Optimal Labeling Conditions Research Articles

113mIn]In-PSMA: high potential agent for SPECT imaging of prostate cancer

Abstract The prevalence rate of prostate cancer is very high and unfortunately causes many deaths worldwide. This study aimed to prepare [113mIn]In-PSMA as a novel agent for single photon emission computed tomography (SPECT) imaging of prostate cancer. 113mIn was eluted from an in-house made 113Sn/113mIn generator in chloride form, and its radionuclide, chemical and radiochemical purities was studied. [113mIn]In-PSMA was prepared with radiochemical purity (RCP) > 99 % under optimal labeling conditions. The biodistribution of the labeled compound was studied in normal rats, showed the major activity in the urine and kidneys as the main excretion route from the body. Considerable uptake was observed in the prostate and salivary glands as the PSMA-expressing organs. These data are in complete agreement with the other published data on PSMA-labeled compounds both in animals and humans. The results demonstrated [113mIn]In-PSMA can be considered as a good option for SPECT imaging of prostate cancer.

Preparation of Radiolabeled Zolbetuximab Targeting CLDN18.2 and Its Preliminary Evaluation for Potential Clinical Applications.

Claudin18.2 (CLDN18.2), due to its high expression in various gastric cancer tissues, is considered an optimal target for antitumor drug molecules. In this study, we obtained the labeled compounds of [125I]I-zolbetuximab using the Iodogen method. Under the optimum labeling conditions, the molar activity of [125I]I-zolbetuximab was 1.75 × 102 GBq/μmol, and the labeling efficiency was more than 99%. The labeled compounds exhibited excellent in vitro stability in both phosphate buffer saline (PBS, pH = 7.4) and fetal bovine serum systems (FBS) (radiochemical purity >90% at 72 h). The uptake percentage of [125I]I-zolbetuximab in MKN45-CLDN18.2 cells is 24.69 ± 0.84% after 6 h. The saturation binding assay and specificity assay further demonstrated the high specificity of [125I]I-zolbetuximab for CLDN18.2. The long retention at the tumor site and rapid metabolic clearance at other organ sites of [125I]I-zolbetuximab were observed in small-animal SPECT-CT imaging. The same trend was also observed in the biodistribution study. Due to the excellent targeting ability of zolbetuximab for CLDN18.2, [125I]I-zolbetuximab exhibits strong specific binding and retention with cells and tumors highly expressing CLDN18.2. However, the balance between mAb's longer cycle time in vivo and targeting binding and retention ability should be intensively considered for using this kind of radiopharmaceutical in the diagnosis and treatment of CLDN18.2-positive gastric cancer.

Synthesis and Ready to Use Kit Formulation of EDTMP for the Preparation of 177Lu-EDTMP as a Bone Palliation Radiopharmaceutical.

With its suitable nuclear decay characteristics and large-scale production feasibility with adequate specific activity, 177Lu is regarded as an excellent radionuclide for developing bone pain palliation agent. Ethylenediamine-tetramethylene phosphonic acid (EDTMP) is a preferred carrier molecule for radiolanthanides, such as 177Lu. The present paper describes the synthesis of EDTMP and the development of a ready-to-use kit for the preparation of 177Lu-EDTMP and its quality control in accordance with the quality and safety criteria required for medicinal use. EDTMP was synthesized by a modified Mannich-type reaction, and the structure was characterized using NMR and IR spectroscopy. Optimization of radiolabeling conditions was done with two different salt forms of EDTMP. The labeling yield was checked by paper chromatography with radiation detection. Kit was developed as a lyophilized mixture of EDTMP and sodium bicarbonate in a maximum volume of 5 mL. Labeling efficiency, radionuclidic purity, radiochemical purity, sterility, and pyrogenicity analysis were performed as the quality control of the labeled kit. The analytical data for the structure determination and purity of the synthesized ligand were in agreement with authentic commercial samples used in radiopharmacy.177Lu-EDTMP complex was prepared using synthesized EDTMP ligand under optimized labeling conditions with high labelling yield (>99%). The radiolabeling yields of the EDTMP kit at room temperature after 30 min and 48 hours were 99.46% and 99.00%. The developed EDTMP kit enables an instant one-step preparation of the radiopharmaceutical of high radiochemical purity (>99%) and has a sufficiently long shelf life. This enables the routine production of the 177Lu-EDTMP in nuclear medicine clinics without requiring experienced staff.

