Proteins play a crucial role in life, taking part in all vital processes in the body. In the past decade, there was increasing interest in delivering active forms of proteins to specific cells and organs. Intracellular protein delivery holds enormous promise for biological and medical applications, including cancer therapy, vaccination, regenerative medicine, treatment for loss-of-function genetic diseases and imaging. This tutorial review surveys recent developments in intracellular protein delivery using various nanocarriers. Methods such as lipid-mediated colloidal systems, polymeric nanocarriers, inorganic systems and protein-mediated carriers are reviewed. Advantages and limitations of current strategies, as well as future opportunities and challenges are also discussed.

An average cell contains thousands of proteins that participate in normal cellular functions, and most diseases are somehow related to the malfunctioning of one or more of these proteins. Protein therapy, which delivers proteins into the cell to replace the dysfunctional protein, is considered the most direct and safe approach for treating disease. However, the effectiveness of this method has been limited by its low delivery efficiency and poor stability against proteases in the cell, which digest the protein. Here, we show a novel delivery platform based on nanocapsules consisting of a protein core and a thin permeable polymeric shell that can be engineered to either degrade or remain stable at different pHs. Non-degradable capsules show long-term stability, whereas the degradable ones break down their shells, enabling the core protein to be active once inside the cells. Multiple proteins can be delivered to cells with high efficiency while maintaining low toxicity, suggesting potential applications in imaging, therapy and cosmetics fields.

Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13°C at 30 µM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5-1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.Engineering of stable enzymes and robust therapeutic proteins is of central importance to the biotechnology and pharmaceutical industries. Although protein engineering provides powerful tools for the enhancement of enzymatic activity and protein stability (1-4), the scope of in vivo engineering methods is limited by the availability of just 20 naturally occurring proteinogenic amino acids (5). Increasing success in the incorporation of noncanonical amino acids into recombinant proteins in vivo has allowed the introduction of novel side-chain functionality into engineered proteins (6-10) and raises prospects of new approaches to the design of peptides and proteins of enhanced activity and/or stability.Leucine zipper peptides are ideal models for the study of protein secondary and tertiary interactions (11-20, and references therein). Such peptides assemble into coiled-coil dimers, trimers, and tetramers in order to exclude solvent at the hydrophobic interfaces between adjacent peptide helices, and the relations between sequence and stability have been carefully examined (21). In particular, the structure (13), dimerization kinetics (20), and thermodynamics (19) of the model peptide GCN4-p1 have been thoroughly described.GCN4-p1 constitutes the dimerization domain of bZip, which is a 56-amino acid DNA binding segment (residues 226-281) of the eukaryotic transcription factor GCN4. The N-terminus of bZip contains a DNA recognition domain rich in the basic residues lysine and arginine. The C-terminal subdomain of bZip contains the GCN4-p1 peptide segment and facilitates dimerization of the protein. While direct contact between DNA and the N-terminal subdomain is important to recognition, protein-protein interactions at the C-terminus also contribute to the specificity and affinity of peptide-DNA binding (22)(23)(24).We present here a successful attempt to stabilize the coiledcoil forms ...