Preparation Of Novel Gene And Drug Loading Microbubble Systems

Objective: To combine lipid microbubbles with cationic nanoliposomes by biotin-avidin system and prepare a new type of gene microbubble vector.Methods: 1,Biotinylated lipid microbubbles (Bio-LMB) were prepared by mechanical shaking with a certain mole ratio of dipalmitoyl phosphatidylcholine (DPPC) and biotinylated polyethylene glycol distearoyl phosphatidylethanolamine (Bio-PEG-DSPE), and trace of fluorescein isothiocyanate (FITC) was added simultaneously. The method of preparing non-biotinylated lipid microbubbles (LMB) was the same with Bio-LMB, but Bio-PEG-DSPE was replaced by distearoyl phosphatidylethanolamine (DSPE).2,Biotinylated cationic nanoliposomes (Bio-CNLP) were prepared by film dispersion method - film extrusion method with a certain mole ratio of hydrogenated soy phosphatidylcholine (HSPC), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and Bio-PEG-DSPE, and trace of rhodamine 6G was added simultaneously. The method of preparing non-biotinylated cationic nanoliposomes (CNLP) was the same with Bio-CNLP, but Bio-PEG-DSPE was not added.3,Bio-LMB were incubated with excessive avidin for 30 min at 4℃. With the suction of lower excessive avidin, the excessive Bio-CNLP were added and incubated for another 30 min at 4℃. LMB and CNLP were combined by the same method.4,The connection effect was observed by laser scanning confocal microscope in experimental group and control group .Results: In Bio-LMB + Bio-CNLP group, it showed that the green Bio-LMB wall was not smooth and a number of red small round Bio-CNLP gathered around Bio-LMB under confocal imaging. In LMB + CNLP group, the LMB wall was smooth and no conjugation of CNLP was observed.Conclusions: Bio-LMB and Bio-CNLP can be combined firmly by avidin-biotin system. It is potential to provide a new train of thought on a gene vector and a new means of tumor targeted gene therapy . Objective: To combine lipid microbubbles with PLGA nanospheres and prepare a novel drug microbubble vector.Methods: 1,Amino lipid microbubbles (NH2-LMB) were prepared by mechanical shaking with a certain mole ratio of dipalmitoyl phosphatidylcholine (DPPC) and amino polyethylene glycol phosphatidylethanolamine (NH2-PEG-DSPE).2,Poly lactic-co-glycolic acid (PLGA) nanospheres (NP) were prepared by double emulsion method.3,Active PLGA nanospheres (NP-COOR) were produced by carbodiimide method to activate the surface carboxyl of NP.4,Then NP and NP-COOR were respectively added to two tubes of NH2-LMB, and they were incubated at 4℃. After 12,24,36,48 h, the connection effect was observed under light microscope.Result: After 12 and 24 hours, no conjugation of nanospheres was observed in both tubes. But after 36 hours, also no conjugation of nanospheres was observed in NP + NH2-LMB group; a small portion of nanospheres gathered around microbubbles in NP-COOR + NH2-LMB group. After 48 hours , the lipid microbubble wall was still smooth, and no conjugation of nanospheres was observed in NP + NH2-LMB group. However, in NP-COOR + NH2-LMB group, the lipid microbubble wall was not smooth and a number of small round nanospheres gathered around microbubbles like garlands.Conclusion: The carbodiimide method enables successful preparation of a novel drug microbubble vector, which is potential to provide a new train of thought on tumor targeted drug therapy.