Lutetium177-Labeled DOTA-Ibandronate: A Novel Radiopharmaceutical for Targeted Treatment of Bone Metastases.

Bone metastases of malignant tumors significantly threaten the patient survival and quality of life. We designed and synthesized a novel bisphosphonate radiopharmaceutical [68Ga- or 177Lu-labeled DOTA-Ibandronate(68Ga/177Lu-DOTA-IBA)] for targeted diagnosis and treatment of bone metastases. This study explored the basic biological characteristics of 177Lu-DOTA-IBA, guiding clinical translation and providing evidence for future clinical applications. The control variable method was used to optimize the optimal labeling conditions. The in vitro properties, biological distribution, and toxicity of 177Lu-DOTA-IBA were studied. Normal mice and tumor-bearing mice were imaged using micro SPECT/CT. With Ethics Committee approval, five volunteers were recruited for a preliminary clinical translation study. 177Lu-DOTA-IBA has a radiochemical purity of more than 98%, with good biological properties and safety. Blood clearance is fast and soft tissue uptake is low. Tracers are excreted mainly through the urinary system, targeting and continuously concentrating in the bones. Three patients experienced significant pain relief within 3 days after 177Lu-DOTA-IBA treatment (740-1110 MBq), lasting more than 2 months, with no toxic side effects. 177Lu-DOTA-IBA is easy to prepare and exhibits good pharmacokinetic characteristics. Low-dose 177Lu-DOTA-IBA is effective, well tolerated, and was associated with no significant adverse reactions. It is a promising radiopharmaceutical for the targeted treatment of bone metastases, controlling the progress of bone metastasis and improving survival and quality of life of patients with advanced bone metastasis.

Fluorescence labeling of C1-oxidized cellulose. Part 1: Method development

A novel method for fluorescence labeling and subsequent profiling of oxidized end groups along the molar mass distribution of cellulose has been developed. The notorious labeling reluctance of this group due to lactone (gluconolactone) formation was overcome by introducing amide-linked rather than ester-linked markers. A coupling method based on carbodiimide chemistry to bind the fluorophore N-(1-naphthyl)ethylenediamine (NED) was used and optimized with the model compound cellobionic acid towards appropriate reaction conditions and complete conversion. The reaction product, NED-labeled cellobionic acid (CBN), was obtained in 98 % yield, isolated by flash chromatography and comprehensively analytically characterized. CBN was used as a standard for HPLC quantification regarding reaction kinetics and stability analysis. The optimized labeling conditions were transferred to the heterogeneous derivatization of cellulose followed by GPC MALLS/IR/fluorescence analysis for molar mass-related profiling of oxidized reduced ends in cellulosic pulps.

Copper-mediated radioiodination and radiobromination via aryl boronic precursor and its application to 125I/77Br–labeled prostate-specific membrane antigen imaging probes

Prostate-specific membrane antigen (PSMA), expressed in prostate cancer cells, is being investigated extensively worldwide as a target for imaging and therapy of prostate cancer. Various radioiodinated PSMA imaging probes have been developed, and their structure has a peptidomimetic urea-based skeleton as a pharmacophore. For direct radioiodination of molecules containing these peptidomimetic structures, prior studies performed radioiododestannylation or electrophilic radioiodination of tyrosine residues. However, although these radiolabeling methods are frequently used, there are some issues with precursor toxicity and by-product production. Therefore, it is required to investigate a radiolabeling method that can be used for the radiosynthesis of radioiodinated PSMA imaging probes with urea-based peptidomimetic structures. We recently reported that copper-mediated radioiodination via a boronic precursor is an effective method for directly labeling a peptide. This radiohalogenation method was expected to be an effective method for radiosynthesis of PSMA imaging probes with a peptidomimetic structure. In this study, to confirm that this labeling method applies to the synthesis of the PSMA imaging probe, we synthesized PSMA imaging probes labeled with 125I and 77Br ([125I]mIB-PS and [77Br]mBrB-PS) using a copper-mediated radiohalogenation via common boronic precursors and investigated optimal boronic precursor and labeling conditions. As a result, the radiochemical yields of [125I]mIB-PS and [77Br]mBrB-PS were improved to > 93% at room temperature by optimizing the structure of the boronic precursor. We demonstrate that copper-mediated nucleophilic radiochemistry using a boronic precursor is a promising radiosynthetic method of PSMA imaging probes. Although we focused on the synthesis of PSMA imaging probes, the results in this study will also be useful for the synthesis of various radioiodine or radiobromine-labeled bioactive molecules.

