A flexible tyrosine-attached chemical lid prevents premature disengagement of growing oligosaccharides and triggers the synthesis of a high molecular weight polymer.
Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve CPPs (cell-penetrating peptides) or protein transduction domains. Most of these require rather high concentrations to elicit cell-penetrating functionality, are non-human, pathogen-derived or synthetic entities, and may therefore not be tolerated or even immunogenic. We identified novel human-protein-derived CPPs by a combination of in silico and experimental analyses: polycationic CPP candidates were identified in an in silico library of all 30-mer peptides of the human proteome. Of these peptides, 60 derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA (small interfering RNA) transfection assays revealed that 20 out of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human NRTN (neurturin), which contains an α-helix, performed the best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.
One approach to further improve the therapeutic efficacy of nanoparticles is employment of active targeting strategies. Bispecific antibodies (bsAbs) that bind to both tumor specific antigens on the cell surface and to haptens such as digoxigenin (Dig) can direct digoxigeninylated payloads to tumor cells. In this study, we investigate the potential of dendritic polyglycerol (dPG) conjugates, which consist of a doxorubicin (DOX) prodrug, Dig moiety, and a poly (ethylene glycol) (PEG) shell, in combination with bsAb, as a novel approach for targeted prodrug delivery. We could show successful binding of the bsAbs to dPGDigMal- DOX-PEG conjugates, as well as binding of these complexes to the cell surface of Lewis Y (LeY) expressing MCF-7 cells. Using flow cytometry, we could show the preferential binding of the targeting complex over the complex of control conjugate lacking Dig moieties. At concentrations that are usually applied for drug delivery, antibodycomplexed nanoparticles (independent of antibody specificity) released cytotoxic compounds into cells to the same degree as unmodified nanoparticles. This indicates that antibody-attachment does not interfere with the inherent cell binding and drug delivery properties of nanoparticles. At low doxorubicin concentrations and short incubation times, however, we were able to see a slightly increased target specific cytotoxicity in vitro which is mediated by complexation of the digoxigeninylated NP with the Dig-binding moiety of a bsAb that in turns direct the complexed bsAb to target cells. This study demonstrates the potential of digoxigeninylated dPG prodrug conjugates in combination with bsAbs as a new platform for targeted prodrug delivery into cancerous tissues. However the nanoparticle design needs to be further optimized for significant targeted delivery.
Site‐specific introduction of bioorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post‐synthetic bioconjugation reactions. Here, we report a ribozyme‐based method for the synthesis of aldehyde‐functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site‐specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5‐amino‐4‐formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde‐reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and tRNA transcripts. Upon fluorogenic condensation with a 2,3,3‐trimethylindole, a novel hemicyanine chromophore was generated directly on the RNA. This work expands the MTR1 ribozyme's area of application from a methyltransferase to a tool for site‐specific late‐stage functionalization of RNA.
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