It is an open question how the chemical structure of prebiotic vesicle-forming amphiphiles complexified to produce robust primitive compartments that could safely host foreign molecules. Previous work suggests that comparingly labile vesicles composed of plausibly prebiotic fatty acids were eventually chemically transformed with glycerol and a suitable phosphate source into phospholipids that would form robust vesicles. Here we show that phosphatidic acid (PA) and phosphatidylethanolamine (PE) lipids can be obtained from racemic dioleoyl glycerol under plausibly prebiotic phosphorylation conditions. Upon in situ hydration of the crude phosphorylation mixtures only those that contained rac-DOPA (not rac-DOPE) generated stable giant vesicles that were capable of encapsulating water-soluble probes, as evidenced by confocal microscopy and flow cytometry. Chemical reaction side-products (identified by IR and MS and quantified by 1H NMR) acted as co-surfactants and facilitated vesicle formation. To mimic the compositional variation of such primitive lipid mixtures, self-assembly of a combinatorial set of the above amphiphiles was tested, revealing that too high dioleoyl glycerol contents inhibited vesicle formation. We conclude that a decisive driving force for the gradual transition from unstable fatty acid vesicles to robust diacylglyceryl phosphate vesicles was to avoid the accumulation of unphosphorylated diacylglycerols in primitive vesicle membranes.

Pretargeted imaging has emerged as an effective multistep strategy aiming to improve imaging contrast and reduce patient radiation exposure through decoupling of the radioactivity from the targeting vector. The inverse electron-demand Diels− Alder (IEDDA) reaction between a trans-cyclooctene (TCO)conjugated antibody and a labeled tetrazine holds great promise for pretargeted imaging applications due to its bioorthogonality, rapid kinetics under mild conditions, and formation of stable products. Herein, we describe the use of functionalized carbonylacrylic reagents for site-specific incorporation of TCO onto a human epidermal growth factor receptor 2 (HER2) antibody (THIO-MAB) containing an engineered unpaired cysteine residue, generating homogeneous conjugates. Precise labeling of THIO-MAB−TCO with a fluorescent or radiolabeled tetrazine revealed the potential of the TCO-functionalized antibody for imaging the HER2 after pretargeting in a cellular context in a HER2 positive breast cancer cell line. Control studies with MDA−MD-231 cells, which do not express HER2, further confirmed the target specificity of the modified antibody. THIOMAB−TCO was also evaluated in vivo after pretargeting and subsequent administration of an 111 In-labeled tetrazine. Biodistribution studies in breast cancer tumorbearing mice showed a significant activity accumulation on HER2 + tumors, which was 2.6-fold higher than in HER2 − tumors. Additionally, biodistribution studies with THIOMAB without the TCO handle also resulted in a decreased uptake of 111 In−DOTA− Tz on HER2 + tumors. Altogether, these results clearly indicate the occurrence of the click reaction at the tumor site, i.e., pretargeting of SK-BR-3 HER2-expressing cells with THIOMAB−TCO and reaction through the TCO moiety present in the antibody. The combined advantages of site-selectivity and stability of TCO tagged-antibodies could allow application of biorthogonal chemistry strategies for pretargeting imaging with minimal side-reactions and background.

Although prebiotic condensations of glycerol, phosphate and fatty acids produce phospholipid esters with a racemic backbone, most experimental studies on vesicles intended as protocell models have been carried out by employing commercial enantiopure phospholipids. Current experimental research on realistic protocell models urgently requires racemic phospholipids and efficient synthetic routes for their production. Here we propose three synthetic pathways starting from glycerol or from racemic solketal (α,β-isopropylidene-dl-glycerol) for the gram-scale production (up to 4 g) of racemic phospholipid ester precursors. We describe and compare these synthetic pathways with literature data. Racemic phosphatidylcholines and phosphatidylethanolamines were obtained in good yields and high purity from 1,2-diacylglycerols. Racemic POPC (rac-POPC, (R,S)-1-palmitoyl-2-oleoyl-3-phosphocholine), was used as a model compound for the preparation of giant vesicles (GVs). Confocal laser scanning fluorescence microscopy was used to compare GVs prepared from enantiopure (R)-POPC), racemic POPC (rac-POPC) and a scalemic mixture (scal-POPC) of (R)-POPC enriched with rac-POPC. Vesicle morphology and size distribution were similar among the different (R)-POPC, rac-POPC and scal-POPC, while calcein entrapments in (R)-POPC and in scal-POPC were significantly distinct by about 10%.

