Pralidoxime is an organophosphate antidote with poor central nervous system distribution due to a high polarity. In the present study, pralidoxime-loaded poly(lactic-co-glycolic acid) nanoparticles were prepared and evaluated as a potential delivery system of the drug into the central nervous system. The nanoparticles were prepared using double emulsion solvent evaporation method. Poly(lactic-co-glycolic acid) (PLGA) in ethyl acetate made the organic phase and pralidoxime in water made the aqueous phase. The system was stabilized by polyvinyl alcohol. Different drug/polymer ratios were used (1 : 1, 1 : 2, and 1 : 4) and the fabricated particles were characterized for encapsulation efficiency using UV-VIS Spectroscopy; particle size distribution, polydispersity index, and zeta potential using photon correlation spectroscopy; and
Organophosphates (OP) intoxication accounts for the highest number of poisoning cases across the globe [
Despite the wide use of organophosphates [
Efforts to overcome the blood brain barrier (BBB) have focused on altering either the barrier integrity and characteristics or the drug properties. Nanoparticles might be a better technique to circumvent the BBB since no BBB or drug manipulation is necessary. Poly(lactic-co-glycolic acid) PLGA nanoparticles have proved to improve the blood brain barrier penetration of a number of drugs that are poorly distributed in the CNS [
Pralidoxime chloride (pyridine-2-aldoxime methochloride) (RD grade), PLGA (50 : 50), ethyl acetate, and polyvinyl alcohol (PVA) were obtained from Sigma Aldrich Company, Germany. Disodium orthophosphate, citric acid, potassium dihydrogen orthophosphate, sodium hydroxide (NaOH), and sodium chloride (NaCl) were obtained from Associated Chemical Enterprise (South Africa). Distilled water was used in all the experiments. The following equipment was used during the study: pH meter (Jenway, UK), vortex mixer (Heidolph Reax 2000, Germany), magnetic stirring plate (Sigma Aldrich, Germany), sonicator (Westwood Ultrasonics, UK), ultracentrifuge (Beckman Optima LE-80k Ultracentrifuge, USA and Hermle Z160M, Germany), UV-VIS Spectrophotometer (Shimadzu, Japan), and Zetasizer (Malvern Zetasizer Nano ZS90, UK).
Pralidoxime-loaded PLGA nanoparticles were prepared using a double emulsion solvent evaporation method. 1.2 mL aqueous pralidoxime solution (25 mg/mL, 50 mg/mL, or 100 mg/mL) was emulsified in 8 mL of ethyl acetate containing PLGA (120 mg) by means of homogenization on an ice bath at a speed of 13000 rpm for 90 seconds. The primary emulsion was further emulsified in 32 mL of 2% (w/v) PVA solution containing 5% (w/v) of NaCl by homogenization at 25000 rpm for 10 minutes on an ice bath. The resultant double emulsion was stirred for 4 hours at 25°C on a magnetic stirring plate at 500 rpm. The nanoparticle suspension was then kept under refrigeration overnight. The nanoparticles were recovered by ultracentrifugation at 13,400 rpm for 30 minutes at 4°C. Following this, nanoparticle sediments were washed thrice with water then lyophilized overnight.
The nanoparticles were destroyed by acetonitrile in 1 mL eppendorf tubes under a vortex mixer, immediately after the washing step of fabrication (before lyophilization). The resultant solution was passed through a membrane filter (0.22
The particle size distribution, polydispersity index, and zeta potential of the pralidoxime-loaded PLGA nanoparticles were determined in triplicates by a photon correlation spectroscopy (PCS) using a zetasizer (Malvern Zetasizer Nano ZS90, UK). Approximately, 1 mg of each sample was dissolved in 1 mL of deionized water. The dissolved sample was sonicated for 30 minutes. The samples were placed in a zetasizer and the particle size, polydispersity index, and zeta potential were then observed.
The
ANOVA was done on the results using GraphPad Prism 5. All the statistical tests were done at 95% level of significance.
Nanoparticles with drug/polymer ratio of 1 : 4 had highest encapsulation efficiency (68.78%) and the encapsulation efficiency decreased with increase in drug/polymer ratio as illustrated in Table
Encapsulation efficiency of pralidoxime-loaded PLGA nanoparticles with different drug/polymer ratios.
