Harnessing the Maltodextrin Transport Mechanism for Targeted Bacterial Imaging: Structural Requirements for Improved in vivo Stability in Tracer Design

Diagnosis and localization of bacterial infections remains a significant clinical challenge. Harnessing bacteria‐specific metabolic pathways, such as the maltodextrin transport mechanism, may allow specific localization and imaging of small or hidden colonies. This requires that the intrabacterial tracer accumulation provided by the transporter is matched by high serum stability of the tracer molecule. Herein, radiolabeled maltodextrins of varying chain lengths and with free nonreducing/reducing ends are reported and their behavior against starch‐degrading enzymes in the blood, which compromise their serum stability, is evaluated. Successful single‐photon emission computed tomography (SPECT) imaging is shown in a footpad infection model in vivo by using the newly developed model tracer, [^99mTc] MB1143 , and the signal is compared with that of ¹⁸F‐fluorodeoxyglucose positron emission tomography ([¹⁸F]FDG‐PET) as a nonbacterial specific marker for inflammation. Although the [^99mTc] MB1143 imaging signal is highly specific, it is low, most probably due to insufficient serum stability of the tracer. A series of stability tests with different ¹⁸F‐labeled maltodextrins finally yielded clear structural guidelines regarding substitution patterns and chain lengths of maltodextrin‐based tracers for nuclear imaging of bacterial infections.     

Synthesis,Characterization, and Initial Biological Evaluation of [99mTc]Tc‐Tricarbonyl‐labeled DPA‐α‐MSH Peptide Derivatives for Potential Melanoma Imaging

α‐Melanocyte stimulating hormone (α‐MSH) derivatives target the melanocortin‐1 receptor (MC1R) specifically and selectively. In this study, the α‐MSH‐derived peptide NAP‐NS1 (Nle‐Asp‐His‐d ‐Phe‐Arg‐Trp‐Gly‐NH₂) with and without linkers was conjugated with 5‐(bis(pyridin‐2‐ylmethyl)amino)pentanoic acid (DPA‐COOH) and labeled with [^99mTc]Tc‐tricarbonyl by two methods. With the one‐pot method the labeling was faster than with the two‐pot method, while obtaining similarly high yields. Negligible trans‐chelation and high stability in physiological solutions was determined for the [^99mTc]Tc‐tricarbonyl–peptide conjugates. Coupling an ethylene glycol (EG)‐based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [^99mTc]Tc‐tricarbonyl–peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the ^99mTc‐labeled peptides for in vivo imaging.     

Synthesis and characterization of poly(HPMA)‐APMA‐DTPA‐99mTc for imaging‐guided drug delivery in hepatocellular carcinoma

To develop a theranostic agent for diagnostic imaging and treatment of  hepatocellular carcinoma (HCC), poly(HPMA)‐APMA‐DTPA‐^99mTc (HPMA: N‐(2‐hydroxypropyl methacrylamide; APMA: N‐(3‐aminopropyl)methacrylamide; DTPA: diethylenetriaminepentaacetic acid) and DTPA‐^99mTc were synthesized and characterized, and their HCC targeting was tested by in vitro cellular uptake and in vivo tumor imaging in this study. Radioactivity of HCC cells incubated with poly(HPMA)‐APMA‐DTPA‐^99mTc was significant higher (16.40%) than that of the cells incubated with DTPA‐^99mTc (2.98%). Scintigraphic images of HCC in mice obtained at 8 h after injection of poly(HPMA)‐APMA‐DTPA‐^99mTc showed increased radioactivity compared with that in mice injected with DTPA‐^99mTc. The results of postmortem tissue radioactivity assay demonstrated higher radioactivity of HCC tumor tissues (2.69 ± 0.15% ID/g) from the tumor‐bearing mice injected with poly(HPMA)‐APMA‐DTPA‐^99mTc compared with that of HCC tumor tissues in the tumor‐bearing mice injected with DTPA‐^99mTc (0.83 ± 0.03 %ID/g), (P <0.001). These results first directly confirm the significant passive hepatocellular tumor targeting of HPMA copolymer. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Novel ether-containing ligands as potential technetium(I) heart agents

