Comparison of PEGylated and non-PEGylated proticles: An in vitro and in vivo study.

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 ±â€¯0.01 % ID/g 1 h p.i.; 111In-proticles: 0.06 ±â€¯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.

Tumor targeting and imaging with dual-peptide conjugated multifunctional liposomal nanoparticles.

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 αvß3 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 (111)In-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.

Design and evaluation of novel radiolabelled VIP derivatives for tumour targeting.

BACKGROUND: Vasoactive 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 METHODS: 1,4,7,10-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. RESULTS: Radiolabelling 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. CONCLUSION: Since 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.

Development of 68Ga-labelled DTPA galactosyl human serum albumin for liver function imaging.

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.

Influence of PEGylation and RGD loading on the targeting properties of radiolabeled liposomal nanoparticles.

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 α(v)ß(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 α(v)ß(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 α(v)ß(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.

Preclinical evaluation of two 68Ga-siderophores as potential radiopharmaceuticals for Aspergillus fumigatus infection imaging.

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.

Targeting properties of peptide-modified radiolabeled liposomal nanoparticles.

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 IC(50) 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.

In vitro and in vivo evaluation of selected 68Ga-siderophores for infection imaging.

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. (68)Ga is a positron emitter with complexing properties comparable to those of Fe(III) and readily available from a generator. Initial studies with (68)Ga-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 (68)Ga as potential radiopharmaceuticals for infection imaging. METHODS: (68)Ga 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 (68)Ga-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, (68)Ga-TAFC and (68)Ga-ferrioxamine E (FOXE) showed rapid renal excretion and low blood values even at a short period after injection; in contrast, (68)Ga-ferricrocin and (68)Ga-ferrichrome revealed high retention in blood and (68)Ga-fusarinine C showed very high kidney retention. CONCLUSIONS: Some of the studied siderophores bind (68)Ga with high affinity and stability, especially (68)Ga-TAFC and (68)Ga-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.

Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607.

PURPOSE: 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. METHODS: Twelve different 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-minigastrin/CCK conjugates were provided within an European COST Action (BM0607) by different laboratories and radiolabelled with (177)Lu. Their in vitro stabilities were tested in fresh human serum. Radiochemical yields (RCY) and intact radioligands for half-life calculations were determined by radio-HPLC. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis of metabolites was performed to identify cleavage products using conjugates labelled with excess stable (nat)Lu, incubated in serum at 37°C. Urine metabolite analysis after injection in normal mice was performed by radio-HPLC analysis. RESULTS: Variable stability in human serum was found for the different peptides with calculated half-lives between 4.5 ± 0.1 h and 198 ± 0.1 h (n = 2). In urine of normal mice only metabolised peptide fragments were detected even at short times after injection for all peptides. MALDI-TOF MS revealed a major cleavage site of all minigastrin derivatives between Asp and Phe-NH(2) at the C-terminal end. CONCLUSION: Development of CCK2 receptor ligands especially for therapeutic purposes in patients with MTC or small cell lung cancer (SCLC) is still ongoing in different laboratories. This comparative study provided valuable insight into the importance of biological stability especially in the context of other results of this comparative trial within the COST Action BM0607.

Influence of biological assay conditions on stability assessment of radiometal-labelled peptides exemplified using a 177Lu-DOTA-minigastrin derivative.

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 ((177)Lu-DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-NIe-Asp-Phe-NH(2)) 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.

68Ga-siderophores for PET imaging of invasive pulmonary aspergillosis: proof of principle.

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.

Radiolabeling of lipid-based nanoparticles for diagnostics and therapeutic applications: a comparison using different radiometals.

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 (111)In and (99m)Tc, for single-photon emission computed tomography (SPECT), (68)Ga for positron emission tomography (PET), and (177)Lu for therapeutic applications. PEGylated DTPA-NP with varying DTPA amounts, different composition, and size were radiolabeled with (111)In, (177)Lu, and (68)Ga, using various buffers. (99m)Tc-labeling was performed directly and by using the carbonyl aquaion, [(99m)Tc(H(2)O)(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 (111)In (>4 MBq/mug liposome), followed by (68)Ga and (177)Lu; for (99m)Tc, high labeling yields and SA were only achieved by using [(99m)Tc(H(2)O)(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 (99m)Tc- and (68)Ga, compared to (111)In-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.

Cyclic minigastrin analogues for gastrin receptor scintigraphy with technetium-99m: preclinical evaluation.

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.