EAK16-II

Herein, we highlight the technical feasibility of generating a functional mini thymus with a novel hydrogel system, based on a peptide-based self-assembly platform that can induce the formation of 3-D thymic epithelial cell (TEC) clusters. Amphiphilic peptide EAK16-II co-assembled with its histidinylated analogue EAKIIH6 into beta-sheet fibrils. When adaptor complexes (recombinant protein A/G molecules loaded with both anti-His and anti-EpCAM IgGs) were added to the mix, TECs were tethered to the hydrogel and formed 3-D mini clusters. TECs bound to the hydrogel composites retained their molecular properties; and when transplanted into athymic nude mice, they supported the development of functional T-cells. These mini thymic units of TECs can be useful in clinical applications to reconstitute T-cell adaptive immunity.

EAK16-II (AEAEAKAKAEAEAKAK) is one of the first building blocks of environmentally responsive materials. This self-assembling peptide undergoes solution-to-gel transition when transferred from a low to high ionic strength environment. Previously we have demonstrated the histidinylated analogue EAKIIH6 (AEAEAKAKAEAEAKAKHHHHHH) coassembles with the parent peptide to render His-tags as a functionalization mechanism in vitro and in vivo. The present study aimed to understand the pathways by which the analogue coassembles with EAK16-II. The results presented herein suggested two competing but not mutually exclusive events in the coassembly. Atomic force microscopic and gel electrophoretic data showed that EAKIIH6 self-sorted to high molecular weight species without EAK16-II. Self-sorting of EAKIIH6 was inhibited by the parent peptide in a concentration dependent manner. Injecting mixtures containing EAKIIH6 subcutaneously rendered His-tags detectable in live mice for at least 312 h, despite diluting the histidinylated analogue by 10-50 folds compared to a previous formulation. The study provided a formulation by which in vivo display of His-tags was attained without excess amphiphilic peptides. By increasing coassembling efficiency, the likelihood of generating immunogenic aggregates outside the main fibrils could be minimized. These findings provide insights for rational functionalization of in situ self-gelling materials.

Atomic force microscopy (AFM) and axisymmetric drop shape analysis-profile (ASDA-P) were used to investigate the mechanism of self-assembly of peptides. The peptides chosen consisted of 16 alternating hydrophobic and hydrophilic amino acids, where the hydrophilic residues possess alternating negative and positive charges. Two types of peptides, AEAEAKAKAEAEAKAK (EAK16-II) and AEAEAEAEAKAKAKAK (EAK16-IV), were investigated in terms of nanostructure formation through self-assembly. The experimental results, which focused on the effects of the amino acid sequence and pH, show that the nanostructures formed by the peptides are dependent on the amino acid sequence and the pH of the solution. For pH conditions around neutrality, one of the peptides used in this study, EAK16-IV, forms globular assemblies and has lower surface tension at air-water interfaces than another peptide, EAK16-II, which forms fibrillar assemblies at the same pH. When the pH is lowered below 6.5 or raised above 7.5, there is a transition from globular to fibrillar structures for EAK16-IV, but EAK16-II does not show any structural transition. Surface tension measurements using ADSA-P showed different surface activities of peptides at air-water interfaces. EAK16-II does not show a significant difference in surface tension for the pH range between 4 and 9. However, EAK16-IV shows a noticeable decrease in surface tension at pH around neutrality, indicating that the formation of globular assemblies is related to the molecular hydrophobicity.