Functional Delivery of Proteins Using Engineered Degradable Polymeric Nanocapsules

Download or read book Functional Delivery of Proteins Using Engineered Degradable Polymeric Nanocapsules written by Anuradha Biswas and published by . This book was released on 2013 with total page 126 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins have distinct intracellular roles which can have widespread therapeutic value in various biological applications. Particularly, transcription factors (TFs) are specialized proteins with the unique ability to direct cell fate which can have important applications in regenerative medicine and design of patient-specific disease models. Polymeric nanocarriers can be used to maintain protein structure and functional integrity while transporting proteins into the cytosol of cells and can be engineered to be intracellularly degradable. The development of a endoprotease-degradable intracellular protein delivery vehicle is described which draws biomimetic inspiration from natural phenomena occurring in response to the intracellular actions of furin. Both in situ and microemulsion polymerization were explored to synthesize degradable NCs. Furin-degradable nanocapsules (NCs) were further optimized to incorporate biologically-compatible polymers which exhibited minimal cytotoxicity in mice and human cell lines. Several nuclear and cytosolic proteins were delivered in active form to various cell lines using furin-degradable or redox-responsive NCs. NCs exhibited stability in response to protease treatment, acidic conditions and storage and high temperatures and retained encapsulated protein as well as mRNA. To demonstrate delivery of an active nuclear protein, the myogenic differentiation TF MyoD was encapsulated in NCs and delivered to myoblast cells. NC-treated cells exhibited differentiation of myoblast cells into myotubes which displayed morphological development and activated gene expression of downstream markers. Active delivery of MyoD confirmed the ability of NCs to retain the activity of encapsulated protein to allow complex downstream actions including DNA binding and association with co-activators. For treatment of type I diabetes, the generation of pancreatic beta-cells using a TF, Pdx1, is a feasible therapeutic option. Pdx1 degradable NCs were delivered to liver and exocrine pancreatic cells to induce transdifferentiation to pancreatic beta-cells. Enhanced nuclear delivery and biological assays indicated functional delivery of Pdx1 to the nuclei of target cells. Morphological differences between untreated and treated exocrine cells indicated that Pdx1 delivered by NCs was able to induce changes in cells. Gene expression analysis also demonstrated slight activation of downstream beta-cell markers. This work represents development of a robust polymeric nanocarrier platform for intracellular delivery and exhibits several examples of functional delivery of proteins to various cell lines.