CAJ | 학술논문

目的：探讨靶向乳腺癌膜相关抗原HAb18G/CD147双模态单光子发射计算机断层成像（SPECT）?MRI显像探针的构建方法，并研究其理化性质及体外生物特性。方法将超顺磁性氧化铁（SPIO）纳米颗粒与HAb18G/CD147的抗体片段HAb18F（ab'）2通过化学偶联法共价结合，得到复合物SPIO?HAb18F（ab'）2，采用Iodogen法对复合物进行125I标记，制得双模态靶向探针125I?SPIO?HAb18F （ab'）2。采用SPIO及125I?HAb18F（ab'）2作对照。对分子探针进行表征，包括记录纳米粒子形态、大小、水合粒径；测定SPIO和125I?SPIO?HAb18F（ab'）2溶液的T2横向弛豫率；测量125I?SPIO?HAb18F（ab'）2及125I?HAb18F（ab'）2的放射化学纯度及脂水分配系数；分析不同Fe2+浓度（1、5、10、20、40μg/ml）的125I?SPIO?HAb18F（ab'）2溶液对乳腺癌细胞系MDA?MB?231（实验组）及MDA?MB?468（对照组）存活率的影响。然后行靶向探针125I?SPIO?HAb18F（ab'）2的体外细胞结合实验，分别行MRI检查和SPECT检查。对与靶向探针125I?SPIO?HAb18F（ab'）2共同体外培养后的实验组、对照组及未作处理的MDA?MB?231细胞悬液（空白组）行MRI检查，测量其信号强度，计算MRI信号变化率。将实验组及对照组细胞悬液，在不同时间（20 min、40 min、1 h、2 h、4 h）经过处理，用γ计数仪测量每管放射性计数，分别计算其细胞结合率。采用单因素方差分析比较不同Fe2+浓度的125I?SPIO?HAb18F（ab'）2溶液对各组细胞增殖的抑制作用及SI值的差异。结果靶向乳腺癌膜相关抗原HAb18G/CD147的双模态SPECT?MRI探针构建成功。SPIO纳米颗粒核心粒径为（10.32±1.30）nm，SPIO及125I?SPIO?HAb18F（ab'）2水合粒径分别为44.80、52.64 nm。SPIO和125I?SPIO?HAb18F（ab'）2溶液的T2横向弛豫率分别为38.79、106.73 mM－1·s－1。125I?SPIO?HAb18F（ab'）2溶液和对照组125I?HAb18F（ab'）2溶液的标记率分别为41.90%和85.50%，二者的放射化学纯度均>95%，在PBS及鼠血清中均具有较好的稳定性。125I?SPIO?HAb18F（ab'）2与125I?HAb18F（ab'）2的脂水分配系数分别为－0.99±0.03和－1.49±0.08，表明二者均为水溶性物质。不同铁浓度（1、5、10、20、40μg/ml）的125I?SPIO?HAb18F（ab'）2与MDA?MB?231和MDA?MB?468细胞孵育72 h后吸光度值差异无统计学意义（F值分别为0.78、0.66，P值分别为0.58、0.66）。体外细胞结合实验：（1）MRI：实验组、对照组及未作处理组的SI值分别为1670±5、1930±8、2349±14，差异有统计学意义（F=4408.48，P=0.000）。实验组平均信号强度变化率为28.87%，对照组为17.78%。（2）SPECT：MDA?MB?231细胞可特异性摄取125I?SPIO?HAb18F（ab'）2，20 min时其结合率为（6.52±0.60）%，4 h时为（10.52±2.04）%。结论靶向乳腺癌膜相关抗原HAb18G/CD147的特异性双模态SPECT?MRI探针125I?SPIO?HAb18F（ab'）2制备成功，该探针具有良好的理化性质和体外生物特性。目的：探討靶嚮乳腺癌膜相關抗原HAb18G/CD147雙模態單光子髮射計算機斷層成像（SPECT）?MRI顯像探針的構建方法，併研究其理化性質及體外生物特性。方法將超順磁性氧化鐵（SPIO）納米顆粒與HAb18G/CD147的抗體片段HAb18F（ab'）2通過化學偶聯法共價結閤，得到複閤物SPIO?HAb18F（ab'）2，採用Iodogen法對複閤物進行125I標記，製得雙模態靶嚮探針125I?SPIO?HAb18F （ab'）2。採用SPIO及125I?HAb18F（ab'）2作對照。對分子探針進行錶徵，包括記錄納米粒子形態、大小、水閤粒徑；測定SPIO和125I?SPIO?HAb18F（ab'）2溶液的T2橫嚮弛豫率；測量125I?SPIO?HAb18F（ab'）2及125I?HAb18F（ab'）2的放射化學純度及脂水分配繫數；分析不同Fe2+濃度（1、5、10、20、40μg/ml）的125I?SPIO?HAb18F（ab'）2溶液對乳腺癌細胞繫MDA?MB?231（實驗組）及MDA?MB?468（對照組）存活率的影響。然後行靶嚮探針125I?SPIO?HAb18F（ab'）2的體外細胞結閤實驗，分彆行MRI檢查和SPECT檢查。對與靶嚮探針125I?SPIO?HAb18F（ab'）2共同體外培養後的實驗組、對照組及未作處理的MDA?MB?231細胞懸液（空白組）行MRI檢查，測量其信號彊度，計算MRI信號變化率。