Good Labeling Efficiency Research Articles

Pressed Solid Target Production of 89Zr and its Application for Antibody Labelling.

Zirconium-89 ( 89Zr, t1/2=3.27d) is an important + emitting radionuclide used in Positron Emission Tomography (PET) immuno studies due to its unique characteristics and increased demand due to simple and cost-effective production capacity. Production of 89Zr is achieved primarily through solid natural yttrium targets via different target preparation methodologies, such as electrodeposition, pressed foils, and spark plasma sintering. In this study, we have investigated the pressed solid target methodology. The Yttrium Oxide (Y2O3) powder was pressed to pellet form and stacked over a different back support plate, such as platinum (Pt), niobium (Nb), and tantalum (Ta). The target was irradiated with approximately 12 MeV proton beam for 10-60 minutes at 20µA current. The irradiated target was purified through a solid phase extraction method via hydroxamate-based resin by manual or automatic approach. The purified 89Zr was analyzed using gamma scintigraphy, and specific activity was calculated through Deferoxamine (DFO) chelation. 89Zr radionuclide via pressed target was effectively produced with a production yield of 20-30 MBq/µA.h, and the purification was achieved in 35 minutes with (87.46)% average recovery and >98% purity while using automated purification, but manual purification took 2 hours with (91 ± 2)% recovery and >98% purity. The production yield was comparable to the reported pressed target approach. Deferoxamine (DFO) chelation with 89Zr-oxalate was performed with purity >98% and specific activity of 25-30 µCi/mmol. In this study, we explored the production of 89Zr by pressed targets and purification via manual or automated methods with good radionuclide purity. The chelation with DFO or its analog was performed with good labeling efficiency and stability</p>.

Abstract 325: Nascent proteome analysis of tumor cells and their microenvironment in cultured human tumor tissues

Abstract Solid tumors are often considered as abnormal organs composed of the cancerous cells and their surrounding tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. The heterotypic interactions between this diversity of cell types within the TME are maintained through a wide variety of secreted proteins, resulting in a favorable milieu for the progression of the malignancy. The interactions between tumor cells and TME are complex and remain poorly understood. Here we investigated this by developing a unique nascent proteomic approach in tumor tissues.Precision cut cancer tissue slices (PCCTS) maintain tissue heterogeneity with different cell types and preserved TME. Cultivation of PCCTS provides an ex vivo model for tumor tissues. We developed an approach for PCCTS's nascent proteome analysis, using pulsed-SILAC (stable isotope labeling with amino acids in cell culture) labeling combined with click-chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon applying a cellular perturbation. It is a powerful tool to selectively enrich secretory proteins from culture media even with presence of serum. Primary human ovarian tumors (phOVT) and patient derived xenografts (PDX) were used to produce the PCCTS with thickness of 150µm to 300µm. The different cell types and extracellular matrix of PCCTS make the depletion period of cells from the amino acids (methionine, lysine and arginine) prior the AHA-SILAC treatment difficult to define. The PCCTS need longer depletion periods than the 2D cell culture, the longer the depletion period the better depletion efficiency. Following, PCCTS were cultured in AHA-SILAC media and treated with cisplatin. PCCTS and culture media (containing secreted proteins) were harvested; newly synthesized proteins were enriched via click-chemistry and analyzed with mass spectrometry. The labeling time of 10 to 12h showed a good labeling efficiency of more than 60%, still further optimizations are needed. Different PCCTS showed various labeling efficiency indicating the patients heterogeneity. The nascent proteome analysis with cisplatin treatment demonstrated different protein regulations in patients suggesting different drug responses. STRING analysis can be applied to predict the protein-protein interactions. The immunohistochemical staining of the same PCCTS can be processed to further validate the results of the proteome analysis. In conclusion, we established a pulsed SILAC-AHA treatment approach for the PCCTS with the TME. This unique approach allows tracking the compositional and dynamic changes within the proteome and monitoring the direct proteome response at rapid time scale. It can be used to reveal a part of the proteome that has been poorly understood in the tumor tissues and contribute to studying cellular communication and finding new therapeutic targets. Citation Format: Meng Dong, Karim Aljakouch, Kathrin Böpple, Bernd Winkler, Julia Schüler, Frank Essmann, Hans-Georg Kopp, Jeroen Krijgsveld, Walter E. Aulitzky. Nascent proteome analysis of tumor cells and their microenvironment in cultured human tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 325.

