Present studies have focused on a novel cyanide antidotal system, on the coencapsulation of a new sulfur donor DTO with rhodanese within sterically stabilized liposomes. The optimal lipid composition for coencapsulation of DTO with rhodanese is the combination of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, cationic lipid (DOTAP), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (with molar ratios of 82.7 : 9.2 : 3.0 : 5.1). With the optimized compositions, prophylactic and therapeutic
The specific treatment of cyanide (CN) intoxication means the use of scavengers (e.g., methemoglobin former sodium nitrite (SN) or cobalt compounds or cyanohydrin formers, hydroxocabalamin (Cyanokit has been approved in the US), cobinamide [
Extensive researches are also focusing on developing effective sulfur-containing compounds serving as sulfur donors for reacting with CN with or without Rh. Thiosulfate (TS) is the classical sulfur compound found to participate in the enzyme reaction [
Earlier investigations were focused on administration of free Rh and the sulfur donor (SD) directly into the bloodstream [
The lipid composition has a significant impact on the encapsulation efficiency of the Rh and/or sulfur compound and on the
Present work deals with a new lipophilic sulfur-containing compound, developed at the US Army Medical Research Institute of Chemical Defense, called DTO. In order to achieve the highest CN antidotal protection, the liposomal encapsulation of DTO with and without Rh was examined.
The objectives of this study are (1) optimization of the liposomal encapsulation for the new sulfur donor, DTO, with superior sulfur donor reactivity to the present therapy TS; (2)
All chemicals employed were of the highest purity commercially available: potassium cyanide, TS, sodium nitrite, phosphate buffer components, ethanol, sodium chloride, concentrated hydrochloric acid, and sodium hydroxide were purchased from J. T. Baker, (Phillipsburg, NJ), formaldehyde and ferric nitrate were purchased from Fisher Scientific (Pittsburgh, PA). The liposome components (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), soy lecithin (LEC), cholesterol (Chol)) and Rh were purchased from Sigma-Aldrich (St. Louis, MO), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (PEG-PE-2000) was purchased from Avanti Polar Lipids (Alabaster, AL). Bio-Rad Protein Kit was purchased from Bio-Rad Life Sciences Laboratories, Hercules, CA.
Male (CD-1) mice (Charles River Breeding Laboratories, Inc., Wilmington, MA) weighing 18–20 g were housed at 21°C and in light-controlled rooms (12 h light/dark, full-spectrum lighting cycle with no twilight), and were furnished with water and 4% Rodent Chow (Teklad HSD, Inc., CITY, WI)
POPC, DOPC, DOTAP, LEC, PEG-PE-2000, and Chol dissolved in ethanol were applied in various molar ratios in order to determine the optimal lipid composition.
The liposomes were prepared by the thin-film hydration method [
The formation of SCN from CN was measured spectrophotometrically (Genesys 10UV, Thermo Electron Corporation, Waltham, MA) by the method of Westley [
Formation of SCN from CN with the investigated sulfur donors of TS and DTO were determined spectrophotometrically by the method of Westley [
Four different Rh concentrations (0.25 mg/mL, 0.50 mg/mL, 1.00 mg/mL, 1.67 mg/mL) were employed with a lipid composition of POPC : Chol : PEG-PE-2000 with and without DOTAP. Percentage of Rh incorporation within the liposomes was determined by the Bradford Assay [
Optimal lipid composition for Rh encapsulation was determined based on the highest enzyme activity achieved by the same encapsulation process with various lipid compositions. Unencapsulated Rh was separated from SL-Rh by gel filtration on a G-100 Sephadex gel column (0.7 cm × 10 cm; GE Healthcare BioSciences AB, Sweden). Measurements were carried out in isotonic phosphate buffer at pH = 7.4. Rh activity for the fractions was determined as described above.
The encapsulation efficiency for the sulfur donor DTO was determined by the Rh assay described above with constant Rh concentration. When Rh concentration was constant, the rate of formation of SCN was directly proportional to the sulfur donor concentration.
Formation of SCN by SL-Rh-DTO with various lipid compositions was measured spectrophotometrically as described above.
