Bioconjugate Supramolecular Pd2+ Metallacages Penetrate the Blood Brain Barrier In Vitro and In Vivo.

The biomedical application of discrete supramolecular metal-based structures, specifically self-assembled metallacages, is still an emergent field of study. Capitalizing on the knowledge gained in recent years on the development of 3-dimensional (3D) metallacages as novel drug delivery systems and theranostic agents, we explore here the possibility to target [Pd2L4]4+ cages (L = 3,5-bis(3-ethynylpyridine)phenyl ligand) to the brain. In detail, a new water-soluble homoleptic cage (CPepH3) tethered to a blood brain barrier (BBB)-translocating peptide was synthesized by a combination of solid-phase peptide synthesis (SPPS) and self-assembly procedures. The cage translocation efficacy was assessed by inductively coupled mass spectrometry (ICP-MS) in a BBB cellular model in vitro. Biodistribution studies of the radiolabeled cage [[99mTcO4]- ⊂ CPepH3] in the CD1 mice model demonstrate its brain penetration properties in vivo. Further DFT studies were conducted to model the structure of the [[99mTcO4]- ⊂ cage] complex. Moreover, the encapsulation capabilities and stability of the cage were investigated using the [ReO4]- anion, the "cold" analogue of [99mTcO4]-, by 1H NMR spectroscopy. Overall, our study constitutes another proof-of-concept of the unique potential of supramolecular coordination complexes for modifying the physiochemical and biodistribution properties of diagnostic species.

In Vivo Pretargeting Based on Cysteine-Selective Antibody Modification with IEDDA Bioorthogonal Handles for Click Chemistry.

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 (THIOMAB) containing an engineered unpaired cysteine residue, generating homogeneous conjugates. Precise labeling of THIOMAB-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 111In-labeled tetrazine. Biodistribution studies in breast cancer tumor-bearing 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 111In-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.

Improved Fmoc-solid-phase peptide synthesis of an extracellular loop of CFTR for antibody selection by the phage display technology.

Cystic fibrosis (CF), a life-shortening genetic disease, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that codes for the CFTR protein, the major chloride channel expressed at the apical membrane of epithelial cells. The development of an imaging probe capable of non-invasively detect CFTR at the cell surface could be of great advantage for the management of CF. With that purpose, we synthesized the first extracellular loop of CFTR protein (ECL1) through fluorenylmethyloxycarbonyl (Fmoc)-based microwave-assisted solid-phase peptide synthesis (SPPS), according to a reported methodology. However, aspartimide formation, a well-characterized side reaction in Fmoc-SPPS, prompted us to adopt a different side-chain protection strategy for aspartic acid residues present in ECL1 sequence. The peptide was subsequently modified via PEGylation and biotinylation, and cyclized through disulfide bridge formation, mimicking the native loop conformation in CFTR protein. Herein, we report improvements in the synthesis of the first extracellular loop of CFTR, including peptide modifications that can be used to improve antigen presentation in phage display for selection of novel antibodies against plasma membrane CFTR.

Targeting of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein with a Technetium-99m Imaging Probe.

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to almost total absence of CFTR at the plasma membrane, a defect potentially corrected via drug-based therapies. Herein, we report the first proof-of-principle study of a noninvasive imaging probe able to detect CFTR at the plasma membrane. We radiolabeled the CFTR inhibitor, CFTRinh -172a, with technetium-99m via a pyrazolyl-diamine chelating unit, yielding a novel 99m Tc(CO)3 complex. A non-radioactive surrogate showed that the structural modifications introduced in the inhibitor did not affect its activity. The radioactive complex was able to detect plasma membrane CFTR, shown by its significantly higher uptake in wild-type versus mutated cells. Furthermore, assessment of F508del CFTR pharmacological correction in human cells using the radioactive complex revealed differences in corrector versus control uptake, recapitulating the biochemical correction observed for the protein.

Technetium-99m complexes of l-arginine derivatives for targeting amino acid transporters.

Although relevant from the clinical point of view, radiotracers targeting cationic amino acid transporters are relatively unexplored and, in particular, no metal-based radiotracers are known. The rare examples of complexes recognized by amino acid transporters, namely by the Na+-independent neutral l-type amino acid transporter 1 (LAT1), are 99mTc(i)/Re(i) compounds. Herein, we describe conjugates comprising a pyrazolyl-diamine chelating unit and the cationic amino acid l-arginine (l-Arg) linked by a propyl (L1) or hexyl linker (L2), which allowed the preparation of stable complexes of the type fac-[99mTc(CO)3(k3-L)]+ (Tc1, L = L1; Tc2, L = L2) and of the respective surrogates Re1 and Re2. Interestingly, complex Tc2 exhibited moderate levels of time-dependent internalization in three human tumoural cell lines, with approximately 3% of total applied activity internalized, corresponding to 21% of the cell-associated activity. A putative mechanism of retention in the cytoplasm of cells could be the interaction of the complex with inducible nitric oxide synthase (iNOS), which is the enzyme responsible for the catalytic oxidation of l-Arg to citrulline and nitric oxide. However, the surrogate complex Re2 does not recognize iNOS, as demonstrated by the in vitro assays with purified iNOS and in studies with lipopolysaccharide(LPS)-activated macrophages. Preliminary mechanistic studies suggest that the internalization of Tc2 is linked to the cationic amino acid transporters, namely system y+. This finding might open the way towards the development of novel families of metal-based radiotracers for probing metabolically active cancer cells.

Isostructural Re(I)/(99m)Tc(I) tricarbonyl complexes for cancer theranostics.

Merging classical organic anticancer drugs with metal-based compounds in one single molecule offers the possibility of exploring new approaches for cancer theranostics, i.e. the combination of diagnostic and therapeutic modalities. For this purpose, we have synthesized and biologically evaluated a series of Re(I)/(99m)Tc(I) tricarbonyl complexes (Re1­Re4 and Tc1­Tc4, respectively) stabilized by a cysteamine-based (N,S,O) chelator and containing 2-(4'-aminophenyl)benzothiazole pharmacophores. With the exception of Re1, all the Re complexes have shown a moderate cytotoxicity in MCF7 and PC3 cancer cells (IC50 values in the 15.9­32.1 µM range after 72 h of incubation). The cytotoxic activity of the Re complexes is well correlated with cellular uptake that was quantified using the isostructural (99m)Tc congeners. There is an augmented cytotoxic effect for Re3 and Re4 (versusRe1 and Re2), and the highest cellular uptake for Tc3 and Tc4, which display a long ether-containing linker to couple the pharmacophore to the (N,S,O)-chelator framework. Moreover, fluorescence microscopy clearly confirmed the cytosolic accumulation of the most cytotoxic compound (Re3). Biodistribution studies of Tc1­Tc4 in mice confirmed that these moderately lipophilic complexes (logDo/w = 1.95­2.32) have a favorable bioavailability. Tc3 and Tc4 presented a faster excretion, as they undergo metabolic transformations, in contrast to complexes Tc1 and Tc2. In summary, our results show that benzothiazole-containing Re(I)/(99m)Tc(I) tricarbonyl complexes stabilized by cysteamine-based (N,S,O)-chelators have potential to be further applied in the design of new tools for cancer theranostics.