Transcriptome analysis unveils the mechanisms of lipid metabolism response to grayanotoxin I stress in Spodoptera litura.

Background: Spodoptera litura (tobacco caterpillar, S. litura) is a pest of great economic importance due to being a polyphagous and world-distributed agricultural pest. However, agricultural practices involving chemical pesticides have caused resistance, resurgence, and residue problems, highlighting the need for new, environmentally friendly methods to control the spread of S. litura. Aim: This study aimed to investigate the gut poisoning of grayanotoxin I, an active compound found in Pieris japonica, on S. litura, and to explore the underlying mechanisms of these effects. Methods: S. litura was cultivated in a laboratory setting, and their survival rate, growth and development, and pupation time were recorded after grayanotoxin I treatment. RNA-Seq was utilized to screen for differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to determine the functions of these DEGs. ELISA was employed to analyze the levels of lipase, 3-hydroxyacyl-CoA dehydrogenase (HOAD), and acetyl-CoA carboxylase (ACC). Hematoxylin and Eosin (H & E) staining was used to detect the development of the fat body. Results: Grayanotoxin I treatment significantly suppressed the survival rate, growth and development, and pupation of S. litura. RNA-Seq analysis revealed 285 DEGs after grayanotoxin I exposure, with over 16 genes related to lipid metabolism. These 285 DEGs were enriched in the categories of cuticle development, larvae longevity, fat digestion and absorption. Grayanotoxin I treatment also inhibited the levels of FFA, lipase, and HOAD in the hemolymph of S. litura. Conclusion: The results of this study demonstrated that grayanotoxin I inhibited the growth and development of S. litura. The mechanisms might, at least partly, be related to the interference of lipid synthesis, lipolysis, and fat body development. These findings provide valuable insights into a new, environmentally-friendly plant-derived insecticide, grayanotoxin I, to control the spread of S. litura.

Salvianolic acid B suppresses hepatic fibrosis by inhibiting ceramide glucosyltransferase in hepatic stellate cells.

UDP-glucose ceramide glucosyltransferase (UGCG) is the first key enzyme in glycosphingolipid (GSL) metabolism that produces glucosylceramide (GlcCer). Increased UGCG synthesis is associated with cell proliferation, invasion and multidrug resistance in human cancers. In this study we investigated the role of UGCG in the pathogenesis of hepatic fibrosis. We first found that UGCG was over-expressed in fibrotic livers and activated hepatic stellate cells (HSCs). In human HSC-LX2 cells, inhibition of UGCG with PDMP or knockdown of UGCG suppressed the expression of the biomarkers of HSC activation (α-SMA and collagen I). Furthermore, pretreatment with PDMP (40 µM) impaired lysosomal homeostasis and blocked the process of autophagy, leading to activation of retinoic acid signaling pathway and accumulation of lipid droplets. After exploring the structure and key catalytic residues of UGCG in the activation of HSCs, we conducted virtual screening, molecular interaction and molecular docking experiments, and demonstrated salvianolic acid B (SAB) from the traditional Chinese medicine Salvia miltiorrhiza as an UGCG inhibitor with an IC50 value of 159 µM. In CCl4-induced mouse liver fibrosis, intraperitoneal administration of SAB (30 mg · kg-1 · d-1, for 4 weeks) significantly alleviated hepatic fibrogenesis by inhibiting the activation of HSCs and collagen deposition. In addition, SAB displayed better anti-inflammatory effects in CCl4-induced liver fibrosis. These results suggest that UGCG may represent a therapeutic target for liver fibrosis; SAB could act as an inhibitor of UGCG, which is expected to be a candidate drug for the treatment of liver fibrosis.

Kaiso represses the expression of glucocorticoid receptor via a methylation-dependent mechanism and attenuates the anti-apoptotic activity of glucocorticoids in breast cancer cells.

