Branched oligopeptides form nanocapsules with lipid vesicle characteristics

Abstract

In a recent article (Gudlur et al. PLOS ONE, 2012, 7 (9) e45374), we described the special properties of a mixed branched peptide assembly in which equimolar bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK self-associate to form bilayer delimited capsules capable of trapping solutes. These polycationic vesicle-like capsules are readily taken up by epithelial cells in culture, escape or evade the endocytic pathway, and accumulate in the perinuclear region where they persist without any apparent degradation. In this report, we examine the lipidlike properties of this system including initial assembly; solute encapsulation and washing; fusion and resizing by membrane extrusion through polycarbonate filters with defined pore sizes. The resized peptide capsules have uniform diameters in nm size ranges. Once resized, the capsules can be maintained at the new size by storing them at 4 °C. Having the ability to prepare stable uniform nanoscale capsules of desired sizes makes them potentially attractive as biocompatible delivery vehicles for various solutes/drugs.

Figure 1

Branched Bilayer Forming Sequences

Figure 2. Scanning Transmission Electron Micrograph (STEM) Hg-Labeled Peptides 24 hr after mixing

Capsules were prepared with 30% Me-Hg label in both peptides at 0.1 mM. The images were captured using annular dark field mode and then inverted to produce the final image.

Figure 3. Time course of capsule formation. S/TEM images of Hg-Labeled peptides taken at the indicated times

Capsules contain 30% Me-Hg label in both the bis(FLIVI) and bis(FLIVIGSII) peptides at 0.1 mM. The images were captured using annular dark field mode and then inverted to produce the final image. The scale bars at the bottom of the micrographs, in nm, are 200, 500, 200, 100, 200, and 500, for the 0, 5, 10, 30, 60 and 120 min time points, respectively.

Figure 4. Snapshots of initial and equilibrated structures of capsule coarse-grained model

The C-terminus group is represented by a yellow sphere; outside peptides are shown as light grey lines, with inside shown as cyan lines. The outside diameter of capsule is ~22nm

Figure 5

TEM image of Me-Hg labeled washed capsules just prior to fusion experiment.

Figure 6. Capsule Fusion Study

Salt washed eosin Y trapped capsules were mixed with water filled capsules in the ratio of 1:20 at RT. A) Five min fluorescence scans of eosin encapsulated vesicles spectra were taken at 5 min intervals for 235 min. The inset shows spectrum of sample stored at 4° C for 6.5 h. The units shown are identical to those in panel A. B) Measured maximum eosin fluorescence intensity as a function of time during the fusion reaction. The t = 0 represents quenched value of salt washed eosin encapsulate in the capsules (2.0 mM). The data was fitted to a second order exponential with the error bars representing the SEM with n = 3.

Figure 7. Filter Resizing Study

TEM images are shown for the A) 24 h control. B) 100 nm membrane extruded material and C) 30 nm membrane extruded material. All figures are displayed as inverse images.