In orthopedic surgery, implant infections are a serious issue and difficult to treat. The aim of this study was to use superparamagnetic nanoporous silica nanoparticles (MNPSNP) as candidates for directed drug delivery. Currently, short blood circulation half-life due to interactions with the host's immune system hinder nanoparticles in general from being clinically used. PEGylation is an approach to reduce these interactions and to enhance blood circulation time. The effect of PEGylation of the used 68Ga-labelled MNPSNP on the distribution and implant accumulation was examined by PET/CT imaging and gamma counting in an implant mouse model.Female Balb/c mice (n=24) received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. On day one, 12 of these mice received an additional clodronate®-injection for macrophage depletion. On the second postoperative day, mice were anaesthetized and MNPSNP (native or PEGylated) injected intravenously, followed by a dynamic PET-scan over 60 minutes, a CT- and a static PET-scan at 120 min. As control, 12 mice received only 68Ga-MNPSNP (native or PEGylated). Gamma counting of inner organs, urine, blood and implant area was performed as further final analysis. Although PEGylation of the nanoparticles already resulted in lower liver uptakes, both variants of 68Ga-labeled MNPSNP accumulated in liver and spleen. Combination of PEGylation with clodronate®-injection led to a highly significant effect whereas clodronate®-injection alone could not reveal significant differences. In gamma counting, a significantly higher %I.D./g was found for the tissue surrounding the magnetic implants compared to the titanium control, although in a low range. PEGylation and/or clodronate®-injection revealed no significant differences regarding nanoparticle accumulation at the implantation site. PEGylation increases circulation time, but MNPSNP accumulation at the implant site was still insufficient for treatment of infections. Additional efforts have to further increase circulation time and local accumulation.Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 280642759).

Background Implant infections caused by biofilm forming bacteria are a major threat in orthopedic surgery. Delivering antibiotics directly to an implant affected by a bacterial biofilm via superparamagnetic nanoporous silica nanoparticles could present a promising approach. Nevertheless, short blood circulation half-life because of rapid interactions of nanoparticles with the host’s immune system hinder them from being clinically used. The aim of this study was to determine the temporal in vivo resolution of magnetic nanoporous silica nanoparticle (MNPSNP) distribution and the effect of PEGylation and clodronate application using PET/CT imaging and gamma counting in an implant mouse model. Methods PEGylated and non-PEGylated MNPSNPs were radiolabeled with gallium-68 ( 68 Ga), implementing the chelator tris(hydroxypyridinone). 36 mice were included in the study, 24 mice received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. MNPSNP pharmacokinetics and implant accumulation was analyzed in dependence on PEGylation and additional clodronate application. Subsequently gamma counting was performed for further final analysis. Results The pharmacokinetics and biodistribution of all radiolabeled nanoparticles could clearly be visualized and followed by dynamic PET/CT imaging. Both variants of 68 Ga-labeled MNPSNP accumulated mainly in liver and spleen. PEGylation of the nanoparticles already resulted in lower liver uptakes. Combination with macrophage depletion led to a highly significant effect whereas macrophage depletion alone could not reveal significant differences. Although MNPSNP accumulation around implants was low in comparison to the inner organs in PET/CT imaging, gamma counting displayed a significantly higher %I.D./g for the tissue surrounding the magnetic implants compared to the titanium control. Additional PEGylation and/or macrophage depletion revealed no significant differences regarding nanoparticle accumulation at the implantation site. Conclusion Tracking of 68 Ga-labeled nanoparticles in a mouse model in the first critical hours post-injection by PET/CT imaging provided a better understanding of MNPSNP distribution, elimination and accumulation. Although PEGylation increases circulation time, nanoparticle accumulation at the implantation site was still insufficient for infection treatment and additional efforts are needed to increase local accumulation.

