Experimental Study Of Different Delivery Methods Of Bifidobacterium Combined With Lipid Nanoparticles On HIFU Treatment Efficiency

Targeted treatment of malignant tumors is the key to achieving precise,safe and effective treatment in non-surgical treatment.At present,the targeted therapy usually combine the carrier and the drug by chemical method,which leads to instability and poor targeting in the body.Focused Ultrasound Ablation Surgery(FUAS)or High Intensity Focused Ultrasound(HIFU)is a method of physical therapy for tumors,and is a non-invasive treatment method for solid tumors.However,during the treatment,as the propagation distance of ultrasonic waves increases,the energy attenuation is severe,which leads to a reduction in the treatment efficiency when treating large or deep tumors,and complications and side effects are likely to occur.To this end,research on HIFU treatment synergists came into being.The previous research of the research group taken Bifidobacterium with anaerobic preference as a carrier for targeting tumors,combined with HIFU synergistic substances and delivered them into the tumor,which made certain progress,starting a new attempt to biologically deliver HIFU synergistic substances.The existing problem is that the electrotransformation method has not succeeded in transferring the synergist into Bifidobacterium;the connection success rate of the chemical bonding method is low and can only be used for the method of ex vivo connection and delivery;although the cation connection method has a higher connection rate,it is less stable in vivo,and some studies believe that cations have the disadvantages of biological toxicity and side effects.In addition,due to the complex tumor microenvironment,including factors such as hypoxia and low p H,the efficiency of the bifidobacterium-loaded HIFU synergistic substance into the tumor is reduced.As a result,the difficulty of targeted therapy for tumors is increased,so it is particularly important to choose a suitable delivery method.To this end,this study attempted to connect a bifidobacterium-streptavidinated bifidobacteria antibody-biotinylated lipid nanoparticles coated with perfluorohexane to prepared a new type of biological targeting HIFU synergist,and discusses the retention of nanoparticles in tumors,HIFU synergism and mechanism in terms of different delivery methods of the synergist.Purpose:To explore the synergistic effect of bifidobacterium combined with lipid nanoparticles on the different delivery ways of HIFU in the treatment of solid tumors.Method:1.PFH/BL-NPs were prepared,and the morphology and distribution were observed by TEM and CLSM.The particle size,surface potential distribution and PFH encapsulation rate were measured.2.Verification of tumor targeting of Bifidobacterium longum(B.longum): 20 tumor rabbits were randomly divided into B.longum group and PBS group,10 in each group.Observe the growth of the colonies in the Petri dish after homogenizing the tumors and important organs of the two groups.3.To verify the biological safety of Streptavidinated Bifidobacterium longum antibody(SBA)+PFH/BL-NPs complex:(1)Normal rabbits were injected with SBA+PFH/BL-NPs complex,and blood samples were collected before injection and1 d,3d,7d,and 14 d after injection for biochemical analysis.(2)After SBA+PFH/BL-NPs complex and human umbilical vein endothelial cells(HUVECs)were incubated together,the cell survival rate was detected by CCK-8 assay.4.In vitro connection of B.longum and PFH/BL-NPs:(1)CLSM observation connection: divide the experiment into a control group and an experimental group,both groups add equal amounts of B.longum,the control group directly add PFH/BL-NPs,and the experimental group add equal amounts of SBA+PFH/BL-NPs Compound,and then use CLSM to observe the connection.(2)Flow cytometry connection rate: divide the experiment into B.longum group,B.longum+PFH/BL-NPs group,B.longum+SBA+PFH/BL-NPs group,and the connection rate of each group were tested by flow cytometry.5.Effects of different delivery methods on tumor targeting and retention of PFH/BL-NPs in vivo:(1)Small animal in vivo fluorescence experiment: 42 VX2 tumor rabbits were randomly divided into(B.longum+SBA+PFH/BL-NPs)group and B.longum+(SBA+PFH/BL-NPs)group,21 rabbits in each group.(B.longum+SBA+PFH/BL-NPs)group of tumor rabbits directly delivered B.longum+SBA+PFH/BL-NPs complex,Tumor rabbits in the B.longum+(SBA+PFH/BL-NPs)group were injected with B.longum first and 7 days later SBA+PFH/BL-NPs complex were injected.The rabbits were sacrificed at different time points(before injection,2h,12 h,24h,48 h,72h,96h),the tumors and important organs were taken out,and the small animal biopsies were taken immediately to measure the fluorescence intensity of tumors and important organs.