Toward Personalized Medicine: One Chelator for Imaging and Therapy with Lutetium-177 and Actinium-225.

We report a nonadentate bispidine (3,7-diazabicyclo[3.3.1]nonane) that unveils the potential to bind theranostically relevant radionuclides, including indium-111, lutetium-177, and actinium-225 under mild labeling conditions. This radiopharmaceutical candidate allows the simultaneous application of imaging and treatment (radionuclide theranostics) without changing the type of the bioconjugate; that is, it allows the strong binding to an imaging and a therapeutic radionuclide by the same chelator. Since sophisticated coordination chemistry is required to achieve high thermodynamic and kinetic stability (inertness), it is not surprising that only a few chelators have been reported that are able to strongly bind several radionuclides to a satisfactory extent. Bispidine-derived ligands have proven to be ideal for di- and trivalent metal ions with generally fast complexation kinetics and high in vitro and in vivo stabilities. The presented (radio)complexes are formed under mild conditions (pH 6, <40 °C) and exhibit thermodynamic stability and inertness in human serum comparable to the corresponding DOTA complexes. The bispidine-based complexing agent was conjugated to a peptide, targeting somatostatin type 2 receptors (SSTR2), overexpressed on neuroendocrine tumors. The 177Lu- and 225Ac-labeled conjugates were investigated, considering their binding to two different SSTR2-positive cell lines, including the human pancreatic carcinoid tumor (BON-SSTR2+) and the murine pheochromocytoma cell line (MPC). The biodistribution and accumulation pattern in MPC tumor-bearing mice was also evaluated. The LuIII and AcIII complexes studied show how ligand structures can be optimized in general by extending the denticity and varying the donor set in order to allow for fast complex formation and medically relevant inertness.

H2pyhox - Octadentate Bis(pyridyloxine).

A new versatile chelating ligand for intermediate size and softness radiometals [64Cu]Cu2+ and [111In]In3+, H2pyhox, was synthesized by introducing pyridine as a new donor moiety to complement 8-hydroxyquinoline on an ethylenediamine backbone. The combination of pyridine and oxine as donor sets was explored through structural analysis, and crystals of the three metal complexes with Cu2+, La3+, and In3+ demonstrate how the ligand adapts to accommodate metal ions of different sizes and charge. Exhaustive in-batch UV solution studies characterized the protonation constants of the free ligand as well as the formation constants of the metal complexes with Cu2+, In3+, and La3+. Preliminary concentration-dependent radiolabeling studies with [111In]In3+ and [64Cu]Cu2+ show the robustness of H2pyhox to successfully coordinate both radiometals under mild conditions (<15 min, room temperature, pH 6). H2pyhox is the first oxinate ligand to successfully radiolabel [225Ac]Ac3+, albeit only at high concentrations (0.1-1 mM) with gentle heating to 37 °C. Whole serum, protein, and ligand challenge assays further demonstrate the kinetic inertness of the [111In]In3+ and [64Cu]Cu2+ radiometal-ligand complexes, confirming H2pyhox to be a promising versatile radiopharmaceutical chelator.

Active Targeting of Dendritic Polyglycerols for Diagnostic Cancer Imaging.

Active tumor targeting involves the decoration of nanomaterials (NMs) with oncotropic vector biomolecules that selectively recognize certain antigens on malignant cells or in the tumor microenvironment. This strategy can facilitate intracellular uptake of NM through specific interactions such as receptor-mediated endocytosis and can lead to prolonged retention in the malignant tissues by preventing rapid efflux from the tumor. Here, the design of actively targeting, renally excretible bimodal dendritic polyglycerols (dPGs) for diagnostic cancer imaging is described. Single-domain antibodies (sdAbs) specifically binding to the epidermal growth factor receptor (EGFR) are employed herein as targeting warheads owing to their small size and high affinity for their corresponding antigen. The dPGs equipped with EGFR-targeting feature are compared head-to-head with their nontargeting counterparts in terms of interaction with EGFR-overexpressing cells in vitro as well as accumulation at receptor-positive tumors in vivo. Experimental results reveal a higher specificity and preferential tumor accumulation for the α-EGFR dPGs, resulting from the introduction of active targeting capabilities on their backbone. These results highlight the potential for improving the tumor uptake properties of dPGs by strategic use of sdAb functionalization, which can ultimately prove useful to the development of ultrasmall NM with highly specific tumor accumulation.

