The development of so-called Proticles opens attractive possibilities for new drug delivery systems. Proticles are nanoparticles (NPs), which are formed by self-assembly of negatively charged oligonucleotides in combination with the positively charged peptide protamine. Polyethylene glycol (PEG) is a widely known pharmaceutical agent to stop particle growth and prolong circulation half-life of drug delivery systems. Therefore, two different NP formulations - one PEGylated and one non-PEGylated - were used in this work to gain information about the biological stability and half-life in circulation of Proticles. Thus, this study presents data of in vitro stability and in vivo pharmacokinetics of both, non-PEGylated and PEGylated Proticles radiolabeled with 111InCl3. The study demonstrated that successful radiolabeling of both Proticle-formulations was performed resulting in high radiochemical yields (> 85 %). Furthermore, the influence of PEGylation on the in vitro stability of 111In-radiolabeled NPs was investigated. No significant difference due to PEGylation was found. Unlike in vitro results, non-PEGylated 111In-Proticles seemed to degrade faster in vivo than PEGylated 111In-proticles, resulting in significantly higher blood values (111In-PEG-proticles: 0.23 ± 0.01 % ID/g 1 h p.i.; 111In-proticles: 0.06 ± 0.01 % ID/g 1 h p.i.; p < 0.05). Visualized by SPECT imaging urinary excretion represented the major pathway of elimination for both NP-formulations. In conclusion, this study provides data indicating a positive influence of PEG-derivatization on the biodistribution and pharmacokinetics of Proticles. These results form the basis for further developments as drug delivery and active drug targeting devices.

Background The significant progress in nanotechnology provides a wide spectrum of nanosized material for various applications, including tumor targeting and molecular imaging. The aim of this study was to evaluate multifunctional liposomal nanoparticles for targeting approaches and detection of tumors using different imaging modalities. The concept of dual-targeting was tested in vitro and in vivo using liposomes derivatized with an arginine-glycine-aspartic acid (RGD) peptide binding to αvβ3 integrin receptors and a substance P peptide binding to neurokinin-1 receptors. Methods For liposome preparation, lipids, polyethylene glycol building blocks, DTPA-derivatized lipids for radiolabeling, lipid-based RGD and substance P building blocks and imaging labels were combined in defined molar ratios. Liposomes were characterized by photon correlation spectroscopy and zeta potential measurements, and in vitro binding properties were tested using fluorescence microscopy. Standardized protocols for radiolabeling were developed to perform biodistribution and micro-single photon emission computed tomography/computed tomography (SPECT/CT) studies in nude mice bearing glioblastoma and/or melanoma tumor xenografts. Additionally, an initial magnetic resonance imaging study was performed. Results Liposomes were radiolabeled with high radiochemical yields. Fluorescence microscopy showed specific cellular interactions with RGD-liposomes and substance P-liposomes. Biodistribution and micro-SPECT/CT imaging of 111In-labeled liposomal nanoparticles revealed low tumor uptake, but in a preliminary magnetic resonance imaging study with a single-targeted RGD-liposome, uptake in the tumor xenografts could be visualized. Conclusion The present study shows the potential of liposomes as multifunctional targeted vehicles for imaging of tumors combining radioactive, fluorescent, and magnetic resonance signaling. Specific in vitro tumor targeting by fluorescence microscopy and radioactivity was achieved. However, biodistribution studies in an animal tumor model revealed only moderate tumor uptake and no additive effect using a dual-targeting approach.

BACKGROUNDVasoactive intestinal peptide (VIP) receptors are overexpressed in a broad variety of tumours. For the detection of these tumours, novel chemically modified and shortened VIP derivatives were designed.MATERIALS AND METHODS1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-derivatised VIP analogues were radiolabelled with (111)In and in vitro and in vivo behaviour was evaluated using stability and internalisation assays, as well as an initial biodistribution study.RESULTSRadiolabelling of the VIP analogues resulted in high radiochemical yields, without need for further purification steps. Stability of the VIP derivatives was variable and cell uptake studies in VIP receptor-positive cell lines revealed that only a limited number of derivatives were internalised. In the tumour mouse model, no specific tumour targeting was shown.CONCLUSIONSince the tested VIP derivatives exhibited impaired in vitro and in vivo characteristics alternative modifications to increase their stability while retaining receptor affinity should be considered to enable the use of synthetic VIP analogues for tumour targeting.

