Reagents
All reagents were purchased from Sigma-Aldrich (Darmstadt, GER) unless otherwise stated. Endothelial cell medium (ECM) was purchased from ScienCell (Carlsbad, CA, USA). Fetal bovine serum and phosphate-buffered saline (PBS) were purchased from Gibco (Waltham, MA, US). Bovine serum albumin was purchased from VWR (Monroeville, PA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Dipalmitoylphosphatidylcholine (DPPC) was purchased from TCI (Tokyo, Japan).
RBMEC study
High activity RBMECs were isolated from 2-week-old Sprague Dawley rats by two-step enzyme digestion, density gradient centrifugation, and differential adhesion in sequence [18, 19]. The extracted primary cells were cultured in T25 culture flasks at a seeding density of 2 × 105 cells/mL, and the medium was changed every 2–3 days.
The growth curve and morphology of the cells were monitored by microscopy and CCK-8 assays daily after seeding in 96-well plates and T25 culture bottles at a density of 1 × 105 cells/mL. Six days later, the cells were collected by separating them from the bottle with a cell scraper and centrifuging for 5 min (1000 rpm, 25 ℃), fixed, dehydrated using 3% paraformaldehyde and alcohol, and observed via transmission electron microscopy (TEM).
Establishment And Characterization Of The Transwell System
The isolated RBMECs were planted on the membrane of the Transwell system at a seeding density of 2 × 105 cells/mL [20]. The growth curve of the cell barrier layer was monitored daily via the transendothelial electrical resistance (TEER) assay using an epithelial volt/ohm meter (Millicell® ERS-2; Millipore, Bedford, MA, USA). The effective resistance was calculated using the following formula:
Effective resistance (Ω·cm2) = (cell pore resistance – blank hole resistance) × membrane area (4.67 cm2).
After a period of time, the barrier layer formed, and a certain amount of ECM was added to both the upper and lower pools of the Transwell chamber to form a level difference of more than 0.5 cm. After 4 h, the liquid level difference was measured to confirm the barrier function of the cell layer.
Optical density (OD) of AChE
The OD of each component (AChE, nerve agent, and antidote) was tested and calculated before it was added to the modified Transwell system. AChE solutions with different concentrations were prepared using an ECM-phenol red free basic solution. The AChE activity was measured with the Ellman method, as previously described [21]. Samples were added to 96-well plates mixed with 8.6% acetylthiocholine (ATCh) and PBS and incubated at 37°C for 30 min. 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) was added to each well for the chromogenic reaction in the last step. ATCh would be degraded by the enzyme of AChE, and the degradation product can react with DTNB for chemiluminescence. The standard curve was calculated using the OD value from the sample plate, and the linear range of the enzyme solution was confirmed.
OD of the nerve agent sarin
AChE concentrations of 0.05, 0.06, and 0.07 mg/mL and sarin concentrations of 10− 3, 10− 4, 10− 5, 10− 6, and 10− 7 v/v were used. AChE (1.6 mL) and sarin (0.4 mL) at each concentration was mixed successively in a six-well plate. After 3 min, samples were collected and detected by the Ellman method. The inhibition ratio was calculated using the following equation:
Inhibition rate of AChE (%) = (1 – inhibition group OD415nm/non-inhibition group OD415nm) × 100% [22].
Study of OD of reactivator
Four different reactivators (asoxime chloride [HI-6], obidoxime [LüH-6], pralidoxime chloride [2-PAM], and methoxime [MMB-4]), at concentrations of 0.05, 0.04, 0.03, 0.02, 0.01, and 0 mg/mL were mixed with AChE (0.07 mg/mL) and sarin (10− 6 v/v). Ten minutes later, samples were collected and tested using the Ellman method. The reactivation rates of the reactivator at different concentrations were calculated using the following equation:
Reactivation rate (%) = (reactivator groupOD415nm – inhibition group OD415nm)/(normal groupOD415nm – inhibition groupOD415nm) × 100% [13].
