Development and Strategies to Improve Cellular Uptake of an Artificial Membraneless Organelle-Based Bioreactor

Liquid-liquid phase separated microcompartments inspired by membraneless organelles is a promising tool in developing bioreactors. They have liquid-like interior, sequestrate biomolecules, exchange materials with external environment and confine biochemical reactions. Adapting the sticker-spacer model of intrinsically disordered proteins, artificial membraneless organelles (aMLOs) were developed using a polymer-oligopeptide hybrid material consists of a dextran backbone and two oligopeptides. The capacity of aMLO to perform biochemical reactions was evaluated by a CRISPR reaction within the microcompartment. For the translation of CRISPR technology for therapeutic use, the core challenge is the development of an effective and safe delivery approaches. The CRISPR reactive complex loaded micron-size bioreactors in cytosol of target cell would be a noninvasive delivery approach with high effectivity. Cas9, sgRNA and target oligo-duplex were enriched within aMLOs with 720-fold, 865-fold and 835-fold increased concentrations, respectively. Consequently, the CRISPR cleavage reaction was achieved within the aMLOs under physiological conditions.<br/>Additionally, strategies of cytosolic delivery of aMLOs were studied. To tap membrane fusion pathway, aMLOs were wrapped with macrophage cell membranes. The wrapping did not change the native enrichment/exclusion functions of the aMLOs but improved the stability from ~9 hours to up to 3 days. Wrapped aMLOs found to be internalized by source cells with intact cargo-molecules. Cellular uptake was further improved by sonoporation with low frequency (40 kHz) ultrasound of mechanical index (MI) and peak pressure of 0.4 and 75 kPa, respectively. Low frequency ultrasound can increase activity of mechanosensitive channels and induce pore formation at membrane protein-lipid interface to improve uptake. In the sonoporation experiment, above 80% cells were viable under continuous ultrasound treatment up to 5 min. Overall, results imply the possibility of developing deliverable bioreactors made of aMLOs and strategies to improve its cellular uptake.