Synthesis and Characterization of an Epidermal Growth Factor Receptor-Selective RuII Polypyridyl-Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy.

There is a current surge of interest in the development of novel photosensitizers (PSs) for photodynamic therapy (PDT), as those currently approved are not completely ideal. Among the tested compounds, we have previously investigated the use of RuII polypyridyl complexes with a [Ru(bipy)2 (dppz)]2+ and [Ru(phen)2 (dppz)]2+ scaffold (bipy=2,2'-bipyridine; dppz=dipyrido[3,2-a:2',3'-c]phenazine; phen=1,10-phenanthroline). These complexes selectively target DNA. However, because DNA is ubiquitous, it would be of great interest to increase the selectivity of our PDT PSs by linking them to a targeting vector in view of targeted PDT. Herein, we present the synthesis, characterization, and in-depth photophysical evaluation of a nanobody-containing RuII polypyridyl conjugate selective for the epidermal growth factor receptor (EGFR) in view of targeted PDT. Using ICP-MS and confocal microscopy, we could demonstrate that our conjugate has high selectivity for the EGFR receptor, which is a crucial oncological target because it is overexpressed and/or deregulated in a variety of solid tumors. However, in contrast to expectations, this conjugate was found to not produce reactive oxygen species (ROS) in cancer cells and is therefore not phototoxic.

Versatile Bispidine-Based Bifunctional Chelators for 64 CuII -Labelling of Biomolecules.

Bifunctional chelators as parts of modular metal-based radiopharmaceuticals are responsible for stable complexation of the radiometal ion and for covalent linkage between the complex and the targeting vector. To avoid loss of complex stability, the bioconjugation strategy should not interfere with the radiometal chelation by occupying coordinating groups. The C9 position of the very stable CuII chelator 3,7-diazabicyclo[3.3.1]nonane (bispidine) is virtually predestined to introduce functional groups for facile bioconjugation as this functionalisation does not disturb the metal binding centre. We describe the preparation and characterisation of a set of novel bispidine derivatives equipped with suitable functional groups for diverse bioconjugation reactions, including common amine coupling strategies (bispidine-isothiocyanate) and the Cu-free strain-promoted alkyne-azide cycloaddition. We demonstrate their functionality and versatility in an exemplary way by conjugation to an antibody-based biomolecule and validate the obtained conjugate in vitro and in vivo.

Chelation in One Fell Swoop: Optimizing Ligands for Smaller Radiometal Ions.

[44/47Sc]Sc3+, [68Ga]Ga3+, and [111In]In3+ are the three most attractive trivalent smaller radiometalnuclides, offering a wide range of distinct properties (emission energies and types) in the toolbox of nuclear medicine. In this study, all three of the metal ions are successfully chelated using a new oxine-based hexadentate ligand, H3glyox, which forms thermodynamically stable neutral complexes with exceptionally high pM values [pIn (34) > pSc (26) > pGa (24.9)]. X-ray diffraction single crystal structures with stable isotopes revealed that the ligand is highly preorganized and has a perfect fit to size cavity to form [Sc(glyox)(H2O)] and [In(glyox)(H2O)] complexes. Quantitative radiolabeling with gallium-68 (RCY > 95%, [L] = 10-5 M) and indium-111 (RCY > 99%, [L] = 10-8 M) was achieved under ambient conditions (RT, pH 7, and 15 min) with very high apparent molar activities of 750 MBq/µmol and 650 MBq/nmol, respectively. Preliminary quantitative radiolabeling of [44Sc]ScCl3 (RCY > 99%, [L] = 10-6 M) was fast at room temperature (pH 7 and 10 min). In vitro experiments revealed exceptional stability of both [68Ga]Ga(glyox) and [111In]In(glyox) complexes against human serum (transchelation <2%) and its suitability for biological applications. Additionally, on chelation with metal ions, H3glyox exhibits enhanced fluorescence, which was employed to determine the stability constants for Sc(glyox) in addition to the in-batch UV-vis spectrophotometric titrations; as a proof-of-concept these complexes were used to obtain fluorescence images of live HeLa cells using Sc(glyox) and Ga(glyox), confirming the viability of the cells. These initial investigations suggest H3glyox to be a valuable chelator for radiometal-based diagnosis (nuclear and optical imaging) and therapy.

