VEGF-loaded graphene oxide as theranostics for multi-modality imaging-monitored targeting therapeutic angiogenesis of ischemic muscle

National Key Basic Research Program (973 Project) [2010CB933901, 2012CB518101, 2013CB733802]; National Science Foundation of China [81272987, 31170961, 51102258, 81090274, 81227901]; Zhejiang Province Foundation [LY12H11011]; Intramural Research Program (IRP) of the National Institute of Biomedical Imaging and Bioengineering (NIBIB); National Institutes of Health (NIH)Herein we report the design and synthesis of multifunctional VEGF-loaded IR800-conjugated graphene oxide (GO-IR800-VEGF) for multi-modality imaging-monitored therapeutic angiogenesis of ischemic muscle. The as-prepared GO-IR800-VEGF positively targets VEGF receptors, maintains an elevated level of VEGF in ischemic tissues for a prolonged time, and finally leads to remarkable therapeutic angiogenesis of ischemic muscle. Although more efforts are required to further understand the in vivo behaviors and the long-term toxicology of GO, our work demonstrates the success of using GO for efficient VEGF delivery in vivo by intravenous administration and suggests the great promise of using graphene oxide in theranostic applications for treating ischemic disease

Kappa-Opioid Agonist U50,488H-Mediated Protection Against Heart Failure Following Myocardial Ischemia/Reperfusion: Dual Roles of Heme Oxygenase-1

Backgrounds/Aims: The selective κ-opioid agonist U50,488H protects heart from myocardial ischemia-reperfusion (MI/R) injury. We examined whether U50,488H is also beneficial for MI/R induced heart failure. Methods: Anesthetized male Sprague-Dawley rats were subjected to 30 min of myocardial ischemia via left anterior descending coronary artery (LAD) occlusion, followed by 4 weeks of reperfusion. Infarct size was examined by Evans blue/triphenyl tetrazolium chloride (TTC) staining. Cardiac function and remodeling were examined by echocardiography and histology. HO-1 gene transcription and expression were measured by RT-PCR and western blot. Results: Compared to vehicle-treated MI/R rats, rats administered a single dose of U50,488H at the beginning of reperfusion exhibited reduced myocardial infarct size, oxidative stress, hypertrophy, and fibrosis, improved mechanical function, and greater neovascularization. U50,488H also increased myocardial heme oxygenase (HO)-1 gene transcription and expression, while pharmacological HO-1 inhibition reversed all protective effects of U50,488H. Furthermore, U50,488H protected control cultured cardiomyoctes against simulated I/R-induced apoptosis but not cultures subjected to shRNA-mediated HO-1 knockdown. Inhibition of HO-1 in the subacute phase of reperfusion reversed the U50,488H-induced increase in neovascularization and suppression of oxidative stress. Finally, U50,488H increased Akt phosphorylation and nuclear translocation of Nrf2, a key HO-1 transcription activator, while inhibition of PI3K-Akt signaling abolished U50,488H-induced Nrf2 nuclear translocation, HO-1 upregulation, and cardioprotection. Conclusion: Activation of HO-1 expression through the PI3K-Akt-Nrf2 pathway may mediate the acute and long-term protective effects of U50,488H against heart failure by enhancing cardiomyocyte survival and neoangiogenesis and by reducing oxidative stress

Noninvasive Evaluation of Angiogenesis and Therapeutic Response after Hindlimb Ischemia with an Integrin-Targeted Tracer by PET