Until now, very few efforts have been made to specifically trace, monitor, and visualize implantations, artificial organs, and bioengineered scaffolds for tissue engineering in vivo. While mainly X-Ray, CT, and MRI methods have been used for this purpose, the applications of more sensitive, quantitative, specific, radiotracer-based nuclear imaging techniques remain a challenge. As the need for biomaterials increases, so does the need for research tools to evaluate host responses. PET (positron emission tomography) and SPECT (single photon emission computer tomography) techniques are promising tools for the clinical translation of such regenerative medicine and tissue engineering efforts. These tracer-based methods offer unique and inevitable support, providing specific, quantitative, visual, non-invasive feedback on implanted biomaterials, devices, or transplanted cells. PET and SPECT can improve and accelerate these studies through biocompatibility, inertivity, and immune-response evaluations over long investigational periods at high sensitivities with low limits of detection. The wide range of radiopharmaceuticals, the newly developed specific bacteria, and the inflammation of specific or fibrosis-specific tracers as well as labeled individual nanomaterials can represent new, valuable tools for implant research. This review aims to summarize the opportunities of nuclear-imaging-supported implant research, including bone, fibrosis, bacteria, nanoparticle, and cell imaging, as well as the latest cutting-edge pretargeting methods.

Ischemic stroke imparts elevated risk of heart failure though the underlying mechanisms remain poorly described. We aimed to characterize the influence of cerebral ischemic injury on cardiac function using multimodality molecular imaging to investigate brain and cardiac morphology and tissue inflammation in two mouse models of variable stroke severity. Transient middle cerebral artery occlusion (MCAo) generated extensive stroke damage (56.31 ± 40.39 mm3). Positron emission tomography imaging of inflammation targeting the mitochondrial translocator protein (TSPO) revealed localized neuroinflammation at 7 days after stroke compared to sham (3.8 ± 0.8 vs 2.6 ± 0.7

In this work, a method for the preparation of the highly lipophilic labeling synthon [89Zr]Zr(oxinate)4 was optimized for the radiolabeling of liposomes and human induced pluripotent stem cells (hiPSCs). The aim was to establish a robust and reliable labeling protocol for enabling up to one week positron emission tomography (PET) tracing of lipid-based nanomedicines and transplanted or injected cells, respectively. [89Zr]Zr(oxinate)4 was prepared from oxine (8-hydroxyquinoline) and [89Zr]Zr(OH)2(C2O4). Earlier introduced liquid–liquid extraction methods were simplified by the optimization of buffering, pH, temperature and reaction times. For quality control, thin-layer chromatography (TLC), size-exclusion chromatography (SEC) and centrifugation were employed. Subsequently, the 89Zr-complex was incorporated into liposome formulations. PET/CT imaging of 89Zr-labeled liposomes was performed in healthy mice. Cell labeling was accomplished in PBS using suspensions of 3 × 106 hiPSCs, each. [89Zr]Zr(oxinate)4 was synthesized in very high radiochemical yields of 98.7% (96.8% ± 2.8%). Similarly, high internalization rates (≥90%) of [89Zr]Zr(oxinate)4 into liposomes were obtained over an 18 h incubation period. MicroPET and biodistribution studies confirmed the labeled nanocarriers’ in vivo stability. Human iPSCs incorporated the labeling agent within 30 min with ~50% efficiency. Prolonged PET imaging is an ideal tool in the development of lipid-based nanocarriers for drug delivery and cell therapies. To this end, a reliable and reproducible 89Zr radiolabeling method was developed and tested successfully in a model liposome system and in hiPSCs alike.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG; Ev Studienwerk Villigst Introduction Cerebral ischemia remains a major contributor to global morbidity and mortality. Beyond neurological dysfunction, cerebral stroke also increases risk of subsequent cardiac events and development of chronic heart failure. The mechanisms underlying this brain-heart communication are poorly characterized, but systemic inflammation is thought to play a role. Purpose We hypothesized that cerebral ischemic stroke evokes systemic inflammation contributing to acute cardiac stress and persistent contractile dysfunction, which may be monitored by serial molecular imaging by positron emission tomography (PET). Methods C57Bl6 mice underwent transient (30min) intraluminal middle cerebral artery occlusion (MCAo, n = 88) to induce stroke or sham surgery (n = 18). Serial 18F-GE180 PET at 24h, 7d, and 21d after injury identified translocator protein (TSPO) in brain microglia and peripheral macrophages as a marker of inflammation. Magnetic resonance imaging (MRI) determined stroke size and characterized cardiac function at 1 and 3wk after MCAo. Imaging signals were confirmed by autoradiography, immunohistochemistry and fluorescence-activated cell sorting. Results MCAo in mice induced cerebral stroke of variable size (median 44.68mm3, range:5.12-139.60mm3). Localized inflammation in the injured hemisphere was observed by TSPO PET covering territory consistent to the stroke location, with marked increase in the neuroinflammatory signal at 7d relative to the contralateral hemisphere (% injected dose (ID)/g max: 3.82 ± 0.84 vs 2.51 ± 0.32, p < 0.001). TSPO signal was similarly elevated in the stroke region compared to sham animals (%ID/g max: 3.82 ± 0.84 vs 2.59 ± 0.72, p < 0.001). Corresponding with neuroinflammation, a heightened global myocardial TSPO signal was observed 7d after MCAo compared to sham (%ID/g: 7.75 ± 2.79 vs 5.61 ± 1.98, p = 0.041). TSPO PET signal remained elevated 21d after stroke in brain (3.20 ± 1.15 vs 2.46 ± 0.37, p = 0.012) and heart (8.62 ± 2.43 vs 5.85 ± 0.66, p = 0.022). Cerebral stroke was associated with reduced contractile function compared to sham at 1wk (ejection fraction, EF: 49.58 ± 16.90 vs 62.17 ± 5.25%, p = 0.025) and 3wk (54.30 ± 5.70 vs 66.06 ± 3.48%, p = 0.009). Larger stroke size identified early after injury by T2 weighted MRI, reflecting the focal injury and penumbra edema, correlated with worse subsequent cardiac function at 21d (r = 0.745, p = 0.009). Despite increased TSPO signal, flow cytometry showed comparable CD45+ leukocyte content in the left ventricle after MCAo compared to sham at 7d (p = 0.749). Conclusions Ischemic stroke evoked parallel cerebral and cardiac TSPO upregulation and persistent contractile dysfunction, beginning early after injury. The extent and severity of brain injury predicts left ventricle dysfunction, but the role of inflammation requires further investigation. Molecular imaging may identify risk and guide novel therapies to improve cardiac outcome after stroke.

