Experimental Study On Magnetized Technique Of Doxorubicin-loaded Erythrocytes

Malignant serious threat to human survival and health, its treatment is surgical resection, the combined chemotherapy, radiotherapy and immune therapy. Chemotherapy because of non-selective, effect on tumor cells while normal cells also have strong side effects, leading to poor effect of chemotherapy. Preparation of the application of good biological compatibility and the anti-cancer drugs targeting vector, by positioning the role of the sustained-release, following the reduction of drug use can and can enhance local drug concentration, reaching to mitigate systemic toxicity is to enhance the development of tumor effect of chemotherapy direction. Red blood cells as anti-cancer drug doxorubicin (Doxorubicin, DOX) vector can reduce the dosage and improve efficacy, But its bad targeting. Idea of using double-red cell containing DOX and nano-Fe3O4 magnetic fluid (Fe3O4 nano-magnetic ferrofluid, MF), through the use of external magnetic field to resolve DOX erythrocyte targeting vector problem. Preparation of cell electroporation drug carrier with high efficiency rate effect. Set up electroporation Preparation method of red blood cell magnetization vector to explore the magnetic fluid magnetization vector Preparation of red blood cells and related optimal experimental conditions and the isolation and identification methods, observation and analysis of erythrocyte magnetization (that is, a single set MF erythrocyte, MF carrier red blood cells) and the magnetization of the magnetic phenomenon DOX carrier red blood cells (ie, dual-containing DOX and MF RBC) of magnetic susceptibility and drug parameters, in vitro biology and physics-related characteristics, compared dual-containing DOX and nano-ferrofluid..(1)Magnetization erythrocyte Experimental Study on Optimization of Preparation Methods:1.To Select the best experimental conditions: different size particles through the magnetic fluid dispersion and the nature of the selection of comparison, select a suitable size and easy to observe the magnetic fluid, set up electroporation Preparation Preparation of carrier erythrocytes MF; study MF concentration and magnetic response relations, RBC and the MF ratio, different voltage, pulse width, shock frequency, electric field strength, hot hatch on the experimental results of the comparison vector optimization of preparation conditions2, select the best separation conditions: To compare the different centrifugation speed and time, selection will be not loaded magnetic fluid of red blood cells removed from the reaction system of the optimal method3, selection of suitable identification methods: To compare the smear techniques, the end product of red blood cell magnetization at the ordinary microscope, fluorescence microscope, staining, electron microscopy and other methods, identification of red blood cell selection has been a suitable detection method magnetization.4, choose a suitable magnetic fluid: comparison of two different modifier dispersion of magnetic fluid and magnetic fluid properties, select the conditions in line with the magnetization of magnetic fluid red blood cell.5, an automatic cell analyzer measured whole blood, washed red blood cells and vector quantity, the average volume (MCV), mean hemoglobin content (MCHC) and other indicators, such as inspection carrier recovery of drug-loaded red blood cell parameters and physiological characteristics of Change. Comparison of magnetization of the storage stability of red blood cells.Results:1, with a fluorescent-labeled magnetic fluid fluidMAG-D and fluidMAG-DP, the diameter of 100nm, as the best experimental magnetization Materials, 500V, 100us, 10P (electric field strength for 2.5KV/cm) for electroporation the best experimental conditions. Red blood cell concentration of 109/ml, magnetic fluid concentration of 15ug/ml, the proportion of red blood cells and the MF for the 1:1, 30min hot hatch can increase the stability of red blood cells.2, through the low-speed centrifuge 800 Ways to 5 minutes to remove the separation package is not included in the magnetic fluid red blood cell.3, through improved quality control methods to observe the experimental end products direct smear, fluorescent microscope, transmission electron microscopy and other methods proved to have been magnetized red blood cells.4, selection with fluorescent markers and fluidMAG-D magnetization fluidMAG-DP as agent, to facilitate observation and quantitative measurement. 5, magnetized red blood cell recovery was (67.50±4.55)%, MCV decreased (4.00±1.06)%, hemoglobin decreased (18.12±3.27)%. Storage of red blood cell stability magnetization up to 5 days.(2)Preparation of DOX magnetization vector optimization erythrocyte experimental studyMethods1, comparison of different concentrations of DOX for electroporation were prepared magnetization vector red blood cell effects of DOX, DOX magnetization vector set up the preparation of red blood cells and the best way to optimize the experimental conditions.2, HPLC Determination of magnetization outside the DOX content of red blood cells, initially identified the package containing the rate of DOX. Automatic cell analyzer measured the number of red blood cell carrier, the average volume (MCV) and other indicators, DOX compared with an average hemoglobin concentration (MCHC), red blood cell carrier recovery and study the relationship between the physiological characteristics of Change. Comparison of magnetization of the storage stability of red blood cells.3, FCM analysis of magnetization DOX red blood cells mean fluorescence intensity in order to semi-quantitatively determine thethe susceptibility of red blood cells.Results:1 to simultaneously electroporation packet containing DOX, MF, red