Background: Peripheral arterial disease (PAD) can severely compromise limb vitality and function. Angiogenesis plays an important role in healing of ischemic lesions. Radiolabeled RGD (Arg-Gly-Asp) peptides specifically targeting αvβ3 integrin are promising tracers for imaging angiogenesis. In this study, we investigated the application of a one-step labeled RGD in evaluation of angiogenesis and therapy response in a mouse model of hindlimb ischemia (HI) by positron emission tomography (PET). Methods: HI was induced by ablation of the femoral artery in mice. PET imaging using 18F-AlF-NOTA-PRGD2 (18F-PRGD2) tracer was performed at day 0 (pre-surgery) and days 3, 7, 14, and 21 after surgery to evaluate hindlimb angiogenesis longitudinally and noninvasively. The control peptide RAD (Arg-Ala-Asp) labeled with a similar procedure and a block agent were used to confirm the specific binding of 18F-PRGD2 to αvβ3 integrin. Ex vivo CD31 staining was performed to detect angiogenesis. In addition, the angiogenic therapy response was monitored with 18F-PRGD2 tracer and immunofluorescence staining to confirm the imaging data. Results: The successful establishment of HI model was confirmed by ultrasound imaging and laser doppler perfusion imaging (LDPI). Specific binding of 18F-PRGD2 to αvβ3 integrin was validated by minimal tracer uptake of the control peptide RAD and significant decrease of tracer accumulation when a block agent was added. Local accumulation of 18F-RRGD2 in ischemic hindlimb was detected as early as 3 days and reached a peak at 7 days after surgery. The temporal change of focal tracer uptake was positively correlated with the pattern of vascular density. Moreover, vascular endothelial growth factor (VEGF) treatment increased the tracer uptake and enhanced angiogenesis, which is consistent with integrin β3 expression. Conclusions: PET imaging of a one-step labeled tracer 18F-PRGD2 targeted to αvβ3integrin allows longitudinal monitoring of ischemia-induced angiogenesis and noninvasive assessment of VEGF treatment response in a mouse model of hindlimb ischemia. The simple synthesis procedure and in vivo performance of this PET tracer enables the feasibility of future clinical translation in ischemic cardiovascular diseases

Comparison of F-18-labeled CXCR4 antagonist peptides for PET imaging of CXCR4 expression

Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH); National Research Council Research Associateship Award at NIH/NIBIBCXCR4 is overexpressed on tumor cells from many types of human cancers. A high level of CXCR4 expression often correlates with poor prognosis, chemotherapy resistance, and metastasis. The development of CXCR4-specific radiotracers for positron emission tomography (PET) imaging will allow in vivo evaluation of receptor expression level for diagnosis or therapeutic evaluation. Two new F-18-labeled radiotracers based on an Ac-TC14012 peptide, [F-18]FP-Ac-TC14012 and [F-18]FB-Ac-TC14012, were synthesized and characterized. The affinities of the 2-fluoropropionate (FP)-conjugated or 4-fluorobenzoate (FB)-conjugated peptides to CXCR4-transfected Chinese hamster ovarian (CHO) cells were evaluated in a competitive binding assay with [I-125]CXCL12 radioligand. The cell uptake and retention of [F-18]FP-labeled and [F-18]FB-labeled peptides were measured. The tumor targetability and pharmacokinetics of these two tracers were also evaluated by microPET imaging and biodistribution studies. The labeled peptides retained high binding affinity to CXCR4 and showed much higher uptake in CXCR4-positive CHO cells than in CXCR4-negative cells in vitro. The smaller and more hydrophilic [F-18]FP prosthetic group resulted in higher affinity and lower nonspecific cell uptake compared to the [F-18]FB-labeled peptide. Both radiotracers showed much higher accumulation in CXCR4-positive than CXCR4-negative tumor xenografts in mice and allowed clear visualization of CXCR4 expression by PET. Among the two, [F-18]FP-Ac-TC14012 showed higher tumor uptake and better tumor-to-background contrast. Unlike their N-terminal 4-F-benzoate analogs, these two tracers had minimal blood retention, likely due to reduced red blood cell binding. Metabolic organs, such as the liver and kidney, also showed high uptake. When blocked with low-dose cold peptide (10 mu g), the tumor uptake was significantly increased, most likely due to the increased concentration in blood circulation, as evidenced by decreased liver uptake. These results demonstrate that the [F-18]FP-labeled Ac-TC14012 peptide with high tumor uptake, low nonspecific binding, and good tumor-to-background contrast promises [F-18]FP-Ac-TC14012 as a PET tracer for in vivo PET imaging of CXCR4 expression

ZP2495 Protects against Myocardial Ischemia/Reperfusion Injury in Diabetic Mice through Improvement of Cardiac Metabolism and Mitochondrial Function: The Possible Involvement of AMPK-FoxO3a Signal Pathway

