Coassembled cytotoxic and pegylated peptide amphiphiles form filamentous nanostructures with potent antitumor activity in models of breast cancer

Abstract

Self-assembled peptide amphiphiles (PAs) consisting of hydrophobic, hydvrogen-bonding, and charged hydrophilic domains form cylindrical nanofibers in physiological conditions and allow for the presentation of a high density of bioactive epitopes on the nanofiber surface. We report here on the use of PAs to form multifunctional nanostructures with tumoricidal activity. The combination of a cationic, membrane-lytic PA coassembled with a serum-protective, pegylated PA was shown to self-assemble into nanofibers. Addition of the pegylated PA to the nanostructure substantially limited degradation of the cytolytic PA by the protease trypsin, with an 8-fold increase in the amount of intact PA observed after digestion. At the same time, addition of up to 50% pegylated PA to the nanofibers did not decrease the in vitro cytotoxicity of the cytolytic PA. Using a fluorescent tag covalently attached to PA nanofibers we were able to track the biodistribution in plasma and tissues of tumor-bearing mice over time after intraperitoneal administration of the nanoscale filaments. Using an orthotopic mouse xenograft model of breast cancer, systemic administration of the cytotoxic pegylated nanostructures significantly reduced tumor cell proliferation and overall tumor growth, demonstrating the potential of multifunctional PA nanostructures as versatile cancer therapeutics.

Figure 1. PA Characterization

(A) Chemical structure of “KLAK PA” with sequence palmitoyl-A₄G₃(KLAKLAK)₂ and “PEG PA” with the sequence PEG₂₀₀₀-E₃G₃A₄K(C₁₂). Cryo-TEM of KLAK PA alone (B) KLAK with PEG (D) show a significant difference in average length. Conventional TEM images show fiber formation for both KLAK alone (C) and KLAK PA with PEG PA (E). SAXS confirms the presence of cylindrical structures in solution for both KLAK mixed with PEG PA (F) and PEG PA alone (G).

Figure 2. DOSY of Co-Assembled Structures

(A) Overlaid DOSY results for KLAK PA alone (red) and KLAK with PEG PA show a shift in the diffusion coefficient. (B) Using the KLAK PA peak at 2.83 ppm, the KLAK PA alone diffusion coefficient shows an increase upon mixing with PEG PA.

Figure 3. Enzymatic Degradation

(A) The percentage of intact KLAK PA, as measured by LC/MS, increases with increasing concentrations of PEG PA. The closed arrow points to the KLAK PA peak, and the open arrow points to the internal standard, kept constant in all runs. (B) Quantification of intact KLAK PA. The addition of PEG PA prevents trypsin degradation. The 50(◆) bar represents a solution of KLAK and PEG PA that were pre-assembled separately prior to mixing and exposing to trypsin. (*** p < 0.001, * p < 0.05).

Figure 4. In Vitro Cytotoxicity

(A) MDA-MB-231, MCF7, and SKBR3 human breast cancer cells were treated as indicated for 24 h and viability was quantified using a MTS assay. MCF7 human breast cancer cells were treated as indicated and intracellular DAPI (B) and Mitotracker red (C) fluorescence was determined by flow cytometry to assess the plasma and mitochondrial membrane integrity.

Figure 5. PA Plasma and Tissue Biodistribution

(A) Quantification of plasma fluorescence, n=6 per time point. (B) Tissue fluorescence following intraperitoneal administration of KLAK PA covalently modified with Alexa 700 to tumor bearing mice; tissues from the 6 h time point are shown. (C), Quantification of fluorescence as average radiant efficiency (n=6 animals per time point) comparing the signal in tissues from animals treated with KLAK PA alone or KLAK/PEG PA; the dashed line represents background fluorescence observed in control untreated animals. Statistical significance in all panels as measured by Student's t test is noted.

Figure 6. In Vivo Anti-Tumor Activity

(A) The growth of MDA-MB-231 human breast cancer orthotopic tumors is inhibited by intraperitoneal treatment (inverted arrows) of KLAK PA nanostructures. Both the KLAK and KLAK/PEG PA treated tumors were statistically smaller as determined by 2-way ANOVA. (B) Mouse weights across groups were similar suggesting the nanostructure treatment was well tolerated. (C) BRDU staining as a measure of cellular proliferation was statistically lower by 1-way ANOVA in the L-KLAK PA treated tumors when co-assembled with PEG PA.