Peptide nanovesicles formed by the self-assembly of branched amphiphilic peptides

Abstract

Peptide-based packaging systems show great potential as safer drug delivery systems. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. Here, we describe a set of 15 & 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. These peptides undergo supramolecular self-assembly and form solvent-filled, bilayer delimited spheres with 50-200 nm diameters as confirmed by TEM, STEM and DLS. Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these all-peptide structures are stabilized potentially by both hydrophobic interactions and hydrogen bonds and remain intact at low micromolar concentrations and higher temperatures. A linear peptide lacking the branch point showed no self-assembly properties. We have observed that these peptide vesicles can trap fluorescent dye molecules within their interior and are taken up by N/N 1003A rabbit lens epithelial cells grown in culture. These assemblies are thus potential drug delivery systems that can overcome some of the key limitations of the current packaging systems.

Figure 1. Assembling and control sequences.

The peptide assemblies are an equimolar mixture of the acetylated bis(h₉)-K-K₄ and bis(h₅)-K-K₄ sequences unless otherwise stated. The linear h₉-K₅ control sequence does not associate to give any type of structure.

Figure 2. A coarse-grained simulation of bis(h₅)-K-K₄ bilayer self-assembly.

All lysine residues are plotted in green ball-and-stick representations, the aromatic sidechains of phenylalanine residues in blue sticks, and all other residues in cyan. The snapshots were taken at 0 ns, 2 ns, 4 ns, 10 ns and 40 ns, respectively. A hypothetical peptide vesicle model is shown to the right.

Figure 3. Transmission Electron Micrograph (TEM) of h₉h₅-vesicles.

These peptides self-assemble into nano-sized vesicles. TEM images of (A) a concentrated and (B) a diluted sample of the peptide mixture, stained with 5% phosphotungstic acid and Osmium tetroxide (OsO4) vapors, respectively (200 nm scale bar).

Figure 4. Scanning Transmission Electron Micrograph (STEM).

Vesicles were prepared with 30% CH₃-Hg label in both the h₅ and h₉ peptides at 0.1 mM concentration were negatively stained using a multi isotope 2% Uranyl acetate (Uranium bis(acetato)-O)dioxo-dihydrate) aqueous solution. The images were captured using annular dark field mode was then inverted to produce the final image.

Figure 5. Dynamic Light Scattering of h₉h₅-vesicles.

Distribution analysis of the data showing two major distributions centered around 1.68 ms and 119.4 ms and a mean correlation time of 1.85 ms and 275.7 ms respectively. These correlation times correspond to a hydrodynamic radius of 80 nm and 10 µm respectively.

Figure 6. Secondary Structural Characterizations.

(A) Circular dichroism (CD) spectroscopy shows that the h₉h₅-vesicles adopt a β-structure (solid line) at pH 7.0 in 5 mM NaHCO₃, a helical structure (dotted line) in 40% TFE and remains unstructured (dashed line) when lacking the lysine branch point (h₉-K₅). (B) FTIR spectroscopy of dried material shows the characteristic 1629 cm⁻¹ maximum corresponding to β- secondary structure.

Figure 7. Differential scanning calorimetry of 1.0 mg/ml peptide in water.

Heat capacity of solution is shown as a function of temperature. The instrument water baseline is shown together with the thermograms obtained during two consecutive upward temperature scans.

Figure 8. Encapsulation studies with 5(6)-Carboxyfluorescein.

(A) 5(6)-Carboxyfluorescein loaded peptide vesicles were prepared in different ethanol concentrations and separated over 30 kDa MWCO filters. Fluorescence intensities of the filtrate (open circles) and retentate (solid circles) were collected after separation and show that encapsulation efficiency decreases with increasing ethanol concentration. (B) h₉h₅-vesicles prepared with the dye present during vesicle formation (solid circles) or added after the vesicles are formed (open circles). Each data point represents an average of 3 separate experiments performed on different days. Data points are connected with straight lines through their midpoints.

Figure 9. Cellular uptake of preloaded h₉h₅-vesicles.

(A–C) h₉h₅-vesicles with one of the peptides labeled (bis(h₅)-K-K₄ labeled with Carboxytetramethylrhodamine) and loaded with 5(6)-Carboxyfluorescein were delivered to N/N 1003A rabbit lens epithelial cells. Green and red filter settings were used for the detection of 5(6)-Carboxyfluorescein (A) and Rhodamine labeled peptide (B) respectively. (C) is a merge of a & b showing co-localization of dye and h₉h₅-vesicles within the cell.