Purpose Tracer kinetic modeling of tissue time activity curves and the individual input function based on arterial blood sampling and metabolite correction is the gold standard for quantitative characterization of microglia activation by PET with the translocator protein (TSPO) ligand 18 F-GE-180. This study tested simplified methods for quantification of 18 F-GE-180 PET. Methods Dynamic 18 F-GE-180 PET with arterial blood sampling and metabolite correction was performed in five healthy volunteers and 20 liver-transplanted patients. Population-based input function templates were generated by averaging individual input functions normalized to the total area under the input function using a leave-one-out approach. Individual population-based input functions were obtained by scaling the input function template with the individual parent activity concentration of 18 F-GE-180 in arterial plasma in a blood sample drawn at 27.5 min or by the individual administered tracer activity, respectively. The total 18 F-GE-180 distribution volume (V T ) was estimated in 12 regions-of-interest (ROIs) by the invasive Logan plot using the measured or the population-based input functions. Late ROI-to-whole-blood and ROI-to-cerebellum ratio were also computed. Results Correlation with the reference V T (with individually measured input function) was very high for V T with the population-based input function scaled with the blood sample and for the ROI-to-whole-blood ratio (Pearson correlation coefficient = 0.989 ± 0.006 and 0.970 ± 0.005). The correlation was only moderate for V T with the population-based input function scaled with tracer activity dose and for the ROI-to-cerebellum ratio (0.653 ± 0.074 and 0.384 ± 0.177). Reference V T , population-based V T with scaling by the blood sample, and ROI-to-whole-blood ratio were sensitive to the TSPO gene polymorphism. Population-based V T with scaling to the administered tracer activity and the ROI-to-cerebellum ratio failed to detect a polymorphism effect. Conclusion These results support the use of a population-based input function scaled with a single blood sample or the ROI-to-whole-blood ratio at a late time point for simplified quantitative analysis of 18 F-GE-180 PET.

