Short peptide motifs for long-lasting anchoring to the cell surface.

A rational design strategy has been developed for the construction of stable peptide-based anchors for the efficient modification of cell surfaces. Six types of peptide composed of five residues with divalent hydrophobic groups have been designed using this new strategy. Among them, a peptide with a sequence of NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH2 (NBD: fluorophore, Lys(X): N-ε-palmitoyl-l-lysine) was found to show the highest modification efficacy and longevity in culture medium. The good performance of this peptide was attributed to (1) its high aqueous solubility, which allowed it to partition from the medium to the cell surface, and (2) the high binding affinity of the saturated palmitoyl groups to the cell membrane. We found that the distribution of the peptide was affected by recycling endosome, which enabled the representation of the peptide following its endocytotic disappearance from the cell membrane. Biotin was also presented on the cell surface using this peptide-based anchor to examine its recognition by streptavidin. The efficacy of the recognition process increased as the length of the oligoethylene glycol spacer increased, indicating that it was necessary for the biotin tag to move away from the membrane glycoproteins on the cell surface to facilitate its efficient recognition by streptavidin.

Folate receptor-specific cell-cell adhesion by using a folate-modified peptide-based anchor.

We report here a folate-modified membrane anchor for cell surface modification to induce cell adhesion to target cells. The membrane anchor region, which was consisted of cationic lysine residues and palmitoyl group-modified residues, was modified with folate through an oligoethlene glycol linker. The peptide anchor was modified on to the cell membrane by using ß-cyclodextrin as a solubilizer of the peptide anchor. After modification, the peptide anchor disappeared from the cell membrane via endocytotic uptake or dissociation from the cell membrane. However, the endocytosed peptide was represented on the cell surface via recycling endosome pathway. The obtained folate-modified cells successfully adhered on to target cells which expressed folate receptor α via ligand-receptor specific interaction and adhesion continued at least 4 hours.

Facilitating the presentation of antigen peptides on dendritic cells for cancer immunotherapy using a polymer-based synthetic receptor.

The introduction of proteins into dendritic cells (DCs) ex vivo is a critical step for the DC-based immunotherapy of cancer. Here, we developed a biotin-modified polymer with multiple hydrophobic membrane anchors for cells that functions as a synthetic receptor for an antigen protein, ovalbumin (OVA), to introduce it efficiently into DCs compared with the conventional pulsing method. Our method showed significant advantages, including the rapid incorporation of OVA and the activation of antigen-specific T cells in a MHC-restricted manner. When mice were immunized by DCs treated with our method, tumor growth was completely suppressed, indicating that our method can be used to prepare adjuvant DCs.

Optimum design of amphiphilic polymers bearing hydrophobic groups for both cell surface ligand presentation and intercellular cross-linking.

Amphiphilic polymers bearing hydrophobic alkyl groups are expected to be applicable for both ligand presentation on the cell surface and intercellular crosslinking. To explore the optimum design for each application, we synthesized eight different acyl-modified dextrans with varying molecular weight, alkyl length, and alkyl modification degree. We found that the behenate-modified polymers retained on the cell surface longer than the palmitate-modified ones. Since the polymers were also modified with biotin, streptavidin can be presented on the cell surface through biotin-streptavidin recognition. The duration of streptavidin on the cell surface is longer in the behenate-modified polymer than the palmitate-modified one. As for the intercellular crosslinking, the palmitate-modified polymers were more efficient than the behenate-modified polymers. The findings in this research will be helpful to design the acyl-modified polymers for the cell surface engineering.

Rapid and serum-insensitive endocytotic delivery of proteins using biotinylated polymers attached via multivalent hydrophobic anchors.

We have designed biotinylated polymers as synthetic receptors that have multiple alkyl groups for endocytotic delivery of target proteins. The polymers were stably attached to a cell surface via multivalent anchoring. The presented biotin was bound to streptavidin (SA) on the cell surface, and, via an endocytotic pathway, the cell rapidly internalized the biotinylated polymer/SA complex. The cell's uptake of the complex was not inhibited by the presence of 10% fetal bovine serum, and its efficacy for the uptake of SA was the highest when compared with commercial reagents and single-anchored-type synthetic receptors. The synthetic receptor-mediated endocytosis can be used generally for other kind of protein by using SA as an adaptor molecule between a target protein and the cell-surface presented biotin.