Experimental Studies Of MRI-Guided Focused Ultrasound Mediated Smart Albumin-Sonosensitizer Nanoassemblies For Sonodynamic Therapy Of Glioma

Objective This study is to design and engineer albumin-nanosensitizer that integrate MRI imaging and therapy functionalities into a single nano-platform for MRI-guided sonodynamic therapy of glioma.Methods?1?HSA-Ce6 were synthesized using human serum albumin?HSA?and chlorin e6?Ce6?.The prepared HSA-Ce6 served as chelating agents to capture Mn²⁺for MRI imaging by forming stable chelates HSA-Ce6-Mn.The average diameter and size distribution of HSA-Ce6-Mn were determined by dynamic light scattering.The morphologic examination was further performed by transmission electron microscope images.The absorption spectra of free Ce6 and HSA-Ce6-Mn were determined using a UV-vis spectrometer and fluorescence spectrometer.To explore the possibility of using HSA-Ce6-Mn as a T1-weighted MRI contrast agent,HSA-Ce6-Mn solutions with different Mn²⁺concentrations were scanned under a 3.0 T clinical MR scanner at room temperature to calculate the final r1 value for HSA-Ce6-Mn.DCFH-DA,a highly sensitive molecular probe for oxygen species?ROS?,was employed to measure the ROS level of HSA-Ce6-Mn during ultrasound irradiation.?2?Fluorescence detection was implemented to evaluating the intracellular uptake of HSA-Ce6-Mn and Ce6,and to estimating the level of ROS in cells after treated by combined sonodynamic therapy and HSA-Ce6-Mn or Ce6.Then,the cell activity of each groups?HSA-Ce6-Mn+US group,HSA-Ce6-Mn group,Ce6+US group Ce6 group,US group and Control group?were determined by standard MTT and Calcein AM and propidiumiodide?PI?co-staining.?3?BALB/C athymic nude mice glioma models were established to acquire fluorescence images and MRI images at different time points.The half-life in blood and 24h tissue biodistribution were determined by ICP-AES.?4?Mice bearing U87 xenografted tumors were randomly divided into four different groups?HSA-Ce6-Mn+HIFU group,HSA-Ce6-Mn group,HIFU group and Saline group?to study the therapy effect of synthesized HSA-Ce6-Mn combined with HIFU.The mice were sacrificed by standard decapitation,and the tumors and major organs were harvested,followed by staining with hematoxylin-eosin?HE?dyes.?5?SPSS19.0 statistical software was used for data analysis.All values were expressed as?Mean±SD?.P<0.05 was considered statistically significant.Results?1?The dynamic light scattering measurement showed that the average hydrodynamic diameter of HSA-Ce6-Mn was 100±2.4nm.Meanwhile,the transmission electron microscopy image showed the average diameter of HSA-Ce6-Mn with 76±3.2nm,which demonstrated a well-defined spherical morphology and homogeneous distribution.There was no difference between the fluorescence spectra of HSA-Ce6-Mn and Ce6 while the UV-vis spectrum of HSA-Ce6-Mn was similar to that of Ce6,but the absorption peak of HSA-Ce6-Mn?0.30?×10⁵ a.u.??were red-shifted at 660nm?2.54?×10⁵ a.u.???P<0.01?.The r1 value for HSA-Ce6-Mn was measured to be 12.2mM^-1S^-1.The fluorescence assay demonstrated that the level of ROS increase with the irradiation time and intensity of ultrasound,which indicating that the ROS release rate induced by HSA-Ce6-Mn is dependent on ultrasound irradiation.?2?The intracellular fluorescence analysis presented that the uptake of HSA-Ce6-Mn?2.97±0.34?was statistical increased than that of Ce6?1.0±0.16??P<0.01?.And the ROS in HSA-Ce6-Mn+US group?2.84±0.07?showed a significant higher level than that in Ce6+US group?1.00±0.20??P<0.01?.It was found that single sensitizer or ultrasound treatment could only induce partial cell death at the current conditions.In marked contrast,the HSA-Ce6-Mn+US group were found to be highly effective in destructing cancer cells and demonstrated a concentration-dependent.The cell viability gradually decreased to?46.12±2.20?%in HSA-Ce6-Mn+US group while?65.01±3.05?%in Ce6+US group at Ce6 concentration of 20?g/L.The live and dead double cell staining results intuitively evidenced the synergistic effects of combined HSA-Ce6-Mn and US.While the dead cells were predominant in HSA-Ce6-Mn+US group,the proportion of live cells prevailed over that of dead cells for the single sensitizer or ultrasound treatment group.?3?Free Ce6 showed a relative weak fluorescence in glioma tissue.After 24 h post-injection,almost no fluorescence signal in the whole body was observed.On the contrary,after i.v.injection of HSA-Ce6-Mn,the fluorescence signal of glioma site could be distinguished from that of surrounding normal tissue at 3 h post-injection,and reached a peak at 24h.The T1 signals of tumor on mice strengthened with the increase of time interval,and reached a peak after 24h post-injection of HSA-Ce6-Mn at orthotopic glioma and subcutaneous tumor.The half-life in blood was 0.98h and the 24h tissue biodistribution showed high tumor retention.?4?For tumors after HSA-Ce6-Mn treatment,their growth was not inhibited.The tumor growth in the HIFU treated group was only partially inhibited.In marked contrast,the HSA-Ce6-Mn+HIFU treatment could greatly inhibit the growth of tumors after treatments.HSA-Ce6-Mn+HIFU group showed a great significant difference with the other groups?P<0.01?.Moreover,neither obvious body weight loss nor noticeable abnormality was found in each group.A significant longer survival period was also observed for group treated with HSA-Ce6-Mn+HIFU than that of the other groups?P<0.01?.In terms of the Saline group and HSA-Ce6-Mn group,the cancer cells still retained their normal morphology with apparent membrane and nuclear structure.The HIFU group showed a section of pyknotic cells or cytolysis,demonstrating part of cancer cells damaged by the HIFU.The HSA-Ce6-Mn+HIFU group indicated that a large number of cancer cells were severely apoptoticed.Moreover,the HE staining images of major organs collected from each group showed that neither obvious damage nor inflammation was observed compared to the Saline group.Conclusion This study has designed and constructed a Ce6-based nanosensitizer,which has a simple structure and shows good biosecurity.By chelating Mn²⁺,the effective integration of the nanosensitizer and imaging function in the nanosystem can be realized.The turmor-targeted nanosensitizer has synergistic effect of imaging guidance and sonodynamic therapy,providing new ideas and strategies for the clinical treatment of cancer.