Killing Effect Of High Intensity Focused Ultrasound Ultrasound-Induced Albendazile Theemosensitive Liposomes On Protoscolices In Vitro

ObjectiveA targeting delivery of albendazole (ABZ) in thermosensitive lip osomes (TSL-ABZ) is induced by the thermal effect of high intensity focused ultrasound (HIFU) for an enhancement of bioavailability of albendazole, which could improve albendazole’s killing effect on the protoscolices in hydatids, the metacestode larvae of Echinococcus gra nulosus.Methods1. Using the single factor experiment to establish a method to det ermine the preparation of TSL-ABZ. Initially identified the formulatio n and process conditions according to inspect the type and amount o f organic solvent, ratio between the synthesis of phospholipids, pH va lue of hydration medium, ratio of lipoid and drug, water bath temper ature.2. The drug carrier were characterized by using transmission electron microscopy, Nano ZS90 Zetasizer particle size measurement, Fourier infrared spectrum, thermogravimetric analysis and in vitro release concentration to demonstrate weather or not that the system was successfully prepared.3. In vitro observation of effects on protoscolices by TSL-ABZ Different proportion of TSL-ABZ were added to cystic fluid containing protoscoleces in different water bath temperature, then detected the vitality of protoscolex under the conditions of different drug concentration and preparation condition.4. In vitro observation the cytotoxic effect of protoscolices by HIFU induced TSL-ABZ. Under the certain high intensity ultrasound doses, different proportion of drugs were added to protoscolices in suspension. Observed the morphological changes and calculated the mortality under the light microscope, inspected the changes of pathological structure of protoscolices in HE staining and under the transmission electron microscope, and inspected the protoscolices nuclear apoptosis condition with TUNEL fluorescence staining.Results1.The preparation prescription was selected and optimized, and th e best one was as follows, lipid ratio:1:40, DPPC:MPPC:DSPG:AB Z at ratio of 66:7:5:3, buffer pH 5.3, ultrasonic time:15 min, tempera ture:38℃, the average encapsulation efficiency of TSL-ABZ was 78. 7%, drug loading rate was 1.91%.2. Under the transmission electron microscope the blank thermosensitive liposomes(TSL) was hollow structure, and TSL-ABZ appear black ball shape; The average particle diameter of TSL-ABZ analyzed by Nano ZS90 Zetasizer was 316 nm, PDI was 0.533, Zeta potential was-9.84 mV. Infrared spectrum showed no chemical force happened between the drug and the carrier. The structure of TSL-ABZ was not changed, moreover, the drug was included in the structure of the liposome. DSC curve indicated that the prepared liposomes had thermal properties, which phase transition temperature was of about 42.1℃.3.In 24h water bath time and 42℃ water bath temperature, the TSL-ABZ 1000 μl group drug releasing induced the greatest extent of protoscolices death, the mortality was higher than that of other groups.4. After the temperature test, chose the HIFU irradiation dose most suitable to the phase change temperature (75W,12s). The mortality of protoscolex in each group showed a growth trend, especially the HIFU+TSL-ABZ 1000 μl group that was the highest. The cortical destruction was observed, the calcium particles disappear, severe vacuolization, hook off, part of the structure or even disappear. The fluorescence staining of protoscolices showed lots of apoptotic nuclei.Conclusion1. The TSL-ABZ preparaed in this experiment method had successfully encapsulated drugs albendazole standardion, while its morphology were regular and under mono dispersed status. The particle size has reached the nanometer level while its morphology were regular and under mono dispersed status.2. Heat-sensitive synthetic phospholipid could control reaction of drug releasing and apply to the experimental object according to the experimental conditions. The drug wouldn’t release under the room temperature and body temperature, but when the temperature exceeds the melting phase change materials, the release got the feature of explosive.3. TSL-ABZ could release the drug at the phase transition temperature, and has the function of killing protoscolices in vitro.4. HIFU could induce TSL-ABZ to enhance in vitro killing effect of protoscolices.