Expermental Study On The Retention Of HIFU Synergistic Substances By Bifidobacterium In The Target Area

BackgroundAs a new non-invasive treatment technology,high-intensity focused ultrasound can treat a variety of diseases including benign and malignant tumors,and its efficacy has been recognized by the majority of patients.The greater the distance the ultrasonic wave travels,the more severe the energy attenuation.When HIFU treats large and deep lesions,the effect is not very satisfactory.Researchers have also been exploring ways to make HIFU safe and efficient for clinical use.The emergence of synergists has brought hope to improve the efficacy and safety of HIFU.Studies have shown that iodized oil,microbubbles,hydroxyapatite,and liquid fluorocarbon-coated nanoparticles can enhance the ablation effect of HIFU.However,these substances have a short residence time in the body and are not highly targeted,and these substances are present in the tumor and its organs.When irradiated with HIFU,normal tissue surrounding the target area is prone to serious complications due to excessive deposition of energy.Therefore,the exploration of HIFU targeting synergists or methods withlong residence time and only to tumor tissues has attracted industry attention.Bifidobacterium are probiotics commonly found in the gut and have proven to be safe and non-toxic.The facultative anaerobic properties of bifidobacteria make it easy to concentrate and multiply in the anaerobic zone of the tumor after entering the blood,showing tumor targeting.Because bifidobacterium tend to be hypoxic,they are targeted to any tumor or inflammatory tissue with a hypoxic environment.In this study,the habit of a probiotic,a bifidobacterium,which is highly safe and easily accessible to the hypoxic zone of the tumor,was used as a carrier of HIFU synergist to try to establish a novel HIFU biotargeting synergist.Observing the retention of HIFU potentiating substances by the bifidobacterium in the tumor target area laid the foundation for further research in this field.ObjectiveThe retention effect of proliferating Bifidobacterium longum ATCC15707 on CL-NPs(HIFU synergist)in tumor tissues was explored.Method1.Bifidobacterium Longum(BF)was cultured,and its morphology was observed by Gram staining,and the surface zeta potential was measured by a Malvern detector.2.Preparation of perfluorohexane-coated cationic lipid nanoparticles(CL-NPs)and observation of their morphology and dispersion.The particle size and surface potential distribution of the Malvern particle size and potential analyzer were measured to observe the effect of temperature increase on the phase transition of CL-NPs.3.In vitro ligation of bifidobacterium with CL-NPs: The experiments were divided into four groups,MDA-MB-231 cells were seeded in designated 1-3 culture dishes,and cells were not seeded in the fourth group.After all the cells were attached,CL-NPs were added to a group of cell culture dishes,and an equal amount of Bifidobacterium was added to the2-4 cell culture dishes.Subsequently,an equal amount of CL-NPs was added to the 3-4th set of cell culture dishes.After 10 min,DAPI fluorescent dyes were added to the culture dishes of groups 1-3,and after 25 min,the connections of the groups were observed under a confocal microscope.Flow cytometry was used to detect the connectivity of Bifidobacterium to CL-NPs in Group.4.Retention experiments of Bifidobacterium on CL-NPs in tumor tissues: 76 tumor-bearing mice were randomly divided into group A and group B(38 in each group).Group A(BF + CL-NP group)mice were injected with 200 ?l of sterile PBS via the tail vein,and Group B(CL-NP group)mice were injected with 200 ?l of Bifidobacterium.After 7 days,two groups of mice were injected with 200 ?L of fluorescently labeled CL-NPs via the tail vein.At 6 time points(2h,6h,24 h,48h,72 h and 96h)after injection of CL-NPs,the in vivo fluoroscopy of small animals was performed to determine the fluorescence intensity of the tumor.48 hours after injection of CL-NPs,5 tumor-bearing mice were randomly selected from the two groups,and the organs(tumor,heart,kidney,spleen,lung and liver)of the tumor-bearing mice were sacrificed.The fluorescence intensity of each isolated organ was detected and sent to a frozen section to observe the distribution of CL-NPs in the tumor at different time points.Finally,each of the excised organ tissues taken out was fixed with formaldehyde and sent for Gram stain to prove the targeting of bifidobacterium.5.Bifidobacterium combined with CL-NPs to enhance the efficacy of HIFU: 40 tumor-bearing mice were randomly divided into 4 groups(10 in each group),and different substances were injected into the tail vein 2times(two days apart).Group A(PBS group)was injected with PBS twice.Group B(Bifidobacterium group)was injected with Bifidobacterium and PBS,respectively,and Group C(CL-NP group)was injected with PBS and CL-NPs,respectively.Group B(Bifidobacterium + CL-NP group)was injected with Bifidobacterium and CL-NPs,respectively.At 48 hours after the completion of the second injection,HIFU irradiation was performed on tumor tissues of tumor-bearing mice,and the gray scale changes of tumor tissues were analyzed.Histological examination was performed 1 day after HIFU irradiation,and the coagulative necrotic volume and energy efficiency factor(EEF)of tumor tissues were measured and compared.Result1.Bifidobacterium longum ATCC15707 has a blue-violet long rod shape with a surface potential of-29 mv.2.The appearance of cationic lipid nano-spheres(CL-NPs)coated with perfluorohexane was a milky white suspension,which was observed to be uniformly distributed under a microscope.The particle size distribution of CL-NPs is 280±60 nm with a positive potential of 38 mV on the surface.At 4 ?,no significant change in the particle size of the nanospheres was observed within 72 h.The CL-NPs remained stable at37?,40?,45?,and 50?.When the temperature rose to 55?,a small amount of CL-NPs began to increase significantly.3.In vitro attachment of Bifidobacterium to CL-NPs: CL-NPs can adhere not only to the surface of negatively charged Bifidobacterium but also to the surface of cell membranes by electrostatic adsorption.When free Bifidobacterium are present around the cell membrane,the number of CL-NPs adhering to the surface of the Bifidobacterium is significantly greater than the number of CL-NPs attached to the cell membrane.The flow results showed that the effective connection rate of Bifidobacterium to CL-NPs was 99.9% under the appropriate ratio.4.Distribution of CL-NPs in tumor-bearing mice: red fluorescence was observed in the tumor areas of both tumor-bearing mice,but the red fluorescence intensity of the tumor area in the CL-NP + BF group wasgreater than that in the CL-NP group(P <0.05),the metabolic rate of fluorescence in the CL-NP + BF group was smaller than that in the CL-NP group(P < 0.05).Gram staining showed that a large number of Bifidobacteria were scattered in the tumor tissues of the BF + CL-NP group,and no bifidobacterium were found in the tumor tissues of the CL-NP group.5.Synergistic effect of Bifidobacterium combined with CL-NPs on HIFU: coagulative necrotic volume of tumor in group D(HIFU + BF + CL-NP)tumor and gray scale change before and after irradiation were the largest in all groups.The EEF value is the smallest.The difference was statistically significant(* P <0.05).And the tumor tissue necrosis of group D tumor-bearing mice was the most thorough.