超声实现离体肿瘤细胞磁性标记
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摘要
细胞的磁性标记是磁共振监测细胞迁移、进行细胞治疗的第一步。超顺磁性纳米氧化铁微粒不能有效跨越层层生物屏障进入胞质内,利用转染剂介导氧化铁标记细胞的方法需要转染剂和相当长的孵育时间,且期间氧化铁降解可能会破坏细胞膜结构、蛋白质和DNA。高频聚焦超声长期在疾病诊断和治疗中因其无创性和操作简单得到关注,而且,超声介导的基因传递使细胞转染率提高几十倍到几百倍,并且无明显毒性。本研究旨在开发安全方便的细胞磁性微粒快速装载的物理新方法-超声磁性微粒装载技术。通过自行设计搭建的超声装载装置,在无需转染剂辅助、细胞无需孵育的情况下,用实验室自制的超顺磁性纳米氧化铁微粒对肿瘤细胞进行了快速磁性标记,并对标记条件进行优化,当所用聚焦换能器共振频率为1.37MHz,当功率放大器输出为2W,所加SPIO浓度为410μg/mL,超声作用120s时,在保证细胞92.8%存活率的基础上,取得最高69.6%的标记率。同时基于微流理论对所用超声条件下微流产生的声辐射力和剪应力进行了估算,并结合细胞力学实验结果,对声孔效应机理进行了分析。具体工作和结论主要包括4个方面:
(1)制备了可用于细胞磁性标记的超顺磁纳米氧化铁微粒肢体。在探寻制备特定尺寸磁性微粒的反应条件和制备工艺的基础上,采用超声辅助的化学共沉淀法,分别合成制备了裸磁性纳米微粒和葡聚糖T-40包覆的超顺磁性纳米粒子。利用X-射线粉末衍射仪(XRD),透射电镜(TEM),原子力显微镜(AFM),样品磁性用振动样品磁强计(VSM)等对其主要物理性质和磁学性质进行了研究,证明制备出的磁性微粒为面心结构的反尖晶石相晶体,裸磁粒平均粒径22nm,磁饱和强度64.396emu/g:葡聚糖包覆的纳米磁微粒核心粒径约为12nm,复合磁性微粒磁饱和强度46.41emu/g。且均具有软磁性特性,具备作为磁共振对比剂的物理性能。
(2)设计搭建适于细胞磁性标记的超声装置。在对超声基因输送技术分析、综合的基础上,通过整合、改进,建立一套由稳压电源、功率放大器、单晶片聚焦探头、除汽水槽等组成的高频聚焦超声-磁性纳米微粒细胞装载实验系统。声场参数经水听器、阻抗分析仪和超声C扫描成像系统标定的。聚焦换能器为实验室自制,共振频率1.37MHz,当放大器输出电功率分别为1W、2W、3W时,超声波压强峰值经针式水听器检测为7.54×10~4,9.34×10~4,1.27×10~5Pa。
(3)首次利用自行设计的超声系统实现了小鼠离体H-22肝癌细胞和S180细胞的磁性标记。在加入不同浓度的SPIO情况下,用不同的剂量对H-22、S180肿瘤细胞悬液进行暴露,提取最佳标记参数:2W输出,持续作用120S,所加铁氧体浓度410μg/mL。用普鲁士蓝染色证实细胞内铁的存在:用台盼蓝拒染法确定标记后细胞的活性原子谱吸收分光光度等方法评估标记细胞内的铁含量。证明最佳传递参数下高频低强度超声有效地将SPIO微粒载入了S180和H-22细胞,且未见明显的细胞损伤与细胞毒性,转染率为69.6%。
(4)基于超声空化的微流理论,从理论上分析估算了稳态空化时微泡附近产生的作用于细胞膜上的剪应力,发现1W、2W、3W的电功率输出时,微泡附近剪应力值分别为568Pa,697Pa,783Pa。估算结果与单细胞微管吸吮技术测试结果量级相近。证实当功率放大器输出为1.37MHz,1-3W时,振荡微泡附近产生的剪应力可以使细胞发生弹性形变,细胞膜上产生可逆性穿孔,进而增大了膜的渗透性,促进细胞对磁性微粒的摄取。
本论文的主要创新点:
(1)在保持细胞活性的前提下,利用超声成功地提高了细胞膜的通透性,首次成功将纳米磁性微粒载入离体肿瘤细胞质内,并使细胞产生了足以满足磁共振成像所需的铁含量。预示一种新的细胞磁性标记技术的诞生。
(2)建立了实现离体肿瘤细胞磁性标记的超声基本参量,且发现利用超声进行细胞标记时,所需纳米磁微粒的最佳浓度为转染剂标记方法的8-16倍,为以后更广泛的研究奠定了基础。
(3)指出高频低强超声稳态空化时微泡附近的剪应力时细胞膜上产生微孔的主要原因。
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.