Kaiso is a Pox Virus and Zinc Finger (POZ-ZF) transcription factor with bi-modal DNA-binding specificity. Here, we demonstrated that Kaiso expression is inversely correlated with glucocorticoid receptor (GR) expression in breast carcinomas. Knockdown of Kaiso increased GR expression, while overexpression of Kaiso inhibited GR expression in breast cancer cells. Furthermore, Kaiso repressed GR proximal promoter-reporter activity in a dose-dependent manner. Remarkably, ChIP experiments demonstrated that endogenous Kaiso was associated with the GR promoter sequence in a methylation-dependent manner. Since glucocorticoids inhibit chemotherapyinduced apoptosis and have been widely used as a co-treatment of patients with breast cancer, we assessed the role of Kasio in GR-mediated anti-apoptotic effects. We found that overexpression of Kaiso attenuated the anti-apoptotic effects of glucocorticoids in breast cancer cells. Our findings suggest that GR is a putative target gene of Kaiso and suggest Kaiso to be a potential therapeutic target in GC-combination chemotherapy in breast cancer. [BMB Reports 2016; 49(3): 167-172].

Small-animal PET of tumors with (64)Cu-labeled RGD-bombesin heterodimer.

UNLABELLED: The overexpression of gastrin-releasing peptide receptor (GRPR) in various tumor types suggests that GRPR is an attractive target for cancer imaging and therapy with radiolabeled bombesin analogs. We recently reported the ability of (18)F-labeled RGD-bombesin heterodimer to be used for dual integrin alpha(v)beta(3)- and GRPR-targeted imaging. To further investigate the synergistic effect of the dual-receptor targeting of peptide heterodimers, we evaluated (64)Cu-labeled RGD-bombesin for PET imaging of tumors. METHODS: RGD-bombesin was coupled with 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and the conjugates were labeled with (64)Cu. The in vitro and in vivo characteristics of (64)Cu-NOTA-RGD-bombesin were compared with those of (64)Cu-NOTA-RGD, (64)Cu-NOTA-bombesin, and (64)Cu-DOTA-RGD-bombesin. RESULTS: (64)Cu-NOTA-RGD-bombesin and (64)Cu-DOTA-RGD-bombesin had comparable dual integrin alpha(v)beta(3)- and GRPR-binding affinities in vitro, both of which were slightly lower than RGD for integrin binding and bombesin for GRPR binding. (64)Cu-NOTA-RGD-bombesin possessed significantly higher tumor uptake than did (64)Cu-NOTA-RGD, (64)Cu-NOTA-bombesin, the mixture of (64)Cu-NOTA-RGD and (64)Cu-NOTA-bombesin, or (64)Cu-DOTA-RGD-bombesin in PC-3 prostate cancer. (64)Cu-NOTA-RGD-bombesin also showed improved in vivo kinetics such as lower liver and intestinal activity accumulation than did the bombesin tracers. (64)Cu-NOTA-RGD-bombesin also outperformed (64)Cu-NOTA-RGD in a 4T1 murine mammary carcinoma model that expresses integrin on tumor vasculature but no GRPR in tumor tissue, which had no uptake of (64)Cu-NOTA-bombesin. CONCLUSION: Compared with other tracers, (64)Cu-NOTA-RGD-bombesin showed favorable in vivo kinetics and enhanced tumor uptake, which warrants its further investigation for targeting tumors that express integrin or GRPR or that coexpress integrin and GRPR for imaging and therapeutic applications. The synergistic effect of RGD-bombesin heterodimers observed in this study also encourages further investigations of novel heterodimers recognizing other cell surface receptors for tumor targeting.

MicroPET, MicroSPECT, and NIR fluorescence imaging of biomolecules in vivo.

Molecular imaging is a newly merged multidisciplinary subject that requires contributions from biology, medical physics, and chemistry/radiochemistry. Integrin alpha(v)beta(3), a cell adhesion molecule, plays pivotal roles in regulating tumor angiogenesis and the growth of new blood vessels. In this chapter, we use the cell adhesion molecule integrin alpha(v)beta(3) as an example to demonstrate how one can synthesize appropriate arginine-glycine-aspartic acid (RGD) peptide-containing probes for visualizing and quantifying the receptor expression in vivo by means of microPET, microSPECT, and NIR fluorescence.

64Cu-labeled PEGylated polyethylenimine for cell trafficking and tumor imaging.

