Theranostics

The kinetic analysis of (11)C-acetate PET provides more information than routine one time-point static imaging. This study aims to investigate the potential of dynamic (11)C-acetate hepatic PET imaging to improve the diagnosis of hepatocellular carcinoma (HCC) and benign liver lesions by using compartmental kinetic modeling and discriminant analysis. 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 (11)C-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. 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%. (11)C-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.

Antisense oligonucleotides (ASOs) have potential as anti-cancer agents by specifically modulating genes involved in tumorigenesis. However, little is known about ASO biodistribution and tissue pharmacokinetics (PKs) in humans, including whether sufficient delivery to target tumor tissue may be achieved. In this preliminary study in human subjects, we used combined positron emission and computed tomography (PET-CT) imaging and subsequent modeling analysis of acquired dynamic data, to examine the in vivo biodistribution and PK properties of LY2181308 - a second generation ASO which targets the apoptosis inhibitor protein survivin. Following radiolabeling of LY2181308 with methylated carbon-11 ([(11)C]methylated-LY2181308), micro-doses (<1mg) were administered to three patients with solid tumors enrolled in a phase I trial. Moderate uptake of [(11)C]methylated-LY2181308 was observed in tumors (mean=32.5ng*h /mL, per mg administered intravenously). Highest uptake was seen in kidney and liver and lowest uptake was seen in lung and muscle. One patient underwent repeat analysis on day 15 of multiple dose therapy, during administration of LY2181308 (750mg), when altered tissue PKs and a favorable change in biodistribution was seen. [(11)C]methylated-LY2181308 exposure increased in tumor, lung and muscle, whereas renal and hepatic exposure decreased. This suggests that biological barriers to ASO tumor uptake seen at micro-doses were overcome by therapeutic dosing. In addition, (18)F-labeled fluorodeoxyglucose (FDG) scans carried out in the same patient before and after treatment showed up to 40% decreased tumor metabolism. For the development of anti-cancer ASOs, the results provide evidence of LY2181308 tumor tissue delivery and add valuable in vivo pharmacological information. For the development of novel therapeutic agents in general, the study exemplifies the merits of applying PET imaging methodology early in clinical investigations.

Aim: To study the long term benefits, toxicity and survival rate in patients with neuroendocrine tumors receiving multiple cycles of high activity In-111 Pentetreotide therapy. Moreover, our secondary aim was to evaluate the value of F-18 FDG PET-CT scan as prognostic indicator in this group of patients. Background: Neuroendocrine tumors are a heterogeneous group of malignancies which are usually metastatic at diagnosis. Standard chemotherapy in these patients is associated with appreciable adverse events and low effectiveness. Since 1990s, Peptide receptor radionuclide therapy (PRRT) with radio-labeled somatostatin analogues has been introduced as a new method of treatment in patients with unresectable and/or metastatic neuroendocrine tumors expressing high levels of Somatostatin receptors. Methods: 112 patients with progressive disseminated and unresectable neuroendocrine tumor (stage III and stage IV) were enrolled in a non-randomized trial in an out-patient setting. High activity In-111 Pentetreotide (500 mCi (18.5 GBq) per cycle) was administered as an intravenous infusion over 3 hours and repeated therapy cycles every 9-12 weeks in eligible patients up to maximum of 4 cycles. Response to therapy was evaluated by clinical imaging using the RECIST criteria, metabolic criteria and patient's quality of life questionnaire. Dosimetry and biodistribution studies were also performed. Finally, Kaplan-Meier survival analysis was performed for patients followed for greater than 12 months. The relationship between pretreatment F-18 FDG PET-CT scan status and survival was determined by two-tailed Student's t-test in 42 patients who underwent pre-therapy PET scans. Results: For an average of 25 (median 18.65) months following the therapy, patients were evaluated for any evidence of toxicity. No significant acute toxicity was observed in patients. Grade II or III hematological toxicity (7.6% of patients), liver toxicity (18.4%) and also grade I renal toxicity (6.1%) was observed in 92 evaluable patients. Radiological responses were evaluated for an average of 29 months following their last cycle of therapy and results were analyzed by the RECIST criteria. Majority (85%) of patients had stable disease (SD), partial response (PR) rate was 7.5% and progressive disease (PD) was observed in 7.5% of patients. The average survival was 24.67 months after 2 cycles of therapy, 30.53 months after 3 cycles of therapy and 30.19 months after 4 cycles of therapy. Of the 42 patients who had pretreatment PET-CT imaging, 31 patients had positive F-18 FDG scans (SUV > 2.5) with an average survival time of 18.9 months (range 1.4-45.8 months) and 11 patients had negative F-18 FDG scans (SUV ≤ 2.5) with an average survival time of 31.8 months (range 7.4-42.9 months). Survival times for FDG negative patients were significantly longer than those for FDG positive patients (p = 0.001 with 95% confidence). Conclusion: High activity In-111 therapy is a safe and effective therapy for patients with progressive disseminated neuroendocrine tumors. No major hematological, renal and hepatic toxicities were observed. There was an increase in survival time particularly in patients with lower degree of liver involvement as well as patients who received three or more cycles of therapy, as compared to historical data. Pre-treatment FDG status may be a predictor of survival following In-111 pentetreotide therapy.

Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Single-chain (sc)-VEGF protein containing an N-terminal Cys-tag has been designed for site-specific modification with a variety of imaging and therapeutic moieties. Site-specific labeling of scVEGF with thiol-reactive prosthetic group, N-[2-(4-18F-fluorobenzamido) ethyl] maleimide ([18F]FBEM) for positron emission tomography (PET) imaging of VEFGR may provide a new tracer which has great potential for clinical translation. Methods: [18F]FBEM-scVEGF was synthesized by site-specific conjugation of 18F-FBEM to a thiol group in Cys-tag of scVEGF at room temperature. The functional activity after labeling was tested by immunofluorescence staining, cellular uptake and efflux. The tumor targeting and in vivo properties were evaluated by biodistribution and microPET studies in tumor-bearing mice. Results: The radiolabeling yield and specific activity of [18F]FBEM-scVEGF were 20.6 ± 15.1% (based on starting [18F]FBEM, uncorrected, n = 5) and 58.8 ± 12.4 GBq/µmol, respectively. Noninvasive microPET and direct tissue sampling experiments demonstrated that [18F]FBEM-scVEGF had VEGFR specific tumor uptake in MDA-MB-435, U87MG and 4T1 xenograft models. The optimal tumor uptake was achieved at 2 h p.i., which can be partially, but significantly blocked by co-injection of non-labeled scVEGF protein. Overall, [18F]FBEM-scVEGF showed VEGFR specific tumor uptake. Conclusion: The scVEGF was site-specifically labeled with 18F via [18F]FBEM prosthetic group and the tracer [18F]FBEM-scVEGF exhibited high receptor binding affinity and tumor targeting efficacy. Further study of [18F] FBEM-scVEGF to evaluate angiogenesis in cancer and other disease types is warranted.

