Effects of Polymer End-Group Chemistry and Order of Deposition on Controlled Protein Delivery from Layer-by-Layer Assembly

Layer-by-layer (LBL) assembly is an attractive platform for controlled release of biologics given its mild fabrication process and versatility in coating substrates of any shape. Proteins can be incorporated into LBL coatings by sequentially depositing oppositely charged polyelectrolytes, which self-assemble into nanoscale films on medical devices or tissue engineering scaffolds. However, previously reported LBL platforms often require the use of a few hundred layers to avoid burst release, which hinders their broad translation due to the lengthy fabrication process, cost, and batch-to-batch variability. Here we report a biodegradable LBL platform composed of only 10 layers with tunable protein release kinetics, which is an order of magnitude less than previously reported LBL platforms. We performed a combinatorial study to examine the effects of polymer chemistry and order of deposition of poly­(β-amino) esters on protein release kinetics under 81 LBL assembly conditions. Using the optimal “polyelectrolyte couples” for constructing the LBL film, basic fibroblast growth factor (bFGF) was released gradually over 14 days with retained biological activity to stimulate cell proliferation. The method reported herein is applicable for coating various substrates including metals, polymers, and ceramics and may be used for a broad range of biomedical and tissue engineering applications

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity

AbstractAlternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.</jats:p

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