Preparation of Benzothiazolyl-Decorated Nanoliposomes

Abstract

Amyloid β (Aβ) species are considered as potential targets for the development of diagnostics/therapeutics towards Alzheimer's disease (AD). Nanoliposomes which are decorated with molecules having high affinity for Aβ species may be considered as potential carriers for AD theragnostics. Herein, benzothiazolyl (BTH) decorated nanoliposomes were prepared for the first time, after synthesis of a lipidic BTH derivative (lipid-BTH). The synthetic pathway included acylation of bis(2-aminophenyl) disulfide with palmitic acid or palmitoyl chloride and subsequent reduction of the oxidized dithiol derivative. The liberated thiols were able to cyclize to the corresponding benzothiazolyl derivatives only after acidification of the reaction mixture. Each step of the procedure was monitored by HPLC analysis in order to identify all the important parameters for the formation of the BTH-group. Finally, the optimal methodology was identified, and was applied for the synthesis of the lipid-BTH derivative. BTH-decorated nanoliposomes were then prepared and characterized for physicochemical properties (size distribution, surface charge, physical stability, and membrane integrity during incubation in presence of buffer and plasma proteins). Pegylated BTH-nanoliposomes were demonstrated to have high integrity in the presence of proteins (in comparison to non-peglated ones) justifying their further exploitation as potential theragnostic systems for AD.

Keywords: amyloid β; benzothiazoles; functionalization; nanoliposomes; targeting.

Scheme 1

Synthesis of bis(2-aminophenyl) disulfide.

Scheme 2

Synthetic method for the formation of Palm-BTH; Compound 3 was used either as palmitic acid (X=OH) when N,N′-diisopropylcarbodiimide (DIC) was used as a condensing agent, or as palmitoyl chloride (X=Cl) when triethylamine (NEt₃) was used as a base.

Figure 1

HPLC analysis during the reaction between bis(2-aminophenyl) disulfide and palmitic acid in tetrahydrofuran (THF), using DIC as condensing agent; (A) t=1d at rt; (B) t=3d at rt; Column: LiChrospher^® 100 RP-18 (5 µm) LiChroCART^® 250-4; Mobile phase: THF/H₂O; Gradient: 50% THF to 100% THF in 30 min; Flow rate: 1ml/min; Detection at 254 nm.

Figure 2

HPLC analysis of (A) the final mixture of 4a+4b after 7 days reaction at rt of bis(2-aminophenyl) disulfide and palmitic acid in THF by using DIC as condensing agent; (B) during the reduction of 4a+4b with NaBH₄; (C) after the reduction of 4a+4b with NaBH₄ without subsequent acidification of the mixture; (D) after the reduction of 4a+4b with NaBH₄ and subsequent acidification with AcOH; Column: LiChrospher^® 100 RP-18 (5 µm) LiChroCART^® 250-4; Mobile phase: THF/H₂O; Gradient: 50% THF to 100% THF in 30 min; Flow rate: 1mL/min; Detection at 254nm.

Figure 3

HPLC analysis of (A) the reaction mixture product between bis(2-aminophenyl) disulfide and palmitoyl chloride in presence of NEt₃; (B) the reaction mixture after reduction with NaBH₄ and acidification with AcOH; Column: LiChrospher^® 100 RP-18 (5 µm) LiChroCART^® 250-4; Mobile phase: THF/H₂O; Gradient: 50% THF to 100% THF in 30 min; Flow rate: 1mL/min; Detection at 254 nm.

Scheme 3

Synthetic method for the formation of Lipid-COOH; R=CH₃-(CH₂)₁₄-.

Scheme 4

Synthetic method for the formation of lipid-BTH.

Figure 4

(A) HPLC analysis of purified lipid-BTH.; Column: LiChrospher^® 100 RP-18 (5 µm) LiChroCART^® 250-4; Mobile phase: THF/H₂O; Gradient: 50% THF to 100% THF in 30 min; Flow rate: 1ml/min; Detection at 254nm; (B) ESI-MS of lipid-BTH.

Figure 5

Size (d-hydrodynamic mean diameter, nm) and pdi values of the LIP1–LIP6 prepared dispersions, in PBS buffer 7.4 at a lipid concentration of 5 mg/mL, during storage at 4 °C. Each value is the mean of at least 5 different measurements and bars are the SD values of each mean.

Figure 6

Integrity of non-pegylated BTH-nanoliposomes (LIP1 and LIP2) and pegylated BTH-nanoliposomes (LIP5 and LIP6) and their corresponding control nanoliposomes (with no BTH on their surface), during incubation in buffer (A) or FCS (80% v/v) (B) at 37 °C. Each data point is the mean of at least 3 different experiments and the bar is the SD of the mean.

Figure 7

DSC thermographs of control nanoliposomes (no BTH) (a); LIP1 (10% BTH) (b); and LIP2 (20% BTH) (c).