Introduction: The GLP-1 receptor plays an important role in glucose homeostasis and thus is a very important target for diabetes therapy. The receptor is also overexpressed in insulinoma, a tumor of pancreatic beta-cells. We previously evaluated two fluorine-18-labeled analogs of exendin-4 prepared by conjugation with [18F]FBEM (N-[2-(4-[18F]fluorobenzamide)ethyl]maleimide). Both compounds demonstrated good tumor uptake, but the synthesis of the radiotracers was time consuming. To overcome this challenge, we developed a NOTA analog and performed radiolabeling using aluminum [18F]fluoride complexation. Methods: Cys40-exendin-4 was conjugated with NOTA mono N-ethylmaleimide. [18F]AlF conjugation was conducted and the radiolabeled product purified by preparative HPLC. Dynamic and static PET imaging scans were conducted on nude mice with established INS-1 xenografts. Uptake of tumor and other major organs in static images was quantitated (%ID/g) and comparison with blocking studies was made. PET quantification was also compared with ex vivo biodistribution results. Results: The radiosynthesis provided [18F]AlF-NOTA-MAL-cys40-exendin-4 in 23.6 ± 2.4 % radiochemical yield (uncorrected, n = 3) after HPLC; the process required about 55 min. The specific activity at time of injection ranged from 19.6 to 31.4 GBq (0.53-0.85 Ci)/µmol. Tumor uptake had reached its maximum (16.09 ± 1.18% ID/g, n = 4) by 5 min and remained nearly constant for the duration of the study. Kidney uptake continued to increase throughout the entire one hour time course. Pre-injection of exendin-4 caused a marked reduction in tissue uptake with the major exception of liver and kidneys, in which uptake was not affected. HPLC analysis of the radioactive components in extracts of the tumor and plasma showed primarily parent compound at 60 min post-injection, whereas extracts of kidney and urine contained exclusively one polar radioactive component. Conclusion: The radiotracer is prepared in a simple one-step procedure and obtained in high specific activity after HPLC purification. [18F]AlF-NOTA-MAL-exendin-4 shows high tumor uptake and highly selective GLP-1 tissue uptake (INS-1 tumor, lung, pancreas), but still suffers from high kidney uptake.

Purpose: Cystine knot (knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of 18F-labeled knottins in a U87MG glioblastoma model. Procedures: Engineered knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-18F-fluoropropionate (18F-NFP). The resulting probes, 18F-FP-2.5D and 18F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies. Results: MicroPET imaging studies with 18F-FP-2.5D and 18F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that 18F-FP-2.5D and 18F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent. Conclusions: 18F-FP-2.5D and 18F-FP-2.5F showed reduced gallbladder uptake compared with previously published 18F-FB-2.5D. 18F-FP-2.5D and 18F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. 18F-FP-2.5D demonstrated more desirable pharmacokinetics compared to 18F-FP-2.5F, and thus has greater potential for clinical translation.

Aim: 18F-DPA-714 is a PET tracer that recognizes macrophage translocator protein (TSPO), and 18F-Alfatide II (18F-AlF-NOTA-E[PEG4-c(RGDfk)]2) is specific for integrin αvβ3. This study aims to apply these two tracers for longitudinal PET imaging of muscular inflammation, and evaluate the value of 18F-DPA-714 in differentiating inflammation from tumor. Methods: RAW264.7 mouse macrophage cells were used for cell uptake analysis of 18F-DPA-714. A mouse hind limb muscular inflammation model was established by intramuscular injection of turpentine oil. For the inflammation model, PET imaging was performed at different days using 18F-DPA-714 and 18F-Alfatide II. The specificity of the imaging probes was tested by co- or pre-injection of PK11195 or unlabeled RGD (Arg-Gly-Asp) peptide. PET imaging using 18F-DPA-714 was performed in A549, HT29, U87MG, INS-1, and 4T1 xenograft models. Immunofluorescence staining was performed to evaluate infiltrated macrophages and angiogenesis in inflammation and/or tumors. Results: Uptake of 18F-DPA-714 in RAW264.7 cells was 45.5% at 1 h after incubation, and could be blocked by PK11195. PET imaging showed increased 18F-DPA-714 and 18F-Alfatide II uptake at inflammatory muscles. Peak uptake of 18F-DPA-714 was seen on day 6 (4.02 ± 0.64 %ID/g), and peak uptake of 18F-Alfatide II was shown on day 12 (1.87 ± 0.35 %ID/g) at 1 h p.i.. Tracer uptakes could be inhibited by PK11195 for 18F-DPA-714 or cold RGD for 18F-Alfatide II. Moreover, macrophage depletion with liposomal clodronate also reduced the local accumulation of both tracers. A549, HT29, U87MG, INS-1, and 4T1 tumor uptakes of 18F-DPA-714 (0.46 ± 0.28, 0.91 ± 0.08, 1.69 ± 0.67, 1.13 ± 0.33, 1.22 ± 0.55 %ID/g at 1 h p.i., respectively) were significantly lower than inflammation uptake (All P < 0.05). Conclusion: PET imaging using 18F-DPA-714 as a TSPO targeting tracer could evaluate the dynamics of macrophage activation and infiltration in different stages of inflammatory diseases. The concomitant longitudinal PET imaging with both 18F-DPA-714 and 18F-Alfatide II matched the causal relationship between macrophage infiltration and angiogenesis. Moreover, we found 18F-DPA-714 uptake in several types of tumors is significantly lower than that in inflammatory muscles, suggesting 18F-DPA-714 PET has the potential for better differentiation of tumor and non-tumor inflammation.

Nanotheranostics is to apply and further develop nanomedicine strategies for advanced theranostics. This review summarizes the various nanocarriers developed so far in the literature for nanotheranostics, which include polymer conjugations, dendrimers, micelles, liposomes, metal and inorganic nanoparticles, carbon nanotubes, and nanoparticles of biodegradable polymers for sustained, controlled and targeted co-delivery of diagnostic and therapeutic agents for better theranostic effects with fewer side effects. The theranostic nanomedicine can achieve systemic circulation, evade host defenses and deliver the drug and diagnostic agents at the targeted site to diagnose and treat the disease at cellular and molecular level. The therapeutic and diagnostic agents are formulated in nanomedicine as a single theranostic platform, which can then be further conjugated to biological ligand for targeting. Nanotheranostics can also promote stimuli-responsive release, synergetic and combinatory therapy, siRNA co-delivery, multimodality therapies, oral delivery, delivery across the blood-brain barrier as well as escape from intracellular autophagy. The fruition of nanotheranostics will be able to provide personalized therapy with bright prognosis, which makes even the fatal diseases curable or at least treatable at the earliest stage.

MicroRNAs (miRs) are small non-coding RNAs that negatively regulate gene expression by binding to the 3` untranslated regions (3`UTR) of their target mRNAs. MiRs were shown to play pivotal roles in tissue development and function and are also involved in the pathogenesis of various diseases including cancer. MicroRNA-206, which belongs to the group of so-called “myomiRs”, is one of the most studied miRs thus far. In addition to being involved in skeletal muscle development and pathology, it has also been established that it is involved in the pathogenesis of numerous diseases including heart failure, chronic obstructive pulmonary disease, Alzheimer's disease and various types of cancers. The aim of this review is to provide a complex overview of microRNA-206, including regulating its expression, a brief description of its known functions in skeletal muscle and a complex overview of its roles in the biology and pathology of other tissues, emphasizing its significant diagnostic and therapeutic potential.

Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials. Gene therapy that utilizes a single enzyme/prodrug combination targeting a single cellular mechanism needs significant overexpression of delivered therapeutic gene in order to achieve therapy response. Hence, to overcome this obstacle we recently developed a dual therapeutic reporter gene fusion that uses two different prodrugs, targeting two distinct cellular mechanisms in order to achieve effective therapy with a limited expression of delivered transgenes. In addition, imaging therapeutic reporter genes offers additional information that indirectly correlates gene delivery, expression, and functional effectiveness as a theranostic approach. In the present study, we evaluate the therapeutic potential of HSV1-sr39TK-NTR fusion dual suicide gene therapy system that we recently developed, in MDA-MB-231 triple negative breast cancer lung-metastatic lesions in a mouse model. We compared the therapeutic potential of HSV1-sr39TK-NTR fusion with respective dual prodrugs GCV-CB1954 with HSV1-sr39TK/GCV and NTR/CB1954 single enzyme prodrug system in this highly resistant metastatic lesion of the lungs. In vitro optimization of dose and duration of exposure to GCV and CB1954 was performed in MDA-MB-231 cells. Drug combinations of 1 μg/ml GCV and 10 μM CB1954 for 3 days was found to be optimal regimen for induction of significant cell death, as assessed by FACS analysis. In vivo therapeutic evaluation in animal models showed a complete ablation of lung metastatic nodules of MDA-MB-231 triple negative breast cancer cells following two consecutive doses of a combination of GCV (40 mg/kg) and CB1954 (40 mg/kg) administered at 5 day intervals. In contrast, the respective treatment condition in animals expressing HSV1-sr39TK or NTR separately, showed minimal or no effect on tumor reduction as measured by bioluminescence (tumor mass) and [(18)F]-FHBG microPET (TK expression) imaging. These highlight the strong therapeutic effect of the dual fusion prodrug therapy and its use in theranostic imaging of tumor monitoring in living animals by multimodality molecular imaging.

Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

Photodynamic therapy (PDT) is a light-based treatment modality in which wavelength specific activation of a photosensitizer (PS) generates cytotoxic response in the irradiated region. PDT response is critically dependent on several parameters including light dose, PS dose, uptake time, fluence rate, and the mode of light delivery. While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response. Here we present a theranostic technique, combining the inherent ability of the PS to serve simultaneously as a therapeutic and imaging agent, with the use of image-based treatment assessment in three dimensional (3D) in vitro tumor models, to comprise a platform to evaluate the impact of PDT parameters on treatment outcomes. We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells. Informed by photobleaching patterns and correlation with cytotoxic response, asymmetric fractionated light delivery at 4 hours BPD uptake was found to be the most effective regimen assessed. Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules. The framework introduced here provides a means for systematic assessment of PDT treatment parameters in biologically relevant 3D tumor models with potential for broader application to other systems.

Purpose: The purpose of this study was to evaluate the capability of u-SPECT-II/CT to monitor the progression of breast cancer lung metastasis using 99mTc-3P-RGD2 as a radiotracer. Methods: The breast cancer lung metastasis model was established by tail-vein injection of 2 x 105 - 1.5 x 106 MDA-MB-231 cells into each athymic nude mouse. SPECT/CT studies were performed at a specified time after inoculation of MDA-MB-231 cells. Histological staining was used to further confirm the presence of lung metastases. Results: We found that both inoculation time and tumor cell load had significant influence on the extent of lung metastasis. For example, if animals were injected with 2 x 105 MDA-MB-231 cells, there were no detectable metastatic breast tumors in the lungs after 8 weeks. If animals were injected with 1 x 106 MDA-MB-231 cells, there were many tumors in both lungs at week 8. When 1.5 x 106 MDA-MB-231 cells were injected, the animal became very weak by week 7. We also found a rare example of breast cancer metastasis in the muscle and mediastinal lymph nodes. The tumor necrotic regions were clearly delineated by u-SPECT-II/CT. Conclusion: This study clearly demonstrated that 99mTc-3P-RGD2 is an excellent radiotracer for noninvasive imaging of metastatic breast tumors in the lungs, mediastinal lymph nodes and muscles. 99mTc-3P-RGD2 SPECT/CT is an outstanding platform for monitoring the progression of breast cancer lung metastases, semi-quantification of breast tumor load in the lungs and delineation of tumor necrosis in small animals.

Purpose: 99mTc-3P-RGD2 is a 99mTc-labeled dimeric cyclic RGD peptide that binds to integrin αvβ3 with high affinity and specificity. The purpose of this study was to demonstrate the utility of 99mTc-3P-RGD2 SPECT/CT (single photon emission computed tomography/computed tomography) as a molecular imaging tool for noninvasive monitoring breast tumor early response to antiangiogenesis therapy with linifanib, and to illustrate its limitations in monitoring the efficacy of anti-αvβ3 treatment. Methods: To support SPECT/CT imaging, biodistribution and therapy studies, the xenografted breast cancer model was established by subcutaneous injection of 5 × 106 MDA-MB-435 cells into the fat pad of each athymic nude mouse. Linifanib (ABT-869) was used as antiangiogenesis agent. The tumor volume was 180 ± 90 mm3 on the day (-1 day) before baseline SPECT/CT. Each animal was treated twice daily with vehicle or 12.5 mg/kg linifanib. Longitudinal 99mTc-3P-RGD2 SPECT/CT imaging was performed on days -1, 1, 4 and 11. Tumors were harvested at each time point for pathological analysis of hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Tumor uptake of 99mTc-3P-RGD2 was calculated from SPECT/CT quantification. When cyclic peptide E[c(RGDfK)]2 (RGD2) was used as the anti-αvβ3 agent, SPECT/CT images were obtained only at 7 and 21 days after last RGD2 dose. Results: The tumor uptake of 99mTc-3P-RGD2 from SPECT/CT quantification was almost identical to that from biodistribution. There was a dramatic reduction in both %ID and %ID/cm3 tumor uptake of 99mTc-3P-RGD2 during the first 24 hours of linifanib therapy. The therapeutic effect of linifanib was on both tumor cells and vasculature, as determined by IHC analysis of integrin αvβ3 and CD31. Changes in tumor vasculature were further confirmed by pathological H&E analysis of tumor tissues. While its %ID tumor uptake increased steadily in vehicle-treated group, the %ID tumor uptake of 99mTc-3P-RGD2 decreased in linifanib-treated group slowly over the 11-day study period. The degree of tumor response to linifanib therapy correlated well to the integrin αvβ3 expression levels before linifanib therapy. Conclusion: 99mTc-3P-RGD2 is an excellent radiotracer for monitoring integrin αvβ3 expression during and after linifanib therapy. 99mTc-3P-RGD2 SPECT/CT is an useful molecular imaging tool for patient selection before antiangiogenic and anti-αvβ3 therapy; but it would be difficult to use 99mTc-3P-RGD2 for accurate and noninvasive monitoring of early tumor response to anti-αvβ3 therapy.

Purpose: Targeted radiotherapy (TRT) is an emerging approach for tumor treatment. Previously, 3PRGD2 (a dimeric RGD peptide with 3 PEG4 linkers) has been demonstrated to be of advantage for integrin αvβ3 targeting. Given the promising results of 99mTc-3PRGD2 for lung cancer detection in human beings, we are encouraged to investigate the radiotherapeutic efficacy of radiolabeled 3PRGD2. The goal of this study was to investigate and optimize the integrin αvβ3 mediated therapeutic effect of 177Lu-3PRGD2 in the animal model. Experimental Design: Biodistribution, gamma imaging and maximum tolerated dose (MTD) studies of 177Lu-3PRGD2 were performed. The targeted radiotherapy (TRT) with single dose and repeated doses as well as the combined therapy of TRT and the anti-angiogenic therapy (AAT) with Endostar were conducted in U87MG tumor model. The hematoxylin and eosin (H&E) staining and immunochemistry (IHC) were performed post-treatment to evaluate the therapeutic effect. Results: The U87MG tumor uptake of 177Lu-3PRGD2 was relatively high (6.03 ± 0.65 %ID/g, 4.62 ± 1.44 %ID/g, 3.55 ± 1.08 %ID/g, and 1.22 ± 0.18 %ID/g at 1 h, 4 h, 24 h, and 72 h postinjection, respectively), and the gamma imaging could visualize the tumors clearly. The MTD of 177Lu-3PRGD2 in nude mice (>111 MBq) was twice to that of 90Y-3PRGD2 (55.5 MBq). U87MG tumor growth was significantly delayed by 177Lu-3PRGD2 TRT. Significantly increased anti-tumor effects were observed in the two doses or combined treatment groups. Conclusion: The two-dose TRT and combined therapy with Endostar potently enhanced the tumor growth inhibition, but the former does not need to inject daily for weeks, avoiding a lot of unnecessary inconvenience and suffering for patients, which could potentially be rapidly translated into clinical practice in the future.

