A nanocarrier containing carboxylic and histamine groups with dual action: acetylcholine hydrolysis and antidote atropine delivery

Abstract

Disruption of cholinesterases and, as a consequence, increased levels of acetylcholine lead to serious disturbances in the functioning of the nervous system, including death. The need for rapid administration of an antidote to restore esterase activity is critical, but practical implementation of this is often difficult. One promising solution may be the development of antidote delivery systems that will release the drug only when acetylcholine levels are elevated. This approach will ensure timely delivery of the antidote and minimize side effects associated with uncontrolled drug release. Here, we describe the creation of a new smart system that serves as a carrier for delivering an antidote (i.e., atropine) and functions as a synthetic esterase to hydrolyze acetylcholine. The nanocarrier was synthesized through microemulsion polycondensation of phenylboronic acid with resorcinarenes containing hydroxy, imidazole, and carboxylic groups on the upper rim. The nanocarrier breaks down acetylcholine into choline and acetic acid. The latter acts on the boronate bonds, dissociating them. This leads to the destruction of the nanocarrier and the release of the antidote. The paper covers the creation of the nanocarrier, its physicochemical and biological properties, encapsulation of the antidote, acetylcholine hydrolysis, and antidote release.

Keywords: acetylcholine; antidote delivery; artificial cholinesterase; atropine; nanocarrier; resorcinarene.

Scheme 1

Synthesis of Atr@p(Hist-CA) and Atr release following ACh hydrolysis.

Figure 1

Data for p(Hist-CA): (a, b) TEM images, (c) distribution diagram of the hydrodynamic diameter, C = 2 mg/mL and (d) Debye plot, C = 0.6–3.3 mg/mL, PB, pH 7.4, 25 °C.

Figure 2

IR spectra of (a) BA, (b) p(Hist-CA), (c) Hist-RA and (d) CA-RA in KBr.

Figure 3

Agglutination assay. Blood samples were observed in the plate wells following the addition of diluted solutions of Hist-RA, CA-RA, and p(Hist-CA) at two- to 128-fold dilutions.

Figure 4

Bright-field microscopy (Nikon Eclipse Ci, 400× magnification). K(−) for intact cells; K(+) for the agglutination control.

Figure 5

The concentration-dependent conductivity of (a) CA-RA, Hist-RA, and the mixture CA-RA + Hist-RA in H₂O, 25 °C, and (b) p(Hist-CA), ACh, and the mixture p(Hist-CA) + ACh (1:1) in H₂O, 25 °C; in the cases of p(Hist-CA) and p(Hist-CA) + ACh, the abscissa represents the RA concentration, while for ACh, it represents the ACh concentration.

Figure 6

(a) UV and fluorescence spectra of Fl (red dotted line) and Fl@p(Hist-CA) (blue line), (b) fluorescence spectra of Fl@p(Hist-CA) over time following the addition of 1.6 mM ACh, and (c) release of Fl from Fl@p(Hist-CA) over time at 517 nm: without ACh (black squares and line), with 0.16 mM ACh (blue triangles and line), and with 1.6 mM ACh (red circles and line), C(Fl) = 0.01 mM; PB, pH 7.4, 37 °C.

Figure 7

¹H NMR spectra for Atr@p(Hist-CA): (a) dialysate after synthesis of Atr@p(Hist-CA), (b) dialysate after addition of ACh, and (c) the rest of dialyzing solution, C(ACh) = 5 mM, m(Atr) = 10 mg, PB, pH 7.4, 25 °C.

Figure 8

Time-dependent Atr release from Atr@p(Hist-CA) without ACh and after addition of ACh 0.04, 4 and 40 mM in dialyzing experiment, V(Atr@p(Hist-CA)) = 3 mL, V(dialysate) = 50 mL, PB, pH 7.4.

Scheme 2

Synthesis of Hist-RA.

Scheme 3

Synthesis of CA-RA.