Supramolecular Assembly of Aminoethylene‐Lipopeptide PMO Conjugates into RNA Splice‐Switching Nanomicelles

Phosphorodiamidate morpholino oligomers (PMOs) are oligonucleotide analogs that can be used for therapeutic modulation of pre‐mRNA splicing. Similar to other classes of nucleic acid‐based therapeutics, PMOs require delivery systems for efficient transport to the intracellular target sites. Here, artificial peptides based on the oligo(ethylenamino) acid succinyl‐tetraethylenpentamine (Stp), hydrophobic modifications, and an azide group are presented, which are used for strain‐promoted azide‐alkyne cycloaddition conjugation with splice‐switching PMOs. By systematically varying the lead structure and formulation, it is determined that the type of contained fatty acid and supramolecular assembly have a critical impact on the delivery efficacy. A compound containing linolenic acid with three cis double bonds exhibits the highest splice‐switching activity and significantly increases functional protein expression in pLuc/705 reporter cells in vitro and after local administration in vivo. Structural and mechanistic studies reveal that the lipopeptide PMO conjugates form nanoparticles, which accelerate cellular uptake and that the content of unsaturated fatty acids enhances endosomal escape. In an in vitro Duchenne muscular dystrophy exon skipping model using H2K‐mdx52 dystrophic skeletal myotubes, the highly potent PMO conjugates mediate significant splice‐switching at very low nanomolar concentrations. The presented aminoethylene‐lipopeptides are thus a promising platform for the generation of PMO‐therapeutics with a favorable activity/toxicity profile

Controlling Nanoparticle Formulation: A Low-Budget Prototype for the Automation of a Microfluidic Platform

Active pharmaceutical ingredients (API) with suboptimal pharmacokinetic properties may require formulation into nanoparticles. In addition to the quality of the excipients, production parameters are crucial for producing nanoparticles which reliably deliver APIs to their target. Microfluidic platforms promise increased control over the formulation process due to the decreased degrees of freedom at the micro- and nanoscale. Publications about these platforms usually provide only limited information about the soft- and hardware required to integrate the microfluidic chip seamlessly into an experimental set-up. We describe a modular, low-budget prototype for microfluidic mixing in detail. The prototype consists of four modules. The control module is a raspberry pi executing customizable python scripts to control the syringe pumps and the fraction collector. The feeding module consists of up to three commercially available, programable syringe pumps. The formulation module can be any macro- or microfluidic chip connectable to syringe pumps. The collection module is a custom-built fraction collector. We describe each feature of the working prototype and demonstrate its power with polyplexes formulated from siRNA and two different oligomers that are fed to the chip at two different stages during the assembly of the nanoparticles