PURPOSE:The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [(99m)Tc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([(99m)Tc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [(68)Ga]GSA for the potential use with positron emission tomography (PET).METHODS:Labelling of GSA with (68)Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [(68)Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [(68)Ga]GSA distribution in Lewis rats was compared with [(99m)Tc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [(68)Ga]GSA and [(99m)Tc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90).RESULTS:[(68)Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free (68)Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98%). Stability after 120 min incubation at 37 °C was high in PBS (>95% intact tracer) and low in human serum (∼27% intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [(68)Ga]GSA compared to [(99m)Tc]GSA. The [(99m)Tc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [(68)Ga]GSA curves. The mean [(68)Ga]GSA t90 was higher than the mean t90 for [(99m)Tc]GSA. The mean [(68)Ga]GSA t50 was not significantly different from the mean t50 for [(99m)Tc]GSA.CONCLUSION:This study provides a promising new (68)Ga-labelled compound based on a commercially used kit for imaging the functional hepatocellular mass.

Purpose: Liposomes have been proposed to be a means of selectively targeting cancer sites for diagnostic and therapeutic applications. The focus of this work was the evaluation of radiolabeled PEGylated liposomes derivatized with varying amounts of a cyclic arginyl-glycyl-aspartic acid (RGD) peptide. RGD peptides are known to bind to alpha(v)beta(3) integrin receptors overexpressed during tumor-induced angiogenesis.Methods: Several liposomal nanoparticles carrying the RGD peptide targeting sequence (RLPs) were synthesized using a combination of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, diethylenetriaminepentaacetic acid-derivatized lipids for radiolabeling, a polyethylene glycol (PEG) building block, and a lipid-based RGD building block. Relative amounts of RGD and PEG building blocks were varied. In vitro binding affinities were determined using isolated alpha(v)beta(3) integrin receptors incubated with different concentrations of RLPs in competition with iodine-125-labeled cyclo-(-RGDyV-). Binding of the indium-111-labeled RLPs was also evaluated. Biodistribution and micro single photon emission computed tomography/computed tomography imaging studies were performed in nude mice using different tumor xenograft models.Results: RLPs were labeled with indium-111 with high radiochemical yields. In vitro binding studies of RLPs with different RGD/PEG loading revealed good binding to isolated receptors, which was dependent on the extent of RGD and PEG loading. Binding increased with higher RGD loading, whereas reduced binding was found with higher PEG loading. Biodistribution showed increased circulating time for PEGylated RLPs, but no dependence on RGD loading. Both biodistribution and micro single photon emission computed tomography/computed tomography imaging studies revealed low, nonspecific tumor uptake values.Conclusion: In this study, RLPs for targeting angiogenesis were described. Even though good binding to alpha(v)beta(3) integrin receptors was found in vitro, the balance between PEGylation and RGD loading clearly requires optimization to achieve targeting in vivo. These data form the basis for future development and provide a platform for the investigation of multimodal approaches.

Introduction: Siderophores are low-molecular-mass iron chelators serving as iron transporters for almost all bacteria, fungi and some plants. Iron is an essential element for majority of organisms and plays an important role in virulence of pathogenic organisms. Ga-68 is a positron emitter with complexing properties comparable to those of Fe(III) and readily available from a generator. Initial studies with Ga-68-triacetylfusarinine C (TAFC) showed excellent targeting properties in a rat infection model. We report here on the in vitro and in vivo evaluation of other siderophores radiolabelled with Ga-68 as potential radiopharmaceuticals for infection imaging.Methods: Ga-68 labelling was performed using acetate buffer. Stability, log P and protein binding values were determined. In vitro uptake was tested using iron-deficient and iron-sufficient Aspergillus fumigatus (A.f.) cultures. Biodistribution of Ga-68-siderophores was studied in Balb/c mice.Results: Significant differences among studied siderophores were observed in labelling efficiency, stability and protein binding. Uptake in A.f. cultures was highly dependent on iron load and type of the siderophore. In mice, Ga-68-TAFC and Ga-68-ferrioxamine E (FOXE) showed rapid renal excretion and low blood values even at a short period after injection; in contrast, Ga-68-ferricrocin and Ga-68-ferrichrome revealed high retention in blood and Ga-68-fusarinine C showed very high kidney retention.Conclusions: Some of the studied siderophores bind Ga-68 with high affinity and stability, especially Ga-68-TAFC and Ga-68-FOXE. Low values of protein binding, high and specific uptake in A.f, and excellent in vivo biodistribution make them favourable agents for Aspergillus infection imaging. (C) 2012 Elsevier Inc. All rights reserved.