Coronary heart disease patients with type 2 diabetes were subject to higher vulnerability for cardiac ischemia-reperfusion (I/R) injury. This study was designed to evaluate the impact of ZP2495 (a glucagon-GLP-1 dual-agonist) on cardiac function and energy metabolism after myocardial I/R injury in db/db mice with a focus on mitochondrial function. C57BLKS/J-lepr+/lepr+ (BKS) and db/db mice received 4-week treatment of glucagon, ZP131 (GLP-1 receptor agonist), or ZP2495, followed by cardiac I/R injury. The results showed that cardiac function, cardiac glucose metabolism, cardiomyocyte apoptosis, cardiac mitochondrial morphology, and energetic transition were improved or ameliorated by ZP2495 to a greater extent than that of glucagon and ZP131. In vitro study showed that ZP2495, rather than glucagon, alleviated mitochondrial depolarization, cytochrome C release, and mitochondria ROS generation in neonatal rat ventricular myocytes subjected to high-glucose and simulated I/R injury conditions, the effects of which were weaker in the ZP131 group. Furthermore, the expressions of Akt, FoxO3a, and AMPK phosphorylation were elevated by ZP2495 to a greater extent than that of ZP131. In conclusion, ZP2495 may contribute to the improvement of cardiac function and energy metabolism in db/db mice after myocardial I/R injury by improving mitochondrial function possibly through Akt/FoxO3a and AMPK/FoxO3a signal pathways

Development of a New Thiol Site-Specific Prosthetic Group and Its Conjugation with [Cys(40)]-exendin-4 for in Vivo Targeting of Insulinomas

National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health; Center for Neuroscience and Regenerative Medicine (CNRM) through Henry Jackson FoundationA new tracer, N-5-[F-18]fluoropentylmaleimide ([F-18]FPenM), for site-specific labeling of free thiol group in proteins and peptides was developed. The tracer was synthesized in three steps (F-18 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/mu mol. [F-18]FPenM showed comparable labeling efficiency with N-[2-(4-[F-18]-fluorobenzamido)ethyl]maleimide ([F-18]FBEM). Its application was demonstrated by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys(40)]-exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution, and metabolic stability of the radiolabeled [F-18]FPenM-[cys(40)]-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, [F-18]FPenM-[cys(40)]-exendin-4, had high tumor uptake (20.32 +/- 4.36%ID/g at 60 min postinjection) and rapid liver and kidney clearance, which was comparable to the imaging results with [F-18]FBEM-[cys(40)]-exendin-4 reported by our group

In vivo multimodality imaging of miRNA-16 iron nanoparticle reversing drug resistance to chemotherapy in a mouse gastric cancer model

miRNA-16 (miR16) plays an important role in modulating the drug resistance of SGC7901 cell lines to adriamycin (ADR). A variety of viral carriers have been designed for miRNA delivery. However, the safety concerns are currently perceived as hampering the clinical application of viral vector-based therapy. Herein a type of magnetic nanoparticles (MNPs) was designed and synthesized using poly(ethylene glycol) (PEG)-coated FeO nanoparticles as a miRNA delivery system for the purpose of reducing drug resistance of gastric cancer cells by enforcing miR16 expression in SGC7901/ADR cells. The MNPs with good biocompatibility were synthesized by thermal decomposition, and then conjugated with miRNA via electrostatic interaction producing miR16/MNPs. After co-culture with miR16/MNPs, ADR-induced apoptosis of SGC7901/ADR was examined by MTT and TUNEL. miR16/MNPs treatment significantly increased cell apoptosis in vitro. SGC7901/ADR tumor-bearing nude mice under ADR therapy were treated with miR16/MNPs by tail vein injection for in vivo study. After intraperitoneal injection of ADR, tumor volume measurement and fluorescence imaging were performed to for the death of SGC7901/ADR cells in vivo. Results showed that miR16/MNPs were able to significantly suppress SGC7901/ADR tumor growth, probably through increasing SGC7901/ADR cells' sensitivity to ADR. Our results suggest the efficient delivery of miR16 by MNPs as a novel therapeutic strategy for drug resistant tumor treatment