Optimize Prescription Process,Characterize Of Anti-MECA79-PBCA-USPIO Nanoparticles And To Differentiate Benign And Malignant Lymph Node In MRI

Malignant tumor is one of the most common diseases that threaten human health, the stage of the tumor diagnosis has implications for clinical program development and patient prognosis, local and distant metastasis of lymph nodes is the main pathway of epithelial malignant route of metastasis, how to properly judge the malignancy patient’s lymph node metastasis is the current focus of the study.Routine clinical use as ultrasound, CT, MR, and PET/CT for lymph nodes judgment has some limitations, which may cause misdiagnosed lymph node metastasis, therefore, to find a new method urgent.MR has a higher soft tissue resolution that can detected lymph nodes well, but Gd-DTPA contrast enhanced imaging similar to CT enhanced in clinical routine, its specificity and sensitivity still poor, ultrasmall superparamagnetic iron oxide contrast agent has been studied now.Currently the USPIO nanoparticles has applied to the clinical abroad, obviously improve the diagnosis of metastatic lymph nodes.The passive uptake of the contrast agent in the body when injected into the body, it could circulate to the lymph nodes’s medullary sinus and phagocytosis by macrophage, resulting in a negative enhancing. Due to metastatic lymph nodes does not have a macrophage, therefore when inject USPIO contrast agent to patient the signal of the lymph nodes does not change or only local signal falls down may be prompted for metastatic lymph nodes. The cells of tumor transfer into the lymph nodes firstly in lymph nodes subcapsular sinus then may be deposited there and hyperplasia and then substitute normal lymph node. while the cortex and cortical hyperplasia in reactive hyperplastic lymph nodes often lead to thickening of the lymph node cortex, USPIO swallow by macrophage that lymph nodes display low signal in lymph node center. Metastatic lymph nodes general performance the localization high signal. L-selectin Which core antigen is MECA-79or called peripheral lymph node vascular addressin, has express in high vascular endothelium, labyrinth in the lymph nodes. MECA-79monoclonal antibody similar to L-selectin ligand that specifically to high endothelial venules, labyrinth in lymph nodes. L-selectin can be used as carrier for superparamagnetic nanopartilces to targeted lymph node. Anti-MECA-79-PBCA-USPIO have been synthesized and successfully targeted lymph nodes nanoparticles in previous work, the purpose of this study is to optimize prescription process and characterize anti-MECA-79-PBCA-USPIO nanoparticles and to differentiate benign and malignant lymph nodes in lymph node model.Purpose1、To synthesize anti-MECA-79-PBCA-USPIO nanoparticles and optimize its prescription process, characterize the property of the nanoparticles and evaluate its toxic effects on the cells in vitro;2、preliminary evaluation of anti-MECA-79-PBCA-USPIO differentiate benign and malignant lymph node in animal models.Method1、Optimize prescription process, characterize of Anti-MECA-79-PBCA-USPIO nanoparticles and evaluate cytotoxicity in vitro1、A three-step synthesis of anti-MECA-79-PBCA-USPIOAt first, ultrasmall superparamagnetic iron oxide nanoparticles be synthesized by coprecipitation, then use the BCA monomer wrap USPIO formate the PBCA-USPIO nanoparticles and prepared anti-MECA-79-PBCA-USPIO by surface modification.1.1Use coprecipitation reaction under alkaline conditions with lauric Synthesis lauric stable USPIO nanoparticles.1.2The synthesis process using orthogonal analysis to the optimize process of the main influence factors of PBCA-USPIO, dextran-70concentration, the BCA concentration, pH value on the synthesis of PBCA-USPIO。1.3It will be obtained under optimized conditions the PBCA-USPIO alkalizate1h Then nanoparticles surface generate carboxyl amino form stable amide bond, and through the EDC role with streptavidin-biotin, the streptavidin and biotinylated the PBCA-USPIO solution adding biotinylated anti-MECA-79monoclonal antibody, anti-MECA-79monoclonal antibody binds by biotin-avidin system so we obtain anti-MECA-79-PBCA-USPIO nanoparticles.2、Characterizate of PBCA-USPIO, anti-MECA-79-PBCA-USPIO nanoparticle2.1The size and dispersity of PBCA-USPIO, anti-MECA-79-PBCA-USPIO nanoparticles be test under Malvern-3000HS laser particle size analyzer Take the two nanoparticle suspension1ml