Optimization of Multimodal Nanoparticles Internalization Process in Mesenchymal Stem Cells for Cell Therapy Studies.

Considering there are several difficulties and limitations in labeling stem cells using multifunctional nanoparticles (MFNP), the purpose of this study was to determine the optimal conditions for labeling human bone marrow mesenchymal stem cells (hBM-MSC), aiming to monitor these cells in vivo. Thus, this study provides information on hBM-MSC direct labeling using multimodal nanoparticles in terms of concentration, magnetic field, and period of incubation while maintaining these cells’ viability and the homing ability for in vivo experiments. The cell labeling process was assessed using 10, 30, and 50 µg Fe/mL of MFNP, with periods of incubation ranging from 4 to 24 h, with or without a magnetic field, using optical microscopy, near-infrared fluorescence (NIRF), and inductively coupled plasma mass spectrometry (ICP-MS). After the determination of optimal labeling conditions, these cells were applied in vivo 24 h after stroke induction, intending to evaluate cell homing and improve NIRF signal detection. In the presence of a magnetic field and utilizing the maximal concentration of MFNP during cell labeling, the iron load assessed by NIRF and ICP-MS was four times higher than what was achieved before. In addition, considering cell viability higher than 98%, the recommended incubation time was 9 h, which corresponded to a 25.4 pg Fe/cell iron load (86% of the iron load internalized in 24 h). The optimization of cellular labeling for application in the in vivo study promoted an increase in the NIRF signal by 215% at 1 h and 201% at 7 h due to the use of a magnetized field during the cellular labeling process. In the case of BLI, the signal does not depend on cell labeling showing no significant differences between unlabeled or labeled cells (with or without a magnetic field). Therefore, the in vitro cellular optimized labeling process using magnetic fields resulted in a shorter period of incubation with efficient iron load internalization using higher MFNP concentration (50 μgFe/mL), leading to significant improvement in cell detection by NIRF technique without compromising cellular viability in the stroke model.

基于精氨酸酶切的蛋白质C端肽段富集方法的优化及评估

基于聚合物的蛋白质C端反向富集策略是用于研究蛋白质C端最为广泛的策略之一。目前,基于胰蛋白酶(trypsin)切割精氨酸残基C端(ArgC型酶切)的蛋白C端组学方法对蛋白质C端的鉴定深度仍有待提高。为解决这一问题,该研究对此方法进行了优化和评估:建立了基于“V型”过滤装置的“一锅法”富集流程,避免了副反应的干扰,缩短了样本的制备时间;优化了蛋白水平乙酰化反应条件,最大限度地降低了丝氨酸、苏氨酸、酪氨酸残基上的副反应,提高了肽段鉴定的可信性;优化了基于固相萃取枪头膜片过滤柱(StageTip柱)的样品分离过程,使C端肽段的鉴定深度增加至原来的4倍。通过以上优化,按照肽段水平错误发现率(FDR)<0.01、离子分数(ion score)≥20,且C端带有乙醇胺修饰的数据筛选标准,从人HEK 293T细胞中共鉴定出696个蛋白质C端。若仅要求肽段水平FDR<0.01,鉴定数目进一步增加到933个,这是基于聚合物富集策略的蛋白质C端组学方法所得的最大数据集之一。探索了胰蛋白酶镜像酶(LysargiNase)切割精氨酸残基N端(ArgN型酶切)与不同肽段N端衍生化修饰组合对蛋白质C端鉴定数目和种类的影响,“LysargiNase酶切+肽段N端乙酰化”新策略在原有“胰蛋白酶酶切+肽段N端二甲基化”策略的基础上将鉴定蛋白质C端的种类提升了47%。综上,该研究通过对基于Arg型酶切的蛋白C端组学方法的优化,提升了C端肽段的鉴定深度,扩大了C端肽段鉴定的覆盖范围。该方法将有望成为系统性表征蛋白质C端的有力工具。

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.

