Construction Of Targted Cooling Nanobubbles And Preliminary Study On Its Treatment Efficacy Of Ischemic Stroke Model

Objective:To investigate whether the new targeted nanobubbles can reach the ischemic lesion area after acute ischemic stroke in mice,and to evaluate the therapeutic effect of nanobubbles on acute ischemic stroke mice using a variety of imaging methods.Method:（1）Preparation of a novel nanobubble contrast agent,targeted cooling nanobubble.Platelets are used as an outer membrane to coat an inert gas with a very low specific heat capacity,helium,which is encapsulated into platelets by ultrasonic oscillation after removing platelet contents,in order to construct nanobubbles and make them have cooling properties.The characteristics of the formed platelet nanobubbles were characterized in various aspects;immunofluorescence was used for morphological characterization,projection electron microscopy was used for physical property characterization,confocal microscopy was used to achieve size characterization,and dynamic light scattering was used for comprehensive characterization of aggregation.Reflect the nature of nanobubbles in the body:near-infrared reflects the targeting performance;photosensitive thermometry evaluates the cooling effect.（2）To monitor the efficacy of targeted cooling nanobubbles in mice with acute ischemic stroke.Male C57BL/6 wild-type mice were selected,they were injected with Rose Bengal 200μL/25g by photochemical embolization to create an acute ischemic animal model,which was evaluated by HE staining.Part of the group were used for targeted experiment and temperature measurement experiment.The remaining mice were divided into three groups,which were acute ischemic stroke model group,platelet transfusion alone group after model establishment,and nanobubble group after model establishment.Some of them were used to observe the changes in lesion area and edema at time points 1 day,3 days,and 7days after the establishment of the stroke model by magnetic resonance scanning imaging（structural phase）,some were used to reflect the improvement of neurological function by the modified neurobehavioral score（mNSS score）,and some were used to evaluate the number of new blood vessels and neuronal changes in and around the ischemic area after the occurrence of stroke by immunohistochemistry to detect the therapeutic effect.The rest mice were fenestrated at the skull level by multispectral imaging equipment to monitor the vascular status in real time to evaluate their therapeutic effects on acute attacks of acute ischemic stroke mice using a multispectral imaging optical imaging system.Results:1)Nanobubbles with diameters ranging from 45nm--75nm were observed by confocal imaging,and a targeted cooling nanobubble system was successfully constructed.2)By near-infrared small animal in vivo imager,a significant increase in fluorescence signal was detected in the ischemic area of mice with acute ischemic stroke,and platelet nanobubbles with fluorescence signal were able to target to the lesion area.Photosensitive infrared thermometry was used to collect the temperature of mice at different time points of 0.5 h,4 h,6 h and 12 h.The temperature of stroke model group increased,the temperature of simple platelet group did not change significantly,the temperature of nanobubbles treatment group decreased significantly(T_0.5h=30.8133±0.58141℃,T_4h=30.3633±0.57187℃,T_6h=29.2767±0.16042℃,T_6h=29.2767±0.16042℃,T_12h=29.1133±0.25325℃).Bra in temperature showed a decreasing trend over time in mice（P=0.007）,and there was a significant difference in brain temperature over time among the three groups（P<0.001）.3)Ischemic animal models were made by photochemical embolization.4)The therapeutic effect of nanobubbles was reflected by magnetic resonance imaging.(S_day1:57.6333±8.22456cm²,S_day3:63.1333±9.12707,S_day7:66.5333±4.26654,P=0.252).The lesion area was reduced in the platelet nanobubble treatment group(S_day1:40.2667±9.42992,S_day3:16.9333±1.49778,S_day7:0.4333±0.32146,P=0.005),and there was a significant difference in infarct size over time between the two groups（P<0.001）,reflecting the presence of a therapeutic effect of microbubbles.The neurological deficit behavior of stroke mice was scored on days1,3,and 7,and the mNSS score increased with time in the stroke model group(mNSS_day1=16±1,mNSS_day3=16.33±0.577,mNSS_day7=18±1,P=0.042);the score did not change significantly with time in the stroke model plus platelet membrane bubble group(mNSS_day1=14.33±0.577,mNSS_day3=13.67±1.155,mNSS_day7=13.67±0.577,P=0.529);the score decreased with time in the stroke model plus platelet microbubble treatment group(mNSS_day1=14.33±0.577,mNSS_day3=11.67±0.577,mNSS_day7=9±1,P=0.001).Immunohistochemistry showed a significant therapeutic effect.The multispectral imaging system reflected that the signal of mice in the acute treatment group was significantly different from that in the model group.Conclusion:Through the preparation of targeted cooling nanobubbles,it can reach the lesion area for cooling and produce a therapeutic effect on ischemic stroke in mice;by observing and measuring the local cortical signal of the multispectral imaging system,it can timely and accurately evaluate the therapeutic effect of nanobubbles on ischemic stroke and preliminarily realize the integration of diagnosis and treatment in the acute phase of ischemic stroke in mice.