Albumin/Thiacalix[4]arene Nanoparticles as Potential Therapeutic Systems: Role of the Macrocycle for Stabilization of Monomeric Protein and Self-Assembly with Ciprofloxacin

Abstract

The therapeutic application of serum albumin is determined by the relative content of the monomeric form compared to dimers, tetramers, hexamers, etc. In this paper, we propose and develop an approach to synthesize the cone stereoisomer of p-tert-butylthiacalix[4]arene with sulfobetaine fragments stabilization of monomeric bovine serum albumin and preventing aggregation. Spectral methods (UV-vis, CD, fluorescent spectroscopy, and dynamic light scattering) established the influence of the synthesized compounds on the content of monomeric and aggregated forms of BSA even without the formation of stable thiacalixarene/protein associates. The effect of thiacalixarenes on the efficiency of protein binding with the antibiotic ciprofloxacin was shown by fluorescence spectroscopy. The binding constant increases in the presence of the macrocycles, likely due to the stabilization of monomeric forms of BSA. Our study clearly shows the potential of this macrocycle design as a platform for the development of the fundamentally new approaches for preventing aggregation.

Keywords: bovine serum albumin; ciprofloxacin; self-assembly; sulfobetaines; thiacalixarene.

Scheme 1

Reagents and conditions: (i)—N,N-diethylethane-1,2-diamine, C₆H₅CH₃/CH₃OH, reflux; (ii)—1,3-propanesultone, CH₃CN, reflux, 72 h; (iii)—1,4-butanesultone, CH₃CN, reflux, 240 h.

Figure 1

Size analysis of the (A) 3 and (B) 3/BSA by TEM (left) and DLS (right). Colored lines represent multiple experiments.

Figure 2

DLS profiles of solutions of BSA in the presence of the 3 (A) and 4 (B).

Figure 3

(A) Fluorescence spectra of BSA in the presence of 4 at 20 °C; (B) the graphs are plotted in the linearization of Stern–Volmer coordinates for 4/BSA system.

Figure 4

Circular dichroism spectra of (A) BSA (3.3 × 10⁻⁷ M) and the associate of 3 (3.3 × 10⁻⁶ M) with BSA (3.3 × 10⁻⁷ M) in buffer at pH 7.4; (B) BSA (3.3 × 10⁻⁷ M) and the associate of 4 (3.3 × 10⁻⁶ M) with BSA (3.3 × 10⁻⁷ M) in buffer at pH 7.4.

Figure 5

Potential sites for binding macrocycle 3 (A) and 4 (B) in BSA molecules calculated by DINC 2.0 web server.

Figure 6

Fluorescence spectra of (A) titration of 3 (1 × 10⁻⁵ M)/BSA (1 × 10⁻⁵ M) with ciprofloxacin (antibiotic concentration changed from 0 to 5 × 10⁻⁵ M) (B) 3 (1 × 10⁻⁵ M), ciprofloxacin (4.17 × 10⁻⁵ M), 3 + BSA system (1/1), 3 + ciprofloxacin system (1/1).