PURPOSE: In this study, we exploited the potential of (64)Cu-labeled polyethylenimine (PEI) for cell trafficking and tumor imaging as compared to copper-64-pyruvaldehyde-bis(N (4)-methylthiosemicarbazone) ((64)Cu-PTSM). PROCEDURES: U87MG cells were labeled with both (64)Cu-PEI and (64)Cu-PTSM, and their in vivo distributions in mice were tracked by positron emission tomography (PET). The tumor imaging ability of (64)Cu-PTSM and (64)Cu-PEI was investigated in U87MG human glioblastoma xenograft model. (64)Cu-PEI-polyethylene glycol (PEG) was also synthesized, and the cell uptake, efflux, cytotoxicity, and the biodistribution were carried out and compared with (64)Cu-PEI. RESULTS: Both (64)Cu-PEI and (64)Cu-PEI-PEG were obtained in high labeling yield without the need of macrocyclic chelating agents. (64)Cu-PEI showed lower cell labeling efficiency than (64)Cu-PTSM. Small-animal PET images of living mice indicate that tail-vein-injected U87MG cells labeled with (64)Cu-PTSM or (64)Cu-PEI traffic to the lungs and liver. In a subcutaneous U87MG xenograft model, (64)Cu-PEI had higher tumor uptake (18.7 +/- 2.2 %ID/g at 24 h) than (64)Cu-PTSM (12.4 +/- 1.7 %ID/g at 24 h). In comparison with (64)Cu-PEI, (64)Cu-PEI-PEG had decreased toxicity and increased cell uptake in cell culture, as well as higher tumor uptake and better tumor-to-background contrast in U87MG xenograft model. CONCLUSION: (64)Cu-labeled polyethylenimine can be used for both cell trafficking and tumor imaging. PEGylation reduces the toxicity of (64)Cu-PEI and improves the tumor imaging ability.

Quantitative PET imaging of VEGF receptor expression.

PURPOSE: Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) signaling pathway plays pivotal roles in regulating tumor angiogenesis. Quantitative positron emission tomography (PET) imaging of VEGFR will facilitate the planning of whether, and when, to start anti-angiogenic treatment and enable more robust and effective monitoring of such treatment. MATERIALS AND METHODS: VEGF(121) was conjugated with DOTA (1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic acid) and then labeled with (64)Cu for PET imaging of mice bearing different-sized human glioblastoma U87MG tumors (n = 15). Western blotting and immunofluorescence staining of tumor tissue was carried out to correlate with/validate the imaging results. RESULTS: The specific activity of (64)Cu-DOTA-VEGF(121) was 3.2 GBq/mg. The uptake of (64)Cu-DOTA-VEGF(121) in the tumor peaked when the tumor size was about 100-250 mm(3). Both small and large tumors had lower tracer uptake indicating a narrow range of tumor size with high VEGFR-2 expression. All tumors had similarly low VEGFR-1 expression. Most importantly, the tumor uptake value obtained from PET imaging had good linear correlation with the relative tumor tissue VEGFR-2 expression as measured by Western blot, where r (2) equals 0.68 based on the PET uptake at 4 h post-injection. Histology of the frozen tumor tissue corroborates well with the imaging results. CONCLUSION: The tumor uptake of (64)Cu-DOTA-VEGF(121) measured by small-animal PET imaging reflects tumor VEGFR-2 expression level in vivo. Such correlation may facilitate future treatment planning and treatment monitoring of cancer and potentially other angiogenesis-related diseases.

Positron emission tomography imaging of poststroke angiogenesis.

BACKGROUND AND PURPOSE: Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles during neurovascular repair after stroke. In this study, we imaged VEGFR expression with positron emission tomography (PET) to noninvasively analyze poststroke angiogenesis. METHODS: Female Sprague-Dawley rats after distal middle cerebral artery occlusion surgery were subjected to weekly MRI, (18)F-FDG PET, and (64)Cu-DOTA-VEGF(121) PET scans. Several control experiments were performed to confirm the VEGFR specificity of (64)Cu-DOTA-VEGF(121) uptake in the stroke border zone. VEGFR, BrdU, lectin staining, and (125)I-VEGF(165) autoradiography on stroke brain tissue slices were performed to validate the in vivo findings. RESULTS: T2-weighed MRI correlated with the "cold spot" on (18)F-FDG PET for rats undergoing distal middle cerebral artery occlusion surgery. The (64)Cu-DOTA-VEGF(121) uptake in the stroke border zone peaked at approximately 10 days after surgery, indicating neovascularization as confirmed by histology (VEGFR-2, BrdU, and lectin staining). VEGFR specificity of (64)Cu-DOTA-VEGF(121) uptake was confirmed by significantly lower uptake of (64)Cu-DOTA-VEGF(mutant) in vivo and intense (125)I-VEGF(165) uptake ex vivo in the stroke border zone. No appreciable uptake of (64)Cu-DOTA-VEGF(121) was observed in the brain of sham-operated rats. CONCLUSIONS: For the first time to our knowledge, we successfully evaluated the VEGFR expression kinetics noninvasively in a rat stroke model. In vivo imaging of VEGFR expression could become a significant clinical tool to plan and monitor therapies aimed at improving poststroke angiogenesis.

64Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium cations as highly selective tumor imaging agents: effects of linkers and chelates on radiotracer biodistribution characteristics.

Radiolabeled organic cations, such as triphenylphosphonium (TPP), represents a new class of radiotracers for imaging cancers and the transport function of multidrug resistance P-glycoproteins (particularly MDR1 Pgp) by single photon emission computed tomography (SPECT) or positron emission tomography (PET). This report presents the synthesis and biological evaluation of (64)Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium (TPEP) cations as novel PET radiotracers for tumor imaging. Biodistribution studies were performed using the athymic nude mice bearing subcutaneous U87MG human glioma xenografts to explore the impact of linkers, bifunctional chelators (BFCs), and chelates on biodistribution characteristics of the (64)Cu-labeled TPEP cations. Metabolism studies were carried out using normal athymic nude mice to determine the metabolic stability of four (64)Cu radiotracers. It was found that most (64)Cu radiotracers described in this study have significant advantages over (99m)Tc-Sestamibi for their high tumor/heart and tumor/muscle ratios. Both BFCs and linkers have significant impact on biological properties of (64)Cu-labeled TPEP cations. For example, (64)Cu(DO3A-xy-TPEP) has much lower liver uptake and better tumor/liver ratios than (64)Cu(DO3A-xy-TPP), suggesting that TPEP is a better mitochondrion-targeting molecule than TPP. Replacing DO3A with DO2A results in (64)Cu(DO2A-xy-TPEP) (+), which has a lower tumor uptake than (64)Cu(DO3A-xy-TPEP). Substitution of DO3A with NOTA-Bn leads to a significant decrease in tumor uptake for (64)Cu(NOTA-Bn-xy-TPEP). The use of DOTA-Bn to replace DO3A has little impact on the tumor uptake, but the tumor/liver ratio of (64)Cu(DOTA-Bn-xy-TPEP) (-) is not as good as that of (64)Cu(DO3A-xy-TPEP), probably due to the aromatic benzene ring in DOTA-Bn. Addition of an extra acetamido group in (64)Cu(DOTA-xy-TPEP) results in a lower liver uptake, but tumor/liver ratios of (64)Cu(DOTA-xy-TPEP) and (64)Cu(DO3A-xy-TPEP) are comparable due to a faster tumor washout of (64)Cu(DOTA-xy-TPEP). Substitution of xylene with the PEG 2 linker also leads to a significant reduction in both tumor and liver uptake. MicroPET imaging studies on (64)Cu(DO3A-xy-TPEP) in athymic nude mice bearing U87MG glioma xenografts showed that the tumor was clearly visualized as early as 1 h postinjection with very high T/B contrast. There was very little metabolite (<2%) detectable in the urine and feces samples for (64)Cu(DO3A-xy-TPEP), (64)Cu(DOTA-Bn-xy-TPEP)(-), and (64)Cu(NOTA-Bn-xy-TPEP). Considering both tumor uptake and T/B ratios (particularly tumor/heart, tumor/liver, and tumor/muscle), it was concluded that (64)Cu(DO3A-xy-TPEP) is a promising PET radiotracer for imaging the MDR-negative tumors.

Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET.