dilution then placed in the test on laser light scattering particle size analyzer, the selected wavelength of the laser light source is633.0nm, the test temperature is25.00+0.05℃, the sample scattered light at90°range varied within different angles. The optical signal with256-channel, high-speed digital correlator for processing.2.2The morphology of PBCA-USPIO and anti-MECA-79-PBCA-USPIO was observed under the transmission electron microscope. Few drops PBCA-USPIO,anti-MECA-79-PBCA-USPIO nanoparticles suspension diluted into copper grids, after drying with phosphotungstic acid negative staining, standing fully dried, observed under the transmission electron microscope.2.3X-ray diffraction analysis the PBCA-USPIO and anti MECA-79-PBCA-USPIO nanoparticlesTwo kinds of nanoparticles after freeze-drying, taking a small amount placed in a grind to powder, placed D/Max-ⅢAX-ray powder diffractometer (polycrystalline) detection of the resulting X-ray diagram with Fe3O4standard chart compared to determine the crystal structure comprising.2.4HH-15vibrating magnetometer be uesed to measure the characteristics of the anti-MECA-79-PBCA-USPIOA small quantity of anti-MECA-79-PBCA-USPIO nanoparticles be vacuum drying, then placed in the HH-15vibrating sample magnetometer (VSM) to determinate the change in intensity of the magnetic saturation of nanoparticles, and draw the hysteresis curve.2.5T2relaxvity of anti MECA-79-PBCA-USPIO nanoparticlesTake a small quantity of anti-MECA-79-PBCA-SPIO nanoparticles suspension be diluted with water in the iron concentration of0.04,0.06,0.08,0.10,0.12,0.14and0.16mmol/L, then put in to seven test tubes and scanned with MR using T2map sequence.Then average T2values of each samples were measured on post-processed,T2relaxvity was calculated3、In vitro evaluate of the anti-MECA-79-PBCA-USPIO cytotoxicityAfter the liver cells (LO2) recovery in culture, Then the cell were seeded in96-well and continued incubating with full media containing uncoated SPIO and anti-MECA-79-PBCA-USPIO with different concentrations respectively. After24h incubation,the viability of the LO2cells was determined by MTT assay2、Differentiate benign and malignant lymph node by anti-MECA-79-PBCA-USPIO in animal models preliminary1、Animal(1) Experimental animals:24healthy adult New Zealand White Rabbits weighted at2.0-2.5kg were used, male or female, were divided into2groups randomly:The reactive hyperplasia group (n=12):the yolk emulsion was used to establish lymph node reactive hyperplasia model; The metastatic lymph node group (n=12):VX2tumor was used to establish lymph node metastases model(2) The development of model.1, The reactive hyperplasia lymph nodes group:New Zealand rabbits anesthesia firstly, then inject0.5ml yolk solution (saline: yolk=1:1) at its side hind limb rectus femoris area, after a repeated injection3days later, the model was successfully created after3days. the swollen lymph node in the popliteal fossa can touch.then reactive hyperplasia lymph node model be builded.2, The metastasis lymph node group model:New Zealand white rabbits were anesthetized then inject approximately0.5ml prepared VX2sarcoma tissue suspension tumors at its side hind limb rectus femoris area,2-3weeks later that can touched popliteal lymph node, the metastasis lymph node model be builded.2、MR imaging1. Pre-scan The model animals were anesthetized, choose the small animal rabbit coil. All the MR imaging was performed with sequences and parameters:T1WI (TR/TE:633/20ms, NEX:2), FsT2WI (TR/TE=5000/65ms, NEX:2), T2WI (TR/TE=6860/62ms, NEX:2). T2*WI (TR/TE=450/12ms, FA=20°, NEX:2). Coronal and sagittal scan, FOV=160mm,slice thickness=1.5nm, spacing=0mm. 2.Enhanced (1) Reactive hyperplasia lymph node group:the reactive hyperplasia lymph node groups of New Zealand white rabbits were divided into the SPIO enhanced group I (n=6), and anti-MECA-79-PBCA-USPIO group enhanced group Ⅱ (n=6).randomly. After the pre-scan the Group I the inject a dose of20umol Fe SPIO and the Group II injection a dose of20umol Fe anti-MECA-79-PBCA-USPIO for the12hours then scan as the same scan parameters;The metastasis lymph node group model:the metastasis lymph node group of New Zealand white rabbits were divided into the SPIO enhanced group Ⅰ (n=6) and anti-MECA-79-PBCA-USPIO group enhanced group Ⅱ (n=6).randomly. After the pre-scan the Group I the inject a dose of20umol Fe SPIO and the Group II injection a dose