: 22784250
Breast cancer; lung metastasis; positron emission tomography (PET); near-infrared fluorescence (NIRF); tumor angiogenesis; CD105 (endoglin); 89Zr
25.  Visualization of Protease Activity In Vivo Using an Activatable Photo-Acoustic Imaging Probe Based on CuS Nanoparticles 
Theranostics  2014;4(2):134-141.
Herein, we for the first time report a novel activatable photoacoustic (PA) imaging nano-probe for in vivo detection of cancer-related matrix metalloproteinases (MMPs). A black hole quencher 3 (BHQ3) which absorbs red light is conjugated to near-infrared (NIR)-absorbing copper sulfide (CuS) nanoparticles via a MMP-cleavable peptide linker. The obtained CuS-peptide-BHQ3 (CPQ) nano-probe exhibits two distinctive absorption peaks at 630 nm and 930 nm. Inside the tumor microenviorment where MMPs present, the MMP-sensitive peptide would be cleaved, releasing BHQ3 from the CuS nanoparticles, the former of which as a small molecule is then rapidly cleared out from the tumor, whereas the latter of which as large nanoparticles would retain inside the tumor for a much longer period of time. As the result, the PA signal at 680 nm which is contributed by BHQ3 would be quickly diminished while that at 930 nm would be largely retained. The PA signal ratio of 680 nm / 930 nm could thus serve as an in vivo indicator of MMPs activity inside the tumor. Our work presents a novel strategy of in vivo sensing of MMPs based on PA imaging, which should offer remarkably improved detection depth compared with traditional optical imaging techniques.
doi:10.7150/thno.7217
PMCID: PMC3900798  PMID: 24465271
Peptide; Photoacoustic imaging; Enzyme cleavage; Copper sulfide; MMPs detection.

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