Background:This work aimed to evaluate the influence of two chelators: DOTA(SCN) and DOTA(NHS) on radioimmunotherapy using(177)Lu-DOTA-Rituximab preparations in murine lymphoma xenograft models. Subsequently, based on animal data, the organ radiation-absorbed doses were extrapolated to humans (adult male). Materials and Methods:Therapeutic efficacy of(177)Lu-DOTA-Rituximab was evaluated in male nude mice bearing either Raji (B lymphocyte, CD20(+)) and Jurkat (T lymphocyte, CD20) xenografts, utilizing an anti-CD20 antibody-Rituximab conjugate with either DOTA(SCN) or DOTA(NHS). The DOTA-Rituximab conjugates were prepared in the form of freeze-dried kits. Results:All radioimmunoconjugates were obtained with high radiolabeling yield (radiochemical purity, RCP > 95%) and specific activity of ca. 0.5 GBq/mg. Therapeutic effects of(177)Lu-DOTA-Rituximab were observed in animals regardless whether DOTA(SCN) or DOTA(NHS) were used for conjugation. Importantly, therapy involving(177)Lu-DOTA-Rituximab was more effective than use of Rituximab alone. Conclusions:The degree of antitumor efficacy was dependent on the type of applied bifunctional chelators conjugated to mAb. However, this difference was not statistically significant. Dosimetry calculations showed that the absorbed radiation doses extrapolated to humans were very low for osteogenic cells regardless of the conjugates. Organs like the liver and spleen, treated with(177)Lu-DOTA(SCN)-Rituximab, showed similar radiation absorbed doses when compared with(177)Lu-DOTA(NHS)-Rituximab.

Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed network-based approaches to prevent epileptogenesis. For proof of concept we combined two drugs (levetiracetam and topiramate) for which in silico analysis of drug-protein interaction networks indicated a synergistic effect on a large functional network of epilepsy-relevant proteins. Using the intrahippocampal kainate mouse model of temporal lobe epilepsy, the drug combination was administered during the latent period before onset of spontaneous recurrent seizures (SRS). When SRS were periodically recorded by video-EEG monitoring after termination of treatment, a significant decrease in incidence and frequency of SRS was determined, indicating antiepileptogenic efficacy. Such efficacy was not observed following single drug treatment. Furthermore, a combination of levetiracetam and phenobarbital, for which in silico analysis of drug-protein interaction networks did not indicate any significant drug-drug interaction, was not effective to modify development of epilepsy. Surprisingly, the promising antiepileptogenic effect of the levetiracetam/topiramate combination was obtained in the absence of any significant neuroprotective or anti-inflammatory effects as indicated by multimodal brain imaging and histopathology. High throughput RNA-sequencing (RNA-seq) of the ipsilateral hippocampus of mice treated with the levetiracetam/topiramate combination showed that several genes that have been linked previously to epileptogenesis, were significantly differentially expressed, providing interesting entry points for future mechanistic studies. Overall, we have discovered a novel combination treatment with promise for prevention of epilepsy.

Leukocyte subtypes bear distinct pro-inflammatory, reparative, and regulatory functions. Imaging inflammation provides information on disease prognosis and may guide therapy, but the cellular basis of the signal remains equivocal. We evaluated leukocyte subtype specificity of characterized clinically relevant inflammation-targeted radiotracers. Leukocyte populations were purified from blood- and THP-1-derived macrophages were polarized into M1-, reparative M2a-, or M2c-macrophages. In vitro uptake assays were conducted using tracers of enhanced glucose or amino acid metabolism and molecular markers of inflammatory cells. Both 18F-deoxyglucose (18F-FDG) and the labeled amino acid 11C-methionine (11C-MET) displayed higher uptake in neutrophils and monocytes compared to other leukocytes (P = 0.005), and markedly higher accumulation in pro-inflammatory M1-macrophages compared to reparative M2a-macrophages (P < 0.001). Molecular tracers 68Ga-DOTATATE targeting the somatostatin receptor type 2 and 68Ga-pentixafor targeting the chemokine receptor type 4 (CXCR4) exhibited broad uptake by leukocyte subpopulations and polarized macrophages with highest uptake in T-cells/natural killer cells and B-cells compared to neutrophils. Mitochondrial translocator protein (TSPO)-targeted 18F-flutriciclamide selectively accumulated in monocytes and pro-inflammatory M1 macrophages (P < 0.001). Uptake by myocytes and fibroblasts tended to be higher for metabolic radiotracers. The different in vitro cellular uptake profiles may allow isolation of distinct phases of the inflammatory pathway with specific inflammation-targeted radiotracers. The pathogenetic cell population in specific inflammatory diseases should be considered in the selection of an appropriate imaging agent.

Identification of patients at risk of developing epilepsy before the first spontaneous seizure may promote the development of preventive treatment providing opportunity to stop or slow down the disease.