of20umol Fe anti-MECA-79-PBCA-USPIO for the12hours then scan as the same scan parameters;3、MRI imaging analysis3.1The measurement of lymph node sizeMeasuring the size of lymph nodes in the MRI post-processing workstation on T1WI, The long and short diameter was measured presented as mean±standard deviation,3.2Pre-scan and enhanced be measure the signal to noise ratio of the lymph nodesThe lymph node signal were measured in the different sequences on the pre-scan, the popliteal lymph nodes in the enhanced signal strength, calculated in each group the lymph nodes of the signal-to-noise ratio (Signal to noise ratio SNR), Evaluation SPIO and anti MECA-79-PBCA-USPIO in differentiating benign from malignant lymph nodes.3.2Histopathological examinationAll experimental animals in the enhanced MRI scan is completed, sacrificed the animal by air in the case of anesthesia, carefully expose popliteal lymph nodes and then remove and measure the short diameter of the lymph node, and calculating the number of lymph nodes. Then with10%neutral formalin soaked, fixed, paraffin-embedded, HE staining and Prussian blue staining to observe the popliteal fossa lymph node reactive hyperplasia and tumor infiltration, histopathology and MRI image in contrast.Result1. orthogonal experimental factorial analysis and analysis results showed that the optimum synthesis process for the anti-MECA-79-PBCA-USPIO nanoparticles was dextran-70concentration15%,the BCA concentration3%, pH value6.0.2. Malvern laser particle size analyzer showed that PBCA-USPIO and anti-MECA-79-PBCA-USPIO’s particle size and polydispersity were90nm and0.20,96nm and0.34nm.3. Transmission electron microscope showed that the PBCA-USPIO, anti-MECA-79-PBCA-USPIO nanoparticles with uniform shape and size, spherical, shell-nuclear structure, the measurement of PBCA-USPIO, anti-MECA-79-PBCA-USPIO particle size approximately25±5nm,30±5nm.4. The characteristic peaks of PBCA-USPIO and anti-MECA-79-PBCA-USPIO nanoparticles in X-ray diffraction the same to the standards X-ray of Fe3O4crystal Spectrum, It proved that the Fe3O4polymorphs of PBCA-USPIO and anti-MECA-79-PBCA-USPIO nanoparticles has no changed by parcels and reaction.5. Anti-MECA-79-PBCA-USPIO has superparamagnetic.Derived from the hysteresis curves, the anti-MECA-79-PBCA-USPIO quality saturation magnetization were22emu/g Fe.6. Anti-MECA-79-PBCA-USPIO nanoparticles relaxation rate results show that anti-MECA-79-PBCA-USPIO T2relaxation rate is0.159×106mol-1s-1.7.1n vitro cytotoxicity experiments show that the anti-MECA-79-PBCA-USPIO has low toxicity to cells, when it reaches1000ug/ml, the cell survival rate is about70%, indicating that it has good biocompatibility and safety.8. MRI pre-scan with different sequences was difficult to identify reactive hyperplasia lymp node from malignant metastatic lymph nodes;9. The prepared anti-MECA79-PBCA-SPIO could targeted lymph nodes similar to SPIO and can be well distributed in the lymph nodes;10. Anti-MECA79-PBCA-SPIO can diagnosis the reactive hyperplasia lymph nodes from metastatic lymph nodes with the correct diagnosis rate of about91.7%, which higher than the SPIO group of diagnostic accuracy rate about83.3%.Conclusions1, With the result of the orthogonal analysis, it confirm the best conditions for the preparation of PBCA-USPIO nanoparticles, and to make sure that the the PBCA-USPIO synthesis of the nanoparticle size, the PBCA-USPIO particle size synthetic uniform, circular shape or round, surface finished, liquid dispersion of the electron microscope particle size approximately25-30nm, water particle size of about90nm, X-Ray diffraction show Fe3O4crystal structure.2,By streptavidin-biotin system successfully Synthesis anti-MECA-79-PBCA-USPIO, it’s superparamagnetic T2magnetization saturation about22emu/g. The X-Ray diffraction show that Fe3O4crystals did not change after wrapped synthesis, anti-MECA-79-USPIO-PBCA fluid particle size is96nm, the T2relaxivity is0.159×106mol-1s-1.3,The cytotoxicity effect of anti-MECA-79-PBCA-USPIO nanoparticles is small,show it is safe and biocompatible.4. MRI pre-scan with different sequences was difficult to identify reactive hyperplasia lymp node from malignant metastatic lymph nodes;5. Anti-MECA79-PBCA-SPIO can diagnosis reactive hyperplasia lymph nodes from metastatic lymph nodes with the correct diagnosis rate of about91.7%, which higher than the SPIO group of diagnostic accuracy rate about83.3%.