De novo design of bioactive protein-resembling nanospheres via dendrimer-templated peptide amphiphile assembly

Abstract

Self-assembling peptide amphiphiles (PAs) have been extensively used in the development of novel biomaterials. Because of their propensity to form cylindrical micelles, their use is limited in applications where small spherical micelles are desired. Here we present a platform method for controlling the self-assembly of biofunctional PAs into spherical 50 nm particles using dendrimers as shape-directing scaffolds. This templating approach results in biocompatible, stable protein-like assemblies displaying peptides with native secondary structure and biofunctionality.

Figure 1

The dendrimers template micelle-forming and vesicle-forming PAs into spherical PRTNs. (a) Schematic of templating process: i. bZip PA cylindrical micelle ii. NLS PA vesicle iii. dendrimer iv. Spherical bZip PRTN v. Spherical NLS PRTN. Cryo-TEM images of self-assemblies: (b) bZip PA cylindrical micelles. (c) NLS PA vesicles. (d) Spherical bZip PRTNs. (e) Spherical NLS PRTNs. Scale bars are 100 nm.

Figure 2

Kinetics experiments show the fraction of aggregates that dissociated over time in the presence of albumin. bZip micelles () dissociate more quickly than bZip PRTNs ().

Figure 3

bZip secondary structure and DNA binding. (a) Circular dichroism of bZip peptide, bZip PA below and above the CMC, and bZip PRTNs. The lipidation of the bZip peptide provides a slight increase in helicity over the free peptide, while aggregation into micelles or on the surface of a dendrimer results in drastic increase in α-helicity indicated by the CD minima at 208 nm and 222 nm. (b) An increase in anisotropy corresponds to an increase in binding of bZip PAs (□) and bZip PRTNs () to rhodamine labeled 30bp DNA strands. The bZip PA curve has higher anisotropy at higher concentrations because the DNA strands are bound by long micelles rather than small PTRNs. Data points are the mean of 10 measurements, and error bars are standard deviation. (c) AFM height image of free ctDNA (3 μm frame depicted). (d) AFM height image of bZip PRTNs (spherical objects indicated by black arrows) bound to ctDNA (6 μm frame). PRTNs are not present on the surface but only appear in areas of DNA, indicating they are bound to the DNA and not attached to free mica. (e, f) AFM height and phase image of PRTNs bound to DNA (3 μm frame),where the spherical geometry is clearly seen. Additional AFM images available in the Supplemental Information.

Figure 4

Formation of multifunctional PRTNs. FRET was observed for the co-assembly of 1:1 ratio of bZip:NLS PAs with dendrimer (), confirming that the PAs formed “mixed' PRTNs. Fluorescein-tagged bZip PRTNs () and rhodamine-labeled NLS PRTNs () were used as controls. These experiments were performed with an excitation wavelength of 475 nm.

Figure 5

In vitro cell uptake of micelles and PRTNs. The green color in the images represents the fluorescein labeled bZip PAs, while the blue is Hoechst nuclear stain. Micelles and PRTNs are both internalized by HeLa cells. (a) Mixed PRTNs (90% bZip-FAM PA and 10% NLS PA) display punctate fluorescence, suggesting they are trapped in endosomes. The fluorescence is located in the bulk volume of the cell near the nucleus, and not observed throughout the cytoplasm to indicate endosomal escape. Intact PRTNs show low fluorescence, presumably due to fluorescence quenching. (b) Mixed micelles (90% bZip-FAM PA and 10% NLS PA) appear to be internalized via endocytosis as seen through the punctate fluorescence, but also show diffuse fluorescence, indicating PA distribution throughout the cell. The exact mechanisms of internalization are still under investigation. Scale bars are 50 μm.