Study Of Tumor-targeted Doxorubicin-loading Ultrasound-sensitive Nanobubbles

Microbubble ultrasound contrast agent (UCA)can only image and exert therapy within tumor vessel and there existed many problems of nanoscale ultrasound contrast agent such as poor imaging and can't therapy. To solve the problem mentioned above, in the base of microbubble and nanobubble UCA, tumor-targeted nanobubble which was sensitive to ultrasound was investigated in this paper.First, the amphipathic polymer, poly (D,L-lactide-co-glycolide) -block-monomethoxy poly(ethylene glycol) (PLGA-PEG) was synthesized and its properties were also investigated. Second, tumor targeted nanobubble was prepared with doxorubicin (DOX) as the model drug, PLGA-PEG as film-former, perfluoropentane(PFP)(boiling point 29℃)as the imaging gas. Then its behavior in vitro and in vivo was investigated The main contents were described in details as follows:1. Synthesis of film-former. The diblock PLGA-PEG was synthesized using poly(D,L-lactide-co-glycolide) (PLGA) as a hydrophobic segment and monomethoxy poly(ethylene glycol) (mPEG) as a hydrophilic segment. By selecting appropriate reaction and purification conditions, PLGA-PEG was obtained and purified.2. Characteristics of film-former. The solubility of PLGA-PEG was determined. PLGA-PEG can dissolve easily in alkylogen, such as dichlormethane(DCM)and chloroform, and dissolve in tetrahydrofuran(THF), acetone, acetoacetate. But it can't dissolve in water and alcohol sovent, such as methanol and ethanol. Monomolecular film properties of PLGA-PEG was determined by Langmuir balance. The results showed that PLGA-PEG had good ability to form monomolecular film and thus certificated that it was amphiphilic. DOX(alkalinized by triethylamine)can form monomolecular film in pH=7.4 PBS and DOX can insert into the monomolecular film of PLGA-PEG. The mixing monomolecular film of DOX and PLGA-PEG had good static and dynamic stability.3. Preparation and Characterization of DOX micelles. HPLC analysis method in vitro of DOX was established. The formulation and preparation methods of DOX micelles were optimized with the particle size distribution, loading amount and encapsulation efficiency as the evaluation index. Finally, the DOX micelles were prepared by acetone injection method at low tempture. The size distribution of DOX micelles obtained was uniform, loading amount and encapsulation efficiency high. The technique reproducibility was very good. Negative staining TEM imaging showed the structure of micelles was globular and core-shell, with hydrophobic domain forming core and hydrophilic domain forming shell. The average size was 61nm.4. Preparation and Characterization of DOX nanobubbles. The preparation methods and technique of DOX nanobubbles were optimized with the size distribution, size change with temperature as the evaluation index. The DOX nanobubbles were prepared by the ultrasound injection method. Its size distribution was uniform, the technique reproducibility good, average size 168nm. After incubated in 50℃water, the structure was observed by light microscope, and the results showed that it was round with red surface. And the fluorescence microscope showed it was core-shell and DOX was localized in the outward hydrophobic compartment.5. Characteristics of DOX nanobubbles. The temperature sensibility, ultrasound sensibility, ultrasound incorporation, ultrasound-induced releasing drug, diluting stability was investigated with the size change as the evaluation index. The results showed that the size of DOX nanobubbles changed with the temperature. Ultrasound could make nanobubbles incorporate into microbubbles, and microbubbles disrupted when ultrasound power was increased, thus releasing DOX rapidly, more easily in pH=6.5 PBS. Nanobubble was stable in short term when stored in 4℃, and could stand dilution of 50 times. Nanobubble was incubated in 37℃water for 15 min after diluted and only 0.79% DOX was released. The stability was very good.6. Pharmacokinetics and tissue distribution of DOX nanobubbles. Subcutaneous tumors model was established using Kunming mice. HPLC analysis method in vivo was established. Pharmacokinetics and tissue distribution of DOX nanobubbles in normal and tumor-bearing mice were investigated, respectively. The elimination t_1/2 in normal and tumor-bearing mice was 1019±655 min and 570±148 min, respectively. DOX nanobubbles were distributed in heart, liver, spleen, lung and kidney in two model animals after iv 0.5h, some in heart,spleen, lung and kidney(0～0.27%), the most in liver(2.77%)of normal mice and tumor(1.11%)of tumor-bearing mice, respectively. Combined with pharmacokinetics results, it demonstrated that DOX nanobubbles had good tumor targeting.7. Pharmacodynamics of DOX nanobubbles. Subcutaneous tumors model was established using Kunming mice. Pharmacodynamics of DOX nanobubbles were investigated using tumor-bearing mice. The tumor weight differentiated significantly between the control and experimental group(p<0.01). There were distinguished differences between the ultrasound and non-ultrasound group (p<0.01). The results showed that DOX nanobubbles had good passive targeting action and ultrasound can facilitate it into tumor cells.Tumor targeting and ultrasound-sensible DOX nanobubbles with uniform size distribution and good stability when stored in 4℃were prepared in this paper. Its core-shell structure was demonstrated and DOX was localized in the hydrophobic wall of the nanobubbles. It had good tumor targeting for tumor- bearing mice after iv, and also exerted good pharmacodynamic action when using ultrasound.