ice batch and when the organic solution was added, the new mixture was also cooled ... the nanoparticles at intervals (cooling the tubes on an ice bath between.
Chitosan modification of PLGA nanoparticles Looking to increase the negative charge density on the surface of the nanoparticles, chitosan was employed. Two schemes were followed; the first included the presence of chitosan during nanoparticle production while the second included the modification of the surface from preformed nanoparticles. Modification during preparation 60 mg of PLGA 5002A were dissolved in 4 ml of dichloromethane along with 0.8 mg of docetaxel and emulsified with 8 ml of PVA (3%, dissolved in 1% acetic acid containing 0 mg/ml of chitosan) (31000 Da, 88% hydrolyzed) at 20-25% for 1 min. The PVA solution was cooled previously in an ice batch and when the organic solution was added, the new mixture was also cooled before the sonication. The same procedure was repeated but chitosan concentrations in the aqueous phase were set to: 0.5, 1.0 and 2.0 mg/ml. After sonication the emulsions were heated to 39 C and vacuum was applied for 0.5 h, then evaporation of the organic solvent was completed at 39 C without vacuum overnight. The resulting solutions were centrifuged for 1.5 h at RT at 12000 rpm (the volume was completed to 8 ml with water). The supernatant was removed and was replaced by cooled water (4 C). The tubes were sonicated to resuspend the nanoparticles at intervals (cooling the tubes on an ice bath between intervals to avoid heating the solution). Five intervals were necessary to completely resuspend the nanoparticles. A 2nd centrifugation was applied at 4 C at 12000 rpm for 1.0 h. The particles were again resuspended in water as before. A 3rd centrifugation was applied at 4 C at 12000 rpm for 0.5 h. The final resuspension was performed using less than 3 ml of water. Then the volume was completed to 3 ml and 6 fractions (0.5 ml) were placed in 1.5 ml centrifuge tubes. To two of the tubes prepared with different concentrations of chitosan, 1 ml of PBS buffer was added and the tubes were centrifuged at 15000 rpm for 15 min, then 1 ml of sample was withdrawn from each tube and replaced by l ml of fresh PBS buffer. Samples were taken at consequent times. The tubes were placed in a water bath at 37 C. To two of the tubes prepared with different concentrations of chitosan, 0.5 ml of water was added and the solution was transferred to 15 ml centrifuge tubes to freeze-dry the samples. Sample
Size, nm
PDI
Z, mV
Encapsulated Nanoparticle drug, µg mass, mg Chitosan 0.0 mg/ml 133.1/134.4 0.050/0.018 -14.9/-16.7 28.8 7.73 Chitosan 0.5 mg/ml 126.2/127.4 0.054/0.052 +2.91/+3.05 32.2 7.91 Chitosan 1.0 mg/ml 126.2/125.2 0.054/0.070 +6.61/+7.45 29.6 6.33 Chitosan 2.0 mg/ml 136.8/137.8 0.008/0.013 +14.9/+14.6 20.4 6.24 For size analysis the original sample (0.5 ml) was diluted 21 times, while for zeta potential analysis it was diluted 2 times. Modification after preparation 60 mg of PLGA 5002A were dissolved in 4 ml of a dichloromethane along with 0.8 mg of docetaxel and emulsified with 8 ml of PVA (3%) (31000 Da, 88% hydrolyzed) at 20-25% for 1 min. The PVA solution was cooled previously in an ice batch and when the organic solution was added, the new mixture was also cooled before the sonication. The same procedure was run twice. After sonication the emulsions were heated to 39 C and vacuum was applied for 0.5 h, then evaporation of the organic solvent was completed at 39 C without vacuum overnight. The resulting solutions were centrifuged for 1.5 h at RT at 12000 rpm (the volume was completed to 8 ml with water). The supernatant was removed and was replaced by cooled water (4 C). The tubes were
sonicated to resuspend the nanoparticles at intervals (cooling the tubes on an ice bath between intervals to avoid heating the solution). Five intervals were necessary to completely resuspend the nanoparticles. A 2nd centrifugation was applied at 4 C at 12000 rpm for 0.75 h. The particles were again resuspended in water as before. A 3rd centrifugation was applied at 4 C at 12000 rpm for 0.5 h. The final resuspension was performed using less than 3 ml of water. Then the volume was completed to 6 ml and 12 fractions (0.5 ml) were placed in 1.5 ml centrifuge tubes. To three tubes 1 ml of water (at 4 C) was added, to three tubes 1 ml of 1% acetic acid (at 4 C) was added, to three tubes 1 ml of 1% acetic acid (at 4 C, containing 0.5 mg/ml of chitosan) was added, and to the final three tubes 1 ml of 1% acetic acid (at 4 C, containing 1.0 mg/ml of chitosan) was added. The tubes were placed in the fridge for 1 h. Afterwards the tubes were centrifuged at 15000 rpm for 15 min, the supernatant was removed and replaced by water (at 4 C). Two more washings were applied to the samples under the previous conditions. The final resuspension was performed in 0.5 ml of water (at 4 C). To four of the tubes (prepared with or without chitosan) 1 ml of PBS buffer was added and the tubes were centrifuged at 15000 rpm for 15 min. 1 ml of supernatant was withdrawn as sample and replaced with 1 ml of fresh PBS buffer. The tubes were placed in a water bath at 37 C and samples were taken at specified times. Sample
Size, nm
PDI
Z, mV
Encapsulated drug, µg Blanck 153.2/155.1 0.093/0.075 -19.3/-19.6 10.2 Chitosan 0.0 mg/ml 172.1/172.3 0.086/0.079 -23.1/-23.0 8.3 Chitosan 0.5 mg/ml 148.4/149.1 0.068/0.069 -12.8/-12.3 7.4 Chitosan 1.0 mg/ml 145.8/147.1 0.050/0.055 -12.5/-11.9 6.8 For size analysis the original sample (0.5 ml) was diluted 21 times, while for zeta potential analysis it was diluted 3 times. Results from drug release are shown in the next Figure.