將實驗組及對照組細胞懸液，在不同時間（20 min、40 min、1 h、2 h、4 h）經過處理，用γ計數儀測量每管放射性計數，分彆計算其細胞結閤率。採用單因素方差分析比較不同Fe2+濃度的125I?SPIO?HAb18F（ab'）2溶液對各組細胞增殖的抑製作用及SI值的差異。結果靶嚮乳腺癌膜相關抗原HAb18G/CD147的雙模態SPECT?MRI探針構建成功。SPIO納米顆粒覈心粒徑為（10.32±1.30）nm，SPIO及125I?SPIO?HAb18F（ab'）2水閤粒徑分彆為44.80、52.64 nm。SPIO和125I?SPIO?HAb18F（ab'）2溶液的T2橫嚮弛豫率分彆為38.79、106.73 mM－1·s－1。125I?SPIO?HAb18F（ab'）2溶液和對照組125I?HAb18F（ab'）2溶液的標記率分彆為41.90%和85.50%，二者的放射化學純度均>95%，在PBS及鼠血清中均具有較好的穩定性。125I?SPIO?HAb18F（ab'）2與125I?HAb18F（ab'）2的脂水分配繫數分彆為－0.99±0.03和－1.49±0.08，錶明二者均為水溶性物質。不同鐵濃度（1、5、10、20、40μg/ml）的125I?SPIO?HAb18F（ab'）2與MDA?MB?231和MDA?MB?468細胞孵育72 h後吸光度值差異無統計學意義（F值分彆為0.78、0.66，P值分彆為0.58、0.66）。體外細胞結閤實驗：（1）MRI：實驗組、對照組及未作處理組的SI值分彆為1670±5、1930±8、2349±14，差異有統計學意義（F=4408.48，P=0.000）。實驗組平均信號彊度變化率為28.87%，對照組為17.78%。（2）SPECT：MDA?MB?231細胞可特異性攝取125I?SPIO?HAb18F（ab'）2，20 min時其結閤率為（6.52±0.60）%，4 h時為（10.52±2.04）%。結論靶嚮乳腺癌膜相關抗原HAb18G/CD147的特異性雙模態SPECT?MRI探針125I?SPIO?HAb18F（ab'）2製備成功，該探針具有良好的理化性質和體外生物特性。
목적：탐토파향유선암막상관항원HAb18G/CD147쌍모태단광자발사계산궤단층성상（SPECT）?MRI현상탐침적구건방법，병연구기이화성질급체외생물특성。방법장초순자성양화철（SPIO）납미과립여HAb18G/CD147적항체편단HAb18F（ab'）2통과화학우련법공개결합，득도복합물SPIO?HAb18F（ab'）2，채용Iodogen법대복합물진행125I표기，제득쌍모태파향탐침125I?SPIO?HAb18F （ab'）2。채용SPIO급125I?HAb18F（ab'）2작대조。대분자탐침진행표정，포괄기록납미입자형태、대소、수합립경；측정SPIO화125I?SPIO?HAb18F（ab'）2용액적T2횡향이예솔；측량125I?SPIO?HAb18F（ab'）2급125I?HAb18F（ab'）2적방사화학순도급지수분배계수；분석불동Fe2+농도（1、5、10、20、40μg/ml）적125I?SPIO?HAb18F（ab'）2용액대유선암세포계MDA?MB?231（실험조）급MDA?MB?468（대조조）존활솔적영향。연후행파향탐침125I?SPIO?HAb18F（ab'）2적체외세포결합실험，분별행MRI검사화SPECT검사。대여파향탐침125I?SPIO?HAb18F（ab'）2공동체외배양후적실험조、대조조급미작처리적MDA?MB?231세포현액（공백조）행MRI검사，측량기신호강도，계산MRI신호변화솔。장실험조급대조조세포현액，재불동시간（20 min、40 min、1 h、2 h、4 h）경과처리，용γ계수의측량매관방사성계수，분별계산기세포결합솔。채용단인소방차분석비교불동Fe2+농도적125I?SPIO?HAb18F（ab'）2용액대각조세포증식적억제작용급SI치적차이。결과파향유선암막상관항원HAb18G/CD147적쌍모태SPECT?MRI탐침구건성공。SPIO납미과립핵심립경위（10.32±1.30）nm，SPIO급125I?SPIO?HAb18F（ab'）2수합립경분별위44.80、52.64 nm。SPIO화125I?SPIO?HAb18F（ab'）2용액적T2횡향이예솔분별위38.79、106.73 mM－1·s－1。125I?SPIO?HAb18F（ab'）2용액화대조조125I?HAb18F（ab'）2용액적표기솔분별위41.90%화85.50%，이자적방사화학순도균>95%，재PBS급서혈청중균구유교호적은정성。125I?SPIO?HAb18F（ab'）2여125I?HAb18F（ab'）2적지수분배계수분별위－0.99±0.03화－1.49±0.08，표명이자균위수용성물질。불동철농도（1、5、10、20、40μg/ml）적125I?SPIO?HAb18F（ab'）2여MDA?MB?231화MDA?MB?468세포부육72 h후흡광도치차이무통계학의의（F치분별위0.78、0.66，P치분별위0.58、0.66）。체외세포결합실험：（1）MRI：실험조、대조조급미작처리조적SI치분별위1670±5、1930±8、2349±14，차이유통계학의의（F=4408.48，P=0.000）。실험조평균신호강도변화솔위28.87%，대조조위17.78%。（2）SPECT：MDA?MB?231세포가특이성섭취125I?SPIO?HAb18F（ab'）2，20 min시기결합솔위（6.52±0.60）%，4 h시위（10.52±2.04）%。결론파향유선암막상관항원HAb18G/CD147적특이성쌍모태SPECT?MRI탐침125I?SPIO?HAb18F（ab'）2제비성공，해탐침구유량호적이화성질화체외생물특성。