Tumor-targeted delivery of cytotoxins presents considerable advantages over their passive transport. Chemical conjugation of cytotoxic module to antibody is limited due to insufficient reproducibility of synthesis, and recombinant immunotoxins are aimed to overcome this disadvantage. We obtained genetically encoded immunophotosensitizer 4D5scFv-miniSOG and evaluated its photocytotoxic effect in vitro. A single-chain variable fragment (scFv) of humanized 4D5 antibody was used as a targeting vehicle for selective recognition of the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) overexpressed in many human carcinomas. As a phototoxic module we used a recently described photoactivated fluorescent flavoprotein miniSOG. We found that recombinant protein 4D5scFv-miniSOG exerts a highly specific photo-induced cytotoxic effect on HER2/neu-positive human breast adenocarcinoma SK-BR-3 cells (IC50= 160 nM). We demonstrated that the 4D5scFv-miniSOG specifically binds to HER2-positive cells and internalizes via receptor-mediated endocytosis. Co-treatment of breast cancer cells with 4D5scFv-miniSOG and Taxol or junction opener protein JO-1 produced remarkable additive effects.

Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable in vivo visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. (64)Cu-T140-2D was evaluated in vitro by migration and binding experiments, and in vivo by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give (64)Cu-T140-2D in a high radiochemical yield of 86 ± 3% (not decay-corrected) and a specific activity of 0.28 - 0.30 mCi/µg (10.36 - 11.1 MBq/µg). (64)Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. In vivo, (64)Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form (64)Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, (64)Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, (64)Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.

Purpose We and others have reported that Sarcophagine-based bifunctional chelators could be effectively used in the syntheses of 64Cu radiopharmaceuticals. The resulted 64Cu-Sarcophagine complexes demonstrated great in vivo stability. The goal of this study was to further derivatize Sarcophagine cage with amino and maleimide functional groups for conjugation with bioligands. Methods Starting from DiAmSar, three novel chelators (AnAnSar, BaMalSar, and Mal2Sar) with two functional groups have been synthesized. Among those, BaMalSar and Mal2Sar have been conjugated with cyclic peptide c(RGDyC) (denoted as RGD) and the resulted conjugates, BaMalSar-RGD and Mal2Sar-RGD2 have been labeled with 64Cu. The tumor targeting efficacy of 64Cu-labeled RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model. Results The conjugates, BaMalSar-RGD and Mal2Sar-RGD2 could be labeled with 64CuCl2 in 10 min with high purity (>98%) and high radiochemical yield (>90%). Both 64Cu-BaMalSar-RGD and 64Cu-Mal2Sar-RGD2 exhibited high tumor uptake and tumor-to-normal tissue ratios. Conclusion Three novel chelators with two functional groups have been developed based on Sarcophagine cage. The platform developed in this study could have broad applications in the design and synthesis of 64Cu-radiopharmaceuticals.

The acronym THERANOSTICS epitomizes the inseparability of diagnosis and therapy, the pillars of medicine and takes into account personalized management of disease for a specific patient. Molecular phenotypes of neoplasms can be determined by molecular imaging with specific probes using positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), or optical methods, so that the treatment is specifically targeted against the tumor and its environment. To meet these demands, we need to define the targets, ligands, coupling and labeling chemistry, the most appropriate radionuclides, biodistribution modifiers, and finally select the right patients for the personalized treatment. THERANOSTICS of neuroendocrine tumors (NETs) using Ga-68 labeled tracers for diagnostics with positron emission tomography/ computed tomography (PET/CT), and using Lu-177 or other metallic radionuclides for radionuclide therapy by applying the same peptide proves that personalized radionuclide therapy today is already a fact and not a fiction.

Human enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) and is associated with several severe neurological complications in the Asia-Pacific region. Here, we evaluated that while passive transfer of neutralizing monoclonal antibody (nMAb) against the VP2 protein protect against lethal EV71 infection in BALB/c mice. Protective nMAb were mapped to residues 141-155 of VP2 by peptide ELISA. High-resolution structural analysis showed that the epitope is part of the VP2 EF loop, which is the "puff" region that forms the "southern rim" of the canyon. Moreover, a three-dimensional structural characterization for the puff region with prior neutralizing epitopes and receptor-binding sites that can serve to inform vaccine strategies. Interestingly, using hepatitis B virus core protein (HBc) as a carrier, we demonstrated that the cross-neutralizing EV71 antibodies were induced, and the VP2 epitope immunized mice serum also conferred 100% in vivo passive protection. The mechanism of in vivo protection conferred by VP2 nMAb is in part attributed to the in vitro neutralizing titer and ability to bind authentic viral particles. Importantly, the anti-VP2(aa141-155) antibodies could inhibit the binding of human serum to EV71 virions showed that the VP2 epitope is immunodominant. Collectively, our results suggest that a broad-spectrum vaccine strategy targeting the high-affinity epitope of VP2 EF loop may elicits effective immune responses against EV71 infection.

Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal α-amines or lysine ε-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[(76)Br]bromo-2,6-dimethoxybenzoate ([(76)Br]SBDMB) was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB) moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [(76)Br]-BrE[c(RGDyK)](2) and [(76)Br]-BrDMB-E[c(RGDyK)](2). An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK) and the brominating agent CH(3)COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK) and E[c(RGDyK)](2) using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [(76)Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK) and c(RGDyK) were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [(76)Br]Br-E[c(RGDyK)](2) and [(76)Br]BrDMB-E[c(RGDyK)](2) showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers was confirmed by decreased tumor uptake after co-administration of unlabeled dimeric RGD peptides. The energy barrier of the transition state of bromination for the dimethoxybenzoyl group was about 9 kcal/mol lower than that for the tyrosine residue. In conclusion, the newly developed N-succinimidyl-2, 6-dimethoxybenzoate molecule can be used either for one step labeling through pre-conjugation or as the precursor for a Br-76 labeled prosthetic group for indirect labeling. Radiobromination on a dimethoxybenzoyl group has selectivity over radiobromination on tyrosine. The energy barrier difference of the transition states of bromination between the dimethoxybenzoyl group and the tyrosine residue may account for the reaction selectivity when both groups are present in the same molecule.

Based on the soil-to-seeds principle, we explored the small-molecular sequential dual-targeting theranostic strategy (SMSDTTS) for prolonged survival and imaging detectability in a xenograft tumor model. Thirty severe combined immunodeficiency (SCID) mice bearing bilateral radiation-induced fibrosarcoma-1 (RIF-1) subcutaneously were divided into group A of SMSDTTS with sequential intravenous injections of combretastatin A4 phosphate (CA4P) and (131)I-iodohypericin ((131)I-Hyp) at a 24 h interval; group B of single targeting control with CA4P and vehicle of (131)I-Hyp; and group C of vehicle control (10 mice per group). Tumoricidal events were monitored by in vivo magnetic resonance imaging (MRI) and planar gamma scintiscan, and validated by ex vivo autoradiography and histopathology. Besides, 9 mice received sequential intravenous injections of CA4P and (131)I-Hyp were subjected to biodistribution analysis at 24, 72 and 120 h. Gamma counting revealed fast clearance of (131)I-Hyp from normal organs but intense accumulation in necrotic tumor over 120 h. After only one treatment, significantly prolonged survival (p<0.001) was found in group A compared to group B and C with median survival of 33, 22, and 21 days respectively. Tumor volume on day 15 was 2.0 ± 0.89, 5.66 ± 1.66, and 5.02 ± 1.0 cm(3) with tumor doubling time 7.8 ± 2.8, 4.4 ± 0.67, and 4.5 ± 0.5 days respectively. SMSDTTS treated tumors were visualized as hot spots on gamma scintiscans, and necrosis over tumor ratio remained consistently high on MRI, autoradiography and histology. The synergistic antitumor effects, multifocal targetability, simultaneous theranostic property, and good tolerance of the SMSDTTS were evident in this experiment, which warrants further development for preclinical and clinical applications.