PURPOSE: Malignant tumors are capable of degrading the surrounding extracellular matrix, resulting in local invasion or metastasis. Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) are central molecules in one of the major protease systems involved in extracellular matrix degradation. Noninvasive imaging of this receptor in vivo with radiolabeled peptides that specifically target uPAR may therefore be useful to decipher the potential invasiveness of malignant lesions. EXPERIMENTAL DESIGN: In this study, we developed a (64)Cu-labeled uPAR-binding peptide for positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled with (64)Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435 human breast cancer (uPAR negative). RESULTS: Surface plasmon resonance measurements show that AE105 with DOTA conjugated at the alpha-amino group (DOTA-AE105) has high affinity toward uPAR. microPET imaging reveals a rapid and high accumulation of (64)Cu-DOTA-AE105 in uPAR-positive U87MG tumors (10.8 +/- 1.5%ID/g at 4.5 hours, n = 3) but not in uPAR-negative MDA-MB-435 tumors (1.2 +/- 0.6%ID/g at 4.5 hours, n = 3). Specificity of this peptide-based imaging of uPAR was validated by further control experiments. First, a nonbinding variant of AE105 carrying a single amino acid replacement (Trp-->Glu) does not target U87MG tumors in vivo. Second, targeting of U87MG tumors by (64)Cu-DOTA-AE105 is specifically inhibited by a nonlabeled antagonist. CONCLUSION: The successful demonstration of the ability of a (64)Cu labeled uPAR-specific probe to visualize uPAR expression in vivo may allow clinical translation of this class of radiopharmaceuticals for uPAR-positive cancer detection and patient stratification for uPA/uPAR system-based cancer therapy.

Dual-modality optical and positron emission tomography imaging of vascular endothelial growth factor receptor on tumor vasculature using quantum dots.

PURPOSE: To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function positron emission tomography (PET)/near-infrared fluorescence (NIRF) probe might allow the accurate assessment of the tumor-targeting efficacy of QDs. MATERIALS AND METHODS: An amine-functionalized QD was conjugated with VEGF protein and DOTA chelator for VEGFR-targeted PET/NIRF imaging after (64)Cu-labeling. The targeting efficacy of this dual functional probe was evaluated in vitro and in vivo through cell-binding assay, cell staining, in vivo optical/PET imaging, ex vivo optical/PET imaging, and histology. RESULTS: The DOTA-QD-VEGF exhibited VEGFR-specific binding in both cell-binding assay and cell staining experiment. Both NIR fluorescence imaging and microPET showed VEGFR-specific delivery of conjugated DOTA-QD-VEGF nanoparticle and prominent reticuloendothelial system uptake. The U87MG tumor uptake of (64)Cu-labeled DOTA-QD was less than one percentage injected dose per gram (%ID/g), significantly lower than that of (64)Cu-labeled DOTA-QD-VEGF (1.52 +/- 0.6%ID/g, 2.81 +/- 0.3%ID/g, 3.84 +/- 0.4%ID/g, and 4.16 +/- 0.5%ID/g at 1, 4, 16, and 24 h post injection, respectively; n = 3). Good correlation was also observed between the results measured by ex vivo PET and NIRF organ imaging. Histologic examination revealed that DOTA-QD-VEGF primarily targets the tumor vasculature through a VEGF-VEGFR interaction. CONCLUSION: We have successfully developed a QD-based nanoprobe for dual PET and NIRF imaging of tumor VEGFR expression. The success of this bifunctional imaging approach may render higher degree of accuracy for the quantitative targeted NIRF imaging in deep tissue.

Evaluation of biodistribution and anti-tumor effect of a dimeric RGD peptide-paclitaxel conjugate in mice with breast cancer.