Pre-targeting approaches based on the inverse-electron-demand Diels-Alder (iEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have emerged in recent years as valid alternatives to classic targeted strategies to improve the diagnostic and therapeutic properties of radioactive probes. To explore these pre-targeting strategies based on in vivo click chemistry, a small family of clickable chelators was synthesized and radiolabelled with medically relevant trivalent radiometals. The structure of the clickable chelators was diversified to modulate the pharmacokinetics of the resulting [111In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [90Y]Y-DOTA-Tz complex was evaluated in a prostate cancer PC3 xenograft by ex-vivo biodistribution studies and Cerenkov luminescence imaging (CLI). The results highlighted a quick elimination through the renal system and no relevant accumulation in non-target organs or non-specific tumor uptake. Furthermore, a clickable bombesin antagonist was injected in PC3 tumor-bearing mice followed by the radiocomplex [90Y]Y-DOTA-Tz, and the mice imaged by CLI at different post-injection times (p.i.). Analysis of the images 15 min and 1 h p.i. pointed out an encouraging quick tumor uptake with a fast washout, providing a preliminary proof of concept of the usefulness of the designed clickable complexes for pre-targeting strategies. To the best of our knowledge, the use of peptide antagonists for this purpose was not explored before. Further investigations are needed to optimize the pre-targeting approach based on this type of biomolecules and evaluate its eventual advantages.

Despite some progress, the overall survival of patients with glioblastoma (GBM) remains extremely poor. In this context, there is a pressing need to develop innovative therapy strategies for GBM, namely those based on nanomedicine approaches. Towards this goal, we have focused on nanoparticles (AuNP-SP and AuNP-SPTyr8) with a small gold core (ca. 4 nm), carrying DOTA chelators and substance P (SP) peptides. These new SP-containing AuNPs were characterized by a variety of analytical techniques, including TEM and DLS measurements and UV-vis and CD spectroscopy, which proved their high in vitro stability and poor tendency to interact with plasma proteins. Their labeling with diagnostic and therapeutic radionuclides was efficiently performed by DOTA complexation with the trivalent radiometals 67Ga and 177Lu or by electrophilic radioiodination with 125I of the tyrosyl residue in AuNP-SPTyr8. Cellular studies of the resulting radiolabeled AuNPs in NKR1-positive GBM cells (U87, T98G and U373) have shown that the presence of the SP peptides has a crucial and positive impact on their internalization by the tumor cells. Consistently, 177Lu-AuNP-SPTyr8 showed more pronounced radiobiological effects in U373 cells when compared with the non-targeted congener 177Lu-AuNP-TDOTA, as assessed by cell viability and clonogenic assays and corroborated by Monte Carlo microdosimetry simulations.

D-Glucosamine was successfully employed as a chiral auxiliary for the enantioselective synthesis of phosphine oxides. The influence of the anomeric position was also investigated and revealed the excellent ability of the α-anomer to perform this transformation in a highly selective fashion. The methodology employed consisted of three steps: diastereoselective formation of the oxazaphospholidine followed by subsequent selective cleavage of P-N and P-O bonds by reaction with two Grignard reagents. P-epimers oxazaphospholidines were prepared switching from a P(v) to a P(III) precursor, thus allowing for the synthesis of enantiomeric phosphine oxides. In addition, the chiral auxiliary could be recovered and efficiently recycled.

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Several gastrin-releasing peptide receptor (GRPR) antagonists with improved in vivo behavior have been recently developed and tested in the clinic. However, despite the generally mild side effects of peptide receptor radionuclide therapy (PRRT), toxicity has been observed due to high doses delivered to nontarget tissues, especially in the kidneys and pancreas. Previous experiences with radiolabeled peptides opened a unique opportunity to explore GRPR pretargeting using clickable bombesin antagonists. Toward this goal, we used clickable DOTA-like radiocomplexes which have been previously evaluated by our group. We functionalized a potent GRPR antagonist with a clickable TCO moiety using two different linkers. These precursors were then studied to select the compound with the highest GRPR binding affinity and the best pharmacokinetics to finally explore the advantages of the devised pretargeting approach. Our results provided an important proof of concept toward the development of bioorthogonal approaches to GRPR-expressing cancers, which are worth investigating further to improve the in vivo results. Moreover, the use of clickable GRPR antagonists and DOTA/DOTAGA derivatives allows for fine-tuning of their pharmacokinetics and metabolic stability, leading to a versatile synthesis of new libraries of (radio)conjugates useful for the development of theranostic tools toward GRPR-expressing tumors.