Objective To prepare dual?modality single?photon emission computed tomography (SPECT)?MRI molecular nanoprobes targeting HAb18G/CD147 expressed on breast cancer cell membranes and investigate the physicochemical and biological properties in vitro. Methods Superparamagnetic iron oxide nanoparticles (SPIOs) were prepared by one?pot reaction method as described. The single?chain antibody fragments HAb18F(ab')2 were conjugated to SPIOs via chemical method and then labeled with 125I using Iodogen method. The final 125I?SPIO?HAbF18(ab')2 nanoprobes were purified. SPIOs or 125I?HAb18F(ab')2 were used as control. We carried preliminary evaluation on their physicochemical properties and biological characteristics in vitro: transmission electron microscope (TEM) and dynamic light scattering (DLS) were used to measure these nanoparticle sizes and the hydrodynamic diameters. The MRI T2 transverse relaxation efficiency of these nanoprobes at different Fe2+concentrations were measured with 1.5 T clinical MR scanner. The 125I?SPIO?HAb18F(ab')2 and 125I?HAb18F(ab')2 radiochemical purity were measured by thin layer chromatography and the radio chemical yield was calculated. We also conducted stability tests in vitro and octanol/water partition coefficient experiments. Two breast tumor cell lines, MDA?MB?231 (HAb18G?overexpressing cells,experimental group) and MDA?MB?468 (control), were used for assessment of cells viability at different Fe2 + concentrations (1, 5, 10, 20, 40 μg/ml) by methyl thiazolyl tetrazolium assay. Specific binding experiments in vitro included two parts:magnetic resonance imaging and radionuclide tests, the above?mentioned breast cancer cell lines were incubated with 125I?SPIO?HAb18F(ab')2 nanoprobes respectively and took MDA?MB?231 cells which were not treated as blank group. First comparing the MR signal intensity differences among experimental group, the control