PURPOSE: Targeting drugs to receptors involved in tumor angiogenesis has been demonstrated as a novel and promising approach to improve cancer treatment. In this study, we evaluated the anti-tumor efficacy of a dimeric RGD peptide-paclitaxel conjugate (RGD2-PTX) in an orthotopic MDA-MB-435 breast cancer model. METHODS: To assess the effect of conjugation and the presence of drug moiety on the MDA-MB-435 tumor and normal tissue uptake, the biodistribution of (3)H-RGD2-PTX was compared with that of (3)H-PTX. The treatment effect of RGD2-PTX and RGD2+PTX was measured by tumor size, (18)F-FDG/PET, (18)F-FLT/PET, and postmortem histopathology. RESULTS: By comparing the biodistribution of (3)H-RGD2-PTX and (3)H-PTX, we found that (3)H-RGD2-PTX had higher initial tumor exposure dose and prolonged tumor retention than (3)H-PTX. Metronomic low-dose treatment of breast cancer indicated that RGD2-PTX is significantly more effective than PTX+RGD2 combination and solvent control. Although in vivo (18)F-FLT/PET imaging and ex vivo Ki67 staining indicated little effect of the PTX-based drug on cell proliferation, (18)F-FDG/PET imaging showed significantly reduced tumor metabolism in the RGD2-PTX-treated mice versus those treated with RGD2+PTX and solvent control. Terminal uridine deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining also showed that RGD2-PTX treatment also had significantly higher cell apoptosis ratio than the other two groups. Moreover, the microvessel density was significantly reduced after RGD2-PTX treatment as determined by CD31 staining. CONCLUSION: Our results demonstrate that integrin-targeted delivery of paclitaxel allows preferential cytotoxicity to integrin-expressing tumor cells and tumor vasculature. The targeted delivery strategies developed in this study may also be applied to other chemotherapeutics for selective tumor killing.

Effects of targeting moiety, linker, bifunctional chelator, and molecular charge on biological properties of 64Cu-labeled triphenylphosphonium cations.

In this report, we present the synthesis and evaluation of six new 64Cu-labeled triphenylphosphonium (TPP) cations. Biodistribution studies were performed using the athymic nude mice bearing U87MG human glioma xenografts to explore the impact of TPP moieties, linkers, bifunctional chelators (BFCs), and molecular charge on biological properties of 64Cu radiotracers. On the basis of the results from this study, it is concluded that (1) mTPP (tris(4-methoxyphenyl)phosphonium) is a better mitochondrion-targeting molecule than TPP and 3mTPP (tris(2,4,6-trimethoxyphenyl)phosphonium); (2) DO3A (1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid) and DO2A (1,4,7,10-tetraazacyclododecane-4,7-diacetic acid) are suitable BFCs for the 64Cu-labeling of TPP cations; (3) NOTA-Bn ( S-2-(4-thioureidobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid) has a significant adverse effect on the radiotracer tumor uptake and tumor-to-background ratios; and (4) monoanionic BFCs should be avoided to ensure that 64Cu chelate has a neutral or negative charge. Considering the tumor uptake and tumor/liver ratios, 64Cu(DO2A-xy-TPP)+ is the best candidate for more extensive evaluations in different tumor-bearing animal models.

Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.

We describe the synthesis and characterization of a 5' conjugate between a 2'-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine-glycine-aspartic acid) peptide that is a high-affinity ligand for the alphavbeta3 integrin. We used alphavbeta3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD-623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while 'free' 623 did not. However, the kinetics of luciferase expression was distinct; the RGD-623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD-623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD-623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide-oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the alphavbeta3 integrin.

18F-labeled BBN-RGD heterodimer for prostate cancer imaging.

UNLABELLED: Both bombesin (BBN) analogs and cyclic RGD peptides have been suitably radiolabeled for prostate cancer imaging. However, the limited expression of gastrin-releasing peptide receptor (GRPR) and integrin alpha(v)beta(3) as well as unfavorable in vivo kinetics limited further applications of these imaging agents. We hypothesize that a peptide ligand recognizing both GRPR and integrin will be advantageous because of its dual-receptor-targeting ability. METHODS: A BBN-RGD heterodimer was synthesized from bombesin(7-14) and c(RGDyK) through a glutamate linker and then labeled with (18)F via the N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) prosthetic group. The receptor-binding characteristics and tumor-targeting efficacy of (18)F-FB-BBN-RGD were tested in vitro and in vivo. RESULTS: FB-BBN-RGD had comparable integrin alpha(v)beta(3)-binding affinity with c(RGDyK) and comparable GRPR-binding affinity with BBN(7-14). (18)F-FB-BBN-RGD had significantly higher tumor uptake compared with monomeric RGD and monomeric BBN peptide tracer analogs at all time points examined. The PC-3 tumor uptake of (18)F-FB-BBN-RGD was inhibited only partially in the presence of an excess amount of unlabeled BBN(7-14) or c(RGDyK) but was blocked completely in the presence of both BBN(7-14) and c(RGDyK). Compared with (18)F-FB-BBN and (18)F-FB-RGD, (18)F-FB-BBN-RGD also had improved pharmacokinetics, resulting in a significantly higher imaging quality. CONCLUSION: Dual integrin alpha(v)beta(3) and GRPR recognition showed significantly improved tumor-targeting efficacy and pharmacokinetics compared with (18)F-labeled RGD and BBN analogs. The same heterodimeric ligand design may also be applicable to other receptor system combinations and other imaging modalities.