Drug/polymer ratio | Loading efficiency (mg/mL) | Encapsulation efficiency (%) |
---|---|---|
1 : 1 | 0.98 | 28.58 ± 0.01 |
1 : 2 | 0.89 | 51.91 ± 0.02 |
1 : 4 | 0.59 | 68.78 ± 0.03 |
Encapsulation efficiency is recorded as mean ± SD.
The results for the particle size, polydispersity, and zeta potential are shown in Table
Mean particle size, polydispersity index (PDI), and zeta potential for pralidoxime-loaded PLGA nanoparticles with different drug : polymer ratio.
Drug/polymer ratio | Particle size (nm) | Zeta potential (mV) | PDI |
---|---|---|---|
1 : 1 | 386.6 ± 15.33 | 5.04 ± 0.35 | 0.323 ± 0.021 |
1 : 2 | 304.7 ± 7.167 | 3.31 ± 0.27 | 0.180 ± 0.032 |
1 : 4 | 322.8 ± 2.193 | 5.98 ± 0.30 | 0.203 ± 0.001 |
Data represented as mean ± SD.
Polydispersity index was less than 0.5 for all the three samples. The particles with drug/polymer ratios 1 : 2 and 1 : 4 had the least PDIs and their difference at 5% standard error was insignificant (
Zeta potential of the produced nanoparticles was significantly different among all the three samples at 5% standard error (
The drug release profile was biphasic for the sample with drug/polymer ratio 1 : 1. This then shifted towards monophasic as the drug/polymer ratio decreased to 1 : 4 (Figure
Graph of drug release profile of pralidoxime-loaded PLGA nanoparticles in comparison with the free drug.
Drug encapsulation into nanoparticles modifies the drug’s pharmacokinetics by masking its physicochemical properties. In turn, the nanoparticle characteristics determine the pharmacokinetics and stability of the drug.
High encapsulation efficiency observed for particles with drug/polymer ratio 1 : 4 (Table
Other studies have however recorded that encapsulation efficiency of water soluble drugs can be as high as 80% to 100% when the double emulsion evaporation method is used [
The decrease in encapsulation efficiency with increase in drug/polymer ratio was in line with the results of studies by Trivedi and colleagues [
Particle size is an important factor to consider in circumvention of the blood brain barrier. Small particles are of great interest as opposed to larger particles that would be trapped in the tight junctions [
The particles produced for all the three samples with different drug/polymer ratios were small (in the nanometer range) and formed monodispersion in water; PDI < 0.5 (Table
Though cell membranes allow a free passage of particles less than 1 nm in diameter [
Much smaller pralidoxime-loaded PLGA nanoparticles could be obtained. One of the reasons that could have resulted in the recorded particles size might be the shearing method used (homogenization) which was recorded to be associated with large particles [
For the three samples with different drug/polymer ratios, zeta potential values were small (less than 20) (Table
On the contrary, small PDI values (close to zero) (Table
The nature of the recorded zeta potential values (small and positively charged) can improve the pharmacokinetics of the drug by increasing the circulation time of the nanoparticles in the blood [
For the drug to be released from the PLGA nanoparticles, the PLGA undergoes degradation by hydrolysis or biodegradation through cleavage of its backbone ester linkage into oligomers and finally monomers [
The first phase (rapid release) is less pronounced for the sample with drug/polymer ratio 1 : 2. The delayed release phase is dominant. This indicates that there is little pralidoxime close to the PLGA surface [
The pralidoxime-loaded PLGA nanoparticles were produced by the double emulsion solvent evaporation method with the highest encapsulation efficiency being 68.78% (for particles with drug/polymer ratio 1 : 4), lowest mean particle size of 304.7 nm (from particles with drug/polymer ratio 1 : 2 though not statistically different from the size recorded by the particles with drug/polymer ratio 1 : 4), and the highest zeta potential being 5.98 mV (recorded for particles with drug/polymer ratio 1 : 4). The
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank technicians at the Western Cape University (South Africa) for assistance in the in particle size, PDI, and zeta potential characterization. The authors also acknowledge the following departments at the University of Zimbabwe for their laboratories and equipment: Department of Biochemistry, Department of Physiology, and Faculty of Veterinary Sciences. We also acknowledge the Innovation and Commercialization (ICF) grant received for the purpose of developing this study.