Experimental animals received KCN after pretreatment with antagonist(s) (sulfur donors and/or Rh and/or SN). Freshly prepared SL-DTO, SL-DTO-TS, SL-DTO-Rh, and SL-DTO-TS-Rh were administered intravenously (iv) by tail vein injection to mice 10 min prior to receiving CN (sc). Using 10 mL/kg doses of (SL-DTO-Rh; Table
Animals received antidotes administered intravenously one min after CN injection (sc). Doses of antidotes were the same as described above for the prophylactic experiments. The animals were evaluated 24 hours after CN exposure for mortality. Results are given as % survival (animals alive/animals total). Total numbers of animals were 6 for each therapeutic experiment for each antidotal system.
These studies focused on the encapsulation optimization for new sulfur donor DTO when encapsulated with Rh and/or TS within sterically stabilized liposomes. The
Comparison of
Rate of CN conversion (mmol SCN/min) | Ratio | |
TS | DTO | DTO/TS |
0.2 | 3.0 | 15.0 |
When encapsulating Rh alone, small amount of the cationic lipid DOTAP proved to be beneficial to enhance encapsulation efficiency (Table
Rh-load optimization with and without DOTAP.
Composition of liposomes | Original Rh concentration (mg/mL) | % of Rh incorporation determined by Bradford |
---|---|---|
POPC : Chol : PEG-PE-2000 = 56.9 : 38: 5.1 | 1.00 | 16.6 |
POPC : Chol : PEG-PE-2000 = 56.9 : 38: 5.1 | 0.25 | 26.9 |
POPC : Chol : PEG-PE-2000 : DOTAP = 82.7 : 9.2 : 5.1 : 3.0 | 1.67 | 18.2 |
POPC : Chol : PEG-PE-2000 : DOTAP = 82.7 : 9.2 : 5.1 : 3.0 | 0.50 | 55.8 |
POPC : Chol : PEG-PE-2000 : DOTAP = 82.7 : 9.2 : 5.1 : 3.0 | 0.25 | 74.0 |
POPC : Chol : PEG-PE-2000 : DOTAP = 82.7 : 9.2 : 5.1 : 3.0 | 1.00 | 52.9 |
For the encapsulation of DTO with a concentration of 2 mM, six different liposomal compositions were examined to rule out the role of lipid composition (Table
Encapsulation efficiencies for DTO in various liposome compositions with and without DOTAP. DTO concentration was 2.0 mM. Total lipid concentration was 10.0 mg/mL.
Liposomal composition (mol%) | Encapsulation efficiency (%) with various lipid components | |||||
Lipid | Chol | PEG-PE-2000 | DOTAP | POPC | DOPC | LEC |
82.71 | 9.19 | 5.1 | 3.0 | 78.4 ± 2.3 | 81.7 ± 3.1 | 64.3 ± 3.0 |
85.41 | 9.49 | 5.1 | — | 60.7 ± 3.0 | 63.8 ± 1.2 | 61.6 ± 1.5 |
Prophylactic protection by various cyanide antidotal combinations. APR denotes antidotal potency ratio, which can be calculated as the ratio of the average LD50 of CN with and without antagonists.
Exp no. | Treatment | LD50 (mg/kg; mean; range) | APR |
---|---|---|---|
1 | Control | 7.8 (4.6–13.1) | 1 |
2 | (SL-DTO) (iv) + CN (sc) | 17.3 (9.8–30.7) | 2.2 |
3 | (SL-DTO-TS) (iv) + CN (sc) | 38.0 (21.5–67.3) | 4.8 |
4 | (SL-DTO-TS) (iv) + SN (ip) + CN (sc) | 52.7 (29.7–93.2) | 6.7 |
5 | (SL-DTO-Rh) (iv) + CN (sc) | 30.7 (14.6–64.0) | 3.9 |
6 | (SL-DTO-TS-Rh) (iv) + CN (sc) | 38.0 (22.6–64.2) | 4.9 |
7 | (SL-DTO-TS-Rh) (iv)+ SN (ip) + CN (sc) | 120.0 (68.2–213.0) | 15.3 |
The liposome compositions including DOTAP were used in further experiments due to the increase in encapsulation efficiency achieved by these films. The effect on encapsulation efficiency by the increase in DTO concentration was evaluated for DOPC and POPC containing liposome compositions with both sets of liposomes containing 3% DOTAP. In order to evaluate the role of DTO concentration on the encapsulation efficiency each set’s films were prepared with DTO concentrations of 10 mM, 20 mM, and 30 mM.
