Synthetic Protein Mimics for Functional Protein Delivery.

The use of proteins as biological tools and therapeutic agents is limited due to the fact that proteins do not effectively cross the plasma membrane of cells. Here, we report a novel class of protein transporter molecules based on protein transduction domain mimics (PTDMs) synthesized via ring opening metathesis polymerization (ROMP). The PTDMs reported here were specifically inspired by amphiphilic peptides known to deliver functional proteins into cells via noncovalent interactions between the peptide and the cargo. This contrasts with peptides like TAT, penetratin, and R9, which often require covalent fusion to their cargoes. Using the easily tunable synthetic ROMP platform, the importance of a longer hydrophobic segment with cationic guanidinium groups was established through the delivery of EGFP into Jurkat T cells. The most efficient of these protein transporters was used to deliver functional Cre Recombinase with ∼80% knockdown efficiency into hard to transfect human T cells. Additionally, a C-terminally deleted form of the transcription factor Runx1 (Runx1.d190) was delivered into primary murine splenocytes, producing a 2-fold increase in c-Myc mRNA production, showcasing the versatility of this platform to deliver biologically active proteins into hard to transfect cell types.

Novel protein transduction domain mimics as nonviral delivery vectors for siRNA targeting NOTCH1 in primary human T cells.

RNA interference technology has recently been highlighted as a powerful research method as well as a potential therapeutic treatment for several diseases. However, the delivery of small interfering RNA (siRNA) into T cell lines and primary blood cells is exceedingly challenging, as they are resistant to transfection by conventional reagents. As a result, there is an unmet need for nonviral, efficient, and easily prepared carriers for siRNA delivery into hard-to-transfect cell types. Here, we report a novel system based on protein transduction domain mimics (PTDMs), generated by ring opening metathesis polymerization, for intracellular delivery of siRNA molecules. PTDM-based siRNA delivery induced efficient NOTCH1 knockdown in Jurkat T cells and human peripheral blood mononuclear cells without any measured toxicity. Furthermore, delivering siRNA to NOTCH1 in human peripheral blood cells modulated cell proliferation and differentiation of T cells into T(H)1 cells.

De novo designed protein transduction domain mimics from simple synthetic polymers.

Protein transduction domains (PTDs) that readily transverse cellular membranes are of great interest and are attractive tools for the intracellular delivery of bioactive molecules. Learning to program synthetic polymers and oligomers with the appropriate chemical information to capture adequately the biological activity of proteins is critical to our improved understanding of how these natural molecules work. In addition, the versatility of these synthetic mimics provides the opportunity to discover analogs with superior properties compared with their native sequences. Here we report the first detailed structure-activity relationship of a new PTD family of polymers based on a completely abiotic backbone. The synthetic approach easily allows doubling the density of guanidine functional groups, which increases the transduction efficiency of the sequences. Cellular uptake studies on three different cell lines (HEK 293T, CHO, and Jurkat T cells) confirm that these synthetic analogs are highly efficient novel protein transduction domain mimics (PTDMs), which are more effective than TAT(49-57) and nonaarginine (R9) and also highlight the usefulness of polymer chemistry at the chemistry-biology interface.