Construction And Evaluation Of Erythrocyte-based Drug Delivery System

Red blood cells are an important part of the human blood.Many substances necessary for life activities in the human body require red blood cells to be transported.With the development of the times,cell preparations have gradually become the focus of research.This thesis builds a nano-drug delivery system based on red blood cells.The anticancer drug doxorubicin hydrochloride（DOX）was packed into the red blood cells,and the biotinylated avidin system was used to modify the biotinylated cyclic peptide RGD（c RGD）and indocyanine green（ICG）to Red blood cell surface.In order to achieve the functions of precise targeting,long blood circulation,fixed-point drug release,and phototherapy and chemotherapy.Optimization and characterization of preparation process of red blood cell drug delivery system.Through orthogonal test,the effect of time and temperature on the erythrocytes during drug loading during the process of low permeability and high permeability packaging was investigated to determine the optimal drug loading conditions.Under the optimal drug loading conditions,determine the optimal dosage of doxorubicin.Then the red blood cell delivery system is characterized.The results showed that the best preparation process of the erythrocyte drug delivery system was 4℃low osmotic analysis for 30 minutes,37℃high osmotic packaging for 30 minutes,and the dosage was 1 mg/m L.The fluorescence spectrophotometer exhibited the fluorescence characteristics of DOX and ICG under the excitation of 485 nm and808 nm.In the ultraviolet spectrophotometer,the same maximum absorption peak as ICG appears at 785 nm.The concentration of S element on the surface in the energy spectrum is6.067%.Confocal microscopy revealed that doxorubicin hydrochloride was encapsulated inside the red blood cells.In the study of physical and chemical properties,through methodological investigation,determine the feasible method of UPLC determination of DOX.The Malvin laser particle size analyzer,SEM and TEM were used to analyze the morphology,particle size and potential of the red blood cell delivery system.Annexin method was used to analyze the exposure of phosphatidylserine on the surface of erythrocytes,and to study the stability and release of erythrocyte drug delivery system.The results showed that the average drug content of the red blood cell delivery system was 0.9555 mg/m L,the average particle size was 3332 nm,the dispersion index was 0.102,and the potential value was-13.3 m V.Theexposure rate of phosphatidylserine analyzed by annexin method was 3.11%.There was no obvious change in the appearance and morphology of red blood cells within 7 days.The release rate was 76.13%within 5 days.With laser irradiation,the release rate reached 87%within 6 minutes,and the temperature reached 47.97℃in 150 s.In the cell experiment,MCF-7,U87,U251,SK-MEL-5 and H460 were administered to verify the targeting and cytotoxicity of the red blood cell delivery system.The results showed that the red blood cells covered the surface of U87,U251,SK-MEL-5 and H460 cells in a large amount,and the fluorescence intensity was higher than that of MCF-7 cells.The IC₅₀values of the five cells in the CCK-8 cytotoxicity experiment were MCF cells 81.64±0.78μg/m L,U87cells 51.31±0.72μg/m L,U251 cells 49.10±0.43μg/m L,SK-MEL-5 cells 52.94±0.98μg/m L,H460 cells 52.58±0.30μg/m L shows that under the targeted effect,the combination of phototherapy and chemotherapy is effective.In the in vivo targeting experiment,the mice were injected with unmodified drug-carrying red blood cells and modified drug-carrying red blood cells.The results showed that laser irradiation was given after injection and the temperature of the tumor site of the modified drug-carrying red blood cell group within 50 s It can reach 48.2℃,and the fluorescence intensity is higher than that of the non-modified group.In summary,this topic successfully prepared a red blood cell delivery system with surface modified c RGD and ICG and loaded with doxorubicin hydrochloride.The preparation process is simple and stable,and does not cause damage to red blood cells.The rapid release of the drug proves to have a good fixed-point release effect.Through a series of related experiments in vivo and in vitro,the near-infrared photothermal characteristics,high drug loading characteristics,and stability characteristics of the red blood cell drug delivery system were investigated.Compared with the single-path cancer treatment path,it is confirmed that the red blood cell delivery system can effectively,multiplely,and actively target the concentrated lesions.Through laser irradiation,phototherapy and chemotherapy show good synergistic inhibition of tumor cell growth and have cancer treatment the value of.This research lays the foundation for the collaborative treatment of cancer with active targeting,hyperthermia and chemotherapy,and provides new ideas for multi-channel cancer cooperative treatment.