Cysteine-Encapsulated Liposome for Investigating Biomolecular Interactions at Lipid Membranes

Abstract

The development of a strategy to investigate interfacial phenomena at lipid membranes is practically useful because most essential biomolecular interactions occur at cell membranes. In this study, a colorimetric method based on cysteine-encapsulated liposomes was examined using gold nanoparticles as a probe to provide a platform to report an enzymatic activity at lipid membranes. The cysteine-encapsulated liposomes were prepared with varying ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol through the hydration of lipid films and extrusions in the presence of cysteine. The size, composition, and stability of resulting liposomes were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, and UV-vis spectrophotometry. The results showed that the increased cholesterol content improved the stability of liposomes, and the liposomes were formulated with 60 mol % cholesterol for the subsequent experiments. Triton X-100 was tested to disrupt the lipid membranes to release the encapsulated cysteine from the liposomes. Cysteine can induce the aggregation of gold nanoparticles accompanying a color change, and the colorimetric response of gold nanoparticles to the released cysteine was investigated in various media. Except in buffer solutions at around pH 5, the cysteine-encapsulated liposomes showed the color change of gold nanoparticles only after being incubated with Triton X-100. Finally, the cysteine-encapsulated liposomal platform was tested to report the enzymatic activity of phospholipase A₂ that hydrolyzes phospholipids in the membrane. The hydrolysis of phospholipids triggered the release of cysteine from the liposomes, and the released cysteine was successfully detected by monitoring the distinct red-to-blue color change of gold nanoparticles. The presence of phospholipase A₂ was also confirmed by the appearance of a peak around 690 nm in the UV-vis spectra, which is caused by the cysteine-induced aggregation of gold nanoparticles. The results demonstrated that the cysteine-encapsulated liposome has the potential to be used to investigate biological interactions occurring at lipid membranes.

Keywords: Triton X-100; colorimetric detection; cysteine; gold nanoparticles; liposome; phospholipase A2.

Scheme 1

(Top) The aggregation and colorimetric response of AuNPs induced by Cys. (Bottom) The release of encapsulated Cys by TX100 or PLA₂ from the liposomes and the subsequent aggregation of AuNPs.

Figure 1

(a) SEM image of Cys-encapsulated liposomes and (b) size distribution of liposomes measured by DLS. The liposomes were formulated with DMPC:Chol molar ratio of 40:60.

Figure 2

¹H NMR spectra of liposomes formulated from the lipid films with DMPC:Chol molar ratios of 40:60 and 60:40. The a indicates the peak at 1.83 ppm from two protons on carbon 1 and one proton on carbon 2, and the b indicates the peak at 0.68 ppm from three protons on carbon 18 in cholesterol.

Figure 3

Colorimetric responses of AuNPs at different concentrations: (a) 0.06 mM, (b), 0.12 mM, (c) 0.24 mM.

Figure 4

The UV–vis spectra of 0.12 mM AuNPs in various conditions and media: neutral pH (a), phosphate buffered saline (pH 7.4) (b), HCl-adjusted water (pH 5) (c), and phosphate buffer saline (pH 5.2) (d). Insets show the corresponding photographs of AuNPs: only AuNPs (i); AuNPs with TX100 (ii); AuNPs with CELP (iii); AuNPs with TX100 and CELP (iv).

Figure 5

The UV–vis spectra of AuNPs (0.12 mM) under activity of PLA₂ in HPLC water at neutral pH 7.0 (a), and phosphate buffered saline (pH 7.4) (b). Insets show the corresponding photographs of AuNPs: (i) AuNPs, (ii) AuNPs with PLA₂, (iii) AuNPs with CELP, (iv) AuNPs with both CELP and PLA₂.

Figure 6

Absorbance ratios of 3 control samples (AuNPs, AuNPs with TX100 or PLA₂, and AuNPs with liposome) and the sample including AuNPs, TX100 (or PLA₂), and liposomes after incubation for 1 h in various buffer media. * indicates the use PLA₂ instead of TX100.