Purpose Invasive pulmonary aspergillosis is mainly caused by Aspergillus fumigatus , and is one of the major causes of morbidity and mortality in immunocompromised patients. The mortality associated with invasive pulmonary aspergillosis remains high, mainly due to the difficulties and limitations in diagnosis. We have shown that siderophores can be labelled with 68 Ga and can be used for PET imaging of A. fumigatus infection in rats. Here we report on the further evaluation of the most promising 68 Ga-siderophore candidates, triacetylfusarinine (TAFC) and ferrioxamine E (FOXE). Methods Siderophores were labelled with 68 Ga using acetate buffer. Log P , protein binding and stability values were determined. Uptake by A. fumigatus was studied in vitro in cultures with high and low iron loads. In vivo biodistribution was determined in normal mice and an infection model was established using neutropenic rats inoculated with A. fumigatus . Static and dynamic μPET imaging was performed and correlated with CT images, and lung infection was evaluated ex vivo. Results 68 Ga-siderophores were labelled with high radiochemical purity and specific activity. 68 Ga-TAFC and 68 Ga-FOXE showed high uptake by A. fumigatus in iron-deficient cultures. In normal mice, 68 Ga-TAFC and 68 Ga-FOXE showed rapid renal excretion with high metabolic stability. In the rat infection model focal lung uptake was detected by μPET with both compounds and increased with severity of the infection, correlating with abnormal CT images. Conclusion 68 Ga-TAFC and 68 Ga-FOXE displayed excellent in vitro stability and high uptake by A. fumigatus . Both compounds showed excellent pharmacokinetics, highly selective accumulation in infected lung tissue and good correlation with severity of disease in a rat infection model, which makes them promising agents for A. fumigatus infection imaging.

Radiolabeled PEGylated liposomal nanoparticles (NPs) open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and monitoring treatment effects. Here we describe the characterization of liposomal NPs (liposomes and micelles) derivatized with the somatostatin analogue tyrosine-3-octreotide as a proof of concept for tumor targeting. NPs were radiolabeled with indium-111, and targeting properties were evaluated in vitro on rat pancreatic tumor cells (AR42J), demonstrating specific binding and IC50 values in the low nanomolar range. Biodistribution studies were performed in Lewis rats and compared to single-photon emission computed tomography images. Moderate tumor uptake was found in xenografted nude mice (<2.5% ID/g tissue) as compared to control. Micelles and liposomes revealed comparable pharmacokinetics and targeting properties. This study provides insight into tumor-targeting characteristics of peptide-derivatized liposomal NPs and can serve as a basis for further improvement of these constructs.From the Clinical Editor: The authors investigated tumor-targeting characteristics of peptide-derivatized liposomal NPs. Similar radiolabeled PEGylated liposomal NPs open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and treatment monitoring. (C) 2012 Elsevier Inc. All rights reserved.

Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites.

Introduction: Lack of correlation between in vitro and in vivo stability is a general problem for the development of radiopeptides especially in the case of minigastrin derivatives for therapeutic applications. In this study, we compared the influence of experimental conditions on radiopeptide stability results in vitro using a model Minigastrin (MG) analogue labelled with Lu-177. Additionally, we attempted to characterize the main serum enzymatic cleavage sites by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) analysis.Methods: In vitro stability of a DOTA-minigastrin derivative (Lu-177-DOTA-His-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Fhe-NH2) was tested in serum, rat tissue homogenates and two different standardised enzymatic mixtures. Quantification of the metabolised radiopeptides at different time intervals was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). Metabolites were characterised by MALDI-TOF-MS. Urine was collected after 15 min p.i. into the mice and compared with in vitro metabolites by RP-HPLC.Results: Faster degradation of the radiopeptide was found in blood in comparison with plasma and serum incubation and in components from rats faster than from human origin. Fast degradation was observed in kidney and liver homogenates as well as in standardised enzymatic mixtures, also revealing variations in the metabolic profile. In urine, no intact peptide was detected already 5 min post injection. MALDI-TOF-MS revealed major cleavage sites at the carboxy terminus of the peptide.Conclusion: Very variable results may be found when different kind of incubation media for testing radiopeptide stabilities is used. Serum incubation studies may overestimate stability; therefore, results should be interpreted with care and combined with alternative in vitro and in vivo investigations. (C) 2011 Elsevier Inc. All rights reserved.