Aneurysm diagnostic is nowadays limited by the lack of technology that enables early detection and rupture risk prediction. New non invasive tools for molecular imaging are still required. In the present study, we present an innovative SPECT diagnostic tool for abdominal aortic aneurysm (AAA) produced from injectable polysaccharide microparticles radiolabeled with technetium 99m (99mTc) and functionalized with fucoidan, a sulfated polysaccharide with the ability to target P-Selectin. P-Selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets which can be found in the thrombus of aneurysms, as well as in other vascular pathologies. Microparticles with a maximum hydrodynamic diameter of 4 µm were obtained by crosslinking the polysaccharides dextran and pullulan. They were functionalized with fucoidan. In vitro interactions with human activated platelets were assessed by flow cytometry that demonstrated a specific affinity of fucoidan functionalized microparticles for P-Selectin expressed by activated platelets. For in vivo AAA imaging, microparticles were radiolabeled with 99mTc and intravenously injected into healthy and AAA rats obtained by elastase perfusion through the aorta wall. Animals were scanned by SPECT imaging. A strong contrast enhancement located in the abdominal aorta of AAA rats was obtained, while no signal was obtained in healthy rats or in AAA rats after injection of non-functionalized control microparticles. Histological studies revealed that functionalized radiolabeled polysaccharide microparticles were localized in the AAA wall, in the same location where P-Selectin was expressed. These microparticles therefore constitute a promising SPECT imaging tool for AAA and potentially for other vascular diseases characterized by P-Selectin expression. Future work will focus on validating the efficiency of the microparticles to diagnose these other pathologies and the different stages of AAA. Incorporation of a therapeutic molecule is also considered.

Pancreas-related complications are some of the most serious ones in abdominal surgery. The goal of this study was to develop and validate novel near-infrared (NIR) fluorophores that would enable real-time pancreas imaging to avoid the intraoperative pancreatic injury. After initial screening of a large NIR fluorophore library, the performance of 3 selected pancreas-targeted 700 nm NIR fluorophores, T700-H, T700-F, and MB, were quantified in mice, rats, and pigs. Dose ranging using 25 and 100 nmol, and 2.5 µmol of T700-F, and its imaging kinetics over a 4 h period were tested in each species. Three different 800 nm NIR fluorophores were employed for dual-channel FLARE™ imaging in pigs: 2 μmol of ZW800-1 for vessels and kidney, 1 μmol of ZW800-3C for lymph nodes, and 2 μmol of ESNF31 for adrenal glands. T700-F demonstrated the highest signal to background ratio (SBR), with peak SBR at 4 h postinjection in mice. In pigs, T700-F produced an SBR ≥ 2 against muscle, spleen, and lymph nodes for up to 8 h after a single intravenous injection. The combination of T700-F with each 800 nm NIR fluorophore provided simultaneous dual-channel intraoperative imaging of pancreas with surrounding organs in real time. Pancreas-targeted NIR fluorophores combined with the FLARE dual-channel imaging system enable the real-time intraoperative pancreas imaging which helps surgeons perform safer and more curative abdominal surgeries.

Imaging guided ablation therapy has been applied in both biomedical research and clinical trials and turned out to be one of the most promising approaches for cancer treatment. Herein, the multifunctional nanocapsules were fabricated through loading perfluorooctylbromide (PFOB) and superparamagnetic iron oxide nanoparticles (SPIOs) into poly(lactic acid) (PLA) nanocapsules (NCs), followed by the formation of PEGylated gold nanoshell on the surface. The resulting multi-component NCs were proved to be able to act as nanotheranostic agent to achieve successful bimodal ultrasound (US)/magnetic resonance imaging (MRI) guided photothermal ablation in human tumor xenograft models non-invasively. Such a single theranostic agent with the combination of real-time US and high-resolution MR imaging would be of great value to offer more comprehensive diagnostic information and dynamics of disease progression for the accurate location of therapeutic focusing spot in the targeted tumor tissue, showing great potential as an effective nanoplatform for contrast imaging guided photothermal therapy.

This paper is an initial work towards developing an image-guided, targeted ultrasound ablation technique by combining histotripsy with nanodroplets that can be selectively delivered to tumor cells. Using extremely short, high-pressure pulses, histotripsy generates a dense cloud of cavitating microbubbles that fractionates tissue. We hypothesize that synthetic nanodroplets that encapsulate a perfluoropentane (PFP) core will transition upon exposure to ultrasound pulses into gas microbubbles, which will rapidly expand and collapse resulting in disruption of cells similar to the histotripsy process but at a significantly lower acoustic pressure. The significantly reduced cavitation threshold will allow histotripsy to be selectively delivered to the tumor tissue and greatly enhance the treatment efficiency while sparing neighboring healthy tissue. To test our hypothesis, we prepared nanodroplets with an average diameter of 204±4.7 nm at 37°C by self-assembly of an amphiphilic triblock copolymer around a PFP core followed by cross-linkage of the polymer shell forming stable nanodroplets. The nanodroplets were embedded in agarose tissue phantoms containing a sheet of red blood cells (RBCs), which were exposed to 2-cycle pulses applied by a 500 kHz focused transducer. Using a high speed camera to monitor microbubble generation, the peak negative pressure threshold needed to generate bubbles >50 μm in agarose phantoms containing nanodroplets was measured to be 10.8 MPa, which is significantly lower than the 28.8 MPa observed using ultrasound pulses alone. High speed images also showed cavitation microbubbles produced from the nanodroplets displayed expansion and collapse similar to histotripsy alone at higher pressures. Nanodroplet-mediated histotripsy created consistent, well-defined fractionation of the RBCs in agarose tissue phantoms at 10 Hz pulse repetition frequency similar to the lesions generated by histotripsy alone but at a significantly lower pressure. These results support our hypothesis and demonstrate the potential of using nanodroplet-mediated histotripsy for targeted cell ablation.

Phthalocyanine-aggregated Pluronic nanoparticles were constructed as a novel type of near-infrared (NIR) absorber for photothermal therapy. Tiny nanoparticles (~ 60 nm, FPc NPs) were prepared by aqueous dispersion of phthalocyanine-aggregated self-assembled nanodomains that were phase-separated from the melt mixture with Pluronic. Under NIR laser irradiation, FPc NPs manifested robust heat generation capability, superior to an individual cyanine dye and cyanine-aggregated nanoparticles. Micro- and macroscopic imaging experiments showed that FPc NPs are capable of internalization into live cancer cells as well as tumor accumulation when intravenously administered into living mice. It is shown here that continuous NIR irradiation of the tumor-targeted FPc NPs can cause phototherapeutic effects in vitro and in vivo through excessive local heating, demonstrating potential of phthalocyanine-aggregated nanoparticles as an all-organic NIR nanoabsorber for hyperthermia.

For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis.

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.

Subtype-targeted therapies can have a dramatic impact on improving the quality and quantity of life for women suffering from breast cancer. Despite an initial therapeutic response, cancer recurrence and acquired drug-resistance are commonplace. Non-invasive imaging probes that identify drug-resistant lesions are urgently needed to aid in the development of novel drugs and the effective utilization of established therapies for breast cancer. The protease receptor urokinase plasminogen activator receptor (uPAR) is a target that can be exploited for non-invasive imaging. The expression of uPAR has been associated with phenotypically aggressive breast cancer and acquired drug-resistance. Acquired drug-resistance was modeled in cell lines from two different breast cancer subtypes, the uPAR negative luminal A subtype and the uPAR positive triple negative subtype cell line MDA-MB-231. MCF-7 cells, cultured to be resistant to tamoxifen (MCF-7 TamR), were found to significantly over-express uPAR compared to the parental cell line. uPAR expression was maintained when resistance was modeled in triple-negative breast cancer by generating doxorubicin and paclitaxel resistant MDA-MB-231 cells (MDA-MB-231 DoxR and MDA-MB-231 TaxR). Using the antagonistic uPAR antibody 2G10, uPAR was imaged in vivo by near-infrared (NIR) optical imaging and (111)In-single photon emission computed tomography (SPECT). Tumor uptake of the (111)In-SPECT probe was high in the three drug-resistant xenografts (> 46 %ID/g) and minimal in uPAR negative xenografts at 72 hours post-injection. This preclinical study demonstrates that uPAR can be targeted for imaging breast cancer models of acquired resistance leading to potential clinical applications.