blood cell concentration of 109/ml, magnetic fluid concentration of 15ug/ml, DOX concentration of 2mg/ml, volume ratio of 1:1:1, after electroporation were incubated 30min, conditions for the production of this magnetization vector DOX the best way to red blood cells.2, when the red blood cells, DOX, MF between the proportion of 1:1:1, package contains the best. Through low-speed centrifugation separated from red blood cells did not pack the magnetic fluid loading. Direct smear, fluorescent microscope, transmission electron microscopy, flow cytometry proved detection methods such as red blood cells has been magnetized.3,DOX magnetization of red blood cells mean fluorescence intensity for 19±5.8, higher than that of single red blood cell containing DOX mean fluorescence intensity of 12± 4.6, the magnetization results are obvious. Conclusion:1 .To electroporation and the magnetic fluid suitable varieties Magnetized Erythrocyte Preparation feasible, set the parameters of good medicine, carrier erythrocyte no significant changes in physiological characteristics, in order to further explore the red blood cell tumor targeted therapy to provide theoretical and experimental basis.2, To simultaneously electroporation packet containing DOX, MF Preparation of carrier MF + DOX viable red blood cells, experimental conditions and a single red blood cell containing the MF are basically the same preparation conditions, red blood cell concentration of 109/ml, magnetic fluid concentration of 15ug/ml, DOX concentration 2mg/ml, red blood cells, DOX, MF volume three is the best when the ratio of 1:1:1 concentration ratio. Dual-containing red blood cells and red blood cell recovery rate of magnetization and physiological characteristics no significant change in red blood cells can be used as anticancer drug carrier for tumor targeting therapy.3, Dual-containing red blood cell MCV decreased (6.20±2.31)%, hemoglobin decreased (25.04±4.55)%, red blood cell recovery rate vector (59.16±6.15)%. DOX magnetization vector at PBS in the stability of red blood cells stored up to 4 days. Dual-containing red blood cell MCV, the stability of the storage time and magnetized red blood cells was no significant difference.(3)Comparison of single red blood cell red blood cells and magnetization vector DOX containing magnetized physical properties of the Experimental Study Preparation of comparative electroporation magnetization magnetization DOX double red blood cell and red blood cell containing the magnetic response, and determine the DOX Preparation of electroporation effects of red blood cell carrier, for further improving the preparation of electroporation magnetization vector of the magnetic susceptibility study of red blood cells and provide a theoretical basis for clinical blood transfusion Cancer targeted therapy to provide a new way of thinking and choices.Methods:1.4℃save regularly used extraction supernatant, HPLC determination of DOX content of the method, the production of red blood cells in vitro drug carrier DOX release curve, while observing the hemolysis, the comparison of the storage stability of red blood cells, the use of VSM Detect DOX vector magnetization hysteresis curve of red blood cells to determine its corresponding magnetic nature.2. By flow cytometry analysis, marked with yellow on the green fluorescence of the magnetic fluid, magnetic magnetization DOX double red blood cell red blood cells containing an analysis of mean fluorescence intensity comparison. DOX on the red blood cells to determine the effects of magnetizationResults:1.DOX vector in vitro characteristics of red blood cell mean corpuscular volume did not change significantly, reduced hemoglobin (25±7.66)%, before the two hours for the drug release (15.64±1.13)%, with time, the release rate increased, the storage stability of up to 4 days. VSM magnetization Detect erythrocyte DOX showed that the effect of RBC success of magnetic fluid, with a ferromagnetic material has a hysteresis phenomenon, changing the size and direction of magnetic field strength, the formation of a closed hysteresis curve. In different size and direction of the magnetic field, the magnetization vector erythrocytes have different magnetization, fully demonstrates that DOX magnetization vector has a certain red blood cells in vitro magnetic responsiveness.2.FCM results confirmed the same red blood cells were successfully magnetization. Dual-containing DOX magnetization of red blood cells mean fluorescence intensity than the single set of high magnetization erythrocytes (26.3±7.89%), single-containing red blood cells magnetization, double red blood cell containing magnetized DOX group and did not contain drugs and magnetization group differences were significant.Conclusion:DOX carrier red blood cells in vitro drug release of the release of drug with the carrier to reduce the content of reduced slow-release effect. Ordinary microscope observation of drug-loaded carrier erythrocytes with no obvious morphological changes of the former, observed under fluorescence microscope, the majority of red blood cells and the edge has weak or moderate intensity of the yellow green fluorescence. Note marked fluorescence magnetic fluid to enter the red blood cell, red blood cells better magnetization VSM magnetization vector determination of red blood cells proved to have obvious hysteresis phenomenon in vitro has a certain magnetic responsiveness. FCM detection of red blood cells with a fluorescent confirmed magnetization, double red blood cells containing DOX vector containing the magnetic susceptibility higher than that of single red blood cells, DOX Preparation of electroporation magnetization vector magnetic susceptibility of red blood cells with enhanced role, and its concentration was positively correlated.