Study On The Synthesis And The Biological Activity Of ¹³¹I-RGDyC-PAMAM

[Objective]Synthesis of 131I-RGDyC-PAMAM and research if it can target to tumor cells and the effect of radiation inhibition in vitro.Meanwhile imaging in a tumor-burdened mouse and monitoring the anti-tumor effect.Evaluating the biological activity of 131I-RGDyC-PAMAM nano probe in vivo of the animal?Lay the foundation of clinical trials for 131I-RGDyC-PAMAM.[Methodology]Statistical method: Both chart and data analysis using GraphPad Prism software.Using t test for means comparison and F test for multiple means comparisonThe preparation of nano probe 131I-RGDyC-PAMAM.Chemical synthesis: PEG(NHS-PEG-MAL)on maleic imide group and RGDyC thiol groups first reacted under the condition of weak acid bifurcation.Reaction products to be moved quickly to dissolve with PAMAM nanoparticles of boric acid buffer(pH 9.0).The purified product to light yellow solid is the product of the aqueous solution of lyophilizing RGDyC-PAMAM.Then using chloramine T method to label RGDyC-PAMAM with radionuclide 131 I and get 131I-RGDyC-PAMAM.Basic properties testing.Using NMR spectrometer to detect the physical manifestations.labeling rate measures by fast thin layer chromatography(ITLC).Detection of the stability.Measure water partition coefficient according to: log P = log(CTS of octanol/CTS of PBS).Cytology test.Cell uptake and elution experiment: The inhibitory effect of 131I-RGDyC-PAMAM on cells.Apoptosis experiment.The zoological experimental.Animal models of construction: Using a tumor-burdened nude mouse to establish animal model of A549 cells with axillary subcutaneous transplantation tumor cells suspension injection method.131I-RGDyC-PAMAM in tumor-burdened rat in vivo: Injected of 131I-RGDyC-PAMAM into a tumor-burdened mouse.A tumor-burdened rat SPECT imaging at different timing after injection of drugs;After injection,analysis the distribution of different organs of radioactive drugs at different timing.after injection of drugs,analyze the inhibit tumor growth in vivo and do weight analysis.immunohistochemically method to detect the heart,liver,spleen,lung,kidney,and internal necrosis of tumor tissue.[Results]The preparation of radioactive nano probe 131I-RGDyC-PAMAM.Synthesis of radioactive marker precursor RGDyC-PAMAM appears pale yellow,confirmed by 1 h NMR(400 MHZ,D2O),ultraviolet absorption spectrum confirmation as the target product,TEM image synthesis RGDyC-PEG-PAMAM as spherical nanometer complex.131 I labeled RGDyC-PAMAM rate at about 86%,specific activity calculation for mCi 0.08 / mg,131I-RGDyC-PAMAM have good stability in different solution(fetal bovine serum,PBS).The water partition coefficient is log P = 1.59 ± 0.09,show that the molecular probe has obvious hydrophilic.Cytology test.Cell uptake and elution experiment: compare to free 131 I,131I-RGDyC-PAMAM nano probe can combine with A549 cell specifically,and its stability is good.The cell inhibition and apoptosis in vitro experimental results show that the 131I-RGDyC-PAMAM has certain inhibitory effect on the A549 cells.In terms of promoting apoptosis,relative to free 131 I,131I-RGDyC-PAMAM is stronger,and is associated with drug dose,under the condition of high dose,131 I-RGDyC-PAMAM has certain effect on promoting apoptosis and destruction of A549.The zoological experimental.Separately injected 131I-RGDyC-PAMAM,free 131 I into different tumor-burdened mice and imaging.The result shows: with the same condition,compared to control group in 131 I,131I-RGDyC-PAMAM in tumor local has a longer retention time,and 131 I was quickly ruled out in vitro.Proved the combination of nanometer probe and tumor has the very good affinity,has the role of specific binding.The biological study in vivo distribution shows in A549 tumor nude mice,after the tumor local injections of 131I-RGDyC-PAMAM,the perturbation values of tumor is significantly higher than the blood,thyroid,heart,spleen,lung,stomach,intestinal,the normal organs,such as muscle and bone.the pharmacodynamics experiment shows: after the 131I-RGDyC-PAMAM injection-tumor intake of PAMAM drug treatment group was obviously inhibition,compared with the control group,with significant difference(P < 0.05).In addition,four groups of a tumor-burdened mice after injection content has no obvious losing weight status,showed no significant side effects.immunohistochemically results showed that 131I-RGDyC-PAMAM drug group has obvious cytotoxic effect to tumor cells,and no obvious side effects of normal organs.[Conclusion]Can successful get 131I-RGDyC-PAMAM and its label rate is higher,its stability is good.Subsequent cell in vitro experimental results showed that 131I-RGDyC-PAMAM on A549 cell has certain inhibitory effect of growth,promoting apoptosis destruction.SPECT imaging of tumor results showed that the 131I-RGDyC-PAMAM can be better gathered in a tumor-burdened mice tumor site and has good tumor targeting property.In subsequent tumor-burdened antitumor effect in mice showed 131I-RGDyC-PAMAM inhibit the growth of A549 tumor,and no obvious side effects to the tumor tissue.