| Part Ⅰ Construction and property evaluation of anti-miR-155 delivery system Objective: To achieve efficient microRNAs therapy for heart transplantation,it is necessary to develop a stable,safe and targeted delivery platform.In this study,we intend to construct an anti-miR-155 delivery system containing antagomir-155 encapsulated lipid nanoparticles(anti-miR-155@LNPs)and gas vesicles(GVs),and characterize its physicochemical properties,to lay a foundation for the targeted delivery of anti-miR-155.Methods: GVs were extracted,isolated and purified from Halobacterium sp.NRC-1 and anti-miR-155@LNPs were prepared by ethanol dilution method.The properties of GVs and anti-miR-155@LNPs were characterized by transmission electron microscopy(TEM),Zeta potential and particle size analyzer,enzyme labeling analyzer and visible nanoparticle analyzer.Results:(1)The GVs were nanostructures with surface negative charge and great dispersion.The mean particle size of GVs was(174.8 ± 2.2)nm,the polydispersion index was(0.03 ±0.02),and the average potential was(-24.47 ± 3.26)m V.(2)After being stored in PBS solution for 180 days,there was no significant difference in particle size,potential and concentration compared with day 0,and no significant change in particle size after being incubated with serum for 2 h,validating their fundamental physical stability.(3)Both LNPs and anti-miR-155@LNPs were nanoscale,with average particle sizes of(125.94 ± 2.18)nm and(139.07 ± 5.03)nm,respectively,and average potential of(26.08 ± 2.13)m V and(-0.39± 0.55)m V,respectively.(4)LNPs and anti-miR-155@LNPs showed great stability,and there was no significant difference in particle size after 28 days of storage.(5)anti-miR-155@LNPs were p H sensitive and had a high encapsulation rate of 96.57% for anti-miR-155.Conclusion: anti-miR-155@LNPs and GVs were successfully prepared,which laid a good foundation for targeted therapy mediated by low-intensity pulsed ultrasound.Part Ⅱ Optimization of targeted delivery parameters for low-intensity pulsed ultrasoundObjective: Targeted ultrasonic delivery of microRNAs needs to not only ensure the safety of cardiac tissue,but also induce cavitation with ultrasonic energy to achieve targeting.Therefore,it is necessary to induce bubble destruction and trigger cavitation at relatively low ultrasonic intensity to minimize potential damage.This part of the study aims to verify that GVs cavitation induced by low-intensity pulsed ultrasound can increase vascular wall permeability,optimize ultrasound parameters,and evaluate the safety of targeted delivery of low-intensity pulsed ultrasound.Methods: To construct a mice model of heterotopic heart transplantation.The sound pressure and irradiation time of low-intensity pulsed ultrasound were set by gradient.The degree of blue staining of mouse heart tissue under different ultrasonic parameters was evaluated by Evans blue staining,so as to screen the optimal parameters.The safety of low intensity pulsed ultrasound was evaluated by blood routine,blood biochemistry and TUNEL staining.Furthermore,the safety of the antimiR-155 delivery system was evaluated by CCK8,blood routine,blood biochemistry,immunogenicity,and HE staining.Results:(1)The model of heterotopic heart transplantation was established successfully.The average survival time of mice in the rejection group was(6.0 ± 1.2)days,while the survival time of mice in the control group was more than 100 days.(2)The optimal lowintensity pulsed ultrasound parameters were selected as follows: when the ultrasonic frequency was 1 MHz,the duty ratio was 20%,and the ultrasonic sound pressure was 360 k Pa,the content of Evans blue in mouse heart tissue was significantly different from that in other groups.(3)Determine the safety of the antimiR-155 delivery system: In the GVs + LNPs + US group,the transplanted blood cells were classified and counted,and there were no obvious abnormalities in liver and kidney function,and no obvious tissue damage was observed in all important organs during HE staining.Conclusion: Low-intensity pulsed ultrasound can induce the destruction of GVs at the frequency of 1 MHz,duty ratio of 20% and ultrasonic sound pressure of 360 k Pa,which can enhance the permeability of cardiac vessels under the premise of ensuring safety.Targeted delivery of liposomes by low-intensity pulsed ultrasound can safely and efficiently open the vascular barrier.Part Ⅲ Evaluation of the therapeutic effect for heart transplant rejectionObjective: To evaluate the therapeutic effect of anti-miR-155 targeted delivery mediated by low-intensity pulse ultrasound on heart transplant rejection.Methods: A mice model of heterotopic heart transplantation was constructed,and the efficiency of targeted delivery of LNPs to the transplanted heart by GVs cavitation induced by low-intensity pulsed ultrasound was evaluated by small animal imaging.Heart transplanted mice were randomly divided into LNPs,anti-miR-155,anti-miR-155@LNPs,anti-miR-155@LNPs+GVs+US groups.7 days after intervention,the expression level of miR-155 in transplanted hearts was evaluated by RT-PCR and fluorescence in situ hybridization.RT-PCR and Western-blot were used to evaluate the expression levels of inflammatory factors and SOCS1.Immunofluorescence and RT-PCR were used to detect the expression of immune cells and their surface markers.Moreover,HE staining and survival period were evaluated.Results:(1)Low-intensity pulsed ultrasound induced GVs cavitation,which could achieve the targeting of LNPs transplant heart.In the LNPs+GVs+US group,the fluorescence intensity of LNPs at 1 h and 24 h was 3.6 and 2.6 times that of the control group,respectively.(2)The expression of miR-155 in anti-miR-155@LNPs+GVs+US group was significantly lower than that in the other three groups,and the expression of SOCS1 was significantly increased.(3)The expression of IL-6,IFN-γ and TNF-α decreased significantly in anti-miR-155@LNPs+GVs+ US group.(4)The number of leukocytes,T cells and macrophages decreased significantly in anti-miR-155@LNPs+GVs+US group,and macrophages were M2-type polarized.(5)The rejection grading of anti-miR-155@LNPs+GVs+US group was significantly reduced,and the survival time was significantly prolonged.Conclusion: Low-intensity pulsed ultrasound mediated anti-miR-155@LNPs can effectively deliver anti-miR-155 to the transplanted heart,reduce miR-155 expression,increase SOCS1 expression,reduce inflammation and down-regulate immune cells,promote macrophage M2 differentiation,thereby significantly reduced the degree of rejection and extend the time of survival of transplantation. |
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