Synthesis and Characterization of Multifunctional Nanovesicles Composed of POPC Lipid Molecules for Nuclear Imaging.

The integration of nuclear imaging analysis with nanomedicine has tremendously grown and represents a valid and powerful tool for the development and clinical translation of drug delivery systems. Among the various types of nanostructures used as drug carriers, nanovesicles represent intriguing platforms due to their capability to entrap both lipophilic and hydrophilic agents, and their well-known biocompatibility and biodegradability. In this respect, here we present the development of a labelling procedure of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)-based liposomes incorporating an ad hoc designed lipophilic NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) analogue, derivatized with an oleic acid residue, able to bind the positron emitter gallium-68(III). Based on POPC features, the optimal conditions for liposome labelling were studied with the aim of optimizing the Ga(III) incorporation and obtaining a significant radiochemical yield. The data presented in this work demonstrate the feasibility of the labelling procedure on POPC liposomes co-formulated with the ad hoc designed NOTA analogue. We thus provided a critical insight into the practical aspects of the development of vesicles for theranostic approaches, which in principle can be extended to other nanosystems exploiting a variety of bioconjugation protocols.

A Fluorescent Probe for the Specific Staining of Cysteine Containing Proteins and Thioredoxin Reductase in SDS-PAGE.

A naphthalimide-based fluorescent probe, Nap-I, with iodoacetamide as the alkylating group, has been synthesized, and its specific fluorescent staining of proteins containing cysteine (Cys) and selenocysteine (Sec) residues in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been evaluated. This molecule shows good fluorescence properties in the labeling of protein Cys/Sec residues, while reducing steric hindrance and minimizing changes in the water solubility of proteins. Reaction parameters, such as labeling time and pH, have been investigated, and the optimal labeling conditions for Cys-containing proteins have been determined. Thioredoxin reductase (TXNRD) is best stained at low pH. The probe Nap-I has been successfully used for the quantification of serum proteins and hemoglobin in Tan sheep serum, and TXNRD in Tan sheep liver and muscle has been labeled at low pH. Based on the probe Nap-I, we have also distinguished TXNRD1 and TXNRD2 by SDS-PAGE. The results showed that, compared with the normal microenvironment in which the protein resides, the lower the pH value, the greater the TXNRD activity.

High Coverage Profiling of Carboxylated Metabolites in HepG2 Cells Using Secondary Amine-Assisted Ultrahigh-Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Carboxylic metabolites are an important class of metabolites, which widely exist in mammals with various types. Chemical isotope labeling liquid chromatography-mass spectrometry (CIL-LC-MS) has been widely used for the detection of carboxylated metabolites. However, high coverage analysis of carboxylated metabolites in biological samples is still challenging due to improper reactivity and selectivity of labeling reagents to carboxylated metabolites. In this study, we used N-methylphenylethylamine (MPEA) to label various types of carboxylated metabolites including short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), long-chain fatty acids (LCFAs), polycarboxylic acids (polyCAs), amino acids (AAs), and aromatic acids. Additionally, metabolites containing other functional groups, such as phenol, sulfhydryl, and phosphate groups, could not be labeled under the conditions of MPEA labeling. After MPEA labeling, the detection sensitivity of carboxylic acids was increased by 1-2 orders of magnitude, and their chromatographic retention on a reversed-phase (RP) column was enhanced (RT > 3 min). Under optimized labeling conditions, we used MPEA and d3-N-methylphenylethylamine (d3-MPEA) for high coverage screening of carboxylated metabolites in HepG2 cells by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). As a result, a total of 403 potential carboxylated metabolites were obtained of which 68 were confirmed based on our established in-house chemically labeled metabolite database (CLMD). SCFAs, MCFAs, LCFAs, polyCAs, AAs, and aromatic acids were all detected in HepG2 cell extracts. Due to the successful identification of AAs, the current method increased the coverage of carboxylated metabolites compared with our previous work. Moreover, 133 and 109 carboxylated metabolites with changed contents were obtained in HepG2 cells incubated with curcumin and R-3-hydroxybutyric acid, respectively. In general, our established method realized high coverage analysis of carboxylated metabolites in HepG2 cells.