BACKGROUND:PET and SPECT imaging methods can be of excellent assistance for the development of new nanoparticle drug delivery systems, and at the same time, these investigations also offer the opportunity to produce exceptional new diagnostic and therapeutic radiopharmaceuticals, as well. With a multifunctional, nano-scaled drug delivery system, the diagnostic (imaging) methods and the therapy (delivering drugs or beta-emitter radionuclides) can be carried out using the same biological and pharmacological mechanisms. By combining therapy and diagnostics in one method or in one specifically targeted nanoparticle system, we can product theranostic pharmaceuticals, and its applications are important elements of personalized medicine.OBJECTIVES:This review takes a short historical look back to the radiocolloids, the great ancestors of (radiolabeled) nanoparticles and then describes the general features of current types of PET and SPECT imaging associated nanoparticle-based products and key radiolabeling methods; entering into details of potential prospective challenges related to radiotheranostic approaches and imaging guided therapy.

Minigastrin (MG) analogues specifically target cholecystokinin-2 receptors (CCK2R) expressed in different tumors and enable targeted radiotherapy of advanced and disseminated disease when radiolabeled with a beta emitter such as 177Lu. Especially truncated MG analogues missing the penta-Glu sequence are associated with low kidney retention and seem therefore most promising for therapeutic use. Based on [d-Glu1,desGlu2-6]MG (MG11) we have designed the two cyclic MG analogues cyclo1,9[γ-d-Glu1,desGlu2-6,d-Lys9]MG (cyclo-MG1) and cyclo1,9[γ-d-Glu1,desGlu2-6,d-Lys9,Nle11]MG (cyclo-MG2). In the present work we have developed and preclinically evaluated a pharmaceutical kit formulation for the labeling with 177Lu of the two DOTA-conjugated cyclic MG analogues. The stability of the kits during storage as well as the stability of the radiolabeled peptides was investigated. A cell line stably transfected with human CCK2R and a control cell line without receptor expression were used for in vitro and in vivo studies with the radioligands prepared from kit formulations. In terms of stability 177Lu-DOTA-cyclo-MG2 showed advantages over 177Lu-DOTA-cyclo-MG1. Still, for both radioligands a high receptor-mediated cell uptake and favorable pharmacokinetic profile combining receptor-specific tumor uptake with low unspecific tissue uptake and low kidney retention were confirmed. Investigating the therapy efficacy and treatment toxicity in xenografted BALB/c nude mice a receptor-specific and comparable therapeutic effect could be demonstrated for both radioligands. A 1.7- to 2.6-fold increase in tumor volume doubling time was observed for receptor-positive tumors in treated versus untreated animals, which was 39-73% higher when compared to receptor-negative tumors. The treatment was connected with transient bone marrow toxicity and minor signs of kidney toxicity. All together the obtained results support further studies for the clinical translation of this new therapeutic approach.

This paper describes the preparation of gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2) nanoparticle (NP) platform of nearly 100nm diameter and its first pharmacokinetic and biodistribution evaluation accomplished with a microPET/CT (μPet/CT) imaging system. Objectives of the investigations were to provide a nanoparticle platform which can be suitable for specific delivery of various therapeutic drugs using surface attached specific molecules as triggering agents, and at the same time, suitable for positron emission tomography (PET) tracing of the prospective drug delivery process. Radiolabeling was accomplished using DOTA bifunctional chelator. DOTA was successfully adsorbed onto the surface of nanoparticles, while the 68Ga-radiolabeling method proved to be simple and effective. In the course of biodistribution studies, the 68Ga-labeled DOTA-ZrNPs showed proper radiolabeling stability in their original suspension and in blood serum. μPet/CT imaging studies confirmed a RES-biodistribution profile indicating stable nano-sized labeled particles in vivo. Results proved that the new method offers the opportunity to examine further specifically targeted and drug payload carrier variants of zirconia NP systems using PET/CT imaging.

Diabetes affects an increasing number of patients worldwide and is responsible for a significant rise in healthcare expenses. Imaging of β-cells in vivo is expected to contribute to an improved understanding of the underlying pathophysiology, improved diagnosis, and development of new treatment options for diabetes. Here, we describe the first radiosyntheses of [3 H]-TAK875 and [18 F]-TAK875 derivatives to be used as β-cell imaging probes addressing the free fatty acid receptor 1 (FFAR1/GPR40). The fluorine-labeled derivative showed similar agonistic activity as TAK875 in a functional assay. The radiosynthesis of the 18 F-labelled tracer 2a was achieved with 16.7 ± 5.7% radiochemical yield in a total synthesis time of 60-70 min.