Effects of Mass Ratio, pH, Temperature, and Reaction Time on Fabrication of Partially Purified Pomegranate Ellagitannin−Gelatin Nanoparticles

Nanoparticles were fabricated using self-assembly between partially purified ellagitannins (PPE) and gelatin. The factors affecting fabrication of nanoparticles, including PPE-to-gelatin mass ratio, pH, temperature, and reaction time, were investigated and the characteristics of formed nanoparticles, including sizes, zeta-potentials, and loading efficiency, were assessed. Nanoparticles that were fabricated using PPE-to-gelatin mass ratio from 1:5 to 6:5 had particle sizes from 121.5 to 129.0 nm. Increasing the ratio to 9:5 caused a drastic increase of particle size (620.7 nm) and was accompanied by formation of precipitation in the colloidal system. Nanoparticles fabricated in the pH range 4.0 to 5.3 (gelatin solution) had particle sizes ranging from 20.6 to 193.9 nm and zeta-potential between +14.7 and +23.8 mV, respectively. Loading efficiency of punicalagin A and B in the nanoparticles under these pH values ranged from 29.5% to 84.3% and from 10.6% to 73.9%, respectively. Extreme pH of gelatin solutions (pH 1.0, 2.0, 3.0, or 11.0) hindered the formation of nanoparticles due to possible hydrolyzation of ellagitannins and weakened affinity between ellagitannins and gelatin. Although gelatin at isoelectric point (pH 6-7) provided more hydrophobic sites to interact with ellagitannins, weakened zeta-potentials resulted in poor stability of nanoparticle suspension. Nanoparticles formed between 25 to 50 °C had particle size below 500 nm, whereas lower temperatures increased the size of nanoparticles. Nanoparticles were formed after 12 h of reaction time, and the nanoparticle colloidal suspension remained stable for 4 days. PPE-gelatin nanoparticles fabricated using PPE-to-gelatin mass ratio below 7:5, pH 4.0, temperature 25-40 °C, and reaction time 1.5 days had smaller particle sizes, higher zeta-potentials, and good loading efficiency.