Novel Ultrasound-Responsive Amyloid Formulation

Abstract

Amyloid aggregates have attracted significant interest in regard to diverse biomedical applications, particularly in the field of drug delivery. Here, we report novel amyloid aggregates based on a 12-amino-acid peptide from the amyloidogenic region of the receptor-interacting kinase 3 (RIP3) protein and a thermoresponsive triblock copolymer, namely, Pluronic F127 (RIP3/F127). Physicochemical characterization was performed to determine the aggregation size, morphology, and stimuli-responsive properties. The potential of the aggregates as a drug depot was assessed in lung cancer cells, using Doxorubicin (Dox) as a model drug. The results show that RIP3 and RIP3/F127 exhibit amyloidogenic properties. Further, the RIP3/F127 amyloids exhibited significant ultrasound-responsive properties compared to amyloid aggregates without Pluronic F127. Moreover, the RIP3/F127/Dox amyloid formulations that were subjected to ultrasound treatment exhibited greater toxicity to lung cancer cells compared to that of Dox alone at equal concentrations. Overall, the results from this proof-of-concept study show that amyloidogenic peptide aggregates with stimuli-responsive properties can be utilized as efficient drug delivery depots.

Keywords: RIP3; amyloid aggregates; drug delivery; thermoresponsive polymer.

Figure 1

(A) Schematic of RIP3 amyloid aggregation formation with and without Pluronic F127. (B) Aggregation of RIP3 peptides with and without F127 characterization was investigated using ThT fluorescence. Measurements were carried out at 24 h (i) and 48 h (ii) after the initiation of aggregation. At least three independent experiments were carried out. * p < 0.05, ** p < 0.01, and **** p < 0.0001. (C) Flow cytometry analysis of Proteostat dye assay of RIP3 and/or RIP3/F127 aggregation. Lysozyme aggregates and monomers were used for comparison. Samples aggregated for 48 h were used for the assay.

Figure 2

(A) (i) The morphologies of amyloid structures were characterized using TEM. Scale bar: 200 nm. (ii) DLS measurements of RIP3 amyloids with and without Pluronic F127. (B) (i) TEM images of the amyloid aggregates before and after subjection to ultrasound stimuli (1.6 W/m², for 3 min). Scale bar: 200 nm. (ii) DLS measurements of the amyloid aggregates before and after they were subjected to ultrasound.

Figure 3

Amyloids with Dox were characterized using (A) ThT and (B) Proteostat assay. Three independent experiments were carried out. **** p < 0.0001. Ultrasound measurements (1.6 W/m² for 3 min) were taken for the amyloid–Dox formulations. DLS measurements (C) and TEM images (scale bar 200 nm) (D) were obtained before and after ultrasound application. Three independent measurements were taken. (E) Dox encapsulation was determined from the dox calibration curve obtained at 470/495 nm excitation/emission. Dox concentrations of 0.5–50 µg/mL were used. Three independent experiments were used to determine encapsulation efficiency.

Figure 4

The cellular uptake of the amyloid aggregates was investigated using confocal microscopy and flow cytometry. A549 cells were treated with amyloid aggregates for 30 min, and then uptake was analyzed. (A). The cellular uptake of the amyloid aggregates was investigated using confocal microscopy. Scale bar 20 µm. (B). Flow cytometry measurements depict the shift in fluorescence with and without aggregate treatment.

Figure 5

(A). Effects of the amyloid drug depots on A549 lung cancer cells were assessed using alamarBlue measurements. A549 lung cancer cells were plated in 96 wells at a density of 10,000 cells/well and treated with free Dox, amyloid aggregates with and without Dox, and/or ultrasound (US) at a concentration of 2.5 µM. Three independent experiments were performed. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (B). Effects of the amyloid drug depots on BEAS-2B normal lung epithelial cells. BEAS-2B lung epithelial cells were plated in 96 wells at a density of 10,000 cells/well and treated with amyloid aggregates, and the metabolic activity was analyzed with alamarBlue after 48 h.