Higher-Order Structure Influences the Kinetics of Diethylpyrocarbonate Covalent Labeling of Proteins.

The combination of covalent labeling (CL) and mass spectrometry (MS) has emerged as a useful tool for studying protein structure due to its good structural coverage, the ability to study proteins in mixtures, and its high sensitivity. Diethylpyrocarbonate (DEPC) is an effective CL reagent that can label N-termini and the side chains of several nucleophilic residues, providing information for about 30% of the residues in the average protein. For DEPC to provide accurate structural information, the extent of labeling must be controlled to minimize label-induced structural perturbations. In this work, we establish a quantitative correlation between general protein structural factors and DEPC reaction rates by measuring the reaction rate coefficients for several model proteins. Using principal component and regression analyses, we find that the solvent accessible surface areas of histidine and lysine residues in proteins are the primary factors that determine a protein's reactivity toward DEPC, despite the fact that other more abundant residues, such as tyrosine, threonine, and serine, are also labeled by DEPC. From the statistical analysis, a model emerges that can be used to predict the reactivity of a protein based on its structure and sequence, allowing the optimal DEPC concentration to be chosen for a given protein. The resulting model is supported by cross-validation studies and by accurately predicting of the reactivity of five test proteins. Overall, our model reveals interesting insight into the reactivity of proteins with DEPC, and it will facilitate identification of optimal DEPC labeling conditions for proteins.

Efficient Labeling Of Mesenchymal Stem Cells For High Sensitivity Long-Term MRI Monitoring In Live Mice Brains.

BackgroundRegenerative medicine field is still lagging due to the lack of adequate knowledge regarding the homing of therapeutic cells towards disease sites, tracking of cells during treatment, and monitoring the biodistribution and fate of cells. Such necessities require labeling of cells with imaging agents that do not alter their biological characteristics, and development of suitable non-invasive imaging modalities.PurposeWe aimed to develop, characterize, and standardize a facile labeling strategy for engineered mesenchymal stem cells without altering their viability, secretion of FGF21 protein (neuroprotective), and differentiation capabilities for non-invasive longitudinal MRI monitoring in live mice brains with high sensitivity.MethodsWe compared the labeling efficiency of different commercial iron oxide nanoparticles towards our stem cells and determined the optimum labeling conditions using prussian blue staining, confocal microscopy, transmission electron microscopy, and flow cytometry. To investigate any change in biological characteristics of labeled cells, we tested their viability by WST-1 assay, expression of FGF21 by Western blot, and adipogenic and osteogenic differentiation capabilities. MRI contrast-enhancing properties of labeled cells were investigated in vitro using cell-agarose phantoms and in mice brains transplanted with the therapeutic stem cells.ResultsWe determined the nanoparticles that showed best labeling efficiency and least extracellular aggregation. We further optimized their labeling conditions (nanoparticles concentration and media supplementation) to achieve high cellular uptake and minimal extracellular aggregation of nanoparticles. Cell viability, expression of FGF21 protein, and differentiation capabilities were not impeded by nanoparticles labeling. Low number of labeled cells produced strong MRI signal decay in phantoms and in live mice brains which were visible for 4 weeks post transplantation.ConclusionWe established a standardized magnetic nanoparticle labeling platform for stem cells that were monitored longitudinally with high sensitivity in mice brains using MRI for regenerative medicine applications.

Detection and phenotyping of extracellular vesicles by size exclusion chromatography coupled with on-line fluorescence detection

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

The elusive actin cytoskeleton of a green alga expressing both conventional and divergent actins.