Towards Utilising Photocrosslinking of Polydiacetylenes for the Preparation of "Stealth" Upconverting Nanoparticles.

We demonstrate a novel strategy for preparing hydrophilic upconverting nanoparticles (UCNPs) by harnessing the photocrosslinking ability of diacetylenes. Replacement of the hydrophobic oleate coating on the UCNPs with 10,12-pentacosadiynoic acid, followed by overcoating with diacetylene phospholipid and subsequent photocrosslinking under 254 nm irradiation produces water-dispersible polydiacetylene-coated UCNPs. These UCNPs resist the formation of a biomolecular corona and show great colloidal stability. Furthermore, amine groups on the diacetylene phospholipid allow for functionalisation of the UCNPs with, for example, radiolabels or targeting moieties. These results demonstrate that this new surface-coating method has great potential for use in the preparation of UCNPs with improved biocompatibility.

New insights into the pretargeting approach to image and treat tumours.

Tumour pretargeting is a promising strategy for cancer diagnosis and therapy allowing for the rational use of long circulating, highly specific monoclonal antibodies (mAbs) for both non-invasive cancer radioimmunodetection (RID) and radioimmunotherapy (RIT). In contrast to conventional RID/RIT where the radionuclides and oncotropic vector molecules are delivered as presynthesised radioimmunoconjugates, the pretargeting approach is a multistep procedure that temporarily separates targeting of certain tumour-associated antigens from delivery of diagnostic or therapeutic radionuclides. In principle, unlabelled, highly tumour antigen specific mAb conjugates are, in a first step, administered into a patient. After injection, sufficient time is allowed for blood circulation, accumulation at the tumour site and subsequent elimination of excess mAb conjugates from the body. The small fast-clearing radiolabelled effector molecules with a complementary functionality directed to the prelocalised mAb conjugates are then administered in a second step. Due to its fast pharmacokinetics, the small effector molecules reach the malignant tissue quickly and bind the local mAb conjugates. Thereby, corresponding radioimmunoconjugates are formed in vivo and, consequently, radiation doses are deposited mainly locally. This procedure results in a much higher tumour/non-tumour (T/NT) ratio and is favourable for cancer diagnosis and therapy as it substantially minimises the radiation damage to non-tumour cells of healthy tissues. The pretargeting approach utilises specific non-covalent interactions (e.g. strept(avidin)/biotin) or covalent bond formations (e.g. inverse electron demand Diels-Alder reaction) between the tumour bound antibody and radiolabelled small molecules. This tutorial review descriptively presents this complex strategy, addresses the historical as well as recent preclinical and clinical advances and discusses the advantages and disadvantages of different available variations.

Ultrasmall inorganic nanoparticles: State-of-the-art and perspectives for biomedical applications.

Ultrasmall nanoparticulate materials with core sizes in the 1-3nm range bridge the gap between single molecules and classical, larger-sized nanomaterials, not only in terms of spatial dimension, but also as regards physicochemical and pharmacokinetic properties. Due to these unique properties, ultrasmall nanoparticles appear to be promising materials for nanomedicinal applications. This review overviews the different synthetic methods of inorganic ultrasmall nanoparticles as well as their properties, characterization, surface modification and toxicity. We moreover summarize the current state of knowledge regarding pharmacokinetics, biodistribution and targeting of nanoscale materials. Aside from addressing the issue of biomolecular corona formation and elaborating on the interactions of ultrasmall nanoparticles with individual cells, we discuss the potential diagnostic, therapeutic and theranostic applications of ultrasmall nanoparticles in the emerging field of nanomedicine in the final part of this review.