A novel pair of lipophilic cationic technetium complexes utilizing the ^99mTc-tricarbonyl core have been developed and evaluated for cardiac uptake. Di-(pyridyl-2-methyl)amine (DPA) and di-(imidazol-2-ylmethyl)amine (DIA) ligands were functionalized using aliphatic or aromatic ether substituents to provide the ligands 3 and 4. Octahedral complexes with the fac {^99mTc(CO)₃(ligand)}⁺ configuration were readily formed by reaction of ^99m[Tc(CO)₃(H₂O)₃]⁺ with the ether-containing tridentate ligands. The ^99mTc-tricarbonyl complexes, formed in >90% RCY and >90% RCP, were stable to transchelation in vitro against molar excesses of cysteine and histidine. Preliminary evaluation in rats after intravenous administration showed that the complexes concentrated in the heart muscle to an extent greater than surrounding tissue and blood. The ^99mTc-complex with derivatized DIA, {^99mTc(CO)₃(4)}⁺ (8), demonstrated 1.8% ID/g in the heart and a 90:1 heart to blood ratio at 120 min post-injection. These initial results provide support for an expanded evaluation of novel cationic ^99mTc-complexes for cardiac imaging.     

Selection and characterization of HER2/neu-binding affibody ligands

Affibody® (affibody) ligands that are specific for the extracellulardomain of human epidermal growth factor receptor 2 (HER2/neu)have been selected by phage display technology from a combinatorialprotein library based on the 58 amino acid residue staphylococcalprotein A-derived Z domain. The predominant variants from thephage selection were produced in Escherichia coli, purifiedby affinity chromatography, and characterized by biosensor analyses.Two affibody variants were shown to selectively bind to theextracellular domain of HER2/neu (HER2-ECD), but not to controlproteins. One of the variants, denoted His₆-Z_HER2/neu:4, wasdemonstrated to bind with nanomolar affinity (     

A New Bifunctional Chelator Enables Facile Biocoupling and Radiolabeling as the Basis for a Bioconjugation Kit

A new tridentate bifunctional chelator, N‐(‐2‐picolyl)(‐4‐hydroxy)(‐3‐amino)benzoic acid (PHAB), was designed to efficiently coordinate the [^99mTc(CO)₃]⁺ core and facilitate coupling reactions to biomolecules. The chelator can be procured in the form of the corresponding benzotriazole ester (PHAB‐OBT), which can be stored and used as a bioconjugation kit. PHAB‐OBT reacts with modified carbohydrates with high selectivity and efficiency in a single step in both aqueous and organic media. As is desirable for a kit, no complicated chemical bench work is required. Glycoconjugate postlabeling resulted in neutral radiolabeled glycans with high radiochemical yields. Prelabeling approaches were assessed by successive reaction of PHAB‐OBT with the [^99mTc(CO)₃]⁺ core and a modified galactose model. The radiolabeled galactose was obtained in 84 % yield as defined by HPLC analysis. Biodistribution of the radioactive ^99mTc‐labeled chelator, as well as the glycoconjugates, were examined in mice. Noticeably different biodistribution patterns were observed that reflect trends in the uptake of carbohydrate analogues by various organs.     

Comparative Assessment of Nanostructured Metal Oxides: A Potential Step Forward to Develop Clinically Useful 99Mo/99mTc Generators using (n,γ)99Mo

While the use of nanostructured metal oxides such as nanotitania, nanozirconia, nanoalumina, and mesoporous alumina, as column matrices constitutes a successful paradigm in the development of chromatographic ⁹⁹Mo/^99mTc generators using (n,γ)⁹⁹Mo, a comparative assessment of their properties is essential not only to determine the merits and bottlenecks of each sorbent but also to evaluate their usefulness commensurate with the specific activity of (n,γ)⁹⁹Mo obtained from different sources. Characteristics which were compared included the sorption capacity, zeta potential, shelf-life of the generator, radioactive concentration and purity of ^99mTc for radiopharmaceutical applications, and recovery of decayed Mo from spent generators. Mesoporous alumina was identified as the most suitable sorbent for ensuring sustainable production of clinical grade ⁹⁹Mo/^99mTc generators using low specific activity ⁹⁹Mo.     

