Biotin‐Functionalized Block Catiomers as an Active Targeting Approach in Gene Delivery

Abstract Recently, biotin (vitamin H) has been described as a ligand for active targeting and it has been found that many cancer cells overexpress the biotin receptor. In this study a biotin‐conjugated block copolymer, biotin‐poly(2‐ethyl‐2‐oxazoline)‐block‐poly{N '‐[N‐(2‐aminoethyl)‐2‐aminoethyl]aspartamide} (biotin‐pEtOx‐b‐pASP(DEA) is synthesized by a living cationic polymerization of the pEtOx‐block followed by the nucleophilic ringopening polymerization of the pASP‐block. The biotin moiety is coupled to the pEtOx‐b‐pASP precursor by a Cu(I) mediated azide‐alkyne click chemistry and finally, the diethylamine (DEA) side chain is introduced by a polymer analogous reaction. The final polymer P1 formed polyplexes in the presence of plasmid DNA that are characterized with respect to N/P ratio, size, zeta potential, and shape compared to a control polymer P2 without biotin. In addition, HEK293 cells are transfected with these polyplexes and the number of fluorescent HEK293 cells is evaluated to assess the influence of polymer nature on the activity of the micelles. Flow cytometric analysis revealed a significantly higher uptake of the biotin‐PEtOx‐PASP(DEA)/pDNA micelle than the PEtOx‐PASP(DEA)/pDNA micelle against HEK293 cells at a low N/P ratio of 20, consistent with the transfection results whereas at higher N/P ratio no difference can be observed anymore between the two polymers

Sulfonated red and far-red rhodamines to visualize SNAP- and Halo-tagged cell surface proteins

The (in)ability to permeate membranes is a key feature of chemical biology probes that defines their suitability for specific applications. Here we report sulfonated rhodamines that endow xanthene dyes with cellular impermeability for analysis of surface proteins. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to benzylguanine and choloralkane substrates for SNAP-tag and Halo-tag labelling. Sulfonated rhodamine-conjugated fluorophores maintain desirable photophysical properties, such as brightness and photostability. While transfected cells with a nuclear localized SNAP-tag remain unlabelled, extracellular exposed tags can be cleanly visualized. By multiplexing with a permeable rhodamine, we are able to differentiate extra- and intracellular SNAP- and Halo-tags, including those installed on the glucagon-like peptide-1 receptor, a prototypical class B G proteincoupled receptor. In more complex biological systems, Sulfo549 and Sulfo646 labelled transfected neurons derived from induced pluripotent stem cells (iPSCs), allowing STED nanoscopy of the axonal membrane. Together, this work provides a new avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags. We anticipate that Sulfo549, Sulfo646 and their congeners will be useful for a number of cell biology applications where labelling of intracellular sites interferes with accurate surface protein analysis