We report on the development of photosensitizer-conjugated gold nanorods (MMP2P-GNR) in which photosensitizers were conjugated onto the surface of gold nanorods (GNR) via a protease-cleavable peptide linker. We hypothesized that fluorescence and phototoxicity of the conjugated photosensitizers would be suppressed in their native state, becoming activated only after cleavage by the target protease matrix metalloprotease-2 (MMP2). Quantitative analysis of the fluorescence and singlet oxygen generation (SOG) demonstrated that the MMP2P-GNR conjugate emitted fluorescence intensity corresponding to 0.4% ± 0.01% and an SOG efficiency of 0.89% ± 1.04% compared to free pyropheophorbide-a. From the in vitro cell studies using HT1080 cells that overexpress MMP2 and BT20 cells that lack MMP2, we observed that fluorescence and SOG was mediated by the presence or absence of MMP2 in these cell lines. This novel activatable photosensitizing system may be useful for protease-mediated fluorescence imaging and subsequent photodynamic therapy for various cancers.

Molecular imaging is a newly emerged multiple disciplinary field that aims to visualize, characterize and quantitatively measure biological processes at cellular and molecular levels in humans and other living systems. A reporter gene is a piece of DNA encoding reporter protein, which presents as a readily measurable phenotype that can be distinguished easily from the background of endogenous protein. After being transferred into cells of organ systems (transgenes), the reporter gene can be utilized to visualize transcriptional and posttranscriptional regulation of gene expression, protein-protein interactions, or trafficking of proteins or cells in living subjects. Herein, we review previous classification of reporter genes and regroup the reporter gene based imaging as basic, inducible and activatable, based on the regulation of reporter gene transcription and post-translational modification of reporter proteins. We then focus on activatable reporters, in which the signal can be activated at the posttranslational level for visualizing protein-protein interactions, protein phosphorylation or tertiary structure changes. The applications of several types of activatable reporters will also be summarized. We conclude that activatable reporter imaging can benefit both basic biomedical research and drug development.

The optical properties of macrophage-targeted theranostic nanoparticles (MacTNP) prepared from a Chlorin e6 (Ce6)-hyaluronic acid (HA) conjugate can be activated by reactive oxygen species (ROS) in macrophage cells. MacTNP are nonfluorescent and nonphototoxic in their native state. However, when treated with ROS, especially peroxynitrite, they become highly fluorescent and phototoxic. In vitro cell studies show that MacTNP emit near-infrared (NIR) fluorescence inside activated macrophages. The NIR fluorescence is quenched in the extracellular environment. MacTNP are nontoxic in macrophages up to a Ce6 concentration of 10 μM in the absence of light. However, MacTNP become phototoxic upon illumination in a light dose-dependent manner. In particular, significantly higher phototoxic effect is observed in the activated macrophage cells compared to human dermal fibroblasts and non-activated macrophages. The ROS-responsive MacTNP, with their high target-to-background ratio, may have a significant potential in selective NIR fluorescence imaging and in subsequent photodynamic therapy of atherosclerosis with minimum side effects.

Targeting particles to sites of inflammation is of considerable interest for applications relating to molecular imaging and drug delivery. We and others have described micron-sized particles of iron oxide (MPIO) that can be directed using specific ligands (e.g. antibodies, peptides and oligosaccharides) to bind to mediators of vascular inflammation in vivo. Since leukocyte binding to these molecules can induce changes in the target cell, an outstanding question has been whether the binding of imaging particles to these mediators induces biologically significant changes in the endothelial cells, potentially initiating or propagating inflammation. Here, we address these questions by looking for changes in endothelial cells following binding of contrast agent. Specifically, we have quantified calcium flux, rearrangement of the actin cytoskeleton, production of reactive oxygen species (ROS), apoptosis and potential secondary changes, such as changes in gene and protein expression follow binding events to primary endothelial cells in vitro. Although leukocytes induced changes to endothelial cell function, we did not see any significant changes to endothelial calcium flux, cytoskeletal organisation, production of ROS or induction of apoptosis in response to antibody-MPIO binding. Furthermore, there were no changes to gene expression monitored via real-time RT-PCR or presentation of protein on the cell surface measured using flow cytometry. Our experiments demonstrate that whilst antibody-targeted microparticles mimic the binding capability of leukocytes to inflamed endothelium, they do not trigger the same cellular responses and do not appear to initiate or compound inflammation. These properties are desirable for targeted therapeutic and diagnostic agents.

In this extensive review, we elucidate the importance of proteases and their role in drug development in various diseases with an emphasis on cancer. First, key proteases are introduced along with their function in disease progression. Next, we link these proteases as targets for the development of prodrugs and provide clinical examples of protease-activatable prodrugs. Finally, we provide significant design considerations needed for the development of the next generation protease-targeted and protease-activatable prodrugs.

Successful integration of diagnostic and therapeutic actions at the level of individual cells requires new materials that combine biological compatibility with functional versatility. This review focuses on the development of liposome-based functional materials, where payload release is activated by light. Methods of sensitizing liposomes to light have progressed from the use of organic molecular moieties to the use of metallic plasmon resonant structures. This development has facilitated application of near infrared light for activation, which is preferred for its deep penetration and low phototoxicity in biological tissues. Presented mechanisms of light-activated liposomal content release enable precise in vitro manipulation of minute amounts of reagents, but their use in clinical diagnostic and therapeutic applications will require demonstration of safety and efficacy.

Despite significant advances in diagnosis and treatment, the prognosis of esophageal adenocarcinoma remains poor highlighting the importance of early detection. Although white light (WL) upper endoscopy can be used for screening of the esophagus, it has limited sensitivity for early stage disease. Thus, development of new imaging technology to improve the diagnostic capabilities of upper GI endoscopy for early detection of esophageal adenocarcinoma is an important unmet need. The goal of this study was to develop a method for the detection of malignant lesions in the esophagus using WL upper endoscopy combined with near infrared (NIR) imaging with a protease activatable probe (Prosense750) selective for cathepsin B (CTSB). An orthotopic murine model for distal esophageal adenocarcinoma was generated through the implantation of OE-33 and OE-19 human esophageal adenocarcinoma lines in immunocompromised mice. The mice were imaged simultaneously for WL and NIR signal using a custom-built dual channel upper GI endoscope. The presence of tumor was confirmed by histology and target to background ratios (TBR) were compared for both WL and NIR imaging. NIR imaging with ProSense750 significantly improved upon the TBRs of esophageal tumor foci, with a TBR of 3.64±0.14 and 4.50±0.11 for the OE-33 and OE-19 tumors respectively, compared to 0.88±0.04 and 0.81±0.02 TBR for WL imaging. The combination of protease probes with novel imaging devices has the potential to improve esophageal tumor detection by fluorescently highlighting neoplastic regions.