Therapeutic effect of the classical reactivator by modified Transwell model
AChE (0.07 mg/mL) and sarin (10− 6 v/v) were simultaneously added to the lower chamber of the Transwell board, and then, the reactivators (0.03 mg/mL) were added to the upper chamber. After allowing time for the reactivator to penetrate the barrier and react with the inhibited enzyme, samples were collected from the lower chamber and detected by the Ellman method for enzyme activity, as previously described.
Preparation And Characterization Of The Liposomal Nanomedicine (LipoHI-6)
The proposed antidote (LipoHI-6) was prepared using an extrusion process [23]. Cholesterol (Chol) and DPPC mixed in a 2:1 molar ratio were dissolved and dispersed in chloroform and dried in a flask to produce a homogeneous lipid film by rotary evaporation. After adding to the HI-6 solution (2.2 mg/mL), ultrasonication was performed for 5 min to separate the film from the flask and encapsulate the HI-6 droplets to form a suspension. The suspension was then extruded successively 10 times through 200-nm and 100-nm polycarbonate filters using an Avanti Mini Extruder (Alabaster, AL, USA). Finally, the un-encapsulated free HI-6 was removed by dialysis with PBS. The size and zeta potential of LipoHI-6 were evaluated by dynamic light scattering (DLS) with a particle analyzer (Nano ZS90; Malvern Panalytical, Malvern, UK). The morphology of the LipoHI-6 nanoparticles was determined using TEM. The concentration of HI-6 loaded into the liposomes was quantified at 280 nm using a C18 column equipped with a UV detector in a high-performance liquid chromatography (HPLC) system (L7100; Hitachi, Tokyo, Japan) [18]. The mobile phase in the HPLC system comprised 15% acetonitrile and 85% H2O at a flow rate of 2.0 mL/min. The encapsulation efficiency (EE%) and loading efficiency (LE%) of LipoHI-6 were evaluated using ultrafiltration/centrifugation. LipoHI-6 solution (1 mL) was sealed in a dialysis bag and suspended in 800 mL of PBS at pH 7.4 in a shaking incubator at 37°C to evaluate the release behavior of the nanomedicine. Samples (1 mL) were collected for the system at a predetermined period and quantified by HPLC.
Permeability and therapeutic effect of LipoHI-6 according to the classical/modified Transwell model
The permeability of LipoHI-6 across the BBB was calculated using the classical Transwell model. LipoHI-6 (0.03 mg/mL) was added to the upper chamber, and 10 min later, samples collected from the lower chamber were quantified using HPLC. The permeability of HI-6 across the BBB was also tested using the same procedure as that for the control.
To evaluate the therapeutic effect of LipoHI-6, it was again added (0.03 mg/mL) to the upper chamber. At the same time, AChE was mixed with sarin in the lower chamber, as described previously. The activity of AChE after 10 min was analyzed using the Ellman method.
Animals
All experiments conformed to the Regulations of the Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China (November 14, 1988). Sprague Dawley rats (2 weeks old; both specific pathogen-free) and Kunming (KM) mice (4 weeks old; both specific pathogen-free) were acquired from Beijing Charles River Laboratory Animal Technology Co, Ltd. (Beijing, China). Rodents had free access to sterilized food and distilled water and were maintained in stainless steel cages filled with hardwood chips in an air-conditioned room on a 12:12 h light/dark cycle.
Therapeutic effect of LipoHI-6 in vivo
The activity of central and peripheral AChE in KM mice was measured after sarin exposure and antidote administration. KM mice were randomly divided into four groups according to their body weight, with 12 mice in each group: normal group (no administration), poisoned group (only sarin poison), control group (poisoned and administered HI-6 [2.2 mg/mL]), and the liposomal nanomedicine group (poisoned and administered LipoHI-6 [2.2 mg/mL]). Mice were injected subcutaneously with sarin (180 µg/kg), followed by a subcutaneous antidote injection (10 µL/g) in the neck. The blood and brain were collected 10 min later, after which the brain tissue supernatant was obtained by centrifugation and diluted 500 times with PBS, and the activity of AChE in the sample was detected using the Ellman method.
Statistical analysis
Data are presented as means ± standard deviation (SD). An unpaired t-test was performed for two groups using GraphPad Prism 7.00 (GraphPad Software; San Diego, CA, USA). Statistical significance was set at p < 0.05.