Application of a macromolecular Sn (II) complex to the preparation of 99mTc radiopharmaceuticals

To develop insoluble Sn²⁺ complexes for the preparation of ^99mTc radiopharmaceuticals, the adsorption of Sn²⁺ to macroreticular chelating ion exchange resins having various functional groups was investigated. Among them, a resin containing aminophosphonic acid groups showed a high adsorption capacity for Sn²⁺, which was bound strongly to the resin by chelation. This macromolecular Sn²⁺ complex was very stable against hydrolysis and oxidation, and could be applied satisfactorily for the reduction of ^99mTc.     

Molecular assembly of multifunctional ⁹⁹(m)Tc radiopharmaceuticals using "clickable" amino acid derivatives

Synthetic strategies that enable the efficient and selective combination of different biologically active entities hold great promise for the development of multifunctional hybrid conjugates useful for biochemical and medical applications. Starting from side‐chain‐functionalized N(α)‐propargyl lysine derivatives, conjugates containing a ^99mTc‐based imaging probe for SPECT and two different moieties (e.g., tumor‐targeting vectors, pharmacological modifiers, affinity tags, or second imaging probes) can be assembled using the Cu^I‐catalyzed alkyne–azide cycloaddition in efficient one‐pot protocols. This strategy was successfully applied to the preparation of a ^99mTc‐labeled conjugate comprising a tumor‐targeting peptide sequence (bombesin(7–14)) and a low‐molecular‐weight albumin binder, a pharmacological modifier that prolongs the blood circulation time of the conjugate. Evaluation of the conjugate in vitro and in vivo provided promising results for its use as an imaging agent for the visualization of tumors positive for the gastrin‐releasing peptide receptor. The methodology presented herein provides an attractive synthetic tool for the preparation of multifunctional ^99mTc‐based radiopharmaceuticals with significant potential for a multitude of applications.     

Preparation and evaluation of nano-crystalline titania as sorbent for 99Mo/99mTc generator

In the presented work, nano-crystalline titania (TiO₂) was synthesized and evaluated as a novel chromatographic sorbent for the preparation of the medical ⁹⁹Mo/^99mTc generator. TiO₂ was synthesized by dissolving TiCl₄.6H₂O with isopropyl alcohol to form a semisolid mass that was extensively dried and washed. The physicochemical parameters and surface area of the synthesized nano-crystalline TiO₂ were evaluated. TiO₂ was packed as a chromatographic column to study the sorption of ⁹⁹Mo-polymolybdate (VI) anionic solution by dynamic equilibrium. The maximum sorption capacity of ⁹⁹Mo was ~141±2mg Mo/g TiO₂, thereby confirming its suitability for using low specific activity ⁹⁹Mo for the preparation of ⁹⁹Mo/^99mTc generator. The elution performance of the prepared ⁹⁹Mo/^99mTc generator based on Ti-⁹⁹Mo was evaluated. It showed high radiochemical purity (≥ 97% TcO₄^?), high radionuclidic purity (≥ 99.99%^99mTc) and high chemical purity; all of these specifications show the suitability for use in nuclear medicine applications.     

Separation of Carrier-Free 99mTc from 99Mo and 144Pr from 144Ce over a Column of Zirconium Arsenate

Abstract

Carrier-free ^99mTc and ¹⁴⁴Pr activities were separated from ⁹⁹Mo and ¹⁴⁴Ce, respectively, by using simple chemical procedures and a column of zirconium arsenate. A crystalline variety of zirconium arsenate was prepared by mixing a proportionate quantity of sodium arsenate solution in water with a solution of zirconium oxychloride in 2N HCl at 70°C. The ratio of zirconium:arsenate was 1:2. γ-Ray spectra of the separated ^99mTc, and β-decay studies of ¹⁴⁴Pr showed that both were of high radionuclidic purity. The overall separation processes were simple, clean, and in each case required less than half an hour, with quantitative yield. After separation, the daughter activities were allowed to form in the column, so that it was possible to obtain these freshly formed species by eluting the column with suitable reagents.     