Preparation of dual-modality yttrium-90 radiolabeled nanoparticles for therapeutic investigation

Abstract Magnetic Fe3O4 nanoparticles with narrow size distribution were synthesized by simple and high yielded co-precipitation technique using ferrous salts with a molar ratio of Fe3+/Fe2+=2. After coating of the nanoparticles with Stöber silica (SiO2@Fe3O4), nanoparticles were functionalized by amine groups. Then chelator molecules diethylenetriaminepentaacetic (DTPA) and tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were coupled to the APTS-SiO2@Fe3O4 to chelating Y-90 radiometal that makes these nanoparticles a suitable agent for therapeutic application as dual-modality PET/MRI imaging. The results show the coupling of DTPA takes place better than DOTA. Synthesis of magnetic nanoparticles (MNPs) was followed by structure identification using XRD, SEM, TGA and IR techniques. In order to trace MNPs biodistribution, the radiolabeled MNPs-DTPA were prepared using 90Y (production of 90Y/90Sr generator) with a good labeling efficiency (about 92%, RTLS method). The biodistribution of the radiolabeled MNPs was checked in normal male rats up to 24 h compared to free Y3+. The data shows that the tracer accumulation is in reticuloendothelial tissue while the stability of the complex is highly retained.

Relative quantification of amine-containing metabolites using isobaric N,N-dimethyl leucine (DiLeu) reagents via LC-ESI-MS/MS and CE-ESI-MS/MS.

Tandem mass spectrometry (MS/MS)-based relative quantification by isobaric labeling is a useful technique to compare different metabolic expression levels in biological systems. For the first time, we have labeled primary and secondary amine-containing small molecules using 4-plex isobaric N,N-dimethyl leucine (DiLeu) to perform relative quantification. Good labeling efficiency and quantification accuracy were demonstrated with a mixture of 12 metabolite standards including amino acids and small molecule neurotransmitters. Labeling amine-containing metabolites with DiLeu reagents also enabled the separation of polar metabolites by nanoRPLC and improved the detection sensitivity by CE-ESI-MS. The 4-plex DiLeu labeling technique combined with LC-MS/MS and CE-MS/MS platforms were applied to profile and quantify amine-containing metabolites in mouse urine. The variability of concentrations of identified metabolites in urine samples from different mouse individuals was illustrated by the ratios of reporter ion intensities acquired from online data-dependent analysis.

Microwave-assisted 18O labeling of Fmoc-protected amino acids.

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of (18)O-labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave-assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency.

In-Vivo Biodistribution and Safety of 99mTc-LLP2A-HYNIC in Canine Non-Hodgkin Lymphoma

Theranostic agents are critical for improving the diagnosis and treatment of non-Hodgkin Lymphoma (NHL). The peptidomimetic LLP2A is a novel peptide receptor radiotherapy candidate for treating NHL that expresses the activated α4β1 integrin. Tumor-bearing dogs are an excellent model of human NHL with similar clinical characteristics, behavior, and compressed clinical course. Canine in vivo imaging studies will provide valuable biodistribution and affinity information that reflects a diverse clinical population of lymphoma. This may also help to determine potential dose-limiting radiotoxicity to organs in human clinical trials. To validate this construct in a naturally occurring model of NHL, we performed in-vivo molecular targeted imaging and biodistribution in 3 normal dogs and 5 NHL bearing dogs. 99mTc-LLP2A-HYNIC-PEG and 99mTc-LLP2A-HYNIC were successfully synthesized and had very good labeling efficiency and radiochemical purity. 99mTc-LLP2A-HYNIC and 99mTc-LLP2A-HYNIC-PEG had biodistribution in keeping with their molecular size, with 99mTc-LLP2A-HYNIC-PEG remaining longer in the circulation, having higher tissue uptake, and having more activity in the liver compared to 99mTc-LLP2A-HYNIC. 99mTc-LLP2A-HYNIC was mainly eliminated through the kidneys with some residual activity. Radioactivity was reduced to near-background levels at 6 hours after injection. In NHL dogs, tumor showed moderately increased activity over background, with tumor activity in B-cell lymphoma dogs decreasing after chemotherapy. This compound is promising in the development of targeted drug-delivery radiopharmaceuticals and may contribute to translational work in people affected by non-Hodgkin lymphoma.

In vivo PET imaging and biodistribution of radiolabeled gold nanoshells in rats with tumor xenografts

Here we report the radiolabeling of gold nanoshells (NSs) for PET imaging in rat tumor model. A conjugation method was developed to attach NSs with the radionuclide, 64Cu. The resulting conjugates showed good labeling efficiency and stability in PBS and serum. The pharmacokinetics of 64Cu-NS and the controls ( 64Cu-DOTA and 64Cu-DOTA-PEG2K) were determined in nude rats with a head and neck squamous cell carcinoma xenograft by radioactive counting. Using PET/CT imaging, we monitored the in vivo distribution of 64Cu-NS and the controls in the tumor-bearing rats at various time points after their intravenous injection. PET images of the rats showed accumulation of 64Cu-NSs in the tumors and other organs with significant difference from the controls. The organ biodistribution of rats at 46 h post-injection was analyzed by radioactive counting and compared between the 64Cu-NS and the controls. Different clearance kinetics was indicated. Neutron activation analysis (NAA) of gold concentration was performed to quantify the amount of NSs in major tissues of the dosed rats and the results showed similar distribution. Overall, PET images with 64Cu had good resolution and therefore can be further applied to guide photothermal treatment of cancer.

99mTc-HMPAO-labeled liposomes: an investigation into the effects of some formulation factors on labeling efficiency and in vitro stability

Technetium-99m complex of hexamethyl-propylene-amineoxime (HMPAO) is used as an efficient agent to label liposomes. For this, 99mTc-HMPAO is incubated with preformed liposomes that contain glutathione (GSH). Effect of GSH and lipid concentration on labeling efficiency, as well as the effect of lipid composition on in vitro stability of labeled liposomes, was investigated in the present study. d,l-HMPAO was synthesized and kits including d,l-HMPAO and SnCl2.2H2O were optimized at 0.5 mg HMPAO, 5.0 microg SnCl2.2H2O and pH 7, and lyophilized. DSPC/CHOL (molar ratio 2:1) liposomes encapsulating GSH were labeled with 99mTc-HMPAO prepared kits. Increase of GSH concentration in hydration buffer from 5 to 200 mM during liposome preparation resulted in a broad labeling efficiency of liposomes ranging from 4.16% to 69.81%. An initial approximate concentration of 100 mM GSH in the hydration buffer seems to be appropriate for a good labeling efficiency. At the optimum concentration of GSH, change of the total initial lipid concentration from 10 to 70 mM did not produce a remarkable difference in labeling efficiency. Study of the effect of lipid composition on the stability of liposomes showed that all three kinds of labeled liposomes composed of DSPC/CHOL, DPPC/CHOL and DMPC/CHOL (molar ratio 2:1) had good in vitro stability in human plasma at 37 degrees C for 48 h; however, employing DSPC resulted in the most stable ones.

A new bifunctional chelate, N-(2-hydroxy-3,5-dimethylbenzyl)-N'-(2-hydroxy-5-(bromoacetamido)benzyl)ethylenediamine-N,N'-diacetic acid (BrMe2HBED): an effective conjugate for radiometals and antibodies

A new bifunctional chelate, N-(2-hydroxy-3,5-dimethylbenzyl)-N{prime}-(2-hydroxy-5-(bromoacetamido)benzyl)ethylenediamine-N,-N{prime}-diacetic acid (BrMe{sub 2}HBED), was designed and synthesized to bind trivalent cationic metals with monoclonal antibodies. The stability constants (log values) for indium complexed with a similar ligand, HBED, were increased over those of more commonly used ligands DTPA and EDTA. Predictably, the increased metal-ligand complex stability would expedite the in vivo clearance from nontarget regions and perhaps enhance the localization of the radiolabeled antibody (Ab). BrMe{sub 2}HBED was conjugated with the Ab (24 h) and then radiolabeled with indium-111 citrate (24 h). Additionally, the Ab was radiolabeled by using conventional methods ({sup 111}In-DTPA and {sup 125}I-lactoperoxidase) and then compared by measuring the in vitro stability, in vitro immunoreactivity(IR), and in vivo distribution and clearance. A 10:1 BrMe{sub 2} HBED:Ab mole ratio resulted in good labeling efficiency with {sup 111}In and more importantly a very high IR. In a hamster tumor model, {sup 111}In-BrMe{sub 2} HBED-labeled monoclonal antibody (1A3) had high uptake in the tumor tissue and preferable blood clearance compared to either of the more conventional radiolabeled 1A3 monoclonal antibodies ({sup 111}In-DTPA or {sup 125}I-lactoperoxidase). 49 refs., 4 figs., 8 tabs.

Backscattered Electron Imaging of Colloidal Gold Markers For Leukocyte Surface Antigens

Markers for immuno-scanning electron microscopy had been, so far, selected for easy identification based on their distinctive shape (for example, haemocyanin, bacteriophage T4). These markers were always viewed in the secondary electron imaging (SEI) mode. Their size was not posing much of a problem of resolution for commercially available SEM, but was likely to prevent good labeling efficiency, due to steric hindrance phenomena. Higher labeling efficiency necessitates the use of markers of smaller size which, unfortunately, can rarely be unambiguously recognized in topographical SE images.To correlate antigenic distribution with small cell surface structures like microvilli or coated pits, markers of smaller size (i.e., in the 20 nm range) must be used and clearly identified on the surface of well preserved cells. With the availability of colloidal gold particles complexed with various ligands this became possible, particularly if the labeled cells are viewed in the backscattered imaging (BEI) mode of the SEM, therefore basing the identification of the marker on its atomic number contrast rather than on its topographical contrast.

A novel approach for scanning electron microscopy of colloidal gold-labeled cell surfaces.

A method is described for the use of scanning electron microscopy on the surface of gold-labeled cells. It includes the use of 45- or 20-nm colloidal gold marker conjugated with Staphylococcal protein A. The marker is best recognized on the basis of its atomic number contrast by using the backscattered electron imaging mode of the scanning electron microscope. When the backscattered electron signal is mixed with the secondary electron signal, an optimum correlation between the distribution of the labeled sites and the cell surface structures is demonstrated. The method is illustrated by its application to the identification of human circulating granulocytes. Its good resolution, high contrast, and good labeling efficiency offers a promising approach to the specific localization of cell surface antigenic sites labeled with particles of colloidal gold.