| BackgroundAs a common type of tumor,ovarian cancer ranks first in mortality rate among all types of gynecologic malignant tumors.There are two main reasons for its high incidence rate and mortality rate: 1)Late detection.Currently,with the existing methods of imaging examination,it is difficult to diagnose ovarian cancer timely in the early stage.Therefore,many patients lose the opportunity to receive surgical treatment after being diagnosed in the late stage;2)Resistance to drug.In most cases,ovarian tumor responds to the initial treatment of platinum drugs,but after stopping treatment for a period,recurrence happens repeatedly with an increasingly short interval.Therefore,it is urgent to find a safe and effective drug to improve the diagnosis and treatment of ovarian cancer.With the rapid development of nanotechnology,new tumor treatment methods,such as photothermal therapy and immunotherapy,appeared on the scene and caught researchers' attention.However,most of these treatments are still in the basic research stage.Among these novel treatment methods,ultrasonic microbubble is a kind of ultrasound contrast agent which has been used in clinical ultrasonic diagnosis.The contrast bubbles vibrate under the action of ultrasound and scatter strong ultrasonic signal to enhance ultrasound imaging.Due to the microbubbles' ability to enhance the cavitation effect and thus promote the biological effect of ultrasound,it is also applied to some fields including ultrasonic thrombolysis,gene transfer mediating,drug delivery and so on.Nevertheless,most of the current ultrasonic microbubbles are micron in size and cannot reach the tumor tissue site through the vascular endothelial space,which greatly restricts their application.In this study,we prepared a type of nanoscale ultrasonic microbubbles(CD44-NBs)and realized the organic combination of ultrasonic diagnosis and sonodynamic therapy.Methods1)Preparation and characterization of ultrasound nanobubbles: CD44-NBs were prepared.The morphology and dispersion of CD44-NBs were observed by transmission electron microscope and optical microscope.The mean particle size and Zeta potential were detected by nanoparticle analyzer.The encapsulation rate and drug release of curcumin were measured by ultraviolet visible spectrophotometer.The phase transition ability was observed after ultrasonic irradiation in vitro.2)In vitro and in vivo targeted evaluation of CD44-NBs: Firstly,the relationship between CD44 expression and clinicopathological characteristics in ovarian cancer tissues was investigated.The expression of CD44 in ovarian cancer cell line was analyzed by flow cytometry.In vivo and in vitro ultrasonic experiments were conducted to explore the effect of ultrasound imaging.3)Experimental study on the targeted treatment of ovarian cancer: Its safety in vitro was tested by CCK-8.Its safety in vivo was detected by blood biochemical indexes and histopathology.The tumor growth and survival of nude mice were observed to explore the therapeutic effect in vivo.4)Study on the killing mechanism of ultrasound nanobubbles for ovarian cancer: To detect the production of reactive oxygen species after ultrasound irradiation of CD44-NBs;To explore the effect of CD44-NBs on the biological behavior of ovarian cancer malignant cells by plate cloning,scratch migration and invasion experiments;To determine the relevant mechanism of reversing drug resistance by Western blot and qRT-PCR.Results1)Observed by electron microscope,the morphology of CD44-NBs were round,the surface was smooth and regular,the distribution was uniform,the measured average particle size was202.4±1.8nm,and the dispersion was good.The encapsulation rate of curcumin was about93.34%,the average drug load was 8.7%,and the encapsulation rate of perfluorohexane was about 77%.No significant changes were found in hydration particle size and encapsulation rate after 1 week at 4 °C,showing good stability.After 2 minutes of treatment on an ultrasound therapy instrument,the average diameter of the microbubbles was 829.8± 5.1nm,and CD44-NBs nanobubbles transformed from nanometer to micron after ultrasonic stimulation.The curcumin was continuously released with the extension of the ultrasonic treatment time measured by ultraviolet spectrophotometer.2)To investigate the relationship between the expression of CD44 and clinicopathological features in ovarian cancer tissues,it was found that the expression of CD44 was correlated with ascites,lymphatic metastasis and disease stage,and the expression rate of CD44 was higher in patients with ascites and lymphatic metastasis.Higher FIGO stages always indicated the stronger CD44 expression.Flow cytometry analysis of the expression of CD44 in ovarian cancer cell lines showed that the expression of CD44 in SKOV3 cells was up to 99%,while CD44 in ES-2 cells was approximately 20%.Flow cytometry and laser confocal detection that CD44-NBs showed good targeting effect on CD44 positive ovarian cancer cell lines.In vivo near-infrared imaging confirmed that CD44-NBs could be concentrated in tumor tissues for a longer time.In vivo and in vitro ultrasonic experiments showed that compared with the anechoic degassing water,CD44-NBs showed strong echo after ultrasonic excitation,which could be used for ultrasonic imaging.3)Safety evaluation experiment of CD44-NBs indicated that CD44-NBs had good cell safety in a certain concentration range in vitro;it had no obvious damage to the tumor bearing mice,no obvious abnormal indexes of blood biochemical,and no other toxic and side effects on nude mice;CCK-8 results showed that the survival rate of CD44-NBs+US group was the lowest among these groups.Thus,CD44-NBs had a good killing effect in vitro.In vivo,CD44-NBs combined with sonodynamic therapy was found to have good tumor suppressive effect by observing tumor changes,survival time and tumor immunohistochemistry in mice.4)The yield of reactive oxygen species(ROS)increased significantly after ultrasonic irradiation of CD44-NBs.By exploring the effect of CD44-NBs on the biological behavior of ovarian cancer cells,it was found that the cell clonal formation rate,migration ability and invasion ability of CD44-NBs combined with sonodynamic therapy group were significantly decreased.Western Blot and qRT-PCR showed that CD44-NBs could reduce the expression of P-gp in cells and increase the expression of p38 MAPK,Beclin 1 and thus reversing the drug resistance to tumor cells.ConclusionsIn this study,CD44 targeted ultrasonic nanobubbles,containing curcumin(a sound sensitive agent)and perfluorohexane(to increase the oxygen content in tumor sites),were successfully prepared.Perfluorohexane undergo a liquid-to-gas transition under low-power ultrasound,which leads to a transformation from nanobubbles to microbubbles,and when the microbubbles burst,the drug contained is released.This method can improve the therapeutic effect and targeting ability of drugs,as well as reduce side effects.Under ultrasonic stimulation,it makes the idea of integrated treatment of diagnosis and treatment a reality.To be more specific,perfluorohexane can be used as ultrasound contrast agents and simultanously provide reactive oxygen species for sonodynamic therapy,thus significantly improving the efficacy of sonodynamic therapy for ovarian cancer.This provides a potentially possible way to improve the clinical diagnosis and treatment of ovarian cancer. |
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