NH2 where NO2A=,4,7-triazacyclononane-1,4,-diacetic acid and X=Paraaminobenzoic acid, 6-aminohexanoic acid, 8-aminooctanoic acid and 9nonanoic acid. All of the new conjugates were radiolabeled and purified by reverse-phase high-performance liquid chromatography. Complete and full characterization of Cu-BBN conjugates was done using electrospray-ionization mass spectrometry. Competitive displacement binding assays indicated that all of the conjugates exhibited IC50s in the nM range in human, prostate PC-3 cells. Biodistribution studies using these new conjugates in rodents bearing PC-3 xenografted tumors indicated rapid clearance of targeting vector from blood with high uptake and retention in tumors. Molecular imaging studies in rodents produced high-contrast, high-quality micro-positron emission tomography images with very high tumor-to-background ratios. doi:10.1016/j.nucmedbio.2010.02.012

UNLABELLED:The diagnosis of invasive pulmonary aspergillosis (IPA) is difficult and lacks specificity and sensitivity. In the pathophysiology of Aspergillus fumigatus, iron plays an essential role as a nutrient during infection. A. fumigatus uses a specific and highly efficient iron uptake mechanism based on iron-complexing ferric ion Fe(III) siderophores, which are a requirement for A. fumigatus virulence. We aimed to evaluate the potential of siderophores radiolabeled with (68)Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA. METHODS:(68)Ga radiolabeling of the A. fumigatus siderophores desferri-triacetylfusarinine C (TAFC) and desferri-ferricrocin (FC) was performed at high specific activity. Stability, protein binding, and log P values were determined. In vitro uptake in A. fumigatus cultures was tested under varying conditions. Biodistribution was studied in healthy noninfected BALB/c mice, and uptake was studied in a model of A. fumigatus infection using immunosuppressed Lewis rats. RESULTS:High-specific-activity (68)Ga labeling could be achieved, and resulting complexes were stable in serum, toward diethylenetriaminepentaacetic acid and Fe(III) challenge. Both siderophores showed hydrophilic properties ((68)Ga-TAFC, log P = -2.59; (68)Ga-FC, log P = -3.17) with low values of protein binding for (68)Ga-TAFC (<2%). Uptake of both siderophores was highly dependent on the mycelial iron load and could be blocked with an excess (10 microM) of siderophore or NaN(3), indicating specific, energy-dependent uptake. In noninfected mice, (68)Ga-TAFC showed rapid renal excretion and low blood values (1.6 +/- 0.37 percentage injected dose per gram [%ID/g] at 30 min); in urine only intact (68)Ga-TAFC was detected. In contrast, (68)Ga-FC revealed high retention in blood (16.1 +/- 1.07 %ID/g at 90 min) and rapid metabolism. In the rat IPA model, lung uptake of (68)Ga-TAFC was dependent on the severity of infection, with less than 0.04 %ID/g in control rats (n = 5) and 0.29 +/- 0.11 %ID/g in mildly infected (n = 3) and 0.95 +/- 0.37 %ID/g in severely infected (n = 4) rats. PET showed focal accumulation in infected lung tissue. CONCLUSION:Both siderophores bound (68)Ga with high affinity, and (68)Ga-TAFC, especially, showed high stability. (68)Ga-TAFC displayed highly selective accumulation by A. fumigatus subspecies in vitro and in vivo. The high and specific uptake by A. fumigatus proves the potential of (68)Ga-labeled siderophores for the specific detection of A. fumigatus during infection. They hold promise as new PET agents for IPA.

Two cyclized minigastrin analogues for gastrin receptor scintigraphy were synthesized and derivatized with HYNIC at the N-terminus for labeling with 99mTc. Radiolabeling efficiency, stability, cell internalization, and receptor binding on CCK-2 receptor expressing AR42J cells were studied and the biodistribution evaluated in tumor bearing nude mice, including NanoSPECT/CT imaging. Metabolites in urine, liver, and kidneys were analyzed by radio-HPLC. Radiolabeled cyclic MG showed high stability in vitro and receptor mediated uptake in AR42J cells. In the animal tumor model, fast renal clearance and low nonspecific uptake in most organs were observed. A tumor uptake >3% was calculated ex vivo 1 h p.i. for both 99mTc-EDDA-HYNIC-cyclo-MG1 and 99mTc-EDDA-HYNIC-cyclo-MG2. In an imaging study with 99mTc-EDDA-HYNIC-cyclo-MG1, the tumor was clearly visualized. The metabolite analysis indicated rapid enzymatic degradation in vivo.

