Self-assembly of thermally responsive amphiphilic diblock copolypeptides into spherical micellar nanoparticles.

As structure–property relationships for protein self-assembly have been elucidated, advances in chemistry and structural biology have facilitated the development of biologically inspired polypeptides through chemical and biosynthetic schemes that have afforded novel protein-based films, fibers, micelles, and gels. In a number of instances, reversible protein self-assembly has been driven by welldefined conformational changes of peptide units induced in response to an external stimulus. Indeed, designed molecular assembly of stimuli-responsive peptides has emerged as a “bottom-up” approach for creating complex, but ordered, hierarchical structures from simple amino acid building blocks. As illustrated by the design of diand triblock polypeptides, microand nanoscale features can be tuned by control of the amino acid sequence, molecular weight, and secondary structure of the peptide. In particular, amphiphilic block copolypeptides can self-assemble into a variety of diverse structures, including rods, cylinders, spheres, and vesicles. Although diblock copolymers consisting of chemically and conformationally distinctive individual polypeptide blocks have been produced by chemical and biosynthetic schemes, to date, relatively few recombinant amphiphilic diblock polypeptides have been synthesized. Given the capacity to incorporate targeting ligands, cell membrane fusion sequences, receptor activating peptides, fluorescent or chelating groups, as well as the ability to tailor pharmacokinetics, biodistribution, and peptide stability, significant opportunities exist for micelles or vesicles produced from recombinant protein block copolymers. Elastin-mimetic polypeptides based on the pentameric repeat sequence (Val-Pro-Gly-Xaa-Gly) undergo thermal and pH-responsive self-assembly in aqueous solution. Spontaneous phase separation of the polypeptide coincides with a conformational rearrangement of local secondary structure above a unique transition temperature (Tt) determined by the chemical identity of the fourth amino acid (Xaa) in the pentapeptide repeat. Recent studies have demonstrated the potential of engineered materials derived from elastin in a broad range of biomedical and biotechnological applications and, in particular, drug delivery. 8] Characteristically, elastin-mimetic blocks that contain hydrophobic amino acids in the fourth amino acid position, such as tyrosine, display a conformational transition from random coil to repetitive type II b turns at temperatures well below 37 8C, whereas blocks that contain a charged amino acid in this position, such as glutamic acid, persist as a random coil throughout the physiologic temperature range. Thus, we postulated that amphiphilic diblock copolymers bearing glutamic acid and tyrosine residues in Nand C-terminal blocks, respectively, would promote micelle formation by temperature-induced self-assembly with a core–shell structure. Moreover, we speculated that at a sufficiently high density of glutamic acid units, charge repulsion would limit the association of the hydrophilic blocks andminimize micelle aggregation. Micelles stabilized by self-assembly alone are typically unstable in a complex environment containing naturally occurring amphiphiles, such as plasma proteins, glycolipids, and lipopeptides. Therefore, by positioning cysteine residues between blocks, we hypothesized that highmolecular-weight protein aggregation or uncontrolled micelle–micelle association would be avoided by nanoparticle stabilization through disulfide cross-linking. These studies represent the first report of thermally responsive and crosslink stabilized protein micelles produced through the tailored design of recombinant amphiphilic diblock copolymers. Two amphiphilic diblock polypeptides (ADP1 and ADP2) were synthesized and self-assembled into micellar structures with consecutive cysteine residues incorporated at the core– shell interface (Scheme 1). Expression of the diblock synthetic genes in E. coli expression strain, BL21(DE3), afforded recombinant protein polymers in high yield after immobilized-metal-affinity chromatography (IMAC) purification from the cell lysate. Mass spectrometry confirmed a correspondence between the observed and expected masses of the respective diblocks with consistent sequence composition by amino acid analysis. The presence of cysteine residues within the polypeptide chain was characterized by the use of a thiolreactive fluorescent dye (see the Supporting Information). Differential scanning calorimetry (DSC) demonstrated an endothermic transition at around 10 8C for both diblock copolymers, which conforms to the established relationship between the position of the transition temperature and the mole fraction of tyrosine in elastin-mimetic protein poly[*] Dr. W. Kim, Dr. E. L. Chaikof Emory University Departments of Biomedical Engineering and Surgery Georgia Institute of Technology, School of Chemical Engineering 101 Woodruff Circle, Rm 5105, Atlanta, GA 30322 (USA) Fax: (+1)404-727-3667 E-mail: echaiko@emory.edu