Zwitterionic-coated "stealth" nanoparticles for biomedical applications: recent advances in countering biomolecular corona formation and uptake by the mononuclear phagocyte system.

Nanoparticles represent highly promising platforms for the development of imaging and therapeutic agents, including those that can either be detected via more than one imaging technique (multi-modal imaging agents) or used for both diagnosis and therapy (theranostics). A major obstacle to their medical application and translation to the clinic, however, is the fact that many accumulate in the liver and spleen as a result of opsonization and scavenging by the mononuclear phagocyte system. This focused review summarizes recent efforts to develop zwitterionic-coatings to counter this issue and render nanoparticles more biocompatible. Such coatings have been found to greatly reduce the rate and/or extent of non-specific adsorption of proteins and lipids to the nanoparticle surface, thereby inhibiting production of the "biomolecular corona" that is proposed to be a universal feature of nanoparticles within a biological environment. Additionally, in vivo studies have demonstrated that larger-sized nanoparticles with a zwitterionic coating have extended circulatory lifetimes, while those with hydrodynamic diameters of ≤5 nm exhibit small-molecule-like pharmacokinetics, remaining sufficiently small to pass through the fenestrae and slit pores during glomerular filtration within the kidneys, and enabling efficient excretion via the urine. The larger particles represent ideal candidates for use as blood pool imaging agents, whilst the small ones provide a highly promising platform for the future development of theranostics with reduced side effect profiles and superior dose delivery and image contrast capabilities.

Bispidine dioxotetraaza macrocycles: a new class of bispidines for (64)Cu PET imaging.

The three new dioxo-tetraazamacrocyclic ligands with a fused, very rigid bispidine (3,7-diazabicyclo[3.3.1]nonane) group connecting the two tertiary amine donors, and ethyl, propyl, or benzene groups connecting the two amide donors are highly preorganized and lead to very stable, uncharged Cu(II) complexes. Solution spectroscopy and solid state structures indicate that these are square pyramidal with a solvent molecule occupying the apical position. Cyclic voltammetry defines a reversible Cu(III/II) couple and a strongly negative irreversible Cu(II/I) couple (ca. -2 V vs Fc/Fc(+)), indicating that the Cu(II) complexes are very stable in solution. This is supported by superoxide dismutase (SOD) and human serum challenge experiments as well as the biodistribution, which all show that the benzene-based ligand has the highest in vitro and in vivo stability and that this was expected on the basis of the macrocycle ring size and shape and the highest degree of preorganization. This ligand is easy to functionalize for a possible coupling to biological vector molecules and/or fluorescence markers for PET (positron emission tomography) and multimodal imaging (i.e., PET and optical imaging).

High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli.

BACKGROUND: For their application in the area of diagnosis and therapy, single-domain antibodies (sdAbs) offer multiple advantages over conventional antibodies and fragments thereof in terms of size, stability, solubility, immunogenicity, production costs as well as tumor uptake and blood clearance. Thus, sdAbs have been identified as valuable next-generation targeting moieties for molecular imaging and drug delivery in the past years. Since these probes are much less complex than conventional antibody fragments, bacterial expression represents a facile method in order to produce sdAbs in large amounts as soluble and functional proteins. RESULTS: By the combined use of high cell density cultivation media with a genetically engineered E. coli mutant strain designed for the cytoplasmic formation of proper disulfide bonds, we achieved high level of intracellular sdAb production (up to 200 mg/L). Due to a carboxyterminal hexahistidine epitope, the soluble recombinant sdAbs could be purified by one-step immobilized metal affinity chromatography to apparent homogeneity and easily radiolabeled with 99mTc within 1 h. The intradomain disulfide bridge being critical for the stability and functionality of the sdAb molecule was shown to be properly formed in ~96% of the purified proteins. In vitro binding studies confirmed the high affinity and specificity of the expressed sdAb 7C12 towards its molecular target. CONCLUSIONS: Our study demonstrates an efficient cultivation and expression strategy for the production of substantial amounts of soluble and functional sdAbs, which may be adopted for high-yield production of other more complex proteins with multiple disulfides as well.