(2)Frozen tumor sections: 18 VX2 tumor rabbits were randomly divided into PFH/BL-NPs group,(B.longum+SBA+PFH/BL-NPs)group,B.longum+(SBA+PFH/BL-NPs)group,6 in each group.48 h after injection,the tumors were prepared into frozen sections and stained with DAPI.Finally,CLSM was used to observe the retention of PFH / BL-NPs in the tumor.6.Experiments on synergistic HIFU with different delivery methods: 50 VX2 tumor rabbits were randomly divided into 5 groups,set up PBS group,B.longum group,PFH/BL-NPs group,(B.longum+SBA+PFH/BL-NPs)group and B.longum+(SBA+PFH/BL-NPs)group,10 animals in each group.HIFU irradiation was performed on the VX2 tumor rabbit tumor tissue under the same conditions with a HIFU dose of 250 w 5s,and the gray scale change of the tumor tissue immediately after ablation was observed.24 hours later,the volume of coagulative necrosis of the tumor tissue was calculated and compared,and the therapeutic effect was evaluated comprehensively by tumor H&E tissue immunostaining and tumor volume and weight changes of the tumor rabbit after HIFU treatment.7.The effect on tumor HIF-1 ? factor expression of B.longum: 18 VX2tumor-bearing rabbits were randomly divided into two groups,9 in each group.Each rabbit in the B.longum group was injected with 2ml of B.longum,and each rabbit in the PBS group was injected with 2ml of PBS.The tumors were dissected and sectioned on days 1,3,and 7 after injection,then immunohistochemical staining was performed to detect the expression of HIF-1? therein.Results:1.The appearance of PFH-encapsulated biotinylated lipid nanoparticles(PFH/BL-NPs)was milky white suspension,which was uniformly spherical under CLSM and TEM.The particle size distribution of PFH/BL-NPs is 364.4±53.49nm(PDI=0.113),and the surface potential is-23.1±5.46 m V.The PFH encapsulation rate is 44.45±1.27%.2.Tumor targeting of B.longum: B.longum colonies only appeared in the tumor culture dish of the B.longum group,indicating that B.longum colonized only in the tumor.3.Biological safety of SBA + PFH / BL-NPs complex:(1)Blood routine and blood biochemical indexes had no significant changes before and after injection(P>0.05).(2)The cell survival rate of CCK-8 detection method is above 85%,and no obvious cytotoxicity is observed.4.The biological safety of SBA + PFH / BL-NPs complex:(1)Blood routine and blood biochemical indicators showed no significant changes before and after injection(P> 0.05).(2)The cell survival rate of CCK-8 detection method is above85%,and no obvious cytotoxicity is observed.5.In vivo PFH/BL-NPs tumor targeting and tumor retention experiments:(1)Small animal in vivo fluorescence experiments showed that red fluorescence can be observed in the tumor area of both groups of VX2 tumor rabbits,and the two groups reached the highest fluorescence intensity at 48 h.However,the red fluorescence intensity of the tumor area in the B.longum+(SBA+PFH/BL-NPs)group was greater than that in the(B.longum+SBA+PFH/BL-NPs)group(P<0.05).(2)The results of frozen tumor sections showed that compared with the first two groups,more PFH/BL-NPs entered the tumor in the B.longum+(SBA+PFH/BL-NPs)group.6.B.longum combined with PFH/BL-NPs different delivery methods to enhance the effect of HIFU: In the B.longum+(SBA+PFH/BL-NPs)group,the coagulative necrosis volume,gray value change and tumor shrinkage before and after irradiation were the largest in all groups,and the difference was statistically significant(P<0.05).H&E tissue immunity results show that a large amount of coagulative necrosis in the tumor has a clear boundary with normal tissue,but no significant changes in important organs.While the coagulative necrosis of the tumor was most obvious in the B.longum+(SBA+PFH/BL-NPs)group.There was no significant change in the weight of tumor rabbits before and after treatment.7.The effect on tumor HIF-1 ? factor expression of B.longum: 3days after injection,the expression of HIF-1? in the B.longum group began to be lower than that in the PBS group,and was statistically significant(p<0.05).7days after injection,the expression of HIF-1 ? in the tumor of the B.longum group was significantly lower than that of the PBS group(p<0.05).Conclusion:The PFH/BL-NPs prepared successfully,then PFH/BL-NPs and B.longum can be effectively connected in vitro by biotin-avidin connection method.Delivery of B.longum first and then(SBA+PFH/BL-NPs)complexes is more effective than direct delivery(B.longum+SBA+PFH/BL-NPs)complexes.It can increase the retention of PFH/BL-NPs in tumors.When HIFU ablated VX2 tumors,the separate delivery method can enhance the HIFU treatment more efficiently.B.longum can reduce the expression of HIF-1? in the tumor,and improve the tumor hypoxic microenvironment may be the reason for this result.