The nuclear factor-κB (NF-κB) signaling pathway plays a critical role in a multitude of cellular processes. Activation of the NF-κB transcription factor family is essential for the initiation of inflammation, immunity, cell proliferation and apoptosis through a list of responsive genes. In hepatic tissue, activation of the NF-κB pathway has been implicated in a number of pathological conditions. Here we described a mouse model for noninvasive quantification of NF-κB activation in the hepatic tissues. Mice were subjected to hydrodynamic delivery with a mixture of pattB-NF-κB-Fluc reporter and φC31o integrase vector. Hepatic expression of φC31o integrase mediated chromosomal integration of the pattB-NF-κB-Fluc reporter, resulting in stable luciferase expression at 300 days post transfection. We applied noninvasive imaging and were able to detect NF-κB activation under acute liver injury and hepatitis conditions. During hepatectomy-induced liver regeneration, NF-κB activation was detected locally in the tissues at the surgery site. Treatment with Sorafenib suppressed NF-κB activation, accompanied with perturbation of liver regeneration. In conclusion, we established a method for stable transfection of the hepatic tissues and applied the transfected mice to longitudinal monitoring of NF-κB activity under pathological conditions. Further exploration of this methodology for establishment of other disease models and for evaluation of novel pharmaceuticals is likely to be fruitful.

Urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein. Besides regulating proteolysis, uPAR could also activate many intracellular signaling pathways that promote cell motility, invasion, proliferation, and survival through cooperating with transmembrane receptors. uPAR is overexpressed across a variety of tumors and is associated with cancer invasion and metastasis. In order to meet the demand for a rapid development and potential clinical application of anti-cancer therapy based on uPA/uPAR system, it is desirable to develop non-invasive imaging methods to visualize and quantify uPAR expression in vivo. In this review, we will discuss recent advances in the development of uPAR-targeted nuclear imaging and radionuclide therapy agents. The successful development of molecular imaging probes to visualize uPAR expression in vivo would not only assist preclinical researches on uPAR function, but also eventually impact patient management.

The Chemokine receptor CXCR4 and its ligand stromal derived factor-1 (SDF-1/CXCL12) are important players involved in cross-talk between leukemia cells and the bone marrow (BM) microenvironment. CXCR4 expression is associated with poor prognosis in AML patients with and without the mutated FLT3 gene. CXCL12 which is constrictively secreted from the BM stroma and AML cells is critical for the survival and retention of AML cells within the BM. In vitro, CXCR4 antagonists were shown to inhibit the migration of AML cells in response to CXCL12. In addition, such antagonists were shown to inhibit the survival and colony forming potential of AML cells and abrogate the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. In vivo, using immune deficient mouse models, CXCR4 antagonists were found to induce the mobilization of AML cells and progenitor cells into the circulation and enhance anti leukemic effects of chemotherapy. The hypothesis that CXCL12/CXCR4 interactions contribute to the resistance of AML cells to signal transduction inhibitor- and chemotherapy-induced apoptosis is currently being tested in a series of Phase I/II studies in humans.

Purpose: A fluorogenic peptide probe-immobilized diagnostic kit was used to analyze MMP activity in the synovial fluids (SFs) from patients with osteoarthritis (OA) and acute inflammatory conditions of the knee joint. Methods: The MMP diagnostic kit containing a polymer-conjugated MMP probe immobilized on a 96-well plate was utilized for high-throughput screening of MMP activity in SFs from OA patients (n = 33) and patients with acute inflammatory conditions of the knee joint (n = 5). Results: Compared to SF from OA patients, SF from patients with acute inflammatory conditions of the knee joint presented stronger NIR fluorescent signals. In gelatin zymography, most samples from patients with acute inflammatory conditions of the knee joint also displayed 92 kDa (pro-form) MMP-9 and faint 84 kDa (active form) MMP-9, while SF from OA patients did not display detectable MMP-9 activity . Conclusion: The presence of a strong fluorescence signal from the MMP diagnostic kit corresponded well with patients with acute inflammatory conditions of the knee joint. The results suggest that our MMP diagnostic kit can be useful in differentiation between early stages of OA and acute inflammatory conditions of the knee joint.

Cardiomyocyte loss via apoptosis plays a crucial role in ventricular remodeling following myocardial infarction (MI). Cell-based therapy approaches using bone marrow derived c-kit+ pluripotent cells may attenuate apoptosis following ischemic injury. We therefore thought to examine the early course of apoptosis following myocardial infarction - in-vivo - and non-invasively determine the effect of c-kit+ bone marrow cells on post-MI remodeling. We studied apoptosis in wild-type Kit+/+, c-kit mutant KitW/KitW-v and KitW/KitW-v mice after cell therapy with bone-marrow derived c-kit+ cells after ischemia-reperfusion injury. Mice were followed by hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) at 6h, 24h and 7 days after ischemia-reperfusion injury using an Annexin V-based fluorescent nanosensor targeting phosphatidylserine. KitW/KitW-v mice showed increased and prolonged apoptosis compared to control Kit+/+ mice while c-kit cell therapy was able to attenuate the altered apoptosis rates. Increased apoptosis was accompanied by severe decline in heart function, determined by cardiac Magnetic Resonance Imaging, and cell therapy was able to rescue the animals from deleterious heart failure. Post-mortem cryoslicing and immunohistochemistry localized the fluorescence signal of the Annexin V sensor within the infarcted myocardium. Flow cytometry of digested infarct specimens identified apoptotic cardiomyocytes as the major source for the in-vivo Annexin V signal. In-vivo molecular imaging using hybrid FMT-XCT reveals increased cardiomyocyte apoptosis in KitW/KitW-v mice and shows that c-kit+ cardioprotective cells are able to attenuate post-MI apoptosis and rescue mice from progressive heart failure.

Theranostic platform integrating diagnostic imaging and therapeutic function into a single system has become a new direction of nanoparticle research. In the process of treatment, therapeutic efficacy is monitored. The use of theranostic nanoparticle can add an additional "layer" to keep track on the therapeutic agent such as the pharmacokinetics and biodistribution. In this report, we have developed quantum rod (QR) based formulations for the delivery of small interfering RNAs (siRNAs) to human neuronal cells. PEGlyated QRs with different surface functional groups (amine and maleimide) were designed for selectively down-regulating the dopaminergic signaling pathway which is associated with the drug abuse behavior. We have demonstrated that the DARPP-32 siRNAs were successfully delivered to dopaminergic neuronal (DAN) cells which led to drastic knockdown of specific gene expression by both the electrostatic and covalent bond conjugation regimes. The PEGlyated surface offered high biocompatibilities and negligible cytotoxicities to the QR formulations that may facilitate the in vivo applications of these nanoparticles.

Competitive inhibition diminishes ligand adhesion as receptor sites become occupied with competing ligands. It is unknown if this effect occurs in ultrasound molecular imaging studies where endothelial binding sites become occupied with adherent bubbles or bubble fragments. The goal of this pilot study was to assess the effect that repeated administration and clearance of targeted agents has on successive adhesion. Two groups of animals were imaged with 3-D ultrasonic molecular imaging. Injections and imaging were performed on Group 1 at time 0 and 60 minutes. Group 2 received injections of microbubbles at 0, 15, 30, 45 and 60 minutes with imaging at 0 and 60 minutes. At 60 minutes, Group 1 targeting relative to baseline was not significantly different from Group 2 (1.06±0.27 vs. 1.08±0.34, p=0.93). Data suggest that multiple injections of targeted microbubbles do not block sufficient binding sites to bias molecular imaging data in serial studies.