Design and Biological Evaluation of Small-Molecule PET-Tracers for Imaging of Programmed Death Ligand 1.

Noninvasive molecular imaging of the PD-1/PD-L1 immune checkpoint is of high clinical relevance for patient stratification and therapy monitoring in cancer patients. Here we report nine small-molecule PD-L1 radiotracers with solubilizing sulfonic acids and a linker-chelator system, designed by molecular docking experiments and synthesized according to a new, convergent synthetic strategy. Binding affinities were determined both in cellular saturation and real-time binding assay (LigandTracer), revealing dissociation constants in the single digit nanomolar range. Incubation in human serum and liver microsomes proved in vitro stability of these compounds. Small animal PET/CT imaging, in mice bearing PD-L1 overexpressing and PD-L1 negative tumors, showed moderate to low uptake. All compounds were cleared primarily through the hepatobiliary excretion route and showed a long circulation time. The latter was attributed to strong blood albumin binding effects, discovered during our binding experiments. Taken together, these compounds are a promising starting point for further development of a new class of PD-L1 targeting radiotracers.

Bis(amido)bis(oxinate)diamine Ligands for theranostic radiometals.

With the interest in radiometal-containing diagnostic and therapeutic pharmaceuticals increasing rapidly, appropriate ligands to coordinate completely and stably said radiometals is essential. Reported here are two novel, bis(amido)bis(oxinate)diamine ligands, H2amidohox (2,2'-(ethane-1,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)azanediyl))diacetamide) and H2amidoC3hox (2,2'-(propane-1,3-diylbis(((8-hydroxyquinolin-2-yl)methyl)azanediyl))diacetamide), that combine two 8-hydroxyquinoline and amide donor groups and differ by one carbon in their 1,2-ethylenediamine vs. 1,3-diaminopropane backbones, respectively. Both ligands have been thoroughly studied via metal complexation, solution thermodynamics and radiolabeling with three radiometal ions: [nat/64Cu]Cu2+, [nat/111In]In3+, and [nat/203Pb]Pb2+. X-ray crystallography determined the structures of the hexacoordinated Cu2+-ligand complexes, indicating a better fit of Cu2+ to the H2amidohox binding pocket. Concentration dependent radiolabeling with [64Cu]Cu2+ was successfully quantitative as low as 1 µM with H2amidohox and 10 µM with H2amidoC3hox within 5 min at room temperature. However, [64Cu][Cu(amidohox)] maintained higher kinetic inertness against a superoxide dismutase enzyme-challenge assay and ligand challenges compared to the [64Cu][Cu(amidoC3hox)] counterpart. Similarly, H2amidohox had significantly higher radiochemical conversion with both [111In]In3+ (97% at 1 µM) and [203Pb]Pb2+ (97% at 100 µM) under mild conditions compared to H2amidoC3hox (76% with [111In]In3+ at 1 µM and 0% with [203Pb]Pb2+). By studying non-radioactive and radioactive complexation with both ligands, a comprehensive understanding of the coordination differences between two- and three­carbon diamine backbones is discussed. Overall, the ethylenediamine backbone of H2amidohox proves to be superior in rapid, mild radiolabeling and kinetic inertness towards competing ligands and proteins.

Modulating the pharmacokinetic profile of Actinium-225-labeled macropa-derived radioconjugates by dual targeting of PSMA and albumin.

