Tuning the Envelope Structure of Enzyme Nanoreactors for In Vivo Detoxification of Organophosphates

Abstract

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 μM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential -8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD₅₀ shift was 23.7xLD₅₀ for prophylaxis and 8xLD₅₀ for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms.

Keywords: enzyme nanoreactor; immune response; organophosphate poisoning; pharmacokinetics; phosphotriesterase; polymersomes; post-exposure treatment; prophylaxis.

Figure 1

Synthesis of PEG-PPS di-block copolymers.

Figure 2

TEM imaging of empty nanoreactors prepared using 1a (A) and 1b (B), C_polymers = 10 μg/mL, Tris-Buffer, pH = 7.4, 25 °C. Scale bars are 2 μm for the main (A) and (B) images, with 500 nm and 200 nm for the insets, respectively.

Figure 3

Release of pNp from 1a and 1b nanoreactors, where control was the absence of nanoreactors, C_1a = C_1b = 0.5% (w/w), C_pNp = 0.1% (w/w), 37 °C, 10 mM Tris-Buffer, pH = 7.4.

Figure 4

TEM imaging (A); size distribution as determined using DLS for the monitoring of stability in vitro conditions in Tris buffer and within 1 h at 37 °C (B); in vitro kinetics of POX detoxification process at λ= 400 nm (C) under second-order conditions: [E] = 0.5 mM and [POX] = 1–5 µM; pNp release (D) of enzyme-loaded nanoreactors 1b, Tris-Buffer, pH = 7.4, 25 °C.

Figure 5

LD₅₀ shifts for acute toxicity of POX s.c., where 1 (reference LD₅₀)—non-treated animals, 2—animals under prophylaxis, and 3—post-exposure treated animals.

Figure 6

Rate of enzyme-catalyzed reaction of POX hydrolysis versus time in mouse plasma after intravenous injection of free enzymes (1) and enzyme-loaded nanoreactors (2, 3), where (2) is the first injection (1 day) and (3) is the second injection (30 days after the first injection). The dose of enzyme was 3.7 mg/kg. Each point represents the mean ± SD in 6 mice.

Figure 7

Development of immune response to free or encapsulated PTE enzyme: 1—negative control, 2—positive control, 3—POX, 4—empty nanoreactor, 5—solvent, 6—PTE, 7—PTE-mPEG−PPS−mPEG nanoreactor (1 injection), 8—PTE-mPEG−PPS−mPEG nanoreactor (2 injections), 9—PTE-mPEG−PPS nanoreactor (1 injection), 10—PTE-mPEG−PPS nanoreactor (2 injections). (A) Serum samples were collected and prepared for indirect ELISA assay to detect IgG against the free and encapsulated enzyme; (B) OD 450 nm values in control and tested serum samples after single paraoxon, empty nanoreactor, or solvent injection; (C) OD 450 nm values in control and tested serum samples after first and second injections of PTE-mPEG−PPS−mPEG nanoreactor; (D) OD450 nm values in control and tested serum samples after first and second injections of PTE-mPEG−PPS nanoreactor; (E) a representative image of the Western blot results for anti-PTE-IgG from 3 independent experiments; (F) the levels of the anti-PTE-IgG in murine serum samples were analyzed by densitometry; (n = 3; *—p ≤ 0.05).