Ultrasound Tumor Cells In Vitro Magnetic Marker

One of the most critical steps for cellular MR imaging clinical applications of cell therapy is the method of intracellular magnetic labeling cell labeling.Traditionally,researchers use transfection agents or antibody/receptor as a vector and prolonged incubation to transfer magnetic nanoparticles into cells.Sonoporation is a technique that permits the transfer of drugs,including genes,into cells. This technique is designed to enhance cell permeability through the use of ultrasound.Its use became common because of its perceived safety,noninvasiveness,and low cost.Further, ultrasound-mediated gene delivery has been enhanced by severafold or hundredfold,with no significant toxicity.Development of nonviral magnetic particles transfer methods would be a valuable addition to the celltherapy armamentarinm,particularly for localized targeting of specific tumor or transplant stem cells.In this study,sonoporation was investigated as an alternative method to achieve instant endosomal labeling with the magnetic particles,which prepared by means of classical coprecipitation in dextran T40 solution in our laboratory,without the need for adjunct agents or initiating cell cultures.H-22 cell labeling efficiency close to 69.6%when ultrasound expose duration 120s at 2W electric power output and the final concentration of added SPIO is 410μg/mL.While the steady shear stress in the vicinity of gaseous microbubble in cells suspension irradiated by continuous ultrasonic field typical of those used in diagnostic practice have been calculated.The main works conducted in my thesis are outlined below.(1) To improve a method of synthesizing dextran-coated superparamagnetic iron oxide nanoparticles and can be used as magnetic markers for Sarcoma 180 and H-22 tumor cells. Magnetite particles were prepared by chemical precipitation method.The dextran coated supermagnetic iron oxide nanoparticles were synthesised by the co-precipitation method.The characters of the particles were investigated by atomic force microscope,transmission electron microscopy,x-ray diffraction analysis,magnetic hysteresis loops and fourier transform infrared spectrometer.The synthesized black power show firrraction pattern typical of single phase spinel oxides.The core size and saturation magnetization Ms of the dextran-coated samples is about 12nm and 46.41emu/g respectively.The mean diameter and Ms of naked particle is 45nm and 64.396emu/g,respectively.The shape of the particle is cubic and the dimension size is (200-300nm)×(400-600nm)×(50-70 nm),no magnetic hysteresis loop was observed.(2) Design a rotating tube sonoporation system to load magnetic particles into cells.Current sonoporation instruments are far from perfect,more needs to been done in improving their performance.We have made improvements and developed a new type of sonoporation system-high frequency at a low intensity levels.The experimental acoustic setup consisted of a 1.37MHz focused single-element transducer(made in our labory) mounded in a water tank(4℃).The system has been tested with respect to its admittance,electroacoustic efficiency and distribution of sound pressure. The frequency used is similar to that used in diagnostic ultrasound,so cell damage is not expected to occur.(3) It has been shown experimentally in cell suspensions that sonporation could be used to deliver the supermagnetic nanoparticles into S180 and H-22 cells in vitro.The superparamagnetic iron oxide(SPIO) particles that prepared in our labory have been used to label these cells.In this study,we investigate the cell labeling efficiencies of two different SPIO nanoparticles.To observe the effects of various physical parameters such as ultrasound exposure duration,acoustic density and the ferum oxide concentration,on both labeling efficiency and cell viability.The sonoporation were performed in 1-3.0W electrical power output from the amplifier and the transducer(diameter is 25 mm) with a resonant frequency of 1.37 MHz in a continuous wave mode.Cellular labeling efficiency is evaluated by Prussian blue staining for iron assessment.the iron content of the labeled cells was assessed by atomic emission spectrometer.The viability of labeled cells is evaluated by trypan blue exclusion test.The results showed that when SPIO was added at 410μg/ml,focused ultrasound sonication at a frequency of 1.37MHz and power from amplifer of 2W,H-22 cells were efficiently labeled at 120s exposure time the labeling efficiency was about 69.6%.Prussian blue staining confirmed iron uptake and showed numerous blue-stained iron particles in the cytoplasm, while more than 92%labeled cells remained viable.The result show ultrasound might be a promising technique for in vitro labeling of the tumor cells.(4) Rapidly oscillating microbubbles genertates a fluid flow over the cell surface.This microstreaming is probably responsible for the disruption of cell membrane by tearing the lipid bilayer.The steady shear stress in the vicinity of gaseous microbubble in cells suspension irradiated by continuous ultrasonic field typical of those used in diagnostic practice have been calculated from a solution of the equation of motion bubble.The values of the shear stresses caused by continuous exposure to ultrasound were found to lie with the range in which biological effects have been reported,it also consistent with the measurement by micropipet technique of HCC tumor cells.The main contributions of this thesis are as follows:(1) This paper developed an experimental technique that enabled us to uptake superparammagnetic particles into tumor cells in vitro during exposure to ultrasound.We report for the first time that a focused sonicator,designed to disrupt cells and homogenize solutions,can be applied to effectively transfer superparamagnetic iron oxide particles into tumor cells in absence of microbubble.(2) The iron oxide concentration used for ultrasound labeling is about 8-to 16-fold higher compared to transfection agents based labeling methods.This fingding should prove useful in further studies to improve the efficiency of magnetic labeling.(3) Numerical calculations have shown that shear stress associated with microstreaming surrounding encapsulated bubbles may be large enough to generate sonoporation at 1-3W of 1.37MHz ultrasound.