Reactive Self-Assembly of Polymers and Proteins to Reversibly Silence a Killer Protein

Abstract

Conjugation of biologically active proteins to polymeric materials is of great interest in the treatment of cancer and other diseases of protein deficiencies. The conjugation of such biomacromolecules is challenging both due to their hydrophilicity and propensity to denature under non-native conditions. We describe a novel reactive self-assembly approach to “wrap” a protein with polymers, simultaneously protecting its delicate folded state and silencing its enzymatic activity. This approach has been demonstrated using caspase-3, an apoptosis-inducing protein, as the first case study. The protein-polymer conjugation is designed to be reversed under the native conditions for caspase-3, i.e. the reducing environment found in the cytosol. The current strategy allowed release and recovery of up to 86% of caspase activity and nanogel-caspase-3 conjugates induced 70–80% apoptotic cell death shortly thereafter. This approach is widely generalizable and should be applicable to the intracellular delivery of a wide range of therapeutic proteins for treatment of complex and genetic diseases.

Utilizing Inverse Emulsion Polymerization To Generate Responsive Nanogels for Cytosolic Protein Delivery

“…To circumvent these challenges, various delivery systems have been developed using nanoparticle-based scaffolds including liposomes, 7, 8 polymers, 9, 10 and nanogels 11–16 . The biomacromolecular cargo in these systems are typically sequestered by entrapment, 12 covalent conjugation 9, 17, 18 or electrostatic complexation.…”

“…The biomacromolecular cargo in these systems are typically sequestered by entrapment, 12 covalent conjugation 9, 17, 18 or electrostatic complexation. 11, 19, 20 Covalent conjugation of PEG polymers, termed PEGylation, 21, 22 has been widely used to increase the circulation lifetimes of biologics and also protect them from degradative enzymes.…”

Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition

“…This altered-specificity caspase, which is the first of its kind, demonstrates the utility of caspases as a platform for development of novel therapeutic proteases that could be delivered by a number of emerging tools. 39–41 The altered-specificity caspase also provides a tool to assess whether exosites exist and play a role in substrate selection.…”

Intracellular Delivery of Proteins By Nanocarriers

“…With further study, FLs can emerge as a powerful vehicle for protein delivery [31] in future. Adapted with permission from [45]. …”

“…However, no diffusion of rhodamine fluorescence was observed inside the nuclei, suggesting further investigation to be done to prove nuclear translocation of the protein. More recently, Thayumanavan and colleagues [45] reported a self-assembly strategy Intracellular delivery of proteins by nanocarriers Review to conjugate active enzymes, such as, caspase-3, to polymeric nanogels. In this system, the enzyme was conjugated on the surface or encapsulated inside the polymeric redox-sensitive nanogel through disulfide linkages as shown in Figure 3A.…”