Probing peptide amphiphile self-assembly in blood serum

Abstract

There has been recent interest in designing smart diagnostic or therapeutic self-assembling peptide or polymeric materials that can selectively undergo morphological transitions to accumulate at a disease site in response to specific stimuli. Developing approaches to probe these self-assembly transitions in environments that accurately amalgamate the diverse plethora of proteins, biomolecules, and salts of blood is essential for creating systems that function in vivo. Here, we have developed a fluorescence anisotropy approach to probe the pH-dependent self-assembly transition of peptide amphiphile (PA) molecules that transform from spherical micelles at pH 7.4 to nanofibers under more acidic pH's in blood serum. By mixing small concentrations of a Ru(bipy)3(2+)-tagged PA with a Gd(DO3A)-tagged PA having the same lipid-peptide sequence, we showed that the pH dependence of self-assembly is minimally affected and can be monitored in mouse blood serum. These PA vehicles can be designed to transition from spherical micelles to nanofibers in the pH range 7.0-7.4 in pure serum. In contrast to the typical notion of serum albumin absorbing isolated surfactant molecules and disrupting self-assembly, our experiments showed that albumin does not bind these anionic PAs and instead promotes nanofibers due to a molecular crowding effect. Finally, we created a medium that replicates the transition pH in serum to within 0.08 pH units and allows probing self-assembly behavior using conventional spectroscopic techniques without conflicting protein signals, thus simplifying the development pathway from test tube to in vivo experimentation for stimuli-responsive materials.

Figure 1

Concentration–pH phase diagram of pure PA1-Gd(DO3A) (red) and the PA1-mix (blue) as determined via CD (solid diamonds) and CAC (hollow diamonds) measurements, respectively. All measurements were done in 150 mM NaCl and 2.2 mM CaCl₂.

Figure 2

(a) pH-dependent FA of 100 μM PA1-mix. The inset shows the fluorescence emission from the PA1-Ru(bipy)₃ in the mixture. (b) pH-dependent ellipticity at 205 nm of 100 μM PA1-mix. (c) pH-dependent CD spectra of 100 μM PA1-mix. The color for each point in panels a–c corresponds to similar (within 0.02 pH units) pH values.

Figure 3

(a) Fluorescence emission from 100 μM PA1-mix in pure serum along with serum autofluorescence background. (b) pH-dependent FA of 100 μM PA1-mix and PA2-mix in salts and pure serum. (c) pH reversibility of the morphology transition in 100 μM PA2-mix in serum. (d) Time-dependent stability of spherical micelles and nanofibers in 100 μM PA2-mix in serum via FA measurements.

Figure 4

(a) pH-dependent FA of 100 μM PA1-mix in 0–4% v/v diluted serum solutions. (b) Kinetics of the morphology switch of 100 μM PA1-mix in 1.5% serum via time-dependent FA measurements. (c, d) TEM images of 100 μM PA1-mix in 1.5% serum at pH 6.85 and 9.21, respectively.

Figure 5

pH-dependent FA of 100 μM PA1-mix in salts (150 mM NaCl, 2.2 mM CaCl₂), 1.5% serum, 7.8 μM MSA, and 26 μM and 1.8 mM PEG.

Figure 6

TEM images of 500 μM PA2-mix in artificial serum at (a) pH 5.5 and (b) pH 8.2. (c) Concentration-dependent CD transition pH values of PA2-mix in artificial (blue) and pure mouse blood serum (red) overlaid on the phase diagram (faded orange) of 100 μM PA2-Gd(DO3A) in artificial serum.

Chart 1

PA Structure and Design