| The purpose of this study is to develop an ultrasound-responsive biomimetic drug carrier with high drug loading rate to achieve the controlled release of chemotherapy drugs.At the same time,the cavitation effect of ultrasound is used to destroy tumor blood vessels and deliver the drug carrier to tumor cells,release drugs at tumor sites and produce reactive oxygen species（ROS）to synergistically inhibit tumors,and achieve multiple targeted therapy of tumors against tumor blood vessels and tumor cells,so as to enhance tumor treatment effects and reduce the toxic and side effects of chemotherapy drugs on the whole body.This study mainly includes the following two parts:Part 1:Preparation and characterization of F-MSN-DOX@RBC nanoparticles Objective:To prepare F-MSN-DOX@RBC nanoparticles and evaluate their properties.Methods:（1）MSN with open cubic channels were synthesized by soft template method,and then modified by PFDTS to synthesize F-MSN.The BET specific surface area and pore size analyzer detects the MSN specific surface area and pore size,and the thermogravimetric method detects the modification of the fluorocarbon chain.（2）The DOX was loaded into F-MSN by solvent evaporation method,and the content of DOX in F-MSN-DOX was detected by the multi-functional microplate reader to calculate the drug encapsulation efficiency and drug loading efficiency.（3）Red blood cell membrane was extracted from blood of C57BL/6 mice,and red cell membrane vesicles with uniform size were prepared by micro liposome extruder.（4）The red blood cell membrane vesicles were wrapped on the surface of F-MSN-DOX by ultrasonic crusher to prepare F-MSN-DOX@RBC nanoparticles,and flow cytometry（FCM）was used to quantify the coating of red blood cell membrane vesicles on F-MSN,SDS-PAGE was used to detect the surface protein expression of red blood cell membrane in different stages.（5）The morphology,particle size,particle concentration and surface potential of the nanoparticles were analyzed by TEM,SEM and particle size potential analyzer.（6）The DOX release behavior was simulated in vitro,and the release of DOX in different groups was detected by multifunctional fluorescent microplate reader.（7）F-MSN-DOX and F-MSN-DOX@RBC with the same DOX concentration were incubated with RM-1 cells or RAW264.7 cells for different times,respectively,and the phagocytosis of nanoparticles was quantified by FCM.Results:（1）Analysis of nitrogen adsorption and desorption isotherm data shows that the BET specific surface area,total pore volume and BJH model pore diameter of MSN were 798.63 m2/g,0.71 cm3/g and 3 nm,respectively.（2）Thermogravimetric analysis measured that the weight loss of MSN and F-MSN was 8.50%and 38.63%during the heating process from room temperature to 800℃,respectively,indicating that the fluorocarbon content in F-MSN was about 30%.（3）The DEE and DLE of F-MSN-DOX were determined by indirect method,and they were 98.54±1.54%and49.63±0.39%,respectively.（4）FCM showed that the efficiency of F-MSN-DOX encapsulated in red blood cell membrane vesicles was 84.57±3.76%.（5）The synthesized MSN,F-MSN,F-MSN-DOX and F-MSN-DOX@RBC had stable properties,with particle sizes of 153.7±86.2 nm、171.1±53.9 nm、211.2±72.4 nm and232.6±78.8 nm,particle concentration of 1.62×1010±2.98×108particles/m L、1.44×1010±2.42×108particles/m L、8.37×109±1.16×108particles/m L and 8.19×109±2.30×108particles/m L,and surface potentials of-17.97±0.60 m V、-19.03±0.62 m V、-21.70±3.18 m V and-35.57±0.74 m V,respectively.（6）TEM showed that various nanoparticles were uniform in size and well dispersed,and MSN has regularly arranged grid-like mesoporous structure;SEM showed that the surface of the nanoparticles was uneven and porous,the surface was smooth after being coated by the red blood cell membrane,and the distribution of nanoparticles was more uniform.（7）The highest drug release rate in DOX group reached 85.58±2.34%in 8 hours,and the highest drug release rates in the MSN-DOX group,F-MSN-DOX group and F-MSN-DOX@RBC group were 43.83±1.42%and 15.68±1.01%and 13.71±1.31%,respectively.