(1)制备了可用于细胞磁性标记的超顺磁纳米氧化铁微粒肢体。在探寻制备特定尺寸磁性微粒的反应条件和制备工艺的基础上,采用超声辅助的化学共沉淀法,分别合成制备了裸磁性纳米微粒和葡聚糖T-40包覆的超顺磁性纳米粒子。利用X-射线粉末衍射仪(XRD),透射电镜(TEM),原子力显微镜(AFM),样品磁性用振动样品磁强计(VSM)等对其主要物理性质和磁学性质进行了研究,证明制备出的磁性微粒为面心结构的反尖晶石相晶体,裸磁粒平均粒径22nm,磁饱和强度64.396emu/g:葡聚糖包覆的纳米磁微粒核心粒径约为12nm,复合磁性微粒磁饱和强度46.41emu/g。且均具有软磁性特性,具备作为磁共振对比剂的物理性能。
(2)设计搭建适于细胞磁性标记的超声装置。在对超声基因输送技术分析、综合的基础上,通过整合、改进,建立一套由稳压电源、功率放大器、单晶片聚焦探头、除汽水槽等组成的高频聚焦超声-磁性纳米微粒细胞装载实验系统。声场参数经水听器、阻抗分析仪和超声C扫描成像系统标定的。聚焦换能器为实验室自制,共振频率1.37MHz,当放大器输出电功率分别为1W、2W、3W时,超声波压强峰值经针式水听器检测为7.54×10~4,9.34×10~4,1.27×10~5Pa。
(3)首次利用自行设计的超声系统实现了小鼠离体H-22肝癌细胞和S180细胞的磁性标记。在加入不同浓度的SPIO情况下,用不同的剂量对H-22、S180肿瘤细胞悬液进行暴露,提取最佳标记参数:2W输出,持续作用120S,所加铁氧体浓度410μg/mL。用普鲁士蓝染色证实细胞内铁的存在:用台盼蓝拒染法确定标记后细胞的活性原子谱吸收分光光度等方法评估标记细胞内的铁含量。证明最佳传递参数下高频低强度超声有效地将SPIO微粒载入了S180和H-22细胞,且未见明显的细胞损伤与细胞毒性,转染率为69.6%。
(4)基于超声空化的微流理论,从理论上分析估算了稳态空化时微泡附近产生的作用于细胞膜上的剪应力,发现1W、2W、3W的电功率输出时,微泡附近剪应力值分别为568Pa,697Pa,783Pa。估算结果与单细胞微管吸吮技术测试结果量级相近。证实当功率放大器输出为1.37MHz,1-3W时,振荡微泡附近产生的剪应力可以使细胞发生弹性形变,细胞膜上产生可逆性穿孔,进而增大了膜的渗透性,促进细胞对磁性微粒的摄取。
本论文的主要创新点:
(1)在保持细胞活性的前提下,利用超声成功地提高了细胞膜的通透性,首次成功将纳米磁性微粒载入离体肿瘤细胞质内,并使细胞产生了足以满足磁共振成像所需的铁含量。预示一种新的细胞磁性标记技术的诞生。
(2)建立了实现离体肿瘤细胞磁性标记的超声基本参量,且发现利用超声进行细胞标记时,所需纳米磁微粒的最佳浓度为转染剂标记方法的8-16倍,为以后更广泛的研究奠定了基础。
(3)指出高频低强超声稳态空化时微泡附近的剪应力时细胞膜上产生微孔的主要原因。
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.
引文
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