Rationale: Small 225Ac-labeled prostate-specific membrane antigen (PSMA)-targeted radioconjugates have been described for targeted alpha therapy of metastatic castration-resistant prostate cancer. Transient binding to serum albumin as a highly abundant, inherent transport protein represents a commonly applied strategy to modulate the tissue distribution profile of such low-molecular-weight radiotherapeutics and to enhance radioactivity uptake into tumor lesions with the ultimate objective of improved therapeutic outcome. Methods: Two ligands mcp-M-alb-PSMA and mcp-D-alb-PSMA were synthesized by combining a macropa-derived chelator with either one or two lysine-ureido-glutamate-based PSMA- and 4-(p-iodophenyl)butyrate albumin-binding entities using multistep peptide-coupling chemistry. Both compounds were labeled with [225Ac]Ac3+ under mild conditions and their reversible binding to serum albumin was analyzed by an ultrafiltration assay as well as microscale thermophoresis measurements. Saturation binding studies and clonogenic survival assays using PSMA-expressing LNCaP cells were performed to evaluate PSMA-mediated cell binding and to assess the cytotoxic potency of the novel radioconjugates [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Biodistributions of both 225Ac-radioconjugates were investigated using LNCaP tumor-bearing SCID mice. Histological examinations of selected organs were performed to analyze the occurrence of necrosis using H&E staining, DNA damage via γH2AX staining and proliferation via Ki67 expression in the tissue samples. Results: Enhanced binding to serum components in general and to human serum albumin in particular was revealed for [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Moreover, the novel derivatives are highly potent PSMA ligands as their KD values in the nanomolar range (23.38 and 11.56 nM) are comparable to the reference radioconjugates [225Ac]Ac-mcp-M-PSMA (30.83 nM) and [225Ac]Ac-mcp-D-PSMA (10.20 nM) without albumin binders. The clonogenic activity of LNCaP cells after treatment with the 225Ac-labeled ligands was affected in a dose- and time-dependent manner, whereas the bivalent radioconjugate [225Ac]Ac-mcp-D-alb-PSMA has a stronger impact on the clonogenic cell survival than its monovalent counterpart [225Ac]Ac-mcp-M-alb-PSMA. Biodistribution studies performed in LNCaP tumor xenografts showed prolonged blood circulation times for both albumin-binding radioconjugates and a substantially increased tumor uptake (46.04 ± 7.77 %ID/g for [225Ac]Ac-mcp-M-alb-PSMA at 128 h p.i. and 153.48 ± 37.76 %ID/g at 168 h p.i. for [225Ac]Ac-mcp-D-alb-PSMA) with favorable tumor-to-background ratios. Consequently, a clear histological indication of DNA damage was discovered in the tumor tissues, whereas DNA double-strand break formation in kidney and liver sections was less pronounced. Conclusion: The modification of the PSMA-based 225Ac-radioconjugates with one or two albumin-binding entities resulted in an improved radiopharmacological behavior including a greatly enhanced tumor accumulation combined with a rather low uptake in most non-targeted organs combined with a high excretion via the kidneys.

Radiolabelled Cyclic Bisarylmercury: High Chemical and in†vivo Stability for Theranostics.

We show the synthesis of an in vivo stable mercury compound with functionality suitable for radiopharmaceuticals. The designed cyclic bisarylmercury was based on the water tolerance of organomercurials, higher bond dissociation energy of Hg-Ph to Hg-S, and the experimental evidence that acyclic structures suffer significant cleavage of one of the Hg-R bonds. The bispidine motif was chosen for its in vivo stability, chemical accessibility, and functionalization properties. Radionuclide production results in 197(m) HgCl2 (aq), so the desired mercury compound was formed via a water-tolerant organotin transmetallation. The Hg-bispidine compound showed high chemical stability in tests with an excess of sulfur-containing competitors and high in vivo stability, without any observable protein interaction by human serum assay, and good organ clearance demonstrated by biodistribution and SPECT studies in rats. In particular, no retention in the kidneys was observed, typical of unstable mercury compounds. The nat Hg analogue allowed full characterization by NMR and HRMS.

Towards Targeted Alpha Therapy with Actinium-225: Chelators for Mild Condition Radiolabeling and Targeting PSMA-A Proof of Concept Study.

