Switching of Self-Assembly in a Peptide Nanostructure with a Specific Enzyme

Abstract

Peptide self-assembly has been shown to be a useful tool for the preparation of bioactive nanostructures, and recent work has demonstrated their potential as therapies for regenerative medicine. In principle, one route to make these nanostructures more biomimetic would be to incorporate in their molecular design the capacity for biological sensing. We report here on the use of a reversible enzymatic trigger to control the assembly and disassembly of peptide amphiphile (PA) nanostructures. The PA used in these studies contained a consensus substrate sequence specific to protein kinase A (PKA), a biological enzyme important for intracellular signaling that has also been shown to be an extracellular cancer biomarker. Upon treatment with PKA, this PA molecule becomes phosphorylated causing the high aspect-ratio filamentous PA nanostructures to disassemble. Treatment with an enzyme to cleave the phosphate group results in reformation of the filamentous nanostructures. We also show that disassembly in the presence of PKA allows the enzyme-triggered release of an encapsulated cancer drug. In addition, these drug-loaded nanostructures were found to induce preferential cytotoxicity in a cancer cell line that is known to secrete high levels of PKA. This ability to control nanostructure through an enzymatic switch could allow for the preparation of highly sophisticated and biomimetic materials that incorporate a biological sensing capability to enable therapeutic specificity.

Figure 1

(A) Chemical structures and reaction scheme for the conversion of peptide amphiphile 1 (PA 1) into its phosphorylated form (PA 2) by protein kinase A and then its conversion back to the de-phosphorylated form (PA 1R) by alkaline phosphatase. (B) Analytical liquid chromatography trace showing the complete conversion of PA 1 to PA 2, as well as the conversion back to PA 1R.

Figure 2

Cryogenic transmission electron microscopy (Cryo-TEM) showing the filamentous nanostructure of (A) PA 1 which is phosphorylated to produce (B) PA 2 and results in a disappearance of the nanostructure. (C) Treatment with alkaline phosphatase to de-phosphorylate the molecule restores the filamentous nanostructure. Scale bars are 200 nm (A and C) and 500 nm (B).

Figure 3

Small angle x-ray scattering (SAXS) showing the raw scattering profiles of PAs 1, 2, and 1R along with a solvent background.

Figure 4

Cartoon depicting the disassembly of PA 1 upon treatment with protein kinase A and its reassembly following treatment with alkaline phosphatase.

Figure 5

Circular dichroism (CD) spectroscopy (A) for PA 1 (black) and PA 2 (red). Vial inversion of a hydrogel (B) formed from PA 1 without exposure to PKA (1) and with exposure to PKA (2).

Figure 6

(A) Release of DOX, measured by dialysate concentration, from solutions of free drug as well as DOX mixed with PA 1 and with PAs 1 and 3 in the presence of PKA. (B) Live(Green)/Dead(Red) cell viability imaging of three different cell types (MDA-MB-231, HUVECs, 3T3) after DOX or either PA 1 or PA 3 mixed with DOX was added to the conditioned media of a confluent cell monolayer.