Because of the more widespread and frequent use of cross-sectional techniques, mainly computed tomography (CT), an increasing number of adrenal tumors are detected as incidental findings ("incidentalomas"). These incidentaloma patients are much more frequent than those undergoing imaging because of symptoms related to adrenal disease. CT and magnetic resonance imaging (MRI) are in most patients sufficient for characterization and follow-up of the incidentaloma. In a minor portion of patients, biochemical screening reveals a functional tumor and further diagnostic work-up and therapy need to be performed according to the type of hormonal overproduction. In oncological patients, especially when the morphological imaging criteria indicate an adrenal metastasis, biopsy of the lesion should be considered after pheochromocytoma is ruled out biochemically. In the minority of patients in whom CT and MRI fail to characterize the tumor and when time is of essence, functional imaging mainly by positron emission tomography (PET) is available using various tracers. The most used PET tracer, [(18)F]fluoro-deoxy-glucose ((18)FDG), is able to differentiate benign from malignant adrenal tumors in many patients. (11)C-metomidate ((11)C-MTO) is a more specialized PET tracer that binds to the 11-beta-hydroxylase enzyme in the adrenal cortex and thus makes it possible to differ adrenal tumors (benign adrenocortical adenoma and adrenocortical cancer) from those of non-adrenocortical origin.

Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance in vivo gene delivery utilizing microbubble contrast agents, namely their ability to 1) improving cell membrane permeability, 2) modulate vascular permeability, and 3) enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve in vivo microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.

A method for highly sensitive and rapid detection of Pseudomonas aeruginosa, based on magnetic enrichment and magnetic separation, is described in this paper. The magnetic nanoparticles (MNPs) were applied to adsorb genome DNA after the sample was lysed. The DNA binding MNPs were directly subjected to polymerase chain reaction (PCR) to amplify gyrB specific sequence of Pseudomonas aeruginosa. The biotin labeled PCR products were detected by chemiluminescence when they were successively incubated with the probes-modified MNPs and alkaline phosphatase (ALP) labeled streptavidin (SA). Agarose gel electrophoresis analyses approved the method of in situ PCR to be highly reliable. The factors which could affect the chemiluminiscence were studied in detail. The results showed that the MNPs of 400 nm in diameter are beneficial to the detection. The sequence length and the binding site of the probe with a target sequence have obvious effects on the detection. The optimal concentration of the probes, hybridization temperature and hybridization time were 10 μM, 60 ºC and 60 mins, respectively. The method of in situ PCR based on MNPs can greatly improve the utilization rate of the DNA template ultimately enhancing the detection sensitivity. Experiment results proved that the primer and probe had high specificity, and Pseudomonas aeruginosa was successfully detected with detection limits as low as 10 cfu/mL by this method, while the detection of a single Pseudomonas aeruginosa can also be achieved.

Successful development of novel electrochemical biosensing interface for ultrasensitive detection of volatile biomarkers of gastric cancer cells is a challenging task. Herein we reported to screen out novel volatile biomarkers associated with gastric cancer cells and develop a novel Au-Ag alloy composites-coated MWCNTs as sensing interface for ultrasensitive detection of volatile biomarkers. MGC-803 gastric cancer cells and GES-1 gastric mucous cells were cultured in serum-free media. The sample preparation approaches and HS-SPME conditions were optimized for screening volatile biomarkers. Volatiles emitted from the headspace of the cells/medium culture were identified using GC-MS. The Au-Ag nanoparticles-coated multiwalled carbon nanotubes were prepared as a sensing interface for detection of volatile biomarkers. Results showed that eight different volatile metabolites were screened out between MGC-803 cells and GES-1 cells. Two compounds such as 3-octanone and butanone were specifically present in the headspace of the MGC-803 cells. Three volatiles such as 4-isopropoxybutanol, nonanol and 4-butoxy 1-butanol coexisted in the headspace of both the MGC-803 cells and the GES-1 cells, their concentrations in the headspace of the GES-1cells were markedly higher than those in the MGC-803 cells, three volatiles such as formic acid propyl ester, 1.4-butanediol and 2, 6, 11-trimethyl dodecane solely existed in the headspace of the GES-1 cells. The nanocomposites of MWNTs loaded with Au-Ag nanoparticles were prepared as a electrochemical sensing interface for detection of two volatile biomarkers, cyclic voltammetry studies showed that the fabricated sensor could detect 3-octanone in the range of 0~0.0025% (v/v) and with a detection limitation of 0.3 ppb, could detect butanone in the range of 0 ~ 0.055% (v/v), and with a detection limitation of 0.5 ppb, and exhibited good selectivity. The novel electrochemical biosensor combined with volatile biomarkers of gastric cancer owns great potential in applications such as early diagnosis and the prognosis of gastric cancer in near future.

The recent advancement of nanotechnology has provided unprecedented opportunities for the development of nanoparticle enabled technologies for detecting and treating cancer. Here, we reported the construction of a PET trackable organic nanoplatform based on phage particle for targeted tumor imaging. Method: The integrin α(v)β(3) targeted phage nanoparticle was constructed by expressing RGD peptides on its surface. The target binding affinity of this engineered phage particle was evaluated in vitro. A bifunctional chelator (BFC) 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) or 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) was then conjugated to the phage surface for (64)Cu(2+) chelation. After (64)Cu radiolabeling, microPET imaging was performed in U87MG tumor model and the receptor specificity was confirmed by blocking experiments. Results: The phage-RGD demonstrated target specificity based on ELISA experiment. According to the TEM images, the morphology of the phage was unchanged after the modification with BFCs. The labeling yield was 25 ± 4% for (64)Cu-DOTA-phage-RGD and 46 ± 5% for (64)Cu-AmBaSar-phage-RGD, respectively. At 1 h time point, (64)Cu-DOTA-phage-RGD and (64)Cu-AmBaSar-phage-RGD have comparable tumor uptake (~ 8%ID/g). However, (64)Cu-AmBaSar-phage-RGD showed significantly higher tumor uptake (13.2 ± 1.5 %ID/g, P<0.05) at late time points compared with (64)Cu-DOTA-phage-RGD (10 ± 1.2 %ID/g). (64)Cu-AmBaSar-phage-RGD also demonstrated significantly lower liver uptake, which could be attributed to the stability difference between these chelators. There is no significant difference between two tracers regarding the uptake in kidney and muscle at all time points tested. In order to confirm the receptor specificity, blocking experiment was performed. In the RGD blocking experiment, the cold RGD peptide was injected 2 min before the administration of (64)Cu-AmBaSar-phage-RGD. Tumor uptake was partially blocked at 1 h time point. Phage-RGD particle was also used as the competitive ligand. In this case, the tumor uptake was significantly reduced and the value was kept at low level consistently. Conclusion: In this report, we constructed a PET trackable nanoplatform based on phage particle and demonstrated the imaging capability of these targeted agents. We also demonstrated that the choice of chelator could have significant impact on imaging results of nano-agents. The method established in this research may be applicable to other receptor/ligand systems for theranostic agent construction, which could have an immediate and profound impact on the field of imaging/therapy and lay the foundation for the construction of next generation cancer specific theranostic agents.

Porphyrin based photosensitizers are useful agents for photodynamic therapy (PDT) and fluorescence imaging of cancer. Porphyrins are also excellent metal chelators forming highly stable metallo-complexes making them efficient delivery vehicles for radioisotopes. Here we investigated the possibility of incorporating (64)Cu into a porphyrin-peptide-folate (PPF) probe developed previously as folate receptor (FR) targeted fluorescent/PDT agent, and evaluated the potential of turning the resulting (64)Cu-PPF into a positron emission tomography (PET) probe for cancer imaging. Noninvasive PET imaging followed by radioassay evaluated the tumor accumulation, pharmacokinetics and biodistribution of (64)Cu-PPF. (64)Cu-PPF uptake in FR-positive tumors was visible on small-animal PET images with high tumor-to-muscle ratio (8.88 ± 3.60) observed after 24 h. Competitive blocking studies confirmed the FR-mediated tracer uptake by the tumor. The ease of efficient (64)Cu-radiolabeling of PPF while retaining its favorable biodistribution, pharmacokinetics and selective tumor uptake, provides a robust strategy to transform tumor-targeted porphyrin-based photosensitizers into PET imaging probes.