Currently, targeted alpha therapy is one of the most investigated topics in radiopharmaceutical cancer management. Especially, the alpha emitter 225Ac has excellent nuclear properties and is gaining increasing popularity for the treatment of various tumor entities. We herein report on the synthesis of two universal 225Ac-chelators for mild condition radiolabeling and binding to conjugate molecules of pharmacological interest via the copper-mediated click chemistry. A convenient radiolabeling procedure was investigated as well as the complex stability proved for both chelators and two PSMA (prostate-specific membrane antigen)-targeting model radioconjugates. Studies regarding affinity and cell survival were performed on LNCaP cells followed by biodistribution studies, which were performed using LNCaP tumor-bearing mice. High efficiency radiolabeling for all conjugates was demonstrated. Cell binding studies revealed a fourfold lower cell affinity for the PSMA radioconjugate with one targeting motif compared to the radioconjugate owing two targeting motifs. Additionally, these differences were verified by in vitro cell survival evaluation and biodistribution studies, both showing a higher cell killing efficiency for the same dose, a higher tumor uptake (15%ID/g) and a rapid whole body clearance after 24 h. The synthesized chelators will overcome obstacles of lacking stability and worse labeling needs regarding 225Ac complexation using the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid) chelator. Moreover, the universal functionalization expands the coverage of these chelators in combination with any sensitive bio(macro)molecule, thus improving treatment of any addressable tumor target.

Protein tyrosine O-glycosylation--a rather unexplored prokaryotic glycosylation system.

Glycosylation is a frequent and heterogeneous posttranslational protein modification occurring in all domains of life. While protein N-glycosylation at asparagine and O-glycosylation at serine, threonine or hydroxyproline residues have been studied in great detail, only few data are available on O-glycosidic attachment of glycans to the amino acid tyrosine. In this study, we describe the identification and characterization of a bacterial protein tyrosine O-glycosylation system. In the Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T), a polysaccharide consisting of [-->3)-beta-d-Galp-(1[alpha-d-Glcp-(1-->6)] -->4)-beta-d-ManpNAc-(1-->] repeating units is O-glycosidically linked via an adaptor with the structure -[GroA-2-->OPO(2)-->4-beta-d-ManpNAc-(1-->4)] -->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->3)-beta-d-Galp-(1--> to specific tyrosine residues of the S-layer protein SpaA. A +AH4-24.3-kb S-layer glycosylation (slg) gene cluster encodes the information necessary for the biosynthesis of this glycan chain within 18 open reading frames (ORF). The corresponding translation products are involved in the biosynthesis of nucleotide-activated monosaccharides, assembly and export as well as in the transfer of the completed polysaccharide chain to the S-layer target protein. All ORFs of the cluster, except those encoding the nucleotide sugar biosynthesis enzymes and the ATP binding cassette (ABC) transporter integral transmembrane proteins, were disrupted by the insertion of the mobile group II intron Ll.LtrB, and S-layer glycoproteins produced in mutant backgrounds were analyzed by mass spectrometry. There is evidence that the glycan chain is synthesized in a process comparable to the ABC-transporter-dependent pathway of the lipopolysaccharide O-polysaccharide biosynthesis. Furthermore, with the protein WsfB, we have identified an O-oligosaccharyl:protein transferase required for the formation of the covalent beta-d-Gal-->Tyr linkage between the glycan chain and the S-layer protein.

Sub-10 nm Radiolabeled Barium Sulfate Nanoparticles as Carriers for Theranostic Applications and Targeted Alpha Therapy.

Invited for this month's cover is the group of Kristof Zarschler at the University of Dresden, Germany. Read the full text of their Full Paper at 10.1002/open.202000126.

Sub-10†nm Radiolabeled Barium Sulfate Nanoparticles as Carriers for Theranostic Applications and Targeted Alpha Therapy.

The treatment of cancer patients with α-particle-emitting therapeutics continues to gain in importance and relevance. The range of radiopharmaceutically relevant α-emitters is limited to a few radionuclides, as stable chelators or carrier systems for safe transport of the radioactive cargo are often lacking. Encapsulation of α-emitters into solid inorganic systems can help to diversify the portfolio of candidate radionuclides, provided, that these nanomaterials effectively retain both the parent and the recoil daughters. We therefore focus on designing stable and defined nanocarrier-based systems for various clinically relevant radionuclides, including the promising α-emitting radionuclide 224Ra. Hence, sub-10 nm barium sulfate nanocontainers were prepared and different radiometals like 89Zr, 111In, 131Ba, 177Lu or 224Ra were incorporated. Our system shows stabilities of >90 % regarding the radiometal release from the BaSO4 matrix. Furthermore, we confirm the presence of surface-exposed amine functionalities as well as the formation of a biomolecular corona.