The encapsulation efficiency remained high for each liposome formulation containing 3% DOTAP for each applied DTO concentrations of 10 mM, 20 mM, and 30 mM. The encapsulation efficiencies of DTO for POPC samples were 69.7 ± 2.3%, 82.8 ± 7.1%, 79.2 ± 8.1%, while for the DOPC samples DTO encapsulation efficiencies of 74.2 ± 2.0%, 86.2 ± 3.9%, and 89.9 ± 4.2% were determined, for 10 mM, 20 mM, and 30 mM DTO concentrations, respectively. For a given DTO concentration there was no significant difference between the encapsulation efficiency values for DOPC or POPC liposomes (
The optimal liposome composition for the encapsulation of Rh was determined in earlier experiments to be the 60 : 40 ratio of POPC to Chol [
For the coencapsulation of DTO and Rh, the combination of POPC, Chol, PEG-PE-2000, and DOTAP (with molar ratios of 82.7 : 9.2 : 5.1 : 3.0) was chosen as the most adequate liposome composition. The mentioned composition of sterically stabilized, positively charged liposomes performed the best in the coencapsulation, with a coencapsulation efficiency for Rh and DTO of 88.6 ± 17.1% (with a Rh load of 0.25 mg/mL and a DTO concentration of 30 mM). As the coencapsulation efficiency was determined on the basis of SCN formation by SL-Rh-DTO; the given value represents the combined effect of Rh and DTO in CN conversion. For the sake of comparison, encapsulation efficiency for the coencapsulated Rh alone—with 0.25 mg/mL concentration—was 74%, while for DTO alone—with 10 mM DTO load—was 57.7 ± 8.1%. Increasing the concentration of DTO produced similar encapsulation efficiencies, than in case of 10 mM. With DTO loads of 20 mM and 30 mM for the coencapsulated DTO encapsulation efficiencies of 55.6 ± 4.0% and 61.6 ± 17.6% were measured, respectively. The conversion of CN to SCN by the coencapsulation of 10 mM, 20 mM, and 30 mM DTO with Rh also proved to remain linear with an
The
SL-DTO alone provided a protection with an APR of 2.2. (Table
Relative prophylactic antidotal potency ratios (RAPRs) to express enhancing effects by Rh, SN, and coencapsulated TS.
Enhancing effects by Rh | RAPR |
Comparison of exps 5 and 2 | 3.9/2.2 = |
Comparison of exps 7 and 4 | 15.3/6.7 = |
Enhancing effects by SN | RAPR |
Comparison of exps 4 and 3 | 6.7/4.8 = |
Comparison of exps 7 and 6 | 15.3/4.9 = |
Enhancement by coencapsulated TS | RAPR |
Comparison of exps 3 and 2 | 4.8/2.2 = |
Comparison of exps 6 and 5 | 4.9/3.9 = |
Table
Therapeutic protection by various CN antidotal combinations. Control: KCN LD50 = 7.8 (4.6–13.1) mg/kg.
Exp no. | KCN dose (mg/kg) | Treatment | Survival (%) |
---|---|---|---|
1 | 15 | (SL-DTO-TS-Rh) | 6/6 (100%) |
2 | 15 | (SL-DTO-TS) + SN | 6/6 (100%) |
3 | 15 | (SL-DTO-TS-Rh) + SN | 6/6 (100%) |
4 | 20 | (SL-DTO-TS) + SN | 4/6 (67%) |
5 | 20 | (SL-DTO-TS-Rh) | 6/6 (100%) |
6 | 20 | (SL-DTO-TS) | 3/6 (50%) |
7 | 20 | (SL-DTO-TS-Rh) + SN | 6/6 (100%) |
The present experiments and results are confirming that the approach of utilizing externally administered, encapsulated, metabolizing rhodanese may have broad implications in cyanide antidotal therapy. The application of this approach has been successfully tested in animal models. In summary, these studies are describing the prophylactic and therapeutic
This paper was supported by NIH: NIAID/USAMRICD Interagency Agreements (W911NF-07-D-0001), the USAMRICD under the auspices of the US Army Research Office Scientific Services Program administered by Battelle (Delivery order 0557, Contract no. TCN 08284). Many thanks for the support by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (M. Budai) and the Robert A. Welch Foundation (x-0011) at Sam Houston State University (Huntsville, Texas, USA).