group and blank group, then calculated the rate of MRI signal changes;Two breast tumor cell lines, MDA?MB?231 and MDA?MB?468 were incubated with 125I?SPIO?HAb18F(ab')2 nanoprobes too, then measured radioactivity counting byγcounter at different time and calculated the cell binding rates, and did statistical analysis by using one?way ANOVA. Results The SPIOs were fairly homogeneous with an average core size of (10.32±1.30) nm;the SPIO and 125I?SPIO?HAb18F(ab')2 hydrodynamic diameter of 44.80 and 52.64 nm, and MRI scanning showed that the transverse relaxation efficiency of SPIO and 125I?SPIO?HAb18F(ab')2 were 38.79 and 106.73 mM-1 · s-1, respectively. The radio chemical yield of 125I?SPIO?HAbF18(ab')2 and 125I?HAb18F(ab')2 were 41.90% and 85.50%, respectively. The radio chemical yield of the two groups were >95%, suggesting well stability in vitro. The lipo?hydro partition coefficient values were -0.99 ± 0.03 and-1.49 ± 0.08, respectively, which demonstrated that they were both water?soluble substances. Different Fe2+concentrations (1,5,10,20,40μg/ml) of 125I?SPIO?HAb18F(ab')2 on breast cancer cell lines MDA?MB?231 and MDA?MB?468 showed no significant inhibition of cell proliferation (F values were 0.78, 0.66; P values were 0.58, 0.66). The cell?specific binding experiment showed: MRI signal intensity values on experimental group, the control group and the blank group were (1 670 ± 5), (1 930 ± 8), (2 349 ± 14), respectively, significant differences existed among these groups (F=4 408.48,P=0.000), the rate of signal intensity change of experimental group and the control group were 28.87%,17.78%. SPECT:MDA?MB?231 could uptake 125I?SPIO?HAb18F(ab')2, the cell binding rates were (6.52 ± 0.60)% and (10.52 ± 2.04)% in 20 min and 4 h, respectively.Conclusions Our results suggested that the dual?modality SPECT?MRI nanoprobes 125I?SPIO?HAb18F(ab')2 were prepared successfully with good physicochemical properties and biological characteristics in vitro. These dual?modality molecular imaging nano?probes may have potential to improvearly detection and diagnosis of HAb18G/CD147?expressing cancers and to facilitate the development of HAb18G/CD147?directed interventions.