(68)Ga-labeled multimeric RGD peptides for microPET imaging of integrin alpha(v)beta (3) expression.

PURPOSE: We and others have reported that (18)F- and (64)Cu-labeled arginine-glycine-aspartate (RGD) peptides allow positron emission tomography (PET) quantification of integrin alpha(v)beta(3) expression in vivo. However, clinical translation of these radiotracers is partially hindered by the necessity of cyclotron facility to produce the PET isotopes. Generator-based PET isotope (68)Ga, with a half-life of 68 min and 89% positron emission, deserves special attention because of its independence of an onsite cyclotron. The goal of this study was to investigate the feasibility of (68)Ga-labeled RGD peptides for tumor imaging. METHODS: Three cyclic RGD peptides, c(RGDyK) (RGD1), E[c(RGDyK)](2) (RGD2), and E{E[c(RGDyK)](2)}(2) (RGD4), were conjugated with macrocyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with (68)Ga. Integrin affinity and specificity of the peptide conjugates were assessed by cell-based receptor binding assay, and the tumor targeting efficacy of (68)Ga-labeled RGD peptides was evaluated in a subcutaneous U87MG glioblastoma xenograft model. RESULTS: U87MG cell-based receptor binding assay using (125)I-echistatin as radioligand showed that integrin affinity followed the order of NOTA-RGD4 > NOTA-RGD2 > NOTA-RGD1. All three NOTA conjugates allowed nearly quantitative (68)Ga-labeling within 10 min (12-17 MBq/nmol). Quantitative microPET imaging studies showed that (68)Ga-NOTA-RGD4 had the highest tumor uptake but also prominent activity accumulation in the kidneys. (68)Ga-NOTA-RGD2 had higher tumor uptake (e.g., 2.8 +/- 0.1%ID/g at 1 h postinjection) and similar pharmacokinetics (4.4 +/- 0.4 tumor/muscle ratio, 2.0 +/- 0.1 tumor/liver ratio, and 1.1 +/- 0.1 tumor/kidney ratio) compared with (68)Ga-NOTA-RGD1. CONCLUSIONS: The dimeric RGD peptide tracer (68)Ga-NOTA-RGD2 with good tumor uptake and favorable pharmacokinetics warrants further investigation for potential clinical translation to image integrin alpha(v)beta(3).

The synthesis of 18F-FDS and its potential application in molecular imaging.

PURPOSE: 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) is the most commonly used positron emission tomography (PET) tracer for oncological and neurological imaging, but it has limitations on detecting tumor or inflammation in brain gray matter. In this study, we describe the development of 2-deoxy-2-[(18)F]fluorosorbitol ((18)F-FDS) and its possible application in lesion detection around brain area. PROCEDURES: (18)F-FDS was obtained by reduction of FDG using NaBH(4) (81 +/- 4% yield in 30 min). Cell uptake/efflux experiments in cell culture and small animal PET imaging on tumor and inflammation models were performed. RESULTS: Despite the low accumulation in cell culture, (18)F-FDS had good tumor uptake and contrast in the subcutaneous U87MG tumor model (4.54%ID/g at 30 min post-injection). Minimal uptake in the normal mouse brain facilitated good tumor contrast in both U87MG and GL-26 orthotopic tumor models. (18)F-FDS also had increased uptake in the inflamed foci of the TPA-induced acute inflammation model. CONCLUSIONS: Because of the ease of synthesis and favorable in vivo kinetics, (18)F-FDS may have potential applications in certain cases where FDG is inadequate (e.g., brain tumor).

Click chemistry for (18)F-labeling of RGD peptides and microPET imaging of tumor integrin alphavbeta3 expression.