The green alga Chlamydomonas reinhardtii is a leading model system to study photosynthesis, cilia, and the generation of biological products. The cytoskeleton plays important roles in all of these cellular processes, but to date, the filamentous actin network within Chlamydomonas has remained elusive. By optimizing labeling conditions, we can now visualize distinct linear actin filaments at the posterior of the nucleus in both live and fixed vegetative cells. Using in situ cryo-electron tomography, we confirmed this localization by directly imaging actin filaments within the native cellular environment. The fluorescently labeled structures are sensitive to the depolymerizing agent latrunculin B (Lat B), demonstrating the specificity of our optimized labeling method. Interestingly, Lat B treatment resulted in the formation of a transient ring-like filamentous actin structure around the nucleus. The assembly of this perinuclear ring is dependent upon a second actin isoform, NAP1, which is strongly up-regulated upon Lat B treatment and is insensitive to Lat B–induced depolymerization. Our study combines orthogonal strategies to provide the first detailed visual characterization of filamentous actins in Chlamydomonas, allowing insights into the coordinated functions of two actin isoforms expressed within the same cell.

The Optimization Method for Synthesis of 99mTc-Rutin as Potential Radiotracer in The Development of Cancer Drugs From Flavonoid

Based on the Basic Health Research Data of Ministry of Health's of Indonesia in 2013, mortality rates from malignant and tumor malignancies in Indonesia are still high with prevalence of cancer is about 1.4%. Chemotherapy is still the primary choice in cancer modality that uses chemotherapeutic drugs to eradicate and inhibit the growth of cancer cells; however the cost this treatment is extremely high. Therefore, patient tends to seek alternative treatment such as consuming traditional herbal medicine for cancer treatment. Rutin is one of the attractive phytochemicals flavonoids because of its antioxidant activities. However, as traditional herbal medicine, its effectiveness is not yet been fully established due to the lack of scientific information. A radiotracer can be defined as a specific radiolabeled molecule that monitors the in vivo behaviour of a functional molecule, and can be used to provide biological information in a living system. Hence, to provide pharmacological information of rutin for cancer treatment, we synthesized radiolabeled flavonoid 99mTc-rutin as radiotracer. The aim of the present study is to develop 99mTc-rutin under varying conditions of rutin quantity, reducing agent concentration and incubation time. Labeling studies were performed by changing the selected parameters one by one and optimum labeling conditions were determined. After observing the conditions for maximum labeling efficiency, 99mTc-rutin was obtained with preparation of 700 μg of rutin with addition of 20 μg of SnCl2.2H2O as reductor and 1-3 mCi 99mTcO4- without any incubation. Radiochemical yield of 99mTc-rutin was determined with radio thin layer chromatography which was found 99.28 ± 0.14% and stable up to 4 hour. From the result of this study, the successfully labeled 99mTc-rutin can be used as a reference for following preclinical study. Furthermore radiolabeled 99mTc-rutin is expected as tools in research and development of rutin as cancer drugs from natural product to obtain detailed information its efficacy.Keywords: radiotracer, 99mTc-rutin, cancer, labeled compounds

An Efficient Automated Radiosynthesis and Bioactivity Confirmation of VMAT2 Tracer [18F]FP-(+)-DTBZ.

The aim of this study was to optimize the radiolabeling method of [18F]fluoropropyl-(+)-dihydrotetrabenazine ([18F]FP-(+)-DTBZ) to fulfill the demand of preclinical and clinical application. Optimized labeling conditions were performed by altering the molar ratio of precursor to base (P/B), base species, solvents, reaction temperature, reaction time, and precursor concentration through manual radiosynthesis of [18F]FP-(+)-DTBZ. The conditions with the highest radiochemical yield (RCY) were applied to automated radiosynthesis, and the crude product was purified with a Sep-Pak Plus C18 cartridge. Quality control and stability of [18F]FP-(+)-DTBZ were carried out by HPLC. In vitro cellular uptake and blocking assays were conducted in human neuroblastoma cell line SH-SY5Y. In vivo imaging with small animal positron emission tomography (microPET) was performed with Sprague-Dawley rats. Under the optimized conditions (P/K2CO3 = 1:8, heating at 120°C for 3min in dimethyl sulfoxide), an RCY of 88.7% was obtained with 1.0mg precursor. The optimized reaction conditions were successfully applied to an automated module and gave a high activity yield (AY) of 30-55% in about 40min with a > 99.0% radiochemical purity (RCP) and a > 44.4GBq/μmol molar activity (Am). Stability test displayed that the RCP retained > 98.0% in 8h in saline and in phosphate buffer saline (PBS, pH7.4). In vitro cellular uptake assay showed accumulation of [18F]FP-(+)-DTBZ in SH-SY5Y cells, which could be significantly inhibited by vesicular monoamine transporter 2 (VMAT2) inhibitor DTBZ. MicroPET images of rat brain displayed that the striatum showed the highest uptake with a standardized uptake value (SUV) of 3.91 ± 0.30 at ~ 70min. Co-injection with DTBZ (1.0mg/kg) resulted in a 75% decrease of the striatal SUV, confirming the specificity of [18F]FP-(+)-DTBZ to VMAT2. We obtained an optimized radiolabeling method of [18F]FP-(+)-DTBZ and successfully applied it to a commercial available module. The automated synthesis gave a high AY and RCP of [18F]FP-(+)-DTBZ with high and specific binding to VMAT2, facilitating its routine application for VMAT2 tracing.

