Normalization Of Tumor Vascular Induced By Oxygen-carrying Perfluoropentane Nanodroplets And Ultrasound To Enhance The Effect Of Chemotherapy

BackgroundChemotherapy resistance is a difficult problem to be solved in clinic.The tumor microenvironment with the features of hypoxia,low PH and high interstitial pressure is responsible for chemotherapy resistance by blocking drug delivery and reducing drug activity.Anti-angiogenesis therapy can induce tumor vessel normalization with enhanced blood perfusion and oxygen level and lowered interstitial pressure to enhance the effect of chemotherapy.However,the short time window of vascular normalization and the phenomenon of drug resistance limit its overall clinical effect.It has the potential of inducing tumor vessel normalization by increasing oxygen level because of rapidly degrading hypoxia inducible factors-1?(HIF-1?)and down regulating vascular endothelial growth factor(VEGF).ObjectiveWe tried to deliver oxygen specifically to tumor using oxygen-carrying nanodroplets(O2-NDs)combined with ultrasound to explore the feasibility of inducing tumor vascular normalization and enhancing the effect of chemotherapy.MethodsO2-NDs were prepared by emulsification and homogenization method,and the particle size,stability,the capacity of oxygen carrying and releasing were characterized.The subcutaneous transplanted tumor model of 4T1 breast cancer in mice was established.When the tumor volume was greater than 100mm3,it was included in the experiment.Twenty five tumor bearing mice were randomly divided into Control,O2-NDs,NDs+US,O2-NDs+US and O2-NDs*2+US(treated twice on day 0 and 9,respectively).Contrast-enhanced ultrasound(CEUS)was used to track the intensity of tumor blood perfusion.For the Control group and O2-NDs+US group,the photoacoustic imaging was used to assess the oxygen saturation of tumor tissues,western blot(WB)was used to detect the expression of HIF-1? and VEGF and masson staining was used to detect the content of collagen fibers.Another twenty five tumor bearing mice were randomly divided into control,DOX,NDs+US+DOX,O2-NDs+US+DOX,O2-NDs*2+US+DOX.The treatment was as follows:doxorubicin(5 mg/kg)was given via tail vein,once every 4 days for 4 times.The tumor size and weight of mice were measured every other day,and tumor tissues were taken and weighed on the 18th day.HE staining was performed on the heart,lung,liver,spleen,kidney of tumor bearing mice to observe the damage.Spss21 and graphpad5 were used for data processing and statistical analysis.The measurement data of repeated tests were expressed as mean ±s.The comparison of measurement data between the two groups was conducted by t-test,and the comparison of measurement data between multiple groups was conducted by one-way ANOVA.Among them,Tukey test or Dunnett test were used for pairwise comparison between groups,and the test level was a=0.05.Results1.The particle size of the O2-NDs was uniform with a mean size of 336±27nm,and kept stable at 4? for 2 weeks.O2-NDs was not vaporized under the condition of 37? and 5MPa sound pressure.It was able to carry large amounts of oxygen,up to 1.48mg of oxygen per milliliter of perfluoropentane liquid and release oxygen sensitively by ultrasound,which was proportional to ultrasound power.2.The peak intensity(PI)of tumor in O2-NDs+US group increased from the second day of treatment and reduced to the level before treatment on the ninth day,while the PI value of control,O2-NDs and NDs+US groups decreased gradually.During the enhanced perfusion,the blood oxygen saturation of tumor tissue and the pericyte coverage of the tumor vessel were increased,the expression of HIF-1? and VEGF and the content of collagen fiber were decreased.PI value of O2-NDs*2+US group began to rise from the second day of treatment,and decreased to the level before treatment on the 14th day,which was longer than that of O2-NDs+US group.3.The tumor inhibition effect of O2-NDs+US and O2-NDs*2+US groups was significantly better than that of control,DOX,NDs+US+DOX groups(P<0.05),and that of O2-NDs*2+US group was better than that of O2-NDs+US group(P<0.05).No obvious damage to heart,lung,liver,spleen,kidney was observed.ConclusionThe O2-NDs prepared in this study have stable properties,high oxygen carrying capacity and good characteristics of oxygen release by ultrasound.O2-NDs combined with ultrasound can successfully induce tumor vascular normalization,and the time window can be prolonged by twice treatments.The inhibitory effect of chemotherapy drugs on tumor growth was enhanced by the treatment of O2-NDs combined with ultrasound,which was better by twice.