Ultrasound-stimulated Lipid Microbubbles For Controllable Oxygen Release And Enhanced Tumor Radiosensitivity

PART?ECHOGENICITY OF OLM AND SCREENING ADMINISTRATION WAYS OF OLM FOR A BREAST TUMOR MODELObjectives To investigate in vivo contrast-enhanced ultrasound imaging performance of OLM compare to 2 available ultrasound contrast agents:Sono Vue and C₃F₈Ms,and to test the effect of two administration ways of OLM on echogenicity of the tumor and tissues aside tumor.Methods In vivo imaging performances were compared in total of 3rabbits?liver?,which were performed with a clinical diagnostic ultrasound system?Mylab Twice?equipped with contrast pulse sequence technology?CnTI?technology?.Each rabbit received a bolus injection of 0.5 mL microbubbles suspension?OLMs,SonoVue and C₃F₈Ms?successively through a 20-gauge catheter in an ear vein.In vivo 2 administration ways of OLM were compared in total of 4 breast tumor xenografts?nude mice?.OLMs?0.1 mL?were injected intratumorally and intravenously in vivo for mice bearing tumors.Quantitative analyses were performed with the US Image Analyzer?DFY-II type?.Results Measured physicochemical characteristics of OLM and TTP?40 s⁴5 s?were in agreement with SonoVue and C₃F₈Ms.In vivo data demonstrated that the performance of OLM was similar to C₃F₈Ms or showed similar contrast enhancement and duration of enhancement,but compared to C₃F₈Ms,peak intensity and duration of enhancement were inferior for OLM.There was a 6.7-fold increase for peak intensity of OLM and 3.8-fold increase for the EI 5 min following microbubble injection when compared to pre-contrast,respectively?P<0.001?.Although the duration of enhancement in the rabbit liver was longer for OLM compared to SonoVue,and quantitative analysis revealed higher enhancement for SonoVue?1.3-fold increase?.For tumor imaging,TTP was between 20s and 25s following injection and peak intensity of tumor with intratumoral injection of OLM was higher than that of intravenous injection?1.4-fold increase?,while the echo intensity of tissues aside tumor for intravenous injection group was 2-fold higher than that for intratumoral injection group.Conclusion Good echogenicity made it possible for OLMs to be guided and targetedly triggered by US in situ.It was superior to intratumal injection for tumor radiosensitivity study of oxygen delivery compared to intravenous injection due to more oxygen payload delivery to tumor and less effects to normal tissues aside tumor.PART II ULTRASOUND-STIMULATED OLM FOR CONTROLLABLE OXYGEN RELEASE FOR ELEVATING TUMOR OXYGENATION LEVELSObjectives To investigate the in vivo feasibility of OLM providing ultrasound enhancement in a VX2 breast tumor,and the ability of enhanced delivering oxygen to tumors using an ultrasound-sensitive oxygen-loaded microbubble by UTMD.Methods The performance of echo-response of OLM was observed in a total of three rabbits inoculated with the VX2 tumor line,which were performed with a LA522 linear transducer equipped with contrast pulse sequence technology?Cn TI? technology?.Each rabbit received a bolus injection of 0.02 m L/kg of the OLMs suspension through a 20-gauge catheter in an ear vein.The tumors' images were continually recorded for 5 min.Quantitative analyses were performed with the US Image Analyzer?DFY-II type?.In vivo proof of concept experiments were performed in three rabbits bearing VX2 tumor xenografts.The study was conducted with C3F8 Ms as the control group compared with OLMs before injection and 5 s after injection without US exposure,or immediately after US triggered after injection.The working electrode was introduced into the center of the tumors before agent injection.The OLM injection was performed after the total destruction of the C3F8 Ms.In brief,the rabbits received an injection of0.5m L microbubbles,then 1.5m L NS.The intra-tumoral oxygen concentration was recorded every 6s for 60 s by an IMP-211 apparatus before injection,similarly recorded 5s after microbubble injection without US exposure and immediately after US-triggered bursting.To record p O2 after US-triggered bursting,every 5s for 30 s after agent injection.The process was guided by US imaging.The value of p O2 was convert the percent change in oxygen saturation by the formula.Results Contrast imaging in vivo indicated a sufficient circulation time and homogenous contrast enhancement in tumors,which can enhance the echogenicity by up to 115.85 ± 34.83 d B?PI?.The time to peak?TTP?was about 20 s.The intensity in the tumor at 20 s after injection was nearly 8-fold the intensity compared to pre-injection?14.03 ± 3.00 d B?.The effective residence time lasted for about 5 min?3.7-fold increase?.After US irradiation,significant enhancement of p O2 in the breast tumor tissue when US stimulated OLMs was generated at 1 min?P< 0.05?.After triggering by US,the p O2 of the tumors in OLMs group were recorded to be 364.37±80.48 mm Hg compared with 54.49±12.05 mm Hg before US triggering.The added presence of US-activated OLMs elicited a nearly 6.7-fold increase in p O2 levels at 1 min compared with that of pre-injection,while the C3F8 Ms microbubbles did not cause such an effect.US-activated OLMs caused an increase oxygen saturation by up to 197%.Conclusion The duration enhancement time of OLM lasts about 5 minutes,which is enough time for UTMD to deliver oxygen payloads.Combined with US,this could become a strong technique used to locally elevate the oxygenation levels in tumors as soon as possible.These results encourage further study with US-activated oxygen release for the radiotherapy sensitization in vivo.PART ? TUMOR RESPONSE TO ULTRASOUND STIMULATED OLM AND RADIATION THERAPY IN VIVOObjectives To investigate the tumor response of ultrasound–stimulated oxygen-loaded-microbubble combined with radiation therapy in a breast tumor model,and to test the in vivo feasibility of ultrasound-stimulated microbubbles to providing synergistic antitumor effects for hypoxic tumor,and to investigate the possible mechanism.Methods Tumor xenografts were treated a single radiation dose of 2Gy alone,or in combination with US alone,USMB,OLM without US,US-stimulated-C3F8 Ms.Tumor cell death were evaluated at 24 h following therapy,using staining using in situ end labelling?ISEL?methods.HIF-1??and Caspase-3 of tumor sections were used to assess cell death and tumor radiosensitivity,using immunohistochemistry?IHC?.Results Results indicated a decrease in the expression of HIF-1? up to 49% and an 1.8-fold increase in the expression of Caspase-3 at 24 h for tumors treated with combined US–stimulated OLM and radiotherapy when compared to RT alone group.At 24 h following treatment,tumors showed an increase in detectable apoptosis compared to the RT alone group.An increase in the apoptotic index was approximately up to 60%?2-fold increase?,while an increase in the apoptotic index was approximately up to 50% for RT combined US–stimulated-C3F8 Ms group.Conclusion The potential of US-stimulated-OLMs therapies has been illustrated to reverse hypoxia-related resistance and induce tumor cell apoptosis to cancer radiation treatment.Tumor responses are promising and suggest that US-stimulated OLMs may be used as a dual-radiosensitizing strategy for cancer.Additional benefits include a potential in dose reduction required to achieve similar radiobiological equivalent outcomes.