Self-assembly of cytotoxic peptide amphiphiles into supramolecular membranes for cancer therapy

Abstract

Peptide amphiphiles (PAs) provide a versatile platform for the design of complex and functional material constructs for biomedical applications. The hierarchical self-assembly of PAs with biopolymers is used to create robust hybrid membranes with molecular order on the micron scale. Fabrication of membranes by assembling hyaluronic acid with positively charged PA nanostructures containing anti-cancer PAs bearing a (KLAKLAK)(2) peptide sequence is reported here. Changes in membrane microstructure as the positively charged PA nanostructures vary from cylindrical nanofibers to spherical aggregates are characterized. Results indicate that formation of highly aligned fibrous membranes requires a threshold concentration of nanofibers in solution. Additionally, variation of PA nanostructure morphology from spherical aggregates to cylindrical nanofibers allows membranes to act either as reservoirs for sustained release of cytotoxicity upon enzymatic degradation or as membranes with surface-bound cytotoxicity, respectively. Thus, the self-assembly processes of these PA-biopolymer membranes can be potentially used to design delivery platforms for anti-cancer therapeutics.

Figure 1

Transmission electron micrographs of PA nanostructures at various co-assembly compositions. High aspect-ratio nanofibers are predominant in aqueous solutions of (a) 100% K₃ PA and (b) 2% KLAK PA. Solutions of (c) 20% KLAK PA display a significant number of small spherical aggregates, while solutions of (d) 50% KLAK PA and (e) 100% KLAK PA contain predominantly spherical aggregates with few short nanofibers. (f) Heating a 50% KLAK PA solution at 80 °C for 8 h leads to nanofiber morphology. Total PA concentration is 5 mm for all samples. Labels indicate the percentage of KLAK PA, with K₃ PA comprising the remaining balance.

Figure 2

Small angle x-ray scattering profiles of various co-assembly compositions. (a) The slope of the Guinier region at low q tends towards 0 as the percentage of KLAK PA is increased, indicating shift towards spherical aggregate nanomorphology. (b-c) Comparisons of co-assembled mixtures with the sum of their individual components suggest mixing on a molecular level and also highlight the bias towards spherical morphology when KLAK PA is present.

Figure 3

Scanning electron micrographs of membranes assembled from hyaluronic acid and various PA co-assemblies. Evidence of diffusion barrier formation and perpendicular fiber growth is seen in the case of (a) 100% K₃ PA, (b) 2% KLAK PA, (c) 20% KLAK PA, and (f) heat treated 50% KLAK PA. Non-fibrous, featureless membrane microstructure is seen for (d) 50% KLAK PA and (e) 100% KLAK PA.

Figure 4

Confocal micrographs showing the distribution of RBITC-labeled K₃ PA and FITC-labeled KLAK PA within the (a) 20% KLAK PA membrane and (b) 50% KLAK PA membrane. Membrane cross-sections are shown in the bottom panels, with the top of the panel corresponding to the side of the membrane that faced biopolymer compartment.

Figure 5

Viability of (a) MDA-MB-231 breast adenocarcinoma cells, (b) SKBR-3 breast adenocarcinoma cells, and (c) MCF10a breast epithelial cells 24 h after addition of membrane degradation products. Membranes assembled with 50% KLAK PA solution do not produce cytotoxic products in FBS but do produce significantly cytotoxic products after two days of enzymatic degradation by hyaluronidase. Non-cancerous MCF10a breast epithelial cells are less sensitive to the cytotoxic effects of degradation products. No other membrane exhibits toxic degradation products even after 4 days of enzymatic degradation. Viability is normalized to degradation products of HA-Tyr gels. Data points represent mean and standard error for n=3 (***P<0.001).

Figure 6

Behavior and viability of fluorescent MB-MDA-231 breast cancer cells seeded onto membranes. Cells on membranes assembled from 20% KLAK PA solution attach to the surface and are rounded (a) immediately after seeding but (b) migrate and eventually die after at later time points. (c) Membranes assembled from 20% KLAK PA and 50% KLAK PA solutions are significantly cytotoxic after only 2 h, while the associated membrane-conditioned media is not cytotoxic.