| Cancer is one common type of diseases characterized by invasion and metastasis.The circulating tumor cells are most likely to metastasis to lung,accounting for 30%～50% of the total metastatic cases.Particularly,the breast tumor is prone to occurrence of lung metastasis.Lung metastasis always emerges in the middle or late stage of primary tumors and multiple nodules can be inspected at high rate.The surgical regimen is not highly recommended and chemotherapy is the alternative method.In the most cases,chemotherapeutics is administrated systemically through oral or intravenous manner,leading to a wide distribution in various organs,low therapeutic efficiency and serious side effects.The local drug concentration in lung tissue could be increased by direct pulmonary administration,avoiding systemic transport through the whole body.Sonodynamic therapy is a new method of cancer treatment.The sonosensitizer generates reactive oxygen species(ROS)under ultrasonic,which can synergistically kill tumor cells with the help of chemotherapy.Here,the cationic liposomal hydroxycamptothecin dry powder inhalers(CLHDs)were prepared and the ultrasound conditions for sonodynamic therapy were optimized thoroughly.In addition,the therapeutic effect of CLHDs and 5-aminolevulinic acid(5-ALA)under ultrasonic was investigated and the mechanism of antitumor was explored.The cationic liposomal hydroxycamptothecin(CLH)were prepared by the thin-film dispersion method.The orthogonal test design was used to optimize the formulation of liposomes.The optimal formulation of liposomes was 20: 5: 2(mol/mol)soybean phospholipid: cholesterol: HCPT.The optimal encapsulation efficiency was 89.71% measured by UV-vis spectrophotometer.Then the CLH was freezedried with mannitol as lyoprotectant to get CLHDs.Scanning electron microscope showed the irregular blocky structure of CLHDs,and transmission electron microscope showed the spherical vesicles structure of re-dissolved CLHDs.The hydrate particle size of re-dissolved CLHDs was 167.57 nm and the zeta potential was 10.10 m V.The average aerodynamic diameter of CLHDs was calculated as 4.85 μm,at which size can reach the bronchioles of the lungs.And the fine particle fraction(FPF)was 19.58%,much higher than 10%,which was suitable for pulmonary administration.In vitro test indicated that CLHDs could inhibit tumor cell proliferation with dependent of concentration,and the efficiency of CLHDs was much higher than free HCPT.The antitumor effect of HCPT was enhanced after encapsulation in liposomes.The viability of BEAS-2B cells was higher than 65% incubation with HCPT or CLHDs at the concentration less than 4 μmol/L.While HCPT showed higher inhibition rate than CLHDs at high concentrations(higher than 4 μmol/L).Incubation with CLHDs or HCPT at concentration of 10 μmol/L,the BEAS-2B viability was 60% and 20%,respectively.Thus,CLHDs showed lower toxicity on normal bronchial cells.Sonodynamic therapy(SDT)is a new approach for cancer treatment.Ultrasound could penetrate the tissue easily,reaching deep lesions site to diagnose and treat diseases.Moreover,ultrasound could locally focus on the malignant tumor site,reducing damage to normal tissues.Therefore,SDT has a promising prospect in the treatment of lung cancer.The ultrasound conditions were optimized on cells,including pre-incubation time of the sensitizer with cells,ultrasonic frequency,and ultrasonic time.The viability of 4T1-luc was the lowest when the preincubation time was 4 h,ultrasonic frequency was 1 MHz,and ultrasonic time was 180 s.It showed that SDT could achieve the best therapeutic effect under the optimal ultrasound conditions.The most protoporphyrin Ⅸ with strong acoustic sensitivity was transformed into the most from 5-ALA when the pre-incubation time was 4 h,and then the content of protoporphyrin Ⅸ gradually decreased.,and then protoporphyrin Ⅸ disappeared at the 8 h of pre-incubation time.It showed that ultrasonic can maximize the anti-tumor effect of sonodynamic therapy when the pre-incubation time was 4 h.The model of metastatic lung cancer was successfully established in female BALB/C mice after 7 days of injection of fluorescently labeled 4T1 cells.CLHDs and 5-ALA were administrated into the lungs of metastatic lung cancer mice through the trachea.After 4 hours,the ultrasound was performed on the lungs for 180 s at a frequency of 1 MHz and a power of 3 W/cm2.Compared with the model group,the tumor of the treatment group mouse was significantly reduced,the lung fluorescence intensity was weak,Caspase-3 positive expression was significantly increased,and Tunel staining showed a significant increase in apoptotic cells,indicating that the treatment group can induce tumor cell apoptosis to play a therapeutic role.The combination of CLHDs and SDT had the strongest anti-tumor effect,which was significantly different from other treatment groups.In this study,the CLHDs were prepared successfully.Combination with sonodynamic therapy,direct drug administration in the lungs has a good therapeutic effect on metastatic lung cancer in mice,which provides new ideas for the clinical treatment of metastatic lung cancer. |
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