Radiolabeling of nanoparticles (NPs) has been performed for a variety of reasons, such as for studying pharmacokinetics, for imaging, or for therapy. Here, we describe the in vitro and in vivo evaluation of DTPA-derivatized lipid-based NP (DTPA-NP) radiolabeled with different radiometals, including In-111 and Tc-99m, for single-photon emission computed tomography (SPECT), Ga-68 for positron emission tomography (PET), and Lu-177 for therapeutic applications. PEGylated DTPA-NP with varying DTPA amounts, different composition, and size were radiolabeled with In-111, Lu-177, and Ga-68, using various buffers. Tc-99m-labeling was performed directly and by using the carbonyl aquaion, [99mTc(H2O)(3) (CO)(3)](+). Stability was tested and biodistribution evaluated. High labeling yields (> 90%) were achieved for all radionuclides and different liposomal formulations. Specific activities (SAs) were highest for In-111 (> 4 MBq/mu g liposome), followed by Ga-68 and Lu-177; for 99mTc, high labeling yields and SA were only achieved by using [99mTc(H2O)(3) (CO)(3)](+). Stability toward DTPA/histidine and in serum was high (> 80 % RCP, 24 hours postpreparation). Biodistribution in Lewis rats revealed no significant differences between NP in terms of DTPA loading and particle composition; however, different uptake patterns were found between the radionuclides used. We observed lower retention in blood (< 3.3 % ID/g) and lower liver uptake (< 2.7 % ID/g) for Tc-99m- and 68Ga, compared to In-111-NP (blood, < 4 % ID/g; liver, < 3.6 % ID/g). Imaging potential was shown by both PET magnetic resonance imaging fusion imaging and SPECT imaging. Overall, our study shows that PEGylated DTPA-NP are suitable for radiolabeling studies with a variety of radiometals, thereby achieving high SA suitable for targeting applications.

322 Objectives Pulmonary aspergillosis (PA) is a life threatening disease and diagnostic options are limited. Iron plays an essential role for the virulence of Aspergillus ssp (A.s.). A.s. employ specific and efficient iron transporter mechanisms based on iron complexing siderophores, which are upregulated during infection. We aimed to evaluate the potential of a peptide siderophore radiolabelled with 68Ga. Methods Triacetylfusarinine (TAFC) was chosen as model siderophore for 68Ga labelling. Radiolabelling was performed at RT. Stability in solution and serum was determined. In vitro uptake in A.s. cultures was tested using varying iron load, blocking with excess siderophore and NaN3. Biodistribution was studied in normal balb/c mice. Specific uptake was studied in a PA model in immunosupressed Sprague Dawley rats. Results High S.A. labelling with quantitative yields could be achieved at RT. 68Ga-TAFC was stable in serum, towards DTPA and Fe(III) challenge. Uptake in A.s. was highly efficient, specific, energy and iron dependent. 68Ga-TAFC showed rapid renal excretion, low blood values and high metabolic stability. In the rat PA model, lung uptake (mean n=3) was Conclusions This work proves the potential of using 68Ga labelled peptide siderophores for specific infection detection. 68Ga-TAFC holds promise as a new PET agent for imaging PA.

1235 Objectives: The development of radiolabeled Minigastrin (MG) derivatives for imaging and therapy of CCK2-receptor expressing tumors such as MTC and SCLC has been hampered by low stability of the native peptide. In this study we describe the evaluation of a new 99mTc-labeled analog stabilized by cyclisation, comparing the organ and tumor uptake in vivo using micro-SPECT/CT imaging with quantification ex vivo. Methods: Cyclisation of MG was achieved by replacing Gly by DLys at Pos 9 allowing cyclisation with DGlu in Pos 1. 99mTc-labelling was carried out by derivatisation with HYNIC at the N-terminal and with EDDA as coligand. Stability and receptor mediated internalization in CCK2-receptor expressing AR4-2J cells were evaluated. Biodistribution was studied in AR4-2J tumor bearing nude mice, including NanoSPECT/CT imaging. Metabolites in urine, liver and kidneys were analyzed by HPLC. Results: Cyclo-MG showed improved radiochemical purity in the labelling process, allowing 99mTc-labelling at high specific activities with high yields. The radioligand revealed high stability in vitro and receptor mediated uptake in AR4-2J cells. In the animal tumor model fast renal clearance and low unspecific uptake in most organs were observed. Tumor uptake 1 h p.i. was 3.26 %ID/g in vivo and 3.61 %ID/g ex vivo. The metabolite analysis indicated rapid conversion into the linear peptide sequence and enzymatic degradation in vivo. Conclusions: Cyclisation of MG only had a limited effect on the stability of radiolabeled MG. Overall biodistribution was comparable to linear analogs. Quantification of the organ and tumor uptake by SPECT/CT image fusion was in accordance with ex vivo data.