Liposome-chaperoned cell-free synthesis for the design of proteoliposomes: Implications for therapeutic delivery

Cell-free (CF) protein synthesis has emerged as a powerful technique platform for efficient protein production in vitro. Liposomes have been widely studied as therapeutic carriers due to their biocompatibility, biodegradability, low toxicity, flexible surface manipulation, easy preparation, and higher cargo encapsulation capability. However, rapid immune clearance, insufficient targeting capacity, and poor cytoplasmic delivery efficiency substantially restrict their clinical application. The incorporation of functional membrane proteins (MPs) or peptides allows the transfer of biological properties to liposomes and imparts them with improved circulation, increased targeting, and efficient intracellular delivery. Liposome-chaperoned CF synthesis enables production of proteoliposomes in one-step reaction, which not only substantially simplifies the production procedure but also keeps protein functionality intact. Building off these observations, proteoliposomes with integrated MPs represent an excellent candidate for therapeutic delivery. In this review, we describe recent advances in CF synthesis with emphasis on detailing key factors for improving CF expression efficiency. Furthermore, we provide insights into strategies for rational design of proteoliposomal nanodelivery systems via CF synthesis. Statement of Significance: Liposome-chaperoned CF synthesis has emerged as a powerful approach for the design of recombinant proteoliposomes in one-step reaction. The incorporation of bioactive MPs or peptides into liposomes via CF synthesis can facilitate the development of proteoliposomal nanodelivery systems with improved circulation, increased targeting, and enhanced cellular delivery capacity. Moreover, by adapting lessons learned from natural delivery vehicles, novel bio-inspired proteoliposomes with enhanced delivery properties could be produced in CF systems. In this review, we first give an overview of CF synthesis with focus on enhancing protein expression in liposome-chaperoned CF systems. Furthermore, we intend to provide insight into harnessing CF-synthesized proteoliposomes for efficient therapeutic delivery.