Multiplexing Angiogenic Receptor Quantification via Quantum Dots.

Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multicell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell characteristics and response in tumor microenvironments. However, existing technologies for quantifying plasma membrane receptor tyrosine kinases (RTKs) lack multiplexing capabilities, limiting detailed characterization. Here, we use the unique spectral properties of quantum dots (Qdots) to optimize and dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, we reduce nonspecific binding between Qdot-conjugated antibodies and cells via buffer optimization. Second, we identify optimal labeling conditions by examining Qdot-conjugated antibody binding to five receptors: VEGFRs (VEGFR1 and VEGFR2), their coreceptor neuropilin1 (NRP1), and platelet-derived growth factor receptor (PDGFRα and PDGFRβ). We establish that 800-20 000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations, we demonstrate measurement of 1 100 VEGFR1 and 6 900 VEGFR2 per HUVEC. We induce ∼90% upregulation of VEGFR1 and ∼30% downregulation of VEGFR2 concentration via 24 h VEGF-A165 treatment. We observe no change in VEGFR1 or VEGFR2 concentration with 24 h VEGF-B167 treatment. We further apply Qdots to analyze HUVEC heterogeneity and observe that 24 h VEGF-A165 treatment induces a ∼15% decrease in VEGFR2 heterogeneity, but little to no change in VEGFR1 heterogeneity. We observe that VEGF-B167 induces little to no change in either VEGFR1 or VEGFR2 heterogeneity. Overall, we demonstrate experimental and analytical strategies for quantifying two or more RTKs at single-level using Qdots, which will help provide new insights into biological systems.

VMAT2 imaging agent, D6-[18F]FP-(+)-DTBZ: Improved radiosynthesis, purification by solid-phase extraction and characterization

ObjectivesRecently, a deuterated tracer, D6-[18F]FP-(+)-DTBZ, 9-O-hexadeutero-3-[18F]fluoropropoxyl-(+)-dihydrotetrabenazine ([18F]9), targeting vesicular monoamine transporter 2 (VMAT2) in the central nervous system, was reported as a useful imaging agent for the diagnosis of Parkinson's disease (PD). The production of [18F]9 was optimized and simplified by using solid-phase extraction (SPE) purification. MethodsThree major nonradioactive impurities were synthesized and characterized. The preparation of [18F]9 was optimized by using different labeling conditions, and an SPE purification method was evaluated. The influence of chemical impurities in the final dose of [18F]9 was assessed by an in vitro binding assay, an assay of the in vivo biodistribution in mice, and ex vivo and in vitro autoradiography of brain sections. ResultsOptimized fluorination conditions for [18F]9 were found – heating at 130 °C for 10 min in DMSO, and a high radiochemical yield and three major chemical impurities were observed. An SPE method involving a Sep-Pak® tC18 Plus Light cartridge with a two-step elution process was successfully implemented. This process gave a good radiochemical yield (38.7 ± 10.5%, decay corrected; radiochemical purity >99%) and low chemical impurities. An in vivo biodistribution study and autoradiography of brain sections showed that there was no significant difference between HPLC-purified and SPE-purified [18F]9. ConclusionA VMAT2 targeting imaging agent, D6-[18F]FP-(+)-DTBZ, [18F]9, was prepared by optimized labeling conditions and an easy SPE purification. This method offers a short preparation time and operational simplicity. In conjunction with PET imaging, this new VMAT2 agent might be a useful clinical tool for diagnosing PD.