The cell adhesion molecule integrin alpha vbeta 3 plays a key role in tumor angiogenesis and metastasis. A series of (18)F-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 (18)F by forming 1,2,3-triazoles. Nucleophilic fluorination of a toluenesulfonic alkyne provided (18)F-alkyne in high yield (nondecay-corrected yield: 65.0 +/- 1.9%, starting from the azeotropically dried (18)F-fluoride), which was then reacted with an RGD azide (nondecay-corrected yield: 52.0 +/- 8.3% within 45 min including HPLC purification). The (18)F-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 (18)F-fluoro-PEG-triazoles-RGD 2 ( (18)F-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 (18)F-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 (18)F labeling method based on click reaction may also be useful for radiolabeling of other biomolecules with azide groups in high yield.

Dual-function probe for PET and near-infrared fluorescence imaging of tumor vasculature.

UNLABELLED: To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function PET/near-infrared fluorescence (NIRF) probe can allow for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs. METHODS: A QD with an amine-functionalized surface was modified with RGD peptides and 1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelators for integrin alpha(v)beta(3)-targeted PET/NIRF imaging. A cell-binding assay and fluorescence cell staining were performed with U87MG human glioblastoma cells (integrin alpha(v)beta(3)-positive). PET/NIRF imaging, tissue homogenate fluorescence measurement, and immunofluorescence staining were performed with U87MG tumor-bearing mice to quantify the probe uptake in the tumor and major organs. RESULTS: There are about 90 RGD peptides per QD particle, and DOTA-QD-RGD exhibited integrin alpha(v)beta(3)-specific binding in cell cultures. The U87MG tumor uptake of (64)Cu-labeled DOTA-QD was less than 1 percentage injected dose per gram (%ID/g), significantly lower than that of (64)Cu-labeled DOTA-QD-RGD (2.2 +/- 0.3 [mean +/- SD] and 4.0 +/- 1.0 %ID/g at 5 and 18 h after injection, respectively; n = 3). Taking into account all measurements, the liver-, spleen-, and kidney-to-muscle ratios for (64)Cu-labeled DOTA-QD-RGD were about 100:1, 40:1, and 1:1, respectively. On the basis of the PET results, the U87MG tumor-to-muscle ratios for DOTA-QD-RGD and DOTA-QD were about 4:1 and 1:1, respectively. Excellent linear correlation was obtained between the results measured by in vivo PET imaging and those measured by ex vivo NIRF imaging and tissue homogenate fluorescence (r(2) = 0.93). Histologic examination revealed that DOTA-QD-RGD targets primarily the tumor vasculature through an RGD-integrin alpha(v)beta(3) interaction, with little extravasation. CONCLUSION: We quantitatively evaluated the tumor-targeting efficacy of a dual-function QD-based probe with PET and NIRF imaging. This dual-function probe has significantly reduced potential toxicity and overcomes the tissue penetration limitation of optical imaging, allowing for quantitative targeted imaging in deep tissue.

64Cu-Labeled triphenylphosphonium and triphenylarsonium cations as highly tumor-selective imaging agents.

This report presents synthesis and evaluation of the 64Cu-labeled triphenylphosphonium (TPP) cations as new radiotracers for imaging tumors by positron emission tomography. Biodistribution properties of 64Cu-L1, 64Cu-L2, 64Cu-L3, and 99mTc-Sestamibi were evaluated in athymic nude mice bearing U87MG human glioma xenografts. The most striking difference is that 64Cu-L1, 64Cu-L2, and 64Cu-L3 have much lower heart uptake (<0.6% ID/g) than 99mTc-Sestamibi ( approximately 18% ID/g) at >30 min p.i. Their tumor/heart ratios increase steadily from approximately 1 at 5 min p.i. to approximately 5 at 120 min p.i. The tumor/heart ratio of 64Cu-L3 is approximately 40 times better than that of 99mTc-Sestamibi at 120 min postinjection. Results from in vitro assays show that 64Cu-L1 is able to localize in tumor mitochondria. The tumor is clearly visualized in the tumor-bearing mice administered with 64Cu-L1 as 30 min postinjection. The 64Cu-labeled TPP/TPA cations are very selective radiotracers that are able to provide the information of mitochondrial bioenergetic function in tumors by monitoring mitochondrial potential in a noninvasive fashion.