Ion Exchange of the Organometallic Aqua‐Ion fac‐[99mTc(CO)3(H2O)3]+ from Aqueous Solutions

Dynamic (column) studies on the ion exchange of triaquatricarbonyltechnetium(I) cation, fac‐[^99mTc(CO)₃(H₂O)₃]⁺ (1), in the system: amphoteric ion exchange resin (Purolite S‐950) ‐ aqueous HNO₃ solutions (0.05–2 M) corroborate the +1 charge of 1. Stability constants of fac‐[^99mTc(CO)₃(H₂O)_3?nCl_n]^1?n complexes, determined from the distribution coefficients in the system anion exchanger (Dowex 1X4) ‐ aqueous HCl solutions (0.1–12 M) differ from the literature values determined at a constant ionic strength. This difference, resulting from the decrease in the activity of water with increasing HCl concentration, may be a measure of the effect of non‐constant ionic strength on ion‐exchange equilibria. Weak acidic properties of 1 in aqueous solution at n.c.a. (no carrier added) level (pK_a ?9) have been demonstrated by paper electrophoresis.     

Evaluation of 5H-Thiazolo[3,2-α]pyrimidin-5-ones as Potential GluN2A PET Tracers

We describe here our efforts to develop a PET tracer for imaging GluN2A-containing NMDA receptors, based on a 5H-thiazolo[3,2-α]pyrimidin-5-one scaffold. The metabolic stability and overall properties could be optimized satisfactorily, although binding affinities remained a limiting factor for in vivo imaging. We nevertheless identified 7-(((2-fluoroethyl)(3-fluorophenyl)amino)-methyl)-3-(2-(hydroxymethyl)cyclopropyl)-2-methyl-5H-thiazolo-[3,2-α]pyrimidin-5-one ([¹⁸F] 7b ) as a radioligand providing good-quality images in autoradiographic studies, as well as a tritiated derivative, 2-(7-(((2-fluoroethyl)(4-fluorophenyl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-α]pyrimidin-3-yl)cyclopropane-1-carbonitrile ([³H₂] 15b ), which was used for the successful development of a radioligand binding assay. These are valuable new tools for the study of GluN2A-containing NMDA receptors, and for the optimization of allosteric modulators binding to the pharmacophore located at the dimer interface of the GluN1-GluN2A ligand-binding domain.     

Blue light-emitting diodes from 2-(10-naphthylanthracene)-spiro[fluorene-7,9′-benzofluorene] host material

A novel, spiro-type host material 2-(10-naphthylanthracene)-spiro[fluorene-7,9′-benzofluorene] was prepared by reacting 2-bromo-spiro[fluorene-7,9′-benzofluorene] with 9-(2-naphthylanthracene)-10-boronic acid via the Suzuki reaction. 2-4′-(Phenyl-4-vinylbenzeneamine)phenyl-spiro[fluorene-7,9′-benzofluorene], 4-[2-naphthyl-4′(phenyl-4-vinylbenzeneamine)]phenyl and diphenyl-[4-(2-[1,1;4,1]terphenyl-4-yl-vinyl)-phenyl]-amine were used as dopant materials. Devices with the configuration of ITO/N,N′-bis[4-(di-m-tolylamino)phenyl]-N,N′-diphenylbiphenyl-4,4′-diamine)/bis[N-(1-naphthyl)-N-phenyl]benzidine/2-(10-naphthylanthracene)-spiro[fluorene-7,9-benzofluorene]:5% dopant/aluminum tris(8-hydroxyquinoline)/Al-LiF showed a maximum power efficiency of 3.7 cd/A at 17.93 mA/cm² and a maximum luminance of 5018 cd/m² at 10 V with a turn-on voltage of 4.5 V.     

In Vivo Albumin-Binding of a C-Functionalized Cyclam Platform for 64Cu-PET/CT Imaging in Breast Cancer Model

An improved glucose-chelator-albumin bioconjugate (GluCAB) derivative, GluCAB-2^Mal, has been synthesized and studied for in vivo ⁶⁴Cu-PET/CT imaging in breast cancer mice models together with its first-generation analogue GluCAB-1^Mal. The radioligand works on the principle of tumor targeting through the enhanced permeability and retention (EPR) effect with a supportive role played by glucose metabolism. [⁶⁴Cu]Cu-GluCAB-2^Mal (99 % RCP) exhibited high serum stability with immediate binding to serum proteins. In vivo experiments for comparison between tumor targeting of [⁶⁴Cu]Cu-GluCAB-2^Mal and previous-generation [⁶⁴Cu]Cu-GluCAB-1^Mal encompassed microPET/CT imaging and biodistribution analysis in an allograft E0771 breast cancer mouse model. Tumor uptake of [⁶⁴Cu]Cu-GluCAB-2^Mal was clearly evident with twice as much accumulation as compared to its predecessor and a tumor/muscle ratio of up to 5 after 24 h. Further comparison indicated a decrease in liver accumulation for [⁶⁴Cu]Cu-Glu-CAB-2^Mal.     