（8）There was no significant difference in the phagocytosis rate of F-MSN-DOX and F-MSN-DOX@RBC by RM-1 cells at 2h,6h,12h,and 24h（P>0.05）,while the phagocytosis rate of F-MSN-DOX@RBC by RAW264.7 cells at each time point was lower than the phagocytosis rate of F-MSN-DOX（P<0.05）.Conclusion:In this study,F-MSN with good superhydrophobic properties was successfully prepared,and F-MSN-DOX@RBC with uniform size,stable properties and high drug loading rate was constructed,which effectively reduced the leakage of DOX and the phagocytosis of nanoparticles by macrophages.Part 2: Evaluation the anti-tumor effect of F-MSN-DOX@RBC with ultrasoundObjective: To observe the biodistribution of F-MSN-DOX and F-MSN-DOX@RBC in tumor-bearing mice,and evaluate the therapeutic effect and toxicity of different treatment methods on subcutaneous prostate cancer in mice.Methods:（1）RM-1 cells were cultured and collected to establish subcutaneous prostate cancer model in C57BL/6 mice.（2）F-MSN-DOX and F-MSN-DOX@RBC with the same DOX concentration were injected into tail vein,and the biodistribution of the two nanoparticles in tumor-bearing mice was observed by in vivo imaging system at 6h、 12 h and 24 h after injection.（3）PBS,F-MSN-DOX,F-MSN@RBC,and F-MSN-DOX@RBC were injected into tail vein,in which the F-MSN@RBC group was given ultrasound,and the F-MSN-DOX@RBC group was divided into the ultrasound group and the non-ultrasound group.The mouse body weight and tumor volume changes were monitored every other day,and the main organs（heart,liver,spleen,lung,kidney）and tumor tissues were taken on the 15 th day of treatment.（4）Paraformaldehyde-fixed specimens of tumor tissues were stained with Tunel fluorescence staining and Ki67 immunofluorescence staining to observe the apoptosis and proliferation of tumor tissues,CD31 immunofluorescence staining was used to observe the blood vessels of tumor tissues,and frozen specimens of tumor tissues were stained with DHE to observe ROS in the tissues,and fixed specimens of main organs were stained with H&E to evaluate the toxicity of different treatments on mice.Results:（1）The subcutaneous prostate cancer model of C57BL/6 mice was successfully constructed.（2）The results of biodistribution experiment showed that the F-MSN-DOX group was mainly distributed in the lungs at 6 h and 12 h,which was significantly higher than that in the F-MSN-DOX@RBC group（P < 0.05）.At 24 h,the accumulation of tumor tissue in the F-MSN-DOX@RBC group was significantly higher than that in the F-MSN-DOX group（P < 0.05）,and the uptake of F-MSN-DOX@RBC by the spleen at 6 h,12 h,and 24 h was significantly lower than that of F-MSN-DOX Group（P < 0.05）.（3）The tumor mass of the ultrasound combined with F-MSN-DOX@RBC group（experimental group）was 212.94±95.21 mg,and the tumor mass of the ultrasound combined with F-MSN@RBC group（ultrasound control group）and the F-MSN-DOX@RBC group（envelope control group）were 409.16±32.27 mg and 428.11±95.18 mg,respectively,the tumor mass of the ultrasound combined with F-MSN-DOX@RBC group was significantly lower than that of the ultrasound combined with F-MSN@RBC group and the F-MSN-DOX@RBC group（P < 0.05）.On the 15 th day,the relative tumor volume change in the experimental group was 3.60±1.21 times,and that of the ultrasound control group and the envelope control group were 5.75±0.64 times and 5.62±0.67 times,respectively,the tumor volume growth of the ultrasound combined with F-MSN-DOX@RBC group was significantly lower than that of the ultrasound combined with F-MSN@RBC group and the F-MSN-DOX@RBC group（P < 0.05）.The tumor inhibition rates of the experimental group,the ultrasound control group and the envelope control group were 71.58±9.54%,54.60±5.03%,and 55.62±5.30%,respectively,the ultrasound combined with F-MSN-DOX@RBC group was significantly better than the ultrasound combined with F-MSN@RBC group and the F-MSN-DOX@RBC group in inhibiting tumor growth（P < 0.05）.The body weight of mice in each group did not change significantly during the treatment period.（4）Compared with other groups,the ultrasound combined with F-MSN-DOX@RBC group significantly reduced tumor proliferation,increased apoptosis and decreased tumor angiogenesis.The ultrasound combined with F-MSN-DOX@RBC group significantly increased the ROS content in tumor tissues than the other groups.The results of H&E sections of main organs showed that all kinds of treatments had no obvious toxic and side effects on mice.Conclusion: Red blood cell membrane wrapping can reduce the phagocytosis of nanoparticles by the mononuclear macrophage system and increase the accumulation of nanoparticles in the tumor site.Ultrasound combined with F-MSN-DOX@RBC can promote drug release,reduce tumor angiogenesis and effectively inhibit tumor growth,without obvious toxic and side effects. |
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