A Click Approach to Structurally Diverse Conjugates Containing a Central Di‐1,2,3‐triazole Metal Chelate

Assemble & chelate : Click chemistry enables the efficient and selective synthesis of structurally diverse conjugates containing a central di‐1,2,3‐triazole chelator for complexation with [^99mTc(CO)₃]⁺. Use of appropriate building blocks allows the modulation of pharmacological relevant characteristics of the conjugate, or the introduction of secondary probes suitable for imaging modalities other than single photon emission computed tomography (SPECT).

     

The “Carbonyl Story” and Beyond; Experiences,Lessons and Implications

The complex [^99mTc(OH₂)₃(CO)₃]⁺ has become a versatile building block in radiopharmaceutical chemistry, applied by many groups worldwide. However, despite widespread efforts, only one compound has made it right the way through clinical trials. Along the way from its discovery to its development into an eventual product, the author experienced issues that he would handle differently in retrospect. In this article, these experiences are turned into “lessons” that might be helpful for young researchers finding themselves in similar situations. Beside issues with patenting and company strategies, the carbonyl story has provided scientific implications beyond its own story, and insights from which any future ^99mTc-based chemistry for radiopharmacy or molecular imaging might benefit.     

In Vitro and in Vivo Evaluation of Lactoferrin-Conjugated Liposomes as a Novel Carrier to Improve the Brain Delivery

In this study, lactoferrin-conjugated PEGylated liposomes (PL), a potential drug carrier for brain delivery, was loaded with radioisotope complex, ^99mTc labeled N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (^99mTc-BMEDA) for in vitro and in vivo evaluations. The hydrophilicity of liposomes was enhanced by PEGylation which was not an ideal brain delivery system for crossing the blood brain barrier (BBB). With the modification of a brain-targeting ligand, lactoferrin (Lf), the PEGylated liposome (PL) might become a potential brain delivery vehicle. In order to test the hypothesis in vitro and in vivo, ^99mTc-BMEDA was loaded into the liposomes as a reporter with or without Lf-conjugation. The mouse brain endothelia cell line, bEnd.3 cells, was cultured to investigate the potential uptake of liposomes in vitro. The in vivo uptake by the mouse brain of the liposomes was detected by tissue biodistribution study. The results indicated that Lf-conjugated PEGylated liposome showed more than three times better uptake efficiency in vitro and two-fold higher of brain uptake in vivo than PEGlyated liposome. With the success of loading the potential Single Photon Emission Tomography (SPECT) imaging probe, ^99mTc-BMEDA, Lf-PL might serve as a promising brain delivery system for loading diagnostics or therapeutics of various brain disorders.     

Effects of serum proteins on intracellular uptake and cytotoxicity of carbon nanoparticles

To explore the effects of the novel properties of carbon nanoparticles (CNPs) on cytotoxicity, the adsorption of serum proteins in cell culture medium on multi-walled carbon nanotubes and three kinds of carbon blacks was investigated. The uptake of CNPs by Hela cells was measured quantitatively using ^99mTc radionuclide labeling and tracing techniques, and the dependence of CNPs uptake on serum proteins was examined. The cytotoxicity of CNPs in medium with and without serum was assayed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. It was found that cellular uptake was much higher in cells exposed to CNPs in serum-free culture medium than those in culture medium with serum. Serum proteins adsorbed on CNPs attenuated the inherent cytotoxicity of CNPs, and the extent of toxicity attenuation increased with increasing amounts of serum proteins adsorbed on CNPs. The possible reasons responsible for the considerable influence of serum proteins on cytotoxicity were indicated.     

Sorption Behavior of Carrier-Free Technetium-99m on Zinc Dust

Abstract

The sorption behavior of ^99mTc on zinc dust was studied as a function of pH. Optimum conditions have been found for the preconcentration of traces of Tc by zinc. The influence of surface-active substances and of